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.

Malignant catarrhal fever virus specific secretory IgA in nasal secretions of wildebeest calves.

Wildebeest IgA was isolated from nasal secretions and precolostrum. It was identified by cross-reaction with anti-human and anti-bovine IgA sera. Nasal secretions collected from wildebeest calves over 3 months old had malignant catarrhal fever virus neutralizing antibody activity. They also contained specific IgA to the virus as detected by indirect immunofluorescence. It is suggested that production of malignant catarrhal fever virus specific IgA in the nasal cavity, contributes to the elimination and cessation of the virus shed in the nasal secretions of wildebeest calves over 3 months. old.

Neutralising antibodies to wildebeest-derived malignant catarrhal fever virus in African wildlife.

A total of 2,722 sera collected between 1963 and 1983, from 43 different species of wildlife in 11 African countries was examined for neutralising antibodies against the wildebeest-derived strain of malignant catarrhal fever (MCF) virus. Antibodies were demonstrated in 10 species of Bovidae which included eight species from the sub-family Hippotraginae and one species each from Bovinae and Antilopinae. Neutralising antibodies were also recorded in hippopotamus. It is suggested that the high prevalence of antibodies recorded in sera from waterbuck and reedbuck indicate infection with MCF. However, titres in other species may be due to antigenically related viruses.

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.

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.

Antibodies to malignant catarrhal fever virus in cattle with non-wildebeest-associated malignant catarrhal fever.

Sera from 48 cattle with non-wildebeest-associated malignant catarrhal fever (MCF) were assayed for antibodies to wildebeest-associated MCF virus. Significant titres were found in 38 sera and these reactions appeared to be specific. Some cases of non-wildebeest-associated MCF may be caused by an agent antigenically related to MCFV.

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.

Observations on two strains of bovine malignant catarrhal fever virus in tissue culture.

Two cell-free strains of bovine malignant catarrhal fever (MCF) virus were examined by fluorescent antibody staining and for cytopathogenicity in secondary bovine thyroid (BTh) and secondary bovine kidney cell cultures, and in a bovine embryo lung cell line. The hartebeest-derived strain (K30) induced syncytia and intra-nuclear inclusions in all three systems, whereas the widebeest-derived strain (WC11) induced intra-nuclear inclusions in all systems, but syncytia in only BTh cells. Fluorescent antibody staining detected virus in tissue culture at least 24 h before the appearance of cytopathic effect.

Influence of temperature on the growth in cell culture of malignant catarrhal fever virus.

The growth characteristics of malignant catarrhal fever (MCF) virus in bovine thyroid cultures were affected by incubation temperature. The cytopathic effect at 37 degrees C was predominantly syncytial and little or no cell-free virus could be detected. At 32 degrees to 34 degrees C foci of rounded refractile cells were observed, and this was accompanied by an increase in the amount of cell-free virus found in culture fluids. Growth curve studies with one low passage isolate of MCF virus showed that optimum yields of cell-free virus were obtained at 32 degrees to 34 degrees C and survival curves at 32 degrees and 37 degrees C indicated that this was a result of the relatively short half life of the virus at the higher temperature. A number of other benefits resulted from the use of lowered incubation temperature and these are discussed with reference to in vitro work with the virus.

A microtitre technique for the assay of malignant catarrhal fever virus and neutralising antibody.

A microtitre technique for the quantal assay of a cell-free strain of malignant catarrhal fever virus was developed, using serially passaged bovine embryonic kidney cells. End-points were determined after 12 days' incubation and the mean titre recorded for a single virus stock stored at -70 degrees C over a six-month period was 10(5.5) +/- 0.2 (SD). In neutralisation tests serum/virus mixtures were best held at 37 degrees C for 1 h in microtitre trays before the addition of cells; assays were highly reproducible, figures of 10(1.5) +/- 0.2 being obtained for a single reference serum.

Isolation of bovine malignant catarrhal fever virus from ocular and nasal secretions of wildebeest calves.

Malignant catarrhal fever (MCF) virus was isolated from ocular secretions of four out of nine free-ranging wildebeest calves, while four out of 11 animals had the virus in nasal secretions. The mean infectivity of virus in ocular and nasal secretions was 10(2.25)TCID50/ml and 10(2.5)TCID50/ml respectively but infectivity of more than 10(3.2)TCID50/ml was found in some ocular and nasal secretions. Thus it appears that MCF virus is excreted via the ocular and nasal routes from wildebeest calves, and the presence of high titre virus supports the suggestion that MCF virus is disseminated by these secretions. This virus was not recovered from urine or saliva.

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.

Antibody response in cattle and rabbits to early antigens of malignant catarrhal fever virus in cultured cells.

Cytosine arabinoside (Ara-C) inhibited the production of late antigens and of infectious virus in monolayers of bovine kidney cells infected with the high-passage, WC-11 strain of malignant catarrhal fever virus. Early antigens were not affected. Using hyperimmune and acute-phase sera from cattle and rabbits in indirect immunofluorescence tests, it was shown that Ara-C treated cultures contained two early antigens; one was diffuse and distributed throughout the cells, the other was particulate and intranuclear. Antibody to early antigens developed later and attained lower titres in infected animals, especially rabbits; only hyperimmune sera reacted with the diffuse early antigen.

Malignant catarrhal fever in an Indian gaur and greater kudu: experimental transmission, isolation, and identification of a herpesvirus.

Herpesviruses were isolated in bovine cell cultures from buffy coat cells obtained from an Indian gaur (Bos gaurus) and a greater kudu (Tragelaphus strepsiceros) with clinical signs of the head and eye form of malignant catarrhal fever (MCF). Both animals were from herds housed in a zoologic park in Oklahoma. Serial transmission of the head and eye form of MCF was accomplished by using whole blood from the gaur into a Hereford-Angus heifer, then whole blood from the heifer into a Holstein calf, and finally, whole blood from the calf into a white-tailed deer (Odocoileus virginianus). A herpesvirus was isolated in bovine cell cultures inoculated with buffy coat cells from the heifer, and 2 deer inoculated with this herpesvirus developed the head and eye form of MCF. A deer inoculated with whole blood from the greater kudu also developed clinical signs of MCF, and a herpesvirus was subsequently recovered from the deer. Clinical signs of MCF included a mucopurulent catarrh, pyrexia (38.8 to 42.1 C), anorexia, and corneal opacity, and death occurred between postinoculation days 15 and 21.

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.

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.

Virologic studies on cattle with naturally occurring and experimentally induced malignant catarrhal fever.

Malignant catarrhal fever is an important disease of cattle and certain wild ruminants. It occurs in sporadic and epizootic forms in Colorado cattle. Specimens from 15 cattle with naturally occurring malignant catarrhal fever and 5 cattle with induced disease were examined for cell-free and cell-associated viruses. Enteroviruses were isolated from leukocytes of 2 cattle with field cases of the disease. A herpesvirus with characteristics of the "Movar"-type, isolated from the spleen of another steer with a field case could be propagated optimally in bovine fetal spleen cells. A cell-associated virus, forming polykaryons, was isolated in adrenal and thyroid cells from 3 cattle with experimentally induced malignant catarrhal fever. It was cultured from leukocytes, ependymal tissue, spleen, lymph node, kidney and thyroid and adrenal glands of affected cattle and remained cell-associated in 48 subsequent passages. It was inactivated by freezing and thawing and by treatment with ultrasound; its polykaryon-forming activity was inhibited by 25 mug of 5 fluoro-2' deoxyuridine/ml. This viral agent replicated in bovine fetal adrenal, thyroid, and spleen cells, but not in primary kidney cells or in MDBK, HeLa, or L cells. A representative isolate was identified electronmicroscopically as an enveloped virus, 120 to 150 nm in diameter. Structural analysis indicated that it had properties of the bovine syncytial viruses. Attempts to induce MCF in cattle with 1 of the isolates failed. A parvovirus was isolated from the jejunal lymph node of calf 72-P-293, which had the experimentally induced disease and was also infected with the syncytial virus.

Viral particles associated with malignant catarrhal fever in deer.

Viral particles associated with malignant catarrhal fever in white-tailed deer (Odocoileus virginianus) and axis deer (Asix axis) are described. Morphologically, the virus resembled the togaviruses and was unlike the herpesvirus which causes wildebeest-associated malignant catarrhal fever (snotsiekte).

Electron microscopic study of the African strain of malignant catarrhal fever virus in bovine cell cultures.

Malignant catarrhal fever (African strain) is a viral disease of ruminants which is considered an exotic disease in the United States. Viral isolates obtained from one clinically ill gaur (Bos gaurus) and a greater kudu (Tragelaphus strepsiceros) located in a zoologic park in Oklahoma, and from one heifer (Bos taurus) and a domestic white-tailed deer (Odocoileus virginianus) experimentally inoculated with the isolated and identified African strain of malignant catarrhal fever virus (MCFV), were each studied in bovine cell cultures by electron microscopy. Certain of the viral isolated were previously characterized as MCFV by serologic and morphologic examinations, their cytopathic effect in cell cultures, and their ability to reproduce disease in a ruminant host. The virions of MCFV (African) examined by electron microscopy were icosahedral similar to herpes-virus, were between 98 and 194 nm, developed in the nucleus and matured in the cytoplasm of the cell, and exhibited budding. The virus in infected cells passed through the nuclear and plasma membranes and also into cytoplasmic vesicles from which it acquired one or more envelopes. Virions of malignant catarrhal fever were closely associated with the cellular endoplasmic reticulum, and aberrant morphologic forms of MCFV were observed in virus-infected cells.

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.