Generation of a nucleic acid probe specific for the alcelaphine herpesvirus 1 and its use for the detection of malignant catarrhal fever virus DNA in blue wildebeest calves (Connochaetes taurinus).

Two WC 11 specific DNA fragments, 3 kb and 2 kb in size, respectively, were cloned and evaluated as probes for their use in diagnostic and epidemiological investigations of malignant catarrhal fever (MCF). Field specimens including blood, ocular fluid, nasal mucus and urine of blue wildebeest (Connochaetes taurinus) calves in the Kruger National Park, South Africa, were tested and found positive for excretion of MCF-virus by slot blot hybridization. In 2 cases MCF-virus DNA was detected in the urine of the calves. No hybridization was observed with DNA from other bovine herpesviruses.

PCR detection of the sheep-associated agent of malignant catarrhal fever.

From a genomic library previously constructed from a lymphoblastoid cell line (LCL) propagated from a bovine case of sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine herpesvirus-2 (OHV-2), several OHV-2 clones were identified and characterised by hybridisation using probes from the unique region of the Alcelaphine herpesvirus-1 (AVH-1) genome. Nucleotide sequence from one clone was generated and the predicted amino acid sequence was found to contain regions of homology with the 140 and 160 kDa tegument proteins of Epstein-Barr virus and herpesvirus saimiri respectively. Oligonucleotide primers were constructed and a polymerase chain reaction (PCR) test was developed for the detection of OHV-2 viral DNA. Amplified product was identified by restriction with RsaI and BmyI. The primers were highly specific for OHV-2 DNA with a limit of detection of 6.4 pg of genomic DNA derived from the parent LCL. This was estimated to correspond to one diploid bovine cell. The PCR was successfully applied to detect OHV-2 DNA in peripheral blood leucocytes (pbl) from clinical cases of SA-MCF and normal sheep.

Studies concerning etiology of sheep-associated malignant catarrhal fever in Europe.

The etiological agent of sheep-associated malignant catarrhal fever (MCF) in Europe has not been isolated directly from sheep. The occurrence of antibodies against the African bovine herpesvirus (BHV-3, WC 11) in cattle and sheep was examined using recently modified indirect immunofluorescence test (IIFT) and neutralization test (NT) methods. Studies revealed that sheep and cattle sera in Europe were free from neutralizing antibodies of BHV-3. The IIFT could not establish the presence of antibodies to African BHV-3 in cattle but revealed that about 22.4% of sheep sera reacted to it. Apart from the well known ovine herpesvirus (BHV-5), occurrence of another herpesvirus in Europe had been expected. This virus is not identical with the WC 11 strain, but it is in antigenic relationship to it. We had for the first time substantial serological evidence to the effect that sheep-associated MCF in Europe is a herpesvirus related to the African strain.

The detection of Alcelaphine herpesvirus-1 DNA by in situ hybridization of tissues from rabbits affected with malignant catarrhal fever.

Tissue sections and cultured lymphocytes from rabbits clinically affected following experimental infection with Alcelaphine herpesvirus-1 (AHV-1) were assessed for the presence of viral DNA by in situ hybridization with the cloned major HindII repeat sequence of this virus. Small numbers of virus-infected cells were consistently detected only in submandibular lymph nodes, while other tissues showed no evidence of viral DNA. Virus titration in culture suggested that there were higher titres of virus in the lymph nodes, spleen and lung of infected animals than in the kidney or peripheral blood lymphocytes and confirmed the low level of virus in these animals. Substantially more viral DNA was detected by in situ hybridization in lymphocytes following at least 24 h of culture, suggesting that viral replication is normally repressed by the host.

Failure of sheep to respond to repeated inoculations with an alcelaphine herpesvirus-1-like virus, isolated from a case of malignant catarrhal fever in American cattle.

The replication of an alcelaphine herpesvirus-1-like virus (707K), isolated from a clinical case of malignant catarrhal fever in American cattle, was studied in sheep. Viraemia was not observed in any of the six sheep repeatedly inoculated with the 707K virus or in four steers susceptible to malignant catarrhal fever which were housed together with these sheep for one year. None of the four steers seroconverted and only two of the six inoculated sheep showed a negligible and short-lived seroconversion. The inability of the sheep to seroconvert adequately after repeated inoculations with the 707K virus, and the failure to recover the agent from them suggests that this agent does not replicate in sheep.

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.

[The etiology of "malignant catarrhal fever" originating in sheep: serological findings in cattle and sheep with ruminant gamma herpesviruses]. / Zur Atiologie des schaforiginären "bösartigen Katarrhalfiebers": serologische Befunde bei Rindern und Schafen mit Wiederkäuer-Gammaherpesviren.

In the case of the wildebeest-derived form of malignant catarrhal fever (WD-MCF) alcelaphine herpesvirus 1 (AlcHV1) is well established as the cause. However, the etiology of the form of the disease circumstantially associated with sheep (SA-MCF) remains equivocal. A serological relationship has been proposed to exist between the unidentified sheep-associated agent causing SA-MCF and AlcHV1 causing WD-MCF. We attempted to confirm this hypothesis. Using an indirect ELISA we found 94 of 100 cattle (94%) and 80 of 90 sheep (89%) to display antibody to AlcHV1. Nine of 10 cattle with SA-MCF showed elevated antibody titers to AlcHV1 when compared with most other animals. However, these findings were of limited diagnostic value, since similar results were also obtained with sera from healthy cattle. When assayed in the presence of antigens of bovine herpesvirus 4 (BHV4), a virus related to AlcHV1 yet without confirmed pathogenicity, 99 cattle sera (99%) and 85 sheep sera (95%) were observed to react specifically with this virus. Together, the results indicated that most domestic cattle and sheep were infected with viruses that are related to AlcHV1 and BHV4. An etiologically meaningful interpretation of the serologic findings does not seem possible at present.

The malignant catarrhal fever complex.

Malignant catarrhal fever (MCF) is defined as a clinicopathological syndrome caused by related herpesviruses and acquired from persistently infected wildebeest and sheep. There is convincing epidemiologic and virologic evidence that Alcelaphine herpesvirus 1 (AHV1) causes the wildebeest-derived disease (WD-MCF). Present knowledge suggests that a herpesvirus related to AHV1 may be associated with some cases of the non-wildebeest-associated disease (NWA-MCF). However, this virus possibly represents a passenger virus not related with the ultimate cause of the disease. Moreover, evidence for the role played by sheep as the reservoir for the agent of NWA-MCF is not convincing and awaits confirmation.

Derivation of a DNA clone corresponding to the viral agent of sheep-associated malignant catarrhal fever.

Malignant catarrhal fever is a fatal lymphoproliferative and degenerative disease of ruminants. One causative agent is the gammaherpesvirus alcelaphine herpesvirus 1 (AHV-1), which produces no disease in its natural host, the wildebeest (Connochaetes species). Epidemiological evidence implicates sheep as the carrier of a similar virus. However, attempts to culture this virus from sheep or from animals affected with sheep-associated malignant catarrhal fever (SA-MCF) have failed. Lymphoblastoid cells have been propagated from cattle, deer and rabbits with SA-MCF. Although these cells show no evidence of viral particles or antigens, hybridisation experiments now show that they contain DNA sequences homologous to those of AHV-1. A genomic library was constructed from one of these lymphoblastoid cell lines and a clone identified which hybridised to cloned AHV-1 DNA. The authors believe that this clone contains part of the SA-MCF viral genome, and that the SA-MCF virus and AHV-1 are closely related gammaherpesviruses.

Evidence that the sheep associated form of malignant catarrhal fever is caused by a herpes virus.

Out of a cow, which was infected with the sheep form of malignant catarrhal fever (MCF), blood and spleen samples were inoculated into rabbits. From the spleen cells of an infected rabbit, which were cocultivated with bovine embryonic gingiva cells, a herpesvirus could be isolated. The isolate showed crossreactions with reference sera against the strain WC 11 (wildebeest form) in the SNT. An immunosuppressed heifer, which was infected with the isolate, contracted typical clinical symptoms of MCF. The isolate was named No. 732.

Nucleotide sequence of a 3.5 kilobase fragment of malignant catarrhal fever virus strain WC11.

A 3.5 kilobase DNA fragment of the malignant catarrhal fever virus (MCFV), strain WC11, was mapped with a number of restriction enzymes, subcloned and sequenced. The fragment was subcloned into plasmid vector, pUC19, for direct sequencing. A complete open reading frame of 2,058 base pairs and a partial open reading frame of 630 base pairs were identified. The sequence of 3,389 nucleotides was compared to other herpesviruses. A 310 base pairs sequence in gene A was 57% homologous to a sequence in reading frame BXLF1 of Epstein-Barr virus strain B95-8.

Comparison of genomes of malignant catarrhal fever-associated herpesviruses by restriction endonuclease analysis.

The restriction endonuclease DNA cleavage patterns of eight isolates of malignant catarrhal fever-associated herpesviruses were examined using the restriction endonucleases HindIII and EcoRI. The eight viruses could be assigned to two distinct groups. Virus isolates from a blue wildebeest, a sika deer and an ibex had restriction endonuclease DNA cleavage patterns that were in general similar to each other. The restriction pattern of these three viruses was distinct from the other five. Of these five, four were isolated from a greater kudu, a white tailed wildebeest, a white bearded wildebeest, and a cape hartebeest. The fifth isolate C500, was isolated from a domestic cow with malignant catarrhal fever. These five viruses had similar DNA cleavage patterns.

Cloning and characterization of a genomic probe for malignant catarrhal fever virus.

A genomic probe specific for malignant catarrhal fever (MCF) virus was cloned by using purified viral DNA from MCF-virus strain WCll. Restriction endonuclease analysis of the purified viral DNA was used to identify the cloned viral genomic fragment. Dot blot hybridization by use of the genomic probe (pRP-5) indicated that the probe hybridized specifically with WCll-MCF virus, as well as with one other isolate of MCF-associated herpesvirus. Hybridization also was observed to a non-MCF virus strain of bovine herpesvirus.

Effect of interferons on the replication of alcelaphine herpesvirus-1 of malignant catarrhal fever.

Four isolates of alcelaphine herpesvirus-1 of malignant catarrhal fever (MCF) were tested for their inducibility of and sensitivity to various interferons. Viral isolates from an Indian gaur (Bos gaurus), a greater kudu (Tragelaphus strepsiceros) and two wildebeest (Connochaetes gnou) calves did not induce measurable interferon (IFN) in bovine fetal kidney cells. However, these low passages of each virus were all highly cell-associated and viral replication was inhibited at these passages by IFN at 14 IFN units/0.05 ml recovered from NDV-infected MDBK cells and at 7.6 IFN units/0.05 ml of IFN from NDV-infected bovine macrophages. The herpesvirus from the Indian gaur and greater kudu and high passages (greater than 50) of the cell-free WC-11 strain of alcelaphine herpesvirus-1 also were inhibited in their replication by recombinant IFN of bovine and human origins as determined by a fluorescent focus unit (FFU) reduction assay. The concentrations of IFN required to produce a 50% reduction in herpesvirus-produced FFU ranged between 6.4 and 480 IFN units. These findings promote the use of IFN as part of the regimens of treatment of captive endangered ruminant species with clinical MCF.

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.

Use of Vero cells for the isolation and propagation of malignant catarrhal fever virus.

Vero cells were compared with primary bovine thyroid (BTh) cultures for the isolation of malignant catarrhal fever virus from infected blood and tissues. Comparative titrations showed Vero cells detected only two-fold less infectivity in rabbit spleen suspensions than BTh cells. Twenty three of 32 bovine buffy coat cell preparations which were positive on BTh cells were also positive on Vero cells. The cytopathic effects (CPE) of virus isolates in Vero cells consisted of syncytia and refractile cytomegalic cells which were as easy to recognise and developed as rapidly as CPE in BTh cells. Two laboratory strains of malignant catarrhal fever virus were readily adapted to and maintained by passage in Vero cells.

Ultrastructure of cellular changes in the replication of the alcelaphine herpesvirus-1 of malignant catarrhal fever.

Cell cultures inoculated with 5 different viral isolates from 4 species of ruminants with clinical signs of malignant catarrhal fever (from the San Diego Wild Animal Park) were examined by electron microscopy. Each had the morphology of a herpesvirus (118 to 220 nm) and was icosahedral, and the nucleocapsid matured in the nucleus of the infected cell. Envelopment of budding occurred with each viral isolate at the nuclear and the plasma membranes. The virions egressed from the cell by budding from the plasma membrane or through channels of the Golgi apparatus or the endoplasmic reticulum. A proposed scheme for the morphogenesis of the herpesvirus of malignant catarrhal fever is presented.

Malignant catarrhal fever.

Malignant catarrhal fever is briefly reviewed and recent findings are described. Initially the disease was observed as a disease of cattle in Europe where, although no cause could be identified, circumstantial evidence implicated sheep as a source of infection and it was thus designated 'sheep-associated' malignant catarrhal fever. Subsequently the disease was observed in Africa where it became evident that a herpesvirus which normally infects wildebeest was the cause. It is now apparent that deer are highly susceptible to both forms of the disease, the sheep-associated form being a serious problem in farmed deer. The wide spectrum of clinical and pathological changes that occur in affected deer are described. A major constraint to studies of sheep-associated malignant catarrhal fever has been the absence of an experimental laboratory system. However, from affected deer it has been possible to transmit the disease to rabbits and thus has allowed detailed pathogenesis studies to be made which are summarised in this paper. It is suggested that the agent of sheep-associated malignant catarrhal fever is a virus and that when a particular subpopulation of T-lymphocytes is infected a profound immunological perturbation results; the lesions of malignant catarrhal fever being explained by a benign T-lymphocyte hyperplasia accompanied by a deregulation of cytotoxic natural killer lymphocytes that gives rise to tissue necrosis.

Transmission and virological studies of a malignant catarrhal fever syndrome in the Indonesian swamp buffalo (Bubalus bubalis).

A malignant catarrhal fever-like syndrome in indonesian swamp buffalo was experimentally transmitted to one of 2 Bos indicus and 3 of 3 Bos javanicus cattle by intravenous inoculation of 250 ml of citrated, whole blood from affected buffaloes. The 4 cattle developed clinical signs of disease on average 32.5 days after receiving the inoculation of blood. The 4 cattle died after a variable period of illness. None of a further 3 B. javanicus cattle inoculated intravenously with a spleen homogenate prepared from another affected buffalo developed the disease. The experimental disease was clinically and pathologically similar to the natural disease in buffaloes although differences were noted. Attempts to adapt the agent to mice, guinea pigs and rabbits failed. A cytopathic agent (Japanese encephalitis virus) was isolated from the spleen of one buffalo with clinical signs but was not considered significant. Sixty-three B. indicus, 7 B. javanicus (and 6 of their crosses), 3 B. taurus and 4 Bubalus bubalis (Murrah buffalo) were kept in the same quarters where 50 of 177 swamp buffaloes died between September 1979 and May 1982. Four of the 7 B. javanicus cattle developed the clinical signs of disease and died. All the other cattle in contact remained healthy.

Characteristics of the herpesvirus of malignant catarrhal fever isolated from captive wildebeest calves.

A herpesvirus was isolated from buffy coat cells from a newborn wildebeest (Connochaetes gnou) and from tissues of a 12-day-old wildebeest during the 1982 calving season of a captive, inbred herd maintained in a zoologic collection. Both wildebeests were clinically healthy, and there was no herd record that malignant catarrhal fever (MCF) existed. Each viral isolate produced cytopathologic changes in bovine kidney cell cultures (intranuclear inclusions and massive syncytia). The viral-infected cell cultures contained antigens of MCF virus detected by immunofluorescence. The morphology of each viral isolate as determined by electron microscopy was that of a herpesvirus. Suspensions of 4 to 5 ml of disrupted cell culture material which contained virus from each wildebeest were inoculated (IV) into white-tailed deer (Odocoileus virginianus). Each deer became clinically ill within 28 days. Both deer had mucoid catarrh and a febrile response (40.5 to 41 C). Each also seroconverted to MCF virus. The histopathologic change in the tissues from the 2 inoculated deer was vasculitis. At 16 to 17 days after the deer were inoculated, a syncytial-forming virus was isolated from each deer from buffy coat cells fused with polyethylene glycol (1000) to bovine fetal kidney cells. The virus was identified as MCF virus by immunofluorescence and production of antibody to MCF virus. The presence of virus in the inbred wildebeest herd established this species as a reservoir or latent carrier of African MCF virus at the zoologic park.