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

African horsesickness virus (No.3922, Type 7), attenuated for the horse by serial passage in suckling mouse brain, was studied at various passage levels to determine whether any change in the biophysical characters of the infectious particles had occurred. during the process of attenuation. Such changes were indeed observed. Propagation of the virus in tissue culture was accomplished only after the modification of standard culture media by the addition of egg white, a complex substance containing a number of proteins including the enzyme lysozyme. Some evidence is presented to show that the presence of egg white materially assisted in the successful cultivation of horsesickness virus; as well as in the formation of plaques in monolayers ·of cultured cells. Electron micrographs of horsesickness virus obtained from these cultured cells, and the results of a study of the fine structure of infected mouse nervous tissue, are presented. A remarkable change in the buoyant density of the infectious particles of this horsesickness virus was found to occur during attenuation. The 'wild' or virulent strain was found to consist mainly of particles of density 1.26 gm/ml. The attenuated strain however proved to be composed of particles with deneities quite different from that of the wild strain, predominantly 1.21 and 1.34 gm/ml. This alteration of the buoyant appeared to be directly related to the degree of attenuation. Studies in electrophoresis using a newly designed apparatus showed that the wild strain of horsesickness virus is homogeneous in its migration in an electric field. The attenuated strain showed a changed electrophoretic pattern· indicating the presence of particles of different mobilities. As in density gradient analysis, electrophoresis showed a fundamental difference between the wild and attenuated strains of this virus. It was possible also to show a correlation between the slowly migrating component of the attenuated strain and the fraction of higher density. The sedimentation coefficient of the infectious particles of the No.3922 strain of horsesickness virus was studied at various stages of attenuation ·and the particle size at three passage levels were calculated. The particle size and other characteristics determined in this way were compared with the results of measurements obtained from ultrafiltration of the virus through collodion membranes. It was found that the diameter of the infectious particles of the attenuated strain is greater than that of the wild strain. This study shows that physical measurements may be used to give

African horsesickness virus (AHSV) is a double-stranded RNA virus belonging to the Orbivirus genus in the Reoviridae family (Bremer et al., 1990; Calisher and Mertens, 1998). The virus is highly pathogenic and its mortality rate in horses, the most susceptible species, may be as high as 95% (House, 1993). S10, the smallest genome segment of AHSV, codes for two proteins (NS3 and NS3A) from in-phase overlapping reading frames. The C-terminal sequences of these proteins are identical, but NS3A lacks the first 10 amino acids present on the N-terminal of NS3 (Van Staden and Huismans, 1991). Nonstructural protein NS3 is a membrane protein, associated with both smooth intracellular membranes and the plasma membrane. NS3 has pleiotropic roles in the viral life cycle including the transport and release of mature virions and viroporin-like alteration of cell membrane permeability. NS3 is cytotoxic when expressed in bacterial or insect cells, and is speculated to play a vital role in viral virulence and disease pathogenesis (Stoltz et al., 1996; Van Staden et al., 1995). A number of different domains that could mediate the membrane interaction or intracellular trafficking of NS3 have been identified. The relative contributions of these domains in insect and mammalian cells are not known, but could differ, as there are distinct differences in NS3 expression levels, cytopathic effects and virus release mechanisms in these two cell types. In order to investigate the subcellular localisation of NS3, a number of full-length, truncated or mutant versions of AHSV-3 NS3 were constructed as C-terminal eGFP (enhanced green fluorescent protein) fusion proteins. These proteins were used to generate recombinant baculoviruses for expression in Spodoptera frugiperda (Sf9) insect cells and were compared in terms of their subcellular localisation by conventional fluorescence microscopy. Confocal laser microscopy was used to investigate co-localisation with the nucleus, the Golgi apparatus and the Endoplasmic Reticulum (ER). Subcellular fractionations and membrane flotation analyses were used to confirm membrane interactions and to identify detergent-resistant membrane fractions. NS3 as well as a C-terminal deletion of NS3 targeting a putative dileucine motif both localised to cellular/nuclear membrane components. In contrast, site-specific mutations to either of the transmembrane domains abolished membrane association and resulted in cytoplasmic localisation. NS3A showed mixed results, displaying both membrane localisation and a cytoplasmic distribution. The 11 amino acid region unique to NS3 and absent from NS3A, which has been shown to bind to cellular exocytosis proteins in bluetongue virus (Beaton et al., 2002), did not display membrane interaction. These results indicate that both of the hydrophobic domains as well as the N11 region are required to be present for NS3 to be properly targeted to the plasma/nuclear membrane. In addition, NS3 was shown to be present in detergent-resistant membrane fractions, indicative of a possible localisation within lipid rafts. The above results indicate that the NS3 protein contains specific signals involved in membrane targeting, confirming a potential role for NS3 in viral localisation and release in the AHSV replication cycle. / Dissertation (MSc (Genetics))--University of Pretoria, 2009. / Genetics / unrestricted

African horsesickness virus (AHSV) is a member of the Orbivirus genus of the Reoviridae family, being a double-layered capsid virus with a genome of ten double-stranded RNA segments. The outer capsid consists of two proteins, VP2 and VP5, which play essential roles in respectively host cell entry and viral release from the endosome into the host cytoplasm for replication. These proteins have best been characterized in the prototype virus Bluetongue virus (BTV), especially with regards to structural and functional characteristics. A cytotoxic effect for BTV VP5 was observed in insect cells and the approximate region conferring this nature identified. For AHSV VP5, a cytotoxic nature has also been observed in bacterial cells in preliminary studies but no region has thus far been mapped for mediating this activity. The main aim of this investigation was thus to map this region of AHSV VP5 using a bacterial expression system. A further aim was to investigate the localization of VP5 within infected cells using the BAC-TO-BACexpression system and the eGFP marker protein fused to VP5. In this way, protein expression could easily be detected and a possible association with cell membranes investigated. Initial cytotoxicity studies in these insect cells were also done to determine if the cytotoxic effect was also present in different host cells. To determine the region conferring the cytotoxic effect, genes encoding full-length VP5 and four truncation mutants of VP5 were cloned into the inducible pET system for expression as GSTfusion proteins within bacterial cells. After confirming protein expression, kinetic studies on the various VP5-fusion proteins were performed. Each protein increased in concentration with time post induction, except for the full-length VP5. Results from the cytotoxicity assay correlated with the expression patterns observed from the kinetic studies. Only GST-VP5 was cytotoxic. The VP5 truncation mutants lacking various N-terminal domains were all non-cytotoxic. Seeing that the only difference between GST-VP5 and GST-VP5Δ1-20 was the presence of amphipathic helix one, the results indicated that it is amphipathic helix one that plays a major role in conferring cytotoxicity. Amphipathic helix two, that is situated directly downstream of amphipathic helix one, seem to still be involved but require the presence of the amphipathic helix one and be expressed in the correct conformation. The role of amphipathic helix two in cytotoxicity could therefore not be inferred from this investigation. To determine its role, further studies involving more truncation mutants would be required. Solubility studies on all bacterial expressed proteins were performed to investigate whether the observed non-cytotoxicity of the truncated mutants might have been influenced by protein aggregation and hence not give a true reflection of the functional properties. Results indicated that a substantial portion of each mutant protein was. However, present in a soluble form and hence expected to be in a functional form. To study protein localization in insect cells using the BAC-TO-BACsystem, genes encoding VP5 and VP5 1-39 were cloned into pFB-eGFP and expressed as eGFP-fusion proteins. The recombinant baculoviruses Bac-VP5-eGFP and Bac-VP5 1-39-eGFP were used to infect insect cells at a high MOI and the green fluorescence signal of the marker monitored using confocal or fluorescent microscopy. No localization to particular cell structures was observed for either proteins and thus no specific association with membranes identified. Initial studies of cytotoxicity within insect cells were performed and the preliminary results indicate that the first two amphipathic helices are responsible for the cytotoxic effect in these cells, correlating with the results obtained in the bacterial system. This study provides the first evidence for the mapping of regions conferring a cytotoxic nature to AHSV VP5. Further characterization of this protein is necessary to obtain a better understanding of its’ role in the viral life cycle and the pathogenesis of AHS. / Dissertation (MSc (Genetics))--University of Pretoria, 2009. / Genetics / unrestricted

No abstract available. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / Unrestricted

African horsesickness virus (AHSV) belongs to the Orbivirus genus within the family Reoviridae. AHSV is transmitted to vertebrates by Culicoides midges and causes an acute disease in horses with a high mortality rate. The virion consists of an outer layer composed of proteins VP2 and VP5, which surround an icosahedral core containing two major proteins, VP3 and VP7, three minor proteins, VP1, VP4 and VP6, and ten segments of double-stranded (ds)RNA. The VP7 protein is not only important in maintaining the structural integrity of the virus particles, but has been reported to play a key role in Culicoides cell entry. The phenomenon of RNA interference (RNAi), which can be used to selectively silence homologous genes post-transcriptionally, has revolutionized approaches to study gene function and it is also projected as a potential tool to inhibit virus replication. In mammalian cells, RNAi can be triggered by the direct introduction of 21-23 nucleotide duplexes of small interfering RNA (siRNA) that specifically and potently inhibits endogenous and heterogeneous gene expression. Consequently, the aims of the investigation were to develop RNAi assays whereby the expression of the AHSV-9 VP7 gene could be suppressed in Spodoptera frugiperda insect cells and in mammalian BHK-21 cell culture, as well as to determine whether gene-specific siRNAs may prevent AHSV-9 infection in cell culture. To investigate RNAi-mediated silencing of an enhanced green fluorescent protein (eGFP) in S.frugiperda cells, the bacmid expression system was used. Transfection of the insect cells with an eGFP-specific siRNA prior to infection with the recombinant bacmid, inhibited eGFP protein expression by 75%, as quantified by fluorometry. Although the results suggest that RNAi could potentially be used as tool to study the function of an expressed transgene in insect cells, the lack of complete inhibition, coupled with the highly cytolytic nature of the bacmid, may complicate interpretation of the gene interference results. To investigate whether siRNAs targeting the AHSV-9 VP7 mRNA is able to silence VP7 protein expression, two siRNAs were designed that targeted different regions on the VP7 mRNA. siVP7-336 and siVP7-441, directed at nucleotides 336-356 and 441-461 on the VP7 coding strand, respectively, were chemically synthesized. The effect of these siRNAs on VP7 protein expression was evaluated by cotransfection of BHK-21 cells with the respective siRNAs and the VP7 expression plasmid pCMVVP7- eGFP. The results indicated that siVP7-336 and siVP7-441 inhibited VP7-eGFP expression by 88% and 75%, respectively. BHK-21 cells were subsequently transfected with the respective siRNAs in separate experiments followed by viral infection. The VP7 mRNA quantities were measured by quantitative reverse transcription PCR and the effect of the siRNAs on viral replication was evaluated by plaque assays. Of the two siRNAs, siVP7-336 was found to be the most effective inhibitor of VP7 transcription and suppressed VP7 mRNA by 93%. The exposure of BHK-21 cells to the VP7 genespecific siRNA, siVP7-336, also led to a 84% reduction in progeny virions, as measured by a plaque assay. Taken together, the results demonstrate that siRNA-mediated gene silencing is an efficient approach for reducing the level of VP7 transcripts and proteins and for inhibiting virus propagation. / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc (Agric))--University of Pretoria, 2005. / Genetics / unrestricted

The aim of this investigation was to clone, characterize and express the gene that encodes the outer capsid protein, VP2, of African horsesickness virus (AHSV), with a view to the evaluation of this protein as a subunit vaccine. The VP2 gene of AHSV serotype 3 (AHSV-3) was cloned as incomplete cDNA fragments of the genome segment 2 double-stranded (ds)RNA, sequenced in its entirety and compared with previously published cognate sequences of AHSV-4, Epizootic hemorrhagic disease virus (EHDV)-l and various bluetongue virus (BTV) serotypes. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50.5% identity to AHSV-4 VP2. Two areas of high variability (approximately 65%) were identified adjacent to the conserved termini. The N-proximal region (amino acids 128-309) exhibited significant hydrophilicity, suggesting a possible role in the determination of the serotype-specific immune response. Orbivirus interserogroup comparisons of VP2 amino acid sequences revealed extreme variability, although an overall structural conservation was demonstrated. Oligonucleotide primers derived from the AHSV-3 genome segment 2 terminal nucleotide sequences were used for PCR amplification and cloning of full length segment 2 cDNA. The cloned gene was expressed in a baculovirus expression system and the expressed VP2 protein was shown to react specifically with anti AHSV-3 serum in Western blots. Although the yields of VP2 in the baculovirus system were low, due to a possible toxic effect on the host cells, sufficient antigen was obtained for further future investigations into the efficacy of VP2 as a possible subunit vaccine against AHSV. / Dissertation (MSc)--University of Pretoria, 2010. / Genetics / unrestricted

African horsesickness virus (AHSV) is a dsRNA virus that belongs to the Orbivirus genus within the Reoviridae family. Each of the ten viral dsRNA segments encodes one virus-specific protein. During its life cycle AHSV replicates both in an insect vector and in a mammalian host, but while it has no detrimental effect on insect cells the virus is highly pathogenic to mammalian cells. It is postulated that this relates to different viral release mechanisms. Currently the main candidate for mediating viral release in both insects and mammals is the viral nonstructural protein NS3. In bluetongue virus (BTV), the prototype virus of the Orbivirus genus, it has been shown that NS3 interacts with both the viral outer capsid protein VP2 and a cellular exocytosis protein. For AHSV, we investigated whether the same mechanism was involved in viral release. This study aimed to identify and map possible protein-protein interaction between AHSV NS3 and VP2, and AHSV NS3 and unknown insect cellular proteins. For investigating the NS3-VP2 interactions a eukaryotic expression system (yeast twohybrid), a column binding assay utilising bacterially expressed NS3 and recombinant baculovirus expressed VP2 as well as a membrane flotation assay utilising recombinant baculovirus expressed VP2 and NS3-GFP, were used. A number of problems were encountered and no conclusive results were obtained. For investigating viral-cellular protein interactions the yeast two-hybrid system was also used, utilising NS3 as bait to screen proteins expressed from a Drosophila cDNA library. Results showed an interaction between the N-terminal region of AHSV NS3 and ubiquitin, an essential protein for the trafficking and degradation of membrane proteins from the endoplasmic reticulum. It also acts as a sorting signal in both the secretory pathway and in endosomes, where it targets proteins into multivesicular bodies in the lumen of vacuoles/lysosomes. It has been shown that ubiquitin could play a role in the pinching off of budding vesicles. An AHSV infected cell could therefore potentially use ubiquitin in its vesicular budding pathway, therefore giving the opportunity for viruses to use this to release them from the cell. The Hsp70 was another protein identified that interacts with AHSV NS3. This protein plays a role in folding reactions, protein translocation across membranes of organelles and protein assembly. It has been reported in other studies done that both ubiquitin and Hsp70 play roles in regulating the bioavailability of viral proteins, which could explain the different levels of NS3, high in insect cells and low in mammalian cells, which indirectly control the viral exit pathway used, budding versus lytic release. These results lay the foundation for explaining the potential role of NS3 in the AHSV life cycle in insect cells. / Dissertation (MS)--University of Pretoria, 2007. / Genetics / unrestricted

Modified Viral Protein 7 (VP7) of African horsesickness virus (AHSV) is being investigated as a peptide display protein. The protein represents a good candidate for recombinant peptide display due to its tertiary structure, which contains flexible hydrophilic loops on the top domain of the protein where small peptides can potentially be inserted. In addition, wild type (WT) AHSV VP7 tends to form hexagonal crystals of predictable shape and size when expressed in a recombinant expression system. Previous research has resulted in a number of AHSV VP7 genes containing modified cloning sites where DNA representing immunologically relevant peptides can be inserted. When these chimeric proteins are expressed the peptides should be displayed on the surface of the VP7 platform. Several studies have tested the ability to insert peptides of varying lengths into these sites and successfully express the chimeric protein. In these past cases, successful expression of a recombinant chimeric protein was gauged by the observation of particles formed by multimers of VP7 proteins that resemble the one formed by WT-VP7. However, little is known about the ability of these chimeric proteins to act as successful peptide presentations vectors. Specifically, it is not known whether the fusion peptides would retain their correct tertiary structure, or indeed be displayed to the surrounding environment in order to generate a specific immune response. Furthermore, there has been no investigation to track these chimeric proteins’ expression in a heterologous expression system. This dissertation attempts to answer several of these questions through the use of a fluorescent protein, enhanced green fluorescent protein (eGFP), as a model peptide. The use of eGFP as a model peptide can prove correct tertiary structure of the fusion peptide via function of the protein (fluorescence), as well as act as a means of monitoring expression of chimeric VP7-eGFP proteins. Chapter 1 of this dissertation reviews literature that is relevant to AHSV VP7 and the use of fluorescent proteins as fluorescent markers. In addition, the recombinant expression of proteins is discussed, with a focus on solubility and expression levels of expressed proteins. Next, a brief overview is given with regards to vaccination strategies that can be undertaken, with a focus on subunit vaccines and their viability as successful alternatives to live-attenuated vaccines. Finally, the progress with regards to using modified AHSV VP7 as a peptide presentation vector is discussed. Chapter 2 investigates the chimeric protein VP7-177-eGFP, including its construction via a recombinant DNA cloning strategy, its expression in Insect cells using a recombinant Baculovirus expression system, and the ability of eGFP to act as a model fusion peptide on the top domain of a modified VP7 protein. Several experiments investigate whether the chimeric protein maintains its tertiary structure under a series of purification steps, and investigate the solubility of the chimeric protein throughout the expression cycle. Finally, purified forms of the chimeric protein are examined for their ability to generate an immune response specific to the fusion protein, eGFP.<p / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted