African horse sickness (AHS) was reported in the south-western Khomas Region, central Namibia (22° 24.063´ S, 17° 01.791´ E; 23° 32.617´ S, 15° 53.870´ E), contrary to expectations that the arid conditions in the area would limit its occurrence. This prompted investigation into the occurrence of AHS in horses, a possible reservoir animal, the Hartmann’s mountain zebra (Equus zebra. hartmannae) and the occurrence of the Culicoides midge vector (Diptera: Ceratopogonidae) of the disease in the area. Questionnaires were used to explore the geographic characteristics of the study area, the occurrence of an expected AHS virus reservoir animal, E. z. hartmannae and AHS in horses in the study area. According to the questionnaire, rainfall patterns seem to follow topography of the area, where the north-east formed the higher rainfall (420 mm/a) high-ground and the south-western formed the lower rainfall (120 mm/a) pediment zone in the south-west. Cases of AHS occurred mostly in mid-rainfall zones. E. z. hartmannae were present throughout the area. They migrated from the southwest towards the north-eastern high-grounds during droughts, presumably along ephemeral river beds. E. z. hartmannae were sampled for blood and tissues and analysed for evidence of African Horse Sickness Virus (AHSV) infection by indirect ELISA, RT-PCR and virus isolation techniques. All useable samples tested positive for anti-AHSV antibodies. Viral RNA was demonstrated in 26% of all the zebra sampled. No viable viruses were isolated from these samples, however further research is required, as difficult sampling conditions may have yielded false-negatives. From 6 July to 21 September 2009, Culicoides midges were collected during the dry winter season in suction UV-light traps installed at five selected sites along a rainfall gradient. In 38 collections, a total of 9091 Culicoides individuals, representing 25 species were collected. The dominance of the proven vector of AHSV, Culicoides imicola Kieffer, varied in dominance from 94% near Windhoek with high altitude and relatively higher annual rainfall, to 12% at the site situated farthest southwest, with the lowest altitude and annual rainfall. From what was observed of the occurrence of AHS in horses, E. z. hartmannae and the distribution and abundance of the AHSV vector (Culicoides spp.), it was concluded that AHS can be maintained in the south-western Khomas Region even in the lowest mean annual rainfall zones. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2012

MA / African horsesickness is a disease endemic in Sub Saharan Africa affecting horses, a non-native species, which are extremely susceptible to this disease. Both the ‘dunkop’ form (with its dramatic high fever, laboured breathing, frothy nasal discharge and sudden death) and the ‘dikkop’ form (with its swollen head and eyes and bleeding in the membranes of the mouth and eyes) have been visited upon equine populations and their human owners in successive epidemics through the earliest colonial settlement until recent times. This thesis traces the development of veterinary science in South Africa and the effect it had on the changing ideas surrounding African horsesickness. It explores not only the veterinary progress in the country but also the impact of the progress on African horsesickness as other diseases received attention. The discussion traces the disease from one of the major epidemics ever encountered in the country, in the mid nineteenth century, to the beginning of the development of veterinary services in South Africa when little was known about African horsesickness. It illustrates the implications of a country's struggle with animal disease, the reasons for a lack of knowledge and the ramifications of the Onderstepoort Veterinary Institute’s interventions. This thesis shows that African horsesickness not only had an impact on the veterinary developments of the country but was also indirectly involved in the South African War, 1899-1902. It demonstrates the impact of disease during wartime while illustrating the importance of horses during such difficult times. Thus, this thesis draws on works on animal diseases and on social history to explore not only the effect African horsesickness had historically on equines, but the effects it had more broadly on southern African society. This study is intended to bring insight into the social history of the disease itself: how it was experienced by livestock owners and also how settler and indigenous efforts were turned towards combating this dramatic disease.

African horse sickness virus (AHSV) affects equine populations around the world. It is the cause of a high rate of morbidity and associated large economic losses in affected regions. The virus is a segmented double stranded RNA virus and a member of Orbivirus genus in the Reoviridae family. The prototype member of the orbiviruses is bluetongue virus (STY) and other members include Chuzan virus and St. Croix River virus. These viruses are all characterized by a genome of ten dsRNA segments that encode at least ten different proteins. Three of the minor core proteins are found within the core of BTV. These are all associated with the RNA transcription complex and the enzymatic activities with which they are associated include an RNA polymerase (VP1), an RNA capping enzyme (VP4) and an RNA helicase (VP6). Genes homologous to the BTV genes that encode these proteins are found in all members of the Orbivirus genus. The aim of this thesis is to characterize VP4 of AHSV, the capping enzyme candidate, and to compare it to other orbivirus capping enzymes. Possible functional motifs and regions of importance within the orbivirus capping enzymes will be identified. The gene will also be expressed and used to perform assays to characterize the different enzymatic activities of VP4. The VP4 cDNA of AHSV serotype 3 was cloned and sequenced. From the full-length verified nucleotide sequence an open reading frame was identified and used to predict the amino acid sequence. These were compared to other orbivirus species including STY, Chuzan virus and St. Croix River virus. These alignments identified a number of highly conserved regions, consisting of four or more amino acids conserved between all the sequences analyzed. A fibronectin type 3-like motif, containing 12 conserved amino acids, was identified which could be responsible for protein binding. This motif contains 12 conserved amino acids making it a good candidate for a functional motif. Conservation does not, however, always predict regions of importance. In BTV a lysine-containing motif was identified to be responsible for GMP binding. This region is not conserved between the different viruses. AHSV has a motif containing a lysine residue similar to the motif identified in rotavirus and reovirus. Two other motifs described in BTV were also not conserved in the other viruses. One of them, a leucine zipper, was shown to dimerize BTV VP4. Phylogenetically, AHSV and Chuzan virus are the most closely related while BTV is more distant and St. Croix River virus forms a distinct out-group when the different VP4 sequences are compared. AHSV-3 VP4 was expressed as a histidine-tagged protein in the baculovirus expression system. Not unexpectedly, the protein was found to be insoluble, similar to BTV VP4 produced by means of the same system. However, whereas BTV VP4 could be solubilized by the addition of salt the AHSV VP4 remained insoluble at high salt concentrations. Several adjustments were made. Cells were lysed in a high salt buffer, the pH of the buffers was adjusted and sucrose cushions were used but none of the methods was found to improve the yield of soluble VP4 significantly. However, the pellet containing VP4 was relatively empty of contaminating protein and, therefore, a number of enzymatic assays were performed with the pellet. Assays for inorganic phosphatase and nucleotide phosphatase were performed. Strikingly, both assays indicated the presence of active phosphatases in the WT and VP4 pellets. Also, an assay was performed for guanylyltransferase activity but no activity was observed for this assay. The sequence data therefore points to VP4 as the probable capping enzyme although it may have a different structural complex. The failure to produce a reliable source of soluble purified AHSV VP4 made it impossible to provide evidence to confirm the associated enzymatic activities. / Dissertation (MSc(Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, causes an acute disease in horses with a high mortality rate. AHSV encodes four nonstructural proteins (NS1, NS2, NS3/NS3A), whose functions in the viral life cycle are not fully understood. The NS1 protein is the most abundantly expressed viral protein during AHSV infection and forms tubular structures within the cell cytoplasm. No function has been ascribed to these tubules to date, although it has been suggested that they may play a role in cellular pathogenesis. Studies aimed at understanding the function of NS1 have been hampered by the lack of a suitable reverse genetics system for AHSV. However, the phenomenon of RNA interference (RNAi) has emerged as a powerful tool whereby the function of individual genes can be studied. In mammalian cells, RNAi can be triggered by exposing cells to double-stranded RNA either via exogenous delivery of chemically synthesized small interfering RNAs (siRNAs) or endogenous expression of short hairpin RNAs (shRNAs). Consequently, the aim of this investigation was to develop a plasmid DNA vector-based RNAi assay whereby expression of the AHSV-6 NS1 gene could be suppressed in BHK-21 cell culture with shRNAs directed to the NS1 gene. To investigate, complementary oligonucleotides corresponding to selected AHSV-6 NS1 gene sequences were chemically synthesized, annealed and cloned into the pSUPER shRNA delivery vector under control of the RNA polymerase III H1 promoter. The plasmid DNA vector-expressed shRNAs targeted sequences within the NS1 gene corresponding to nucleotides 710 to 728 (shNS1-710) and 1464 to 1482 (shNS1-1464), respectively. A NS1- eGFP chimeric gene was constructed and used towards establishing a simple assay whereby the gene silencing efficiency of different RNAi effector molecules could be evaluated by analysis of the protein level visually and quantitatively by fluorometry. The effect of the NS1- directed shRNAs on AHSV-6 NS1 protein expression was subsequently evaluated by cotransfection of BHK-21 cells with the respective recombinant pSUPER shRNA delivery vectors and the NS1 reporter plasmid pCMV-NS1-eGFP. The results indicated that shNS1- 710 and shNS1-1464 suppressed NS1-eGFP expression by 19% and 9%, respectively. The potential of the NS1-directed shRNAs to suppress NS1 mRNA expression was investigated by transfection of BHK-21 cells with the respective recombinant pSUPER shRNA delivery vectors, followed by transfection with the recombinant mammalian expression vector pCMVNS1 or infection with AHSV-6. Results obtained by semi-quantitative real-time PCR assays indicated that both NS1-directed shRNAs interfered with NS1 mRNA expression, albeit to different extents in the respective assays. Taken together, these results demonstrated that AHSV-6 NS1 gene expression can be suppressed in BHK-21 cells by plasmid DNA vectorderived shRNAs and suggests that this approach may, with further optimization, be useful in determining the function of the NS1 protein in virus-infected cells. / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Novel vaccine strategies for the presentation of immunologically important epitopes to the immune system are continuously being developed. Two such systems include the particulate protein and live viral vector delivery systems. In his study the long-term objective is to explore the African horsesickness virus (AHSV) serotype 9 viral protein 7 (VP7) and the Lumpy skin disease (LSDV) viral vector as two different vaccine strategies, particularly in view of the development of an HIV-1 vaccine. Consequently two very specific objectives were outlined in this study. The first was to express the HIV¬1 subtype C strain Du 151 gp41 epitopes ALDSWK and RVLAIERYLKD on the surface of the AHSV-9 VP7 particulate protein crystalline structures. A longer-term aim is to synthesise large quantities of these chimeric VP7 crystals in order to assess the immune response against the inserted epitopes. Secondly, the efficiency of the LSDV bi-directional promoter pA7LA8R in expressing chimeric VP7 proteins was to be evaluated by utilising the late element of this promoter to determine expression levels. Nucleotide sequences encoding the ALDSWK and RVLAIERYLKD epitopes were amplified from the HIV-1 subtype C strain Du 151 gp160 gene utilising PCR. These sequences were cloned individually as well as in combination into a multiple cloning site (549-566bp) present in the AHSV-9 VP7 gene. Recombinant pFASTBAC vectors PFASTBAC-VP7-MT 177-RVLAIERYLKD, PFASTBAC-VP7-MT 177-ALDSWK AND PFASTBAC-VP7-MT-177-RVLAIERYLKD-ALDSWK were identified, sequenced and used in the generation of recombinant baculoviruses utilising the BAC-to-BAC™ Baculovirus expression system. Expression of all three chimeric proteins, VP7-ALDSWK, VP7-RVLAIERYLKD and VP7- RVLAIERYLKD-ALDSWK was detected in infected Sf9 insect cells utilising SDS-PAGE. Further investigations will involve high-level expression of these proteins, which in turn will allow their characterisation as well as solubility, scanning electron and immunogenicity studies. In order to evaluate the efficiency of the LSDV bi-directional promoter, the AHSV-9 VP7 gene was cloned under the control of the late element (pA7L) of this promoter. The recombinant pHSsgpt-VP7 transfer vector was subsequently transfected into lamb testis cells infected with wild type LSDV in order to generate recombinant LSDV-VP7. Several rounds of recombinant virus selection in the presence of mycophenolic acid resulted in the loss of the LSDV-VP7 recombinant. Due to this unforeseen result, the expression of the VP7 protein from the late element of the pA7LA8R bi¬directional promoter could not be quantified and the efficiency of this promoter was not determined. The loss of LSDV recombinants, which contain a gene under the control of the late promoter element pA7L, has occurred previously and is suspected to be because of the instability of these recombinants. Due to the difficulties inherent in working with the LSDV viral vector delivery system, it has subsequently been decided to explore an alternate poxviral vector system. The focus in this study is now being shifted onto the promising Modified Vaccinia Ankara (MVA) viral vector system. / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

Historically African Horsesickness (AHS) outbreaks are rare occurrences in the Western Cape Province. The 2004 outbreak was particularly troubling since it followed only five years after the previous outbreak and even before any cases were reported further inland, which is traditionally the source of infection for the southern (non-endemic) parts of the country. Following confirmation of the diagnosis, control measures were immediately instituted and an epidemiological investigation was initiated. The investigation revealed, inter alia, that serological profiles of case horses were inconsistent. A case was subsequently defined as a horse showing typical symptoms of AHS and from which virus could be isolated. The disease pattern for both the 2004 and 1999 outbreaks can be classified as sporadic epidemics. This type of epidemic pattern is to be expected in a vector borne disease and it is typical in a disease situation where some of the animals are immune. The temporal pattern revealed that the level of immunity in the equine population of the affected area was higher during the 2004 outbreak than during the 1999 outbreak. In addition, it showed a clustering of cases during the initial stages of both the 2004 and 1999 outbreaks. This illustrated the efficacy of the control measures (including movement and vector control), which was instituted immediately after the diagnosis of the first case. The analysis of the spatial pattern during both the 2004 and 1999 outbreaks identified the Eerste-river-valley as a high-risk area for the outbreak of AHS in the surveillance zone. The population pattern during the 2004 outbreak illustrated that the risk of dying of AHS was higher in horses of 5 years and younger (p<0.10). It was shown that vaccination and stabling offers the best protection against the risk of dying as a result of AHS infection in an exposed population (p<0.05). A questionnaire survey was conducted as part of the epidemiological investigation and it revealed that only 12.4% of equine holdings in the affected area practiced vector control, while a high percentage of horses (69.6%) were protected by means of vaccination, which impacts negatively on the purpose of a surveillance zone. The number of Culicoides imicola midges in the area where the outbreak was detected was extremely high, constituting 94.6% of the Culicoides midge population. This is comparable with the 1999 outbreak when 96.0% of the midges collected were identified as C. imicola. However, during a similar survey in 1996, C. imicola comprised only 11.3% of the population (Neville et al. 1988, Venter, G., personal communication 2004). Furthermore, the outbreak was detected even before significant rainfall was recorded in the region and transmission occurred at average minimum temperatures below 15 °C. The virus responsible for the 2004 outbreak was typed as AHS serotype 1, while AHS serotype 7 was identified as the cause of the 1999 outbreak. The source of the infection in the 1999 outbreak was the illegal movement of two horses from the Free State Province in the infected zone into the surveillance zone. Although no absolute proof could be obtained, there is strong evidence that the source of the 2004 outbreak was again the movement of horses, this time from Namibia, accentuating that horse movements constitutes the highest risk to the integrity of the free zone. Since the ability to control an outbreak successfully is directly dependant on rapid detection and given the large number of vaccinated horses as a result of the outbreaks and the AHS movement control policy, amendments to the export policy and legislation are recommended. AHS outbreaks in the control area of South Africa cause substantial financial loss to the horse industry and the controlling authorities. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2006. / Production Animal Studies / unrestricted

Equine encephalosis virus (EEV) is the cause of equine encephalosis. The disease is similar to mild forms of African horse sickness (AHS) and the two diseases are easily confused. Laboratory identification and serotyping of EEV is based on viral isolation in BHK-21 cells and a viral plaque inhibition neutralization test (Erasmus <i<et al., 1970). These procedures require long durations to confirm results and it was desirable that a rapid diagnostic assay was developed to distinguish EEV from African horse sickness virus (AHSV). A PCR test developed for AHSV (Quan et al., 2008) formed the basis for development of a similar assay for EEV. The aim was to develop and evaluate a real time PCR assay for the detection of EEV in the blood and organs of horses. FastPCR software was used to design primers to amplify and sequence the EEV S7 (VP7) gene. RNA was extracted from EEV tissue culture isolates, representing all seven serotypes, using a MagMaxTM Express Particle Processor and MagMaxTM-96 Total RNA Isolation kits. A one step reverse transcription PCR (RT-PCR) was carried out to amplify the EEV S7 gene using a GeneAmp Gold RNA PCR core kit. Sequence reactions were carried out using a BigDye terminal v3.1 sequencing kit and analyzed with an ABI 3130xl Genetic Analyzer. After sequences alignment using BioEdit software, conserved regions were identified and Primer Express 3.0 software was used to design EEV primers and TaqMan® MGBTM hydrolysis probes for real-time RT-PCR assay. The EEV real-time RT-PCR assay was specific and did not detect AHSV nor bluetongue virus (BTV). The real-time format was selected because of its convenience, sensitivity and ability to produce results rapidly. Validation of the assay is the next step in establishing it as a routine diagnostic assay. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Veterinary Tropical Diseases / unrestricted

VP6 is one of the minor structural core proteins of African horsesickness virus. The minor core proteins VP1, VP4 and VP6 are presumed to constitute the dsRNA dependent RNA polymerase transcription complex of the virus. In the Orbivirus prototype bluetongue virus (BTV), VP6 has a helicase activity. The aim of this investigation was to characterize the primary structure and nucleic acid binding function of the inner core protein VP6 of African horsesickness virus (AHSV). To characterize the primary structure of AHSV VP6, VP6 genes of serotypes 3 and 6 were cloned and sequenced. Both genes encode a 369 amino acid polypeptide. A comparison to the VP6 proteins of other Orbiviruses indicated that in all cases the proteins are rich in basic residues and in glycine. The proteins are highly conserved within serogroups but the conservation between serogroups is low. VP6 of AHSV-3 and AHSV-6 have 93.5% identity and 96% similarity in amino acid residues. AHSV-6 VP6 has 27% identical and 46% similar amino acid residues to BTV-10 VP6. Phylogenetic analysis of four orbivirus VP6 genes indicated that AHSV and BTV are most closely related to each other. Motifs characteristic of known helicases were identified by sequence analysis. Glycine rich protein motifs and a N-glycosylation signal were present. No nucleic acid binding motifs identified in other proteins were found in AHSV VP6. To characterize the VP6 protein of AHSV VP6, the genes were expressed using both a baculovirus and a bacterial expression system. Proteins were found to be soluble and the VP6 expressed in insect cells was found to be N-glycosylated. The nucleic acid binding function of AHSV VP6 was investigated. Bacterially expressed VP6 was demonstrated to bind nucleic acids by electrophoretic mobility shift assays. Baculovirus expressed VP6 bound double and single-stranded RNA and DNA in nucleic acid overlay protein blot assays. Competition assays indicated that VP6 may have a preference for binding to RNA rather than DNA. Glycosylation was found to play no direct role in nucleic acid binding but the binding is strongly dependent on the NaCl concentration. A series of truncated VP6 peptides were produced to investigate the importance of localized regions in nucleic acid binding. Two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. Binding appeared to be influenced by charge as reflected by the isoelectric points (pI) of the peptides and experiments indicating the effect of pH on the binding activity. However, only peptides containing amino acid residues 190 to 289 showed binding activity. This region corresponded to the region on BTV VP6 that contains two binding domains. It is proposed that the dsRNA binding domain in AHSV VP6 is a sequence of positively charged amino acids constituting a domain that determines the nucleic acid binding characteristics of the peptide. The mechanism of binding of baculovirus expressed VP6 in a nucleic acid overlay protein blot is proposed to be charge related. / Thesis (PhD (Genetics))--University of Pretoria, 2007. / Genetics / unrestricted

African horse sickness (AHS) is a viral disease with high mortality rates, vectored by the Culicoides midge and affecting members of the Equidae family. AHS is endemic to South Africa, and, as a result, affects export and international competitiveness in equine trade, and impacts significantly on the South African racehorse and performance horse industries. AHS also has devastating consequences for rural and subsistence equine ownership. The protocol developed in this dissertation has the potential to serotype and confirm the AHS virus within a few hours at significantly less cost than current methods. It will ease the financial and time constraints of studying an outbreak in real time and has the potential to solve many of the unknown factors surrounding AHS, particularly and most importantly, the role that each serotype plays in outbreaks and the form of the disease contracted by horses. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

No abstract available. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.