Comparative study of two strains of Bovid herpesvirus-4

Two strains of Bovid herepesvirus-4 (BHV-4), i.e. the prototype strain DN-599, obtained from a steer suffering of a respiratory disease, and the strain 85/BH 16TV, originated from a cow with vulvovaginitis, were compared in studies which included restriction endonuclease analysis, experimental infection and reciprocal cross protection tests. The restriction endonuclease analysis revealed that the resultant DNA patterns of the isolates were generally similar with only a difference in one fragment. The two strains were capable of causing respiratory tract infection in calves, even if they displayed a different level of virulence: the strain 85/BH 16TV being the most virulent while the strain DN-599 the least. The two viral strains were mutually protective in that the calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. Finally, both viral strains failed to evoke the production of neutralizing antibody in the experimental calves.     

Bovine abortion associated with mixed Movar 33/63 type herpesvirus and bovine viral diarrhea virus infection.

A herpesvirus and cytopathogenic bovine viral diarrhea virus were isolated from a bovine fetus aborted in the 6th month of gestation. The herpesvirus was serologically indistinguishable from bovine herpesviruses DN-599 and Movar 33/63.     

Genetic relatedness of disease-associated field isolates of bovid herpesvirus type 4

Eight field isolates of bovid herpesvirus type 4 (BHV-4) were examined by restriction analysis and Southern blot hybridization with respect to their relatedness to one another and to the BHV-4 prototype strain DN-599. Isolates were obtained from cattle exhibiting a range of disease states including abortion, pneumonia, enteritis, metritis, and vaginal blisters. Initial growth studies of all 9 viruses were performed and revealed that the overall rate of virus growth was slow when compared with that of other herpesviruses. Infection with each virus also resulted in the formation of large fused cells, which in addition to the slow growth rate, indicated that the isolates were of the cytomegalovirus type. Further studies to characterize and compare the various BHV-4 isolates were undertaken by obtaining cell-free virus from infected cell populations. Viral isolates were purified and used as a source of BHV-4 DNA. Purified DNA, representing each of the 8 field isolates and the prototype strain DN-599, were each cleaved with 3 restriction enzymes and were separated by agarose-gel electrophoresis, and the resultant fragment patterns were compared. In general, genomic fragments of the field isolates corresponded to those generated by cleavage of DN-599 DNA, with the exception of the abortion-associated isolate 83-3572. Additional minor differences were also seen between DN-599 DNA and DNA from the other field isolates, but the overall restriction patterns were similar. To confirm that all isolates were members of the BHV-4 type, hybridization studies were performed using DN-599.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of some biologic properties of Bovid herpesvirus-4.

This article summarizes the results of a study on several strains of Bovid herpesvirus-4 (BHV-4), isolated from cattle. The study had several objectives, namely, to verify (a) the disease-causing potential of the virus, (b) the possibility by BHV-4 to induce a latent infection in the natural host and (c) the entity of the relationships among strains of the virus isolated from different disease syndromes. The following data were obtained: (1) All strains tested were able to replicate in experimentally infected calves; however, only one strain (85/BH 16TV) caused an overt systemic disease. (2) The nervous system as well as the lymphoid structures appeared to be the target organs for replication of the virus. (3) BHV-4, like other herpesviruses, was able to establish latent infection in cattle. (4) When two strains of the virus, isolated from cattle affected by different disease syndromes, i.e. respiratory disease (strain DN-599) or vulvovaginitis (strain 85/BH 16TV), respectively, they resulted to be closely related to each other. In particular, they revealed a similar DNA pattern and both strains were able to cause respiratory disease in calves. Moreover, the two viral strains were mutually protective in that calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. (5) All BHV-4 strains tested generally failed to evoke a significant production of neutralizing antibody in the experimental calves.     

Immunoferritin and immune electron microscopic study of bovine herpesvirus strain DN-599.

Intracellular antigens of strain DN-599 bovine herpesvirus were detected in the cytoplasm and the nucleus of infected bovine embryonic kidney cells by the indirect immunoferritin (IF) technique. Specific tagging was observed in viral envelope and capsids. Aggregates of viral particles heavily coated with antibody were seen by immune electron microscopy (IEM).     

Propagation and quantitation of animal herpesviruses in eight cell culture systems

A comparative study was carried out to determine the relative sensitivities of eight different cell culture systems to six different herpesviruses of animals. The cells used were: OFL (ovine fetal lung), ML (mink lung), FK (ferret kidney), PTK-2 (potoroo kidney), TEK (turkey embryo kidney), ED (equine dermal), BT (bovine turbinate), and PK15 (porcine kidney). The viruses tested were: PRV (pseudorabies) of swine, CPHV (caprine herpesvirus), IBRV (infectious bovine rhinotracheitis virus), DN-599 strain of bovine herpesvirus type 4, EHV-1 (equine herpesvirus), and CHV (canine herpesvirus). On the basis of virus titers obtained and the time of appearance of CPE (cytopathic effects), ML cells were found to be the most useful because of their sensitivity to all six viruses tested. BT and OFL cells were also found to be highly sensitive to all viruses with the exception of CHV.     

Experimental infection of calves with strains of Bovid herpesvirus-4

Fourteen calves were inoculated intranasally (i.n.) with the viral isolates as follows: 5 with 85/BH 16TV, 1 with 85/BH 17TV, 1 with 85/BH 18TV, 2 with 85/BH 231TN and 5 with 85/BH 232TN. Strain 85/BH 16TV was the only one which caused overt respiratory-like disease in all inoculated calves. Onset of the disease was observed after 7-8 days of incubation and was characterized by fever, depression, nasal discharge and coughing. Virus was isolated from the nasal swabbings of calves obtained from post-infection day (PID) 2-10. The other viral strains did not cause any sign of disease although virus was isolated regularly from the nasal swabbings of the inoculated calves. Virus was recovered from central nervous system tissues of calves that were infected with 85/BH 16TV or 85/BH 232TN strains and were killed on PID 4 or 8. Virus was also isolated from other tissues, such as lymph node, nasal mucosa (PID 8), or lung (PID 4). It was speculated that the nervous system could be one of the target areas of the virus of the naturally occurring infection by BHV-4. This might indicate a possible role of the nervous system (site of latency?) in the pathogenesis of BHV-4 as is the case in certain herpesviral infections of man and the lower animals.     

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.     

Bovine herpesvirus 4 isolates: a comparison of three major glycoproteins.

Twenty-four Belgian field isolates of bovine herpesvirus 4 (BHV-4), together with four reference strains were compared by radio-immunoprecipitation and western blotting using a polyvalent antiserum and monoclonal antibodies raised against major glycoproteins. Most of these strains showed the same protein profile as the European reference strain Movar 33/63. For two strains the molecular weight of gp 6, p (gp 10/gp 17) and gp 10 were the same as those of the American reference strain DN 599. No relationship could be established between the protein profiles and origin of the isolates or with the restriction patterns. This study provides a view of the molecular weight variations of the major BHV-4 glycoproteins among field isolates.     

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)     

Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer)

Eleven virus isolations were made from the blood of 45 free living healthy African buffaloes by long term cocultivation of their leucocytes with bovine thymus or spleen cells. The isolates were indistinguishable from each other or from herpesviruses isolated from a severely ill buffalo calf and from a dead buffalo. These viruses possessed the characteristics of the bovine herpesvirus-3 (BHV-3) group and were indistinguishable by serology and restriction endonuclease analysis from the BHV-3 type strains Movar 33/63 and DN599. There was a 93.6 per cent prevalence of indirect immunofluorescent antibody to BHV-3 in the sera of 94 buffaloes in the sample population. No clinical signs or viraemia were detected in five cattle inoculated with 10(8.7) log10 TCID50 of the isolate from the sick buffalo calf. Two of three cattle hyperimmunised with this virus resisted challenge with malignant catarrhal fever herpesvirus, which proved fatal for the other immunised animal and for three control cattle.     

Experimental inoculation of cattle with bovine herpesvirus-4: evidence for a lymphoid-associated persistent infection

A strain of bovine herpesvirus-4 isolated from cows with mammary pustular dermatitis was used for experimental inoculation of cattle. This strain is serologically indistinguishable from the group prototype Movar 33/63 and from strain DN-599. Seronegative cattle were inoculated IV or by simultaneous intranasal, IV, intramammary (via teat channel), and intradermal inoculations. All inoculated cattle seroconverted. Clinical signs of disease or lesions were not evident, except for a dermal lesion corresponding with one intradermal inoculation site. Virus was recovered from the dermal lesion and was excreted in the milk for 17 days. Virus was recovered from esophagopharyngeal fluid at 9 and 13 days after inoculation. At different times of euthanasia (2 to 14 months after inoculation), virus was recovered from cocultures with bovine lung cells and/or explant cultures of lymph nodes, spleen, tonsils, and, in one case, kidney. In 2 animals, the virus was recovered repeatedly during 1 year from peripheral blood leukocytes by cocultivation with bovine lung cells. The number of infectious leukocytes, as determined by infectious center assay, ranged from less than 1 to 6 infectious cells/10(7) leukocytes.     

Molecular characterisation of a field strain of bubaline herpesvirus isolated from buffaloes (Bubalus bubalis) after pharmacological reactivation

Two healthy buffaloes (Bubalus bubalis) in a herd which had not been vaccinated against infectious bovine rhinotracheitis (IBR), were selected for their seropositivity for anti-bovine herpesvirus type 1 (BoHV-1) glycoprotein E antibodies, and injected intramuscularly daily with dexamethasone for five consecutive days (day 1 to day 5) to reactivate any latent herpesvirus. Blood samples and nasal and vaginal swabs were collected daily from day 5 to day 15 from each buffalo for virological examination. All the vaginal swabs and blood samples were negative, but 13 of the 22 nasal swabs were positive; a cytopathic effect was observed in primary cultures of bovine fetal lung cells, and the viral isolates were identified as a herpesvirus by PCR. The viral strains were characterised by the sequence analysis of the genes coding for glycoproteins D and B, and the gene sequences were then used for phylogenetic analysis. The isolates from both buffaloes appeared identical at the level of the two genes, and were more closely related to bovine herpesvirus type 5 than to BoHV-1.     

Characterization of herpesviruses isolated from lactating dairy cows with mammary pustular dermatitis.

Herpesviruses serologically indistinguishable from the DN599 strain of bovine herpesvirus were isolated from 2 separate enzootics of mammary pustular dermatitis in lactating Holstein-Friesian cows.     

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.     

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.     

Pathogenicity of a caprine herpesvirus.

A herpesvirus isolated from neonatal Angora kids (Capra hircus) with a relatively severe generalized infection was shown to be infective for adult goats as well as for kids. However, the virus lacked pathogenicity for either lambs or calves. Although a nonreciprocating serologic overlap exists between the caprine virus and the antibody to bovine herpesvirus (BHV-1), results of cross immunity tests in calves indicated that the 2 viruses are immunologically distinct. On the basis of these findings, the caprine virus seems to be a specific pathogen of goats. Accordingly, the designation of Herpesvirus caprae or caprine herpesvirus 1 seems taxonomically appropriate.     

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.     

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.     

Susceptibility of bovine macrophage and tracheal-ring cultures to bovine viruses.

Cultures of macrophages initiated from peripheral blood monocytes and organ cultures of tracheal rings were tested for their susceptibility to bovine viruses. With several notable exceptions, viruses cytopathogenic for bovine embryonic lung cultures were cytopathogenic for macrophages. Although cowpox virus replicated in macrophages, pseudocowpox did not, and although pseudorabies virus replicated within macrophages, infectious bovine rhinotracheitis and DN-599 herpesviruses did not. Bluetongue virus established an interesting relationship with macrophages. Whereas bluetongue virus was initially cytopathogenic for macrophages, it lost its cytopathogenicity on repeated passage, although it was capable of continued replication in macrophages. When subsequently passaged onto bovine embryonic lung cultures, it regained its cytopathogenicity. Parainfluenza-3, bovine viral diarrhea, and infectious bovine rhinotracheitis viruses readily destroyed ciliary activity in tracheal-ring cultures, as contrasted with the inability of bovine respiratory syncytial virus to destroy ciliary activity, even though bovine respiratory syncytial virus was able to replicate within ciliated epithelial cells of tracheal rings.