Characterization of bovine herpesviruses isolated from six sheep and four goats by restriction endonuclease analysis and radioimmunoprecipitation

Viral DNA from 10 herpesviruses isolated from 6 sheep and 4 goats were examined by restriction endonuclease analysis with respect to their relatedness to one another; to bovine herpesvirus type 6 (BHV-6), also known as caprine herpesvirus; and to 2 strains of bovine herpesvirus type 1 (BHV-1), known as infectious bovine rhinotracheitis virus (IBRV) and infectious pustular vulvovaginitis virus (IPVV). Viral proteins from the isolates were examined by radioimmunoprecipitation with anti-BHV-1/IBRV gnotobiotic calf (bovine) serum, anti-BHV-1/IBRV bovine hyperimmune serum, and anti-BHV-6 rabbit serum to evaluate their antigenic relatedness to each other. The goat isolates were obtained from animals with various disease conditions including respiratory tract disorders, vulvovaginitis, and wart-like lesions on the eyelid. The other isolates were from domestic sheep and came from aborted fetuses or from sheep with fatal pneumonia or proliferative lesions around lips and nose. All of the goats and 4 of the sheep from which the viral isolates were obtained had comingled with cattle. Purified DNA from each of the 10 field isolates and from BHV-1/IBRV, BHV-1/IPVV, and BHV-6 caprine herpesvirus was cleaved with restriction endonuclease Pst I. Five of 6 sheep isolates and 3 of 4 goat isolates yielded unique restriction patterns, ie, patterns that differed from each other by one or more bands. Sheep isolate DNA patterns were different from goat isolate patterns, and all restriction endonuclease analysis patterns were similar to the pattern for BHV-1/IBRV, but different from that for BHV-1/IPVV or for BHV-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesviruses types 1 and 5

Twelve Brazilian isolates and three reference strains of bovine herpesviruses (BHVs) were subjected to restriction endonuclease analysis (REA) and monoclonal antibody (MAb) analysis. Viral DNA was cleaved with BamHI, BstEII, EcoRI, HindIII and PstI. The monoclonal antibody panel allowed the differentiation between types 1 and 5 viruses, while REA with BstEII and HindIII showed the distinction between BHV-1 and -5 subtypes. Typical 1.1 and 1.2a patterns were observed with two isolates from respiratory disease. An isolate from semen of a clinically healthy bull displayed 1.2b profile, whereas another displayed a clear 5a pattern, which was never reported before. Seven out of nine Brazilian type 5 (BHV-5) isolates displayed REA patterns similar to the Australian BHV-5 strain N569 (BHV-5a), and differing from the Argentinean A663 strain (BHV-5b) virus. Another two BHV-5 isolates, which displayed an unusual MAb pattern of reactivity, showed a BstEII profile different from both reference strains of BHV-5. These two viruses were considered BHV-5 "non-a/non-b" subtype.     

Isolation and differentiation of bovine mammary gland progenitor cell populations

OBJECTIVE: To isolate bovine mammary gland cells with stem cell characteristics. SAMPLE POPULATION: Monolayers of bovine mammary gland cells. PROCEDURE: Mammary gland cell populations were separated by use of selected media supplements. Phenotypic characteristics were examined via light and transmission electron microscopy. Cellular expression of casein and connexin 43 was identified immunohistochemically. A scrape-loading and dye transfer assay was used to examine the mammary gland cell populations for homogenous gap junctional intercellular communication (GJIC). RESULTS: Subpopulations of mammary gland cells grown in vitro are classified on the basis of their distinct morphologic features and ability to communicate via gap junctions. Ultrastructurally, 2 morphologically distinct cell types were classified as type I and II cells. Type I cells were small light undiffertiated cells and large light undifferentiated cells that were deficient in functional gap junctions (as is characteristic of stem cells). Type II cells included large light differentiated cells and terminally differentiated cells; GJIC was functional in type II cells. Type II cells had cytoplasmic expression of connexin 43, whereas, type I cells did not. All cells expressed casein. CONCLUSIONS AND CLINICAL RELEVANCE: Subpopulations of bovine mammary gland cells with stem cell characteristics were identified. Phenotypic differences are observed among type I bovine mammary gland cells with stem cell characteristics. Gap junctional intercellular communication may be necessary for the differentiation of stem cells. Characterization of bovine mammary gland stem cells and their progeny may provide a new tool with which to study mammary gland health.     

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

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

Seroprevalence of infectious bovine rhinotracheitis and bovine viral diarrhea virus type 1 and type 2 in non-vaccinated cattle herds in the Pacific Region of Central Costa Rica

The objectives of this cross-sectional study were to estimate the seroprevalence of infectious bovine rhinotracheitis (IBR, BHV-1) and bovine viral diarrhea virus (BVDV) in a population of non-vaccinated, double purpose, dairy and beef herds in the Pacific Region of Central Costa Rica. Blood samples were collected from a total of 496 animals from 35 herds. Sera were tested for antibodies against BHV-1(IBR) and BVDV types 1 and 2 using serum neutralization test. The average number of animals tested in each herd for each of the viruses was 14. Overall individual seroprevalence was 48%, 27%, and 19% for IBR, BVDV type 1, and BVDV type 2, respectively. Median within-herd seroprevalence for IBR, BVDV type 1 and type 2 were 43%, 27%, and 24%, respectively.     

Molecular cloning of DNA from a bovine herpesvirus 1 strain isolated in Hungary

Molecular cloning of the HindIII fragments of bovine herpesvirus 1 (BHV-1) strain HB144, isolated from infectious bovine rhinotracheitis (IBR) in Hungary, and of an infectious pustular vulvovaginitis (IPV) reference strain (K22) is reported. So far 52% of the IBR viral genome and 28% of the IPV viral genome have been cloned. The analysis of differences between the strains is currently in progress.     

Molecular diagnosis of alcelaphine herpesvirus (malignant catarrhal fever) infections by nested amplification of viral DNA in bovine blood buffy coat specimens

A fragment of alcelaphine herpesvirus-1 (AHV-1; malignant catarrhal fever) DNA was subcloned into pUC 18 and sequenced. The subclone hybridized strongly to AHV-1 DNA, weakly to alcelaphine herpesvirus-2 (AHV-2) DNA, and not at all to DNA from bovine herpesvirus-1 (BHV-1; infectious bovine rhinotracheitis [IBR] virus), bovine herpesvirus-2 (BHV-2; bovine herpes mamillitis [BHM] virus), and bovine herpesvirus-4 (BHV-4; isolate DN599). A 2-stage (nested) polymerase chain reaction (PCR) diagnostic test was devised based on a portion of the subcloned AHV-1 DNA sequence. First and second stage amplified AHV-1 DNA targets were 487 and 172 base pairs (bp) in length, respectively. Unique Pvu II and Stu I restriction endonuclease cleavage sites confirmed the identity of amplified AHV-1 DNA. Five AHV-1 and 2 AHV-2 isolates were identically and specifically PCR positive. BHV-1, BHV-2, and BHV-4 viruses were negative by the same procedure. As little as 0.01 TCID50 AHV-1 was detected using the nested amplification procedure. Simple methods of buffy coat isolation from bovine blood were employed to prepare specimens for PCR. An AHV-1-infected calf was PCR positive from 3 to 77 days postinoculation (PI), with rising seroconversion first noted 14 days PI. The AHV-1 DNA sequence was 62% homologous to a portion of the Epstein-Barr virus genome. The nested PCR procedure may improve the viral diagnosis of clinical and subclinical alcelaphine herpesvirus infections.     

Fatal, generalized bovine herpesvirus type-1 infection associated with a modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine administered to neonatal calves.

Generalized bovine herpesvirus 1 (BHV-1) infection was diagnosed in six Salers calves from the same herd. The calves had received an intramuscular injection of modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine between birth and three days of age. The purpose of this study was to determine if the outbreak was associated with the vaccine strain of BHV-1. Analysis of epidemiological data and BHV-1 DNA for restriction fragment length polymorphism was undertaken. Multifocal necrosis in multiple organs was observed on pathological examination, and the presence of BHV-1 in tissues was confirmed by immunohistochemistry. Forty-three calves (aged birth to thirty days) were vaccinated over an 11-day interval. The 10 deaths recorded for vaccinated calves were clustered over a subsequent 14-day interval. Mortality in calves vaccinated between birth and three days of age was significantly higher than in nonvaccinated calves (chi-square test; p < or = 0.025), and this mortality was characterized by a greater age at death and duration of illness for vaccinated calves (t test; p < or = 0.001). The patterns of the restriction fragments, generated by six restriction endonucleases, of BHV-1 isolated from a necropsied calf and from the vaccine were identical, and different from that of a laboratory strain of BHV-1 (P8-2). These findings support the conclusion that newborn calves were susceptible to an intramuscularly injected vaccine strain of BHV-1, and that administration of an intramuscular modified-live infectious bovine rhinotracheitis parainfluenza-3 vaccine to neonatal calves may not be an innocuous procedure.     

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

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

DNA restriction enzyme analysis of a bovine herpesvirus 1 strain isolated from encephalitis in Hungary

The DNA of a bovine herpesvirus 1 (BHV-1) strain isolated from calf encephalitis in Hungary was analysed with restriction enzymes. The cleavage pattern of the encephalitis strain Na/67 differed from those of all the other Hungarian BHV-1 isolates investigated so far. The EcoRI and HindIII cleavage patterns of virus strain Na/67 were found to be similar to the patterns of two other encephalitis strains (N569 and A663 from Australia and Argentina, respectively) characterized earlier. Strain Na/67 is the first isolate in Europe which showed the restriction enzyme pattern of BHV-1.3 previously supposed to be characteristic of encephalitis strains.     

传染性牛鼻气管炎病毒分离鉴定及特性分析

传染性牛鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)是牛的重要传染病病原,除能引起呼吸道疾病外,还可引起结膜炎、流产、脑炎和全身性感染。本试验从广东某牛场疑似IBRV感染牛中采取鼻拭子样品,用PCR和荧光PCR方法,初步诊断病牛感染IBRV。用MDBK细胞对初诊阳性病料进行病毒分离和鉴定,分离出1株传染性牛鼻气管炎病毒,命名为GD0109。细胞感染试验表明,该株病毒可以使MDBK细胞产生典型的圆缩、呈葡萄样聚集以及空洞等细胞病变。将GD0109与强毒的ATCCVR-2112TM株感染细胞裂解液分别接种MDBK细胞并盲传5代,对5代、10代、15代的病毒分别进行TCID50测定及中和试验,结果表明分离株和参考株VR-2112TM相似,提示该分离株为强毒株,并且具有良好的传代稳定性,为进一步疫苗生产打下良好的基础。     

Persistent infection of bovine herpesvirus type 4 in bovine endothelial cell cultures

Herpesviruses can establish a persistent infection in the cells and tissues of their natural hosts and thus may produce diseases due to cytolytic infections. We have isolated a herpesvirus from a bovine vascular endothelial cell culture after continuous subculturing. Typical cytopathic changes were observed in bovine endothelial cell cultures 2 days after inoculation of the virus. The virus had an icosahedral nucleocapsid of 100-150 nm in diameter and an envelope. The sequences of some DNA fragments of the virus were highly homologous to those of the bovine herpesvirus type 4 (BHV-4) strains. The DNA restriction maps of the virus and the reference strains of BHV-4, DN 599 and Movar 33/63 were very similar but not identical. Therefore, the newly isolated virus has been designated Taiwan strain. The presence of BHV-4 DNA in apparently normal bovine endothelial cell cultures was shown by Southern blot hybridization with the BamHI fragment of the newly isolated BHV-4 and was further confirmed by digestion of the DNA with BamHI plus AccI. In conclusion, we have demonstrated that BHV-4 persisted in the bovine endothelial cell cultures and continuous subcultures could lead to the production of infectious viral particles.     

A serological comparison of some animal herpesviruses

Bovine herpesvirus 1 (BHV-1) isolates (Cooper-type strain 4975 and Oxford) were compared in neutralization tests with the bovine herpesvirus 4 (BHV-4) isolate (85/16 TV) and the herpesviruses of red deer (D2839/1) and goats (E/CH). Hyperimmune antiserum was prepared in rabbits against the plaque-selected viruses and endpoint and kinetic neutralization test were made. BHV-4 was clearly different from the other four viruses. The closely-related BHV-1 strains were also related in these tests to the red deer herpesvirus. The Oxford strain seemed rather closer antigenically than the Cooper-type strain to the red deer herpesvirus. Antiserum to the caprine herpesvirus failed to neutralize either BHV-1 strain or red deer virus, but antiserum to the Cooper-type and red deer herpesviruses did neutralize caprine virus to a limited extent.     

Comparison of the herpesviruses of cattle by DNA restriction endonuclease analysis and serologic analysis

Reference strains and field isolates of herpesviruses recovered from cattle in the United States were compared by restriction endonuclease (RE) analysis and the indirect fluorescent antibody test. As a result of these comparisons, 5 major biotypes of bovine herpesvirus (BHV) were defined. These types were (i) infectious bovine rhinotracheitis virus (BHV-1), (ii) bovine herpes mammillitis virus (BHV-2), (iii) malignant catarrhal fever (MCF) virus (herpesvirus alcelaphinae), (iv) the group of slow-growth isolates represented by the prototype strain Movar 33/63 (bovine cytomegalovirus candidate), and (v) the syncytia-forming Pennsylvania 47 strain. Bovine herpesvirus-1 and BHV-2 did not cross-react serologically with any other type of BHV tested. A low, but consistent level of serologic cross-reactivity was detected among MCF virus, the Movar group, and Pennsylvania 47. Several nonsyncytial, slow-growth strains, which were recovered from dissimilar clinical syndromes and were serologically related to Movar 33/63, exhibited similar DNA RE cleavage patterns, confirming their identity as members of a single type. There was no isolate from American domestic cattle similar to the African MCF virus, which has been sporadically isolated from exotic ruminants in the United States. The African MCF virus isolated during a MCF epizootic in a United States zoo exhibited some DNA RE cleavage differences in comparison with the MCF virus world prototype strain WC 11, indicating that strain diversity exists within this biotype.     

A retrospective analysis of the infectious bovine rhinotracheitis (bovine herpes virus-1) surveillance program in Norway using Monte Carlo simulation models.

Serological surveillance for antibodies against bovine herpes virus type I (BHV-1) which causes infectious bovine rhinotracheitis and infectious pustular vulvovaginitis has been carried out since 1992 in Norway. Since 1993 (when a single infected herd was detected) all bulk-milk and pooled-serum samples have been negative for BHV-1 antibodies. This paper describes the use of Monte Carlo simulation models for the analysis and interpretation of the results of the surveillance and provides support for the contention that the Norwegian cattle population is not infected by BHV-1.     

Biology of bovine herpesviruses

Cattle are the natural host of herpesviruses. Since now four different bovine viruses have been described as members of the family Herpesviridae. The prototype of the bovine herpesviruses, Bovine Herpesvirus type 1 (BHV-1), is the causative agent of infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV) and infectious balanoposthitis (IBP). The related BHV-5 is an exotic neurovirulent agent and like BHV-1 a member of the genus Varicellovirus, within the subfamily Alphaherpesvirinae. BHV-2, also an alphaherpesvirus but grouped into the genus Simplexvirus is the causative agent of bovine herpes mammilitis and pseudolumpy skin disease. In contrast, BHV-4, a member of the subfamily Gammaherpesvirinae, is not known to cause any disease. Beside bovine herpesviruses there are few other herpesviruses which can infect cattle. Infections of cattle with these herpesviruses have either clinical or diagnostic importance, based on a close antigenic relationship to BHV-1 of some ruminant herpesviruses. This article deals with the molecular virology of bovine herpesviruses and the pathogenesis of bovine herpesvirus infections and provides an overview over herpesviruses that can infect cattle.     

Experimental infection of rabbits with bovine herpesvirus-4: acute and persistent infection

A strain of bovine herpesvirus-4 (BHV-4) isolated from bovine cases of mammary pustular dermatitis was used for experimental infection of rabbits. The strain is serologically indistinguishable from the group prototype Movar 33/63 and from the American isolate DN599. Groups of rabbits were inoculated by various routes. Intravaginal and conjunctival inoculations resulted in vulvovaginitis and conjunctivitis, respectively, and in shedding of virus. The rabbits seroconverted for the virus, with high titers of antibodies (indirect fluorescent antibody test) that persisted throughout the experiment. Treatment with dexamethasone, beyond the acute infection, did not produce recrudescence of disease or shedding of the virus. Rabbits were killed at various times, from 3 to 6 months post-infection, and the virus was recovered from explant cultures of spleen and by cocultivation of spleen cells with bovine lung cells. These results demonstrate the usefulness of the rabbit as a model for studying the pathogenesis of BHV-4 infection in cattle.     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in argentina

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.     

A comparison of polypeptides and restriction endonuclease sites of BHV-1 isolates and identification of IPV virus in Japan

Bovine herpesvirus 1 (BHV-1) isolates from respiratory tract and from vagina of bovine in Japan were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the DNA restriction endonuclease cleavage pattern, and compared with European BHV-1 strains. Both protein profile and DNA cleavaged pattern of BHV-1 isolates from respiratory tract were the same as those of European infectious bovine rhinotracheitis (IBR) virus, whereas the protein profile and DNA cleavage patterns of one isolate (M1) from vagina was the same as those of the European infectious pustular vulvovaginitis (IPV) virus. The facts indicate that IPV virus has existed in Japan.     

Construction and characterization of a glycoprotein E gene-deleted bovine herpesvirus type 1 recombinant

OBJECTIVE: To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1). PROCEDURE: The BHV-1 gEgene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus, gEdelta3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests. RESULTS AND CONCLUSIONS: The gEdelta3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (beta-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEdelta3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves.