The prevalence of ovine herpesvirus-2 in 4 sheep breeds from different regions in South Africa.

About 90% of bovine malignant catarrhal fever (BMCF) PCR-positive cases in South Africa are caused by alcelaphine herpesvirus-1 (AlHV-1) and the other 10% by ovine herpesvirus-2 (OvHV-2). The prevalence of OvHV-2 in different sheep breeds in South Africa was determined in order to investigate whether the lower incidence of BMCF caused by OvHV-2 in comparison with AlHV-1 can be ascribed to a low incidence of the virus in sheep. A single-tube hemi-nested PCR was developed, evaluated and applied to detect OvHV-2 DNA. The prevalence of the virus in 4 sheep breeds from various regions in South Africa was shown to be 77%. No statistically significant difference was found amongst the sheep breeds tested.

Discrimination between sheep-associated and wildebeest-associated malignant catarrhal fever virus by means of a single-tube duplex nested PCR.

A single-tube duplex nested polymerase chain reaction (sdn-PCR) was developed for the detection of and discrimination between ovine herpesvirus-2 (OvHV-2) and alcelaphine herpesvirus-1 (AIHV-1). These viruses respectively cause sheep- and wildebeest-associated malignant catarrhal fever (SA-MCF and WA-MCF). In the first step of the sdn-PCR, two primers with high annealing temperatures based on conserved regions of the tegument genes were used for DNA amplification. In the second step, two primer sets based on variable regions of the respective OvHV-2 and AIHV-1 genes and with annealing temperatures > 11 degrees C below the primers used in the first step, were used. Internal regions of different sizes from amplicons produced in the first step were amplified. This single-tube test obviates the need for two separate assays to detect both viral types, thereby reducing time, labour and cost.

The prevalence of different African horsesickness virus serotypes in the Onderstepoort area near Pretoria, during an outbreak of African horsesickness in South Africa in 1995/1996.

During 1995/1996 parts of South Africa experienced exceptionally high rainfall. Large numbers of Culicoides midges were seen and an outbreak of African horsesickness (AHS) followed. In the Onderstepoort area, near Pretoria in Gauteng, a number of horses died of suspected AHS. Virus isolation and typing was done from blood and/or organ samples of 21 suspected cases as well as from five zebra which were kept in the area. Virus was isolated from 14 of the 21 suspected cases but not from the zebra. The neutralizing antibody response of the zebra to the nine different African horsesickness virus (AHSV) serotypes was determined. Results indicated the highest prevalence of serotypes 2 and 4 followed by serotypes 1, 6 and 9. Reverse transcription polymerase chain reaction (RT-PCR) was performed on total RNA extracted from blood samples of the zebra. AHSV RNA was indicated in three of five zebra by agarose gel electrophoresis analysis of amplicons and in four of five zebra after Southern blot hybridization using a 32P-labelled probe. RT-PCR can be used together with serological techniques in studies of AHS to further clarify the epizootiology of the disease.

Detection of African horsesickness virus and discrimination between two equine orbivirus serogroups by reverse transcription polymerase chain reaction.

A reverse transcription polymerase chain reaction (RT-PCR), based on the gene encoding the NS2 protein of African horsesickness virus (AHSV), was developed for rapid serogroup-specific detection of AHSV. The specificity of RT-PCR products was confirmed by Southern blot hybridization using a radioactively labelled cDNA probe specific for the NS2 gene. This RT-PCR could discriminate between all known members of the AHSV and equine encephalosis virus serogroups. AHSV RNA was detected in a sample representing 0.005 plaque forming units in a dilution series made of infected cell culture material. In an immune horse which had been vaccinated with a baculovirus expressed AHSV (serotype 4) VP2 subunit vaccine, viral RNA could be detected for up to 22 weeks post challenge. AHSV RNA was detected in various organs of an infected horse. Viral RNA was also detected by RT-PCR in nine suspected field cases of African horsesickness while virus isolation was successfully performed on eight of these cases.

Baculovirus expression of non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 and their use as antigens in an indirect ELISA.

Non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 (AHSV3) were expressed in Spodoptera frugiperda cells by recombinant baculoviruses containing the relevant genes. These proteins were purified and analysed by polyacrylamide gel electrophoresis and Western blot. NS2 and VP7 were used separately as antigens in an indirect ELISA for the detection of AHSV antibodies. Both antigens cross-reacted with hyperimmune guinea-pig antisera to infected cell lysates of all nine known AHSV serotypes and to antisera obtained from horses immunized with attenuated virus of seven AHSV serotypes.

An indirect sandwich ELISA utilising F(ab')2 fragments for the detection of African horsesickness virus.

African horsesickness virus (AHSV), an important disease of equines is caused by an orbivirus. Because of the need to contain the spread of the disease, it is often essential to make a rapid diagnosis. For this purpose, an ELISA capable of detecting viral antigen in animal tissue and in cell culture fluid was developed. Immobilised F(ab')2 fragments prepared by digestion of AHSV-specific IgG with pepsin were used to trap virus from tissue homogenates or cell culture supernatant. After addition of intact IgG as detecting antibody, Staphylococcus aureus protein A labelled with horseradish peroxidase was added to allow visualisation of the reaction. Polyclonal antibodies directed against either whole AHSV or viral core particles were suitable as detecting antibodies. On the other hand, a monoclonal antibody that was specific for a major core protein, VP7, gave a much weaker signal in the ELISA. All known AHSV serotypes were recognised in the F(ab')2-ELISA by polyclonal antisera against either whole virus particles or viral cores. Immunoprecipitation of AHSV structural polypeptides showed that such antisera contained populations of antibodies directed against core proteins. The F(ab')2-ELISA has potential as a diagnostic technique for AHSV infections.

Characterization and cloning of the African horsesickness virus genome.

The dsRNA profiles of all nine African horsesickness virus (AHSV) serotypes were compared by agarose gel electrophoresis and PAGE. The agarose profiles were identical, but a unique profile was obtained for each of the nine serotypes by PAGE. Nine of the 10 dsRNA genome segments of AHSV-3 were cloned and the clones were used in dot-spot and Northern blot hybridization experiments to determine intra- and inter-serogroup nucleic acid similarities. Segments 1, 3, 4, 5, 7 and 8 were highly conserved in the AHSV serogroup and no genetic relationship with any of the other orbiviruses was observed. Of these segments 3, 5 and 8 showed the largest degree of cross-hybridization to the cognate genes of all the serotypes. These clones did not cross-hybridize to other orbiviruses such as epizootic haemorrhagic disease virus, bluetongue virus or equine encephalosis virus and are therefore recommended for use as group-specific probes for the identification of the AHSV serogroup. Genome segments 6 and 10 showed an intermediate degree of conservation, whereas segment 2 is serotype-specific and therefore probably codes for the outer capsid protein VP2.

A comparison of an australian bluetongue virus isolate (CSIRO 19) with other bluetongue virus serotypes by cross-hybridization and cross-immune precipitation.

No major differences in size were observed when both the double-stranded RNA and the polypeptides of the Australian bluetongue virus (BTV) isolate CSIRO 19 (BTV-20) were compared with those of other BTV serotypes such as BTV-10 and BTV-4. Minor capsid polypeptide P6 of both BTV-20 and BTV-4, which electrophoreses as a single band on continuous phosphate buffered gels, in separated into 2 distinct bands on discontinuous glycine-buffered gels. This was not the case with BTV-10. Cross-immune precipitation of BTV-20 with BTV-10, BTV-17, BTV-4 and BTV-3 indicated strong immunological cross-reaction of the group-specific antigen P7 of the different serotypes. There was also some cross-immune precipitation of the serotype-specific polypeptide P2 of BTV-20 and BTV-4. This result is in agreement with the observed cross neutralization of these 2 viruses. The main distinction between BTV-20 and the other BTV serotypes was observed in cross-hybridization experiments. The homology between the nucleic acid of BTV-20 and other BTV serotypes was less than 30%, whereas homology normally found between BTV serotypes is at least 70%. The hybridization products of the different BTV serotypes were analysed by electrophoresis and fluorography. Two main hybrid segments were observed in all heterologous hybridizations with BTV-20 as a compared with 7 hybrid segments in hybridizations between BTV-4 and BTV-10. In order to determine from which genome segment of BTV-20 these 2 hybrid segments were derived, the hybridizations were carried out with individually purified double-stranded RNA segments. These results indicate that the 2 segments of BTV-20 that show the largest homology to corresponding segments of a heterologous BTV serotype are No. 7 and 10.

The nucleic acid and proteins of epizootic haemorrhagic disease virus.

Purified epizootic haemorrhagic disease virus (EHDV) was shown to contain 10 double-stranded RNA segments and a double-layered protein capsid with 4 major and 4 minor polypeptides. The virus differed from bluetongue virus (BTV), the orbivirus prototype, in that EHDV had an additional minor polypeptide component. This component, together with the major polypeptides P2 and P5, formed the outer capsid layer of the virus. The extra polypeptide apparently stabilizes this layer since, unlike BTV, EHDV was quite stable on CsCl gradients at both pH 7,0 and 8,0. EHD virions were found to have a density of 1,36 g/microliter, while particles without the outer capsid layer were isolated and had a density of 1,40 g/microliter. Two non-capsid polypeptides, P5A and P6A, were identified in addition to the 8 capsid polypeptides. Polypeptide P5A was synthesized in excess of all the others. There was little homology between the nucleic acids of EHDV and BTV with only 5-10% cross-hybridization. No hybrid double-stranded RNA segments were identified. We found by cross-immune precipitation that the major core polypeptides of the 2 viruses (P7 and P3) have common antigenic determinants.

A gel electrophoretic study of the protein and nucleic acid components of African horsesickness virus.

The physico-chemical structure of African horsesickness virus (AHSV) is compared with that of some of the other members of the Reoviridae, and in particular with that of bluetongue virus (BTV), the type strain of the orbivirus genus. This study adduces evidence of a great similarity between the gel electrophoretic patterns of the polypeptides of AHSV and BTV. The molecular mass values of the 7 AHSV polypeptides range between 0,30 x 10(5) and 1,46 x 10(5) dalton, a variation similar to that of BTV. The close relation between AHSV and BTV is further affirmed by the gel electrophoretic resolution of the AHSV double-stranded RNA genome into 10 segments.