African horsesickness (AHS), caused by the African horsesickness virus (AHSV) (Coetzer&Erasmus, 1994), has 10 segments of double stranded RNA (Verwoerd et al., 1970). These encode seven structural proteins, namely VP1, VP2, VP3, VP4, VP5, VP6 and VP7 (Verwoerd et al., 1972; Huismans&Van Dijk, 1990) as well as four nonstructural proteins (NS1, NS2, NS3 and NS3A) (Van Dijk&Huismans, 1988). NS3 is cytotoxic when expressed in insect cells causing membrane permeabilisation and cell death (Van Staden et al., 1995). This characteristic was postulated to be associated with the two hydrophobic domains (Van Staden et al., 1998; Van Niekerk et al., 2001(a)). Although NS3 has the same basic structure and function in all orbiviruses it differs greatly on nucleic acid sequence level as well as in the degree of variation that is found within the protein (Sailleau et al., 1997; Huismans et al., 2004). Therefore the main aim of this study was to identify which regions or amino acids remain conserved and might therefore play a role in the structure or function of NS3. The focus was mainly on the two hydrophobic domains as well as the spacer region between the hydrophobic domains. The sequence variation analyses between AHSV, BTV and EHDV NS3 revealed that there is a conserved threonine-serine motif in HD1, a conserved aspartic acid at the beginning of the spacer region as well as a conserved asparagine in HD2. The conservation of these residues between the different serogroups indicates that they might have some functional or structural role in the NS3 protein. NS3 protein sequences in the AHSV ã phylogenetic cluster has three extra amino acids in the spacer region at aa 149-151. There is also a significant difference in the spacer region length between EEV NS3 and AHSV NS3, which might have an effect on the topology of the respective proteins. In the spacer region of AHSV NS3, three regions were targeted for mutation analyses: substitution of the conserved isoleucine (aa 141) with a neutral amino acid, deletion of three amino acids (aa 149-151), and insertion of 15 additional amino acids. Three mutants were generated and assayed: the IÄS mutant, the KGDdel mutant and the 15 aa insert mutant. The cytotoxicity of the IÄS and KGD deletion mutants was exactly the same as the native NS3 protein, while the 15 aa insertion resulted in a slightly decreased cytotoxicity. This was the same as the EEV NS3 cytotoxicity level in insect cells. The fluorescent localisation and subcellular fractionation studies showed that both the IÄS mutation and 15 aa insertion affected the localisation of NS3 within the cell and also increased the solubility of the protein. This indicated that even though the membrane localisation of NS3 was disrupted it still had a cytotoxic role in the cell. It could be that wild type NS3 has both a membrane associated and a soluble fraction within cells, and that the soluble fraction is indeed responsible for the observed cytotoxicity and not the membrane fraction as assumed previously. It is therefore postulated that NS3 might have different domains that have various roles within the cell. / Dissertation (MSc)--University of Pretoria, 2009. / Genetics / unrestricted

Polymerase chain reaction (PCR) sequencing of specific viral gene segments was used to investigate the phylogenetic relationships among the orbiviruses. Sequence comparisons of the bluetongue virus (BTV) RNA3 from different regions of the world (North America, South Africa, India, Indonesian, Malaysia, Australia and the Caribbean region) showed that geographic separation had resulted in significant divergence, consistent with the evolution of distinct viral populations. There were at least 3 topotypes (Gould, 1987); the Australasian, African - American and another topotype represented by BTV 15 isolated in Australia in 1986. The topotypes of BTV had RNA3 nucleotide sequences that differed by approximately 20 per cent. Analysis of BTV-specific gene segments from animal and insect specimens showed that bluetongue viruses had entered northern Australia from South East Asia, possibly by wind-borne vectors. Nucleotide sequence comparisons were used to show the close genetic relationship between BTV 2 (Ona-A strain) from Florida and BTV 12 from Jamaica, and to investigate the reassortment of BTV genome segments in nature. The mutation rates of the BTV RNA2 and RNA3 segments were estimated to be of the order of 10(-4) nucleotide changes/site/year, similar in magnitude to that reported for other RNA viruses.

African horse sickness (AHS) is a life threatening disease of equids caused by African horse sickness virus (AHSV), a member of the genus Orbivirus in the family Reoviridae. The virus is transmitted to horses by midges (Culicoides spp.) and the disease is most prevalent during the time of year, and in areas where the Culicoides spp. are most abundant, namely in late summer in the summer rainfall areas of the country. Whilst the clinical signs and presentation of the disease were well documented by Sir Arnold Theiler (1921), very little is known or documented about AHSV dynamics or the clinical pathological and serological responses of horses to natural infection with AHSV. This dissertation describes the history and current knowledge on AHS, and the methods and results of a prospective study on natural AHSV infection of horses, undertaken between 2009 and 2010 by the Equine Research Centre (ERC) at the University of Pretoria, Faculty of Veterinary Science, Onderstepoort. This study is the first documented study of its nature and included animals of various ages and therefore variable vaccination status. The objectives of the study were to describe the viral dynamics of AHSV infection in horses, to gain a better understanding of the clinical pathological and serological responses to natural AHS infection and to demonstrate early detection of AHS infection in horses under field conditions. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

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