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

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

African horsesickness, a disease of equines caused by African horsesickness virus (AHSV), is often fatal, although the pathogenic effect in different animals is variable. Current AHSV vaccines are live attenuated viruses generated by serial passage in cell culture. This process affects virus plaque size, which has been considered an indicator of AHSV virulence (Erasmus, 1966; Coetzer and Guthrie, 2004). The most likely AHSV proteins to be involved in viral virulence and attenuation are the outer capsid proteins, VP2 and VP5, due to their role in attachment of viral particles to cells and early stages of viral replication. Nonstructural protein NS3 may play an equally important role due to its function in release of viral particles from cells. Two viruses were obtained for this study, AHSV-4(1) and AHSV-4(13). The thirteenth passage virus, AHSV-4(13), originated from the primary isolate AHSV-4(1). The three most variable AHSV proteins are VP2, VP5 and NS3. The question of sequence variation of these proteins between AHSV-4(1) and AHSV-4(13) arising during the attenuation process was addressed. The subject of plaque size variation between these viruses was also investigated. Some of the sequence variation observed in NS3, VP2 and VP5, between AHSV-4(1) and AHSV-4(13), occurred in protein regions that may be involved in virus entry into and exit from cells. The sequence information also indicated that AHSV-4(1) and AHSV-4(13) consist of genetically heterogeneous viral pools. The plaque size of AHSV-4(1) was variable, with small to relatively large plaques, whereas the plaques of AHSV-4(13) were mostly large. During serial plaque purification of AHSV-4(1) plaque size increased and became homogenous in size. No sequence variation in NS3 or VP5 of any of the plaque variants could be linked to variation or change in plaque size. NS3 and VP5 have a possible role in the AHSV virulence phenotype, and exhibit cytotoxic properties in bacterial and insect cells. As these proteins have not been studied in mammalian cells, an aim of this study was to express them in Vero cells and investigate their cytotoxic and membrane permeabilization properties within these cells. The NS3 and VP5 genes of AHSV-4(1) and AHSV-4(13) were successfully inserted into a mammalian expression vector and transiently expressed in Vero cells. The transfection procedure was optimized using eGFP, but expression levels were still low. When NS3 and VP5 were expressed, no obvious signs of cytotoxicity were observed. Cell viability and membrane integrity assays were performed and expression of NS3 and VP5 in Vero cells had no detectable effect on cell viability or membrane integrity. Low expression levels may have resulted in protein levels too low to cause membrane damage or affect cell viability. As Vero cells support AHSV replication, low levels of NS3 and VP5 may not be cytotoxic in these cells. NS3 was further investigated by expressing an NS3-eGFP fusion protein in Vero cells. Putative localization with membranous components and possible perinuclear localization of the fusion protein was observed. These observations may be confirmed with more sensitive microscopic techniques for a better assessment of the localization. / Dissertation (MSc (Genetics))--University of Pretoria, 2009. / Genetics / unrestricted

African horsesickness (AHS), caused by African horsesickness virus (AHSV), is an infectious, non-contagious, insect-borne viral disease that affects members of the Equidae family. AHSV is a non-enveloped virus, consisting of 10 segments of double stranded RNA (dsRNA) encoding seven structural and four non-structural proteins. Infection of mammalian cell cultures with AHSV leads to severe cellular pathogenesis effects (CPE), whereas insect cells show no noticeable CPE. Differences are also apparent between different serotypes of AHSV with regards to viral production, viral release, membrane permeabilisation and CPE. In this study we investigated different aspects of the AHSV life cycle in cell culture. The first aim of this study was the development of a real-time RT-PCR assay to quantify and monitor dsRNA from AHSV-infected cells. The dsRNA was used to quantify viral production, as dsRNA (one copy of each segment) is found only within viral particles and is not free within the cytoplasm of infected cells, thus giving a true representation of the amount of virus. This was achieved by cloning genome segment 5, optimising the extraction and purification of dsRNA, optimising the cDNA synthesis reaction, as well as the establishment and standardisation of the real-time PCR reaction. The second part of the study investigated and compared viral production and viral release between three different serotypes of AHSV in either mammalian or insect cell lines. The amount of dsRNA, which represented cell associated virus from AHSV-3- and AHSV-4-infected BHK cells over a 48 hr time period, was monitored by real-time RT-PCR and revealed a second wave of dsRNA production. These findings possibly suggest that a second round of infection of released viruses is re-entering previously uninfected or infected cells to replicate further. AHSV production was monitored in KC cells and indicated no production of progeny virions. However, an improvement was obtained when AHSV was first passaged on KC cells before being used for infections. The results from this study are in agreement with the fact that for a particular virus to replicate efficiently in a specific cell line, it should first be adapted to those cells. The dsRNA was quantified from samples representing equivalent amounts of infectious virus (i.e. same titre values) of AHSV serotypes 2, 3 or 4. The amount of dsRNA was approximately four-fold higher from serotype 2 than from serotypes 3 and 4. When the percentage of viral entry into cells was analysed, the majority (approximately 90%) of virus from serotypes 3 and 4 entered the cells, whereas serotype 2 showed viral entry of only about 50%. These findings suggested that a large amount of virus from serotype 2 was non-infectious, while the majority of virus from serotypes 3 and 4 was infectious. However, serotype 2 was a great deal more cytotoxic to cells (e.g. earlier onset and severity of CPE) when compared to cells infected with either serotypes 3 or 4. / Dissertation (MSc)--University of Pretoria, 2010. / Genetics / unrestricted