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) belongs to the Orbivirus genus in the family Reoviridae. This non-enveloped virus consists of an outer capsid formed by two structural proteins, VP2 and VP5, and an inner core formed by structural proteins VP7 and VP3. Three additional structural proteins associated with viral replication, as well as ten dsRNA molecules responsible for replication, are found inside the core. VP7 is the smallest of the structural proteins and each monomer consist of two domains, a hydrophilic top and hydrophobic bottom domain. Upon expression of VP7, the protein spontaneously assembles into trimers. Recombinant expression of the core protein VP7 results in large hexagonal structures formed by a double layer of these VP7 trimers with the hydrophobic bottom domains on the inside and hydrophilic top domains on the outside. The use of these crystal structures as a general display system for the display of foreign peptides/epitopes is being investigated in our group. In this regard, sites for the insertion of foreign peptides/epitopes were constructed at amino acid positions 177, 144 and 200 of the top domain of the VP7 protein and the resultant proteins named vectors AHSV-9 VP7-177, AHSV-9 VP7-144 and AHSV-9 VP7-200. Various inserts ranging from the HIV-1 ELDKWA epitope and FMDV VP1 epitopes to the eGFP peptide were inserted and subsequently analysed for immunogenicity. Results showed that a significant immune response was only elicited if the soluble trimer component of a chimeric VP7 protein was used for inoculation purposes. The crystal particles initially investigated as a display system did not result in any immune response. These results emphasized the importance of protein solubility for eliciting a significant immune response. The importance of solubility prompted an investigation into the use of the Bluetongue virus (BTV) VP7 protein as a vaccine display system. This protein is inherently more soluble than AHSV VP7 and does not result in crystal hexagonal structures if recombinantly expressed. An insertion site analogous to that of the AHSV-9 VP7-177 vector, located at amino acid 177 within an RGD loop in the top domain of VP7 was constructed. This new BTV VP7 vector, BTV-10 VP7-169, was characterised with regard to solubility and the ability to form trimers. In order to investigate the effect on solubility and trimerisation, FMDV VP1 epitope and eGFP were inserted into the BTV-10 VP7-169 vector. Results showed that following the construction of the insertion site, the vector was largely insoluble compared to the AHSV VP7 vectors and that insertion of the abovementioned peptides/epitopes did not have a significant effect on solubility. Although trimers were present, the yield was low compared to that of the AHSV chimeric VP7 proteins. Methods of improving the solubility of the chimeric VP7 proteins were investigated by treatment with solubilisation agents, sarkosyl and L-arginine. The results indicated that a strong denaturant such as sarkosyl can solubilise the particulate component of all chimeric VP7 proteins whereas Larginine had limited effect. The effect of these agents on the folding of the proteins were evaluated using fluorescence, since the ability to fluoresce is regarded as an indicator of correct folding. A comparison of the different VP7-eGFP proteins treated with these solubilisation agents showed that the sarkosyl solubilised proteins were not necessarily correctly folded. These results combined with the previously performed solubility assays indicated that a large proportion of correctly folded chimeric VP7 proteins associate with the particulate fraction. Investigation showed that expression of a large amount of correctly folded chimeric proteins results in the aggregation of these proteins within the expressing host cell. Once harvested these proteins remain associated with the insoluble fraction but can be solubilised by arginine treatment, or in some cases mere resuspension in a low-salt buffer, and used for vaccination purposes. In conclusion, the comparative analyses of solubility and trimerisation for the display vectors indicated that AHSV-9 VP7-144 vector may be most suitable for the display of foreign epitopes/peptides as it consistently yielded the largest component of correctly folded proteins. Furthermore, considering that large amounts of correctly folded chimeric VP7 proteins occurred in the insoluble component of each the VP7 display proteins, this study emphasize that use of solubility assays alone does not provide adequate information regarding the potential of a display vector for vaccination purposes. / Dissertation (MSc)--University of Pretoria, 2013. / 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

The aim of this study was to standardize and validate an immunohistochemical test for the routine diagnosis of African horsesickness in horses. Hamblin developed the primary anti- African horsesickness virus serum that I used and the avidin-biotin complex detection system was employed. During the standardization process I demonstrate that lung, heart and spleen samples are the most reliable. I also show that it is not necessary to take multiple samples per organ, because the AHSV-positive signal is generally widespread throughout the lung and heart, in particular. In order to validate the technique, samples from 118 negative and 128 positive horse cases, including all nine known serotypes, were immunostained. All of the positive cases were confirmed by means of virus isolation. Negative horse samples were obtained from countries where African horsesickness does not occur. None of the negative cases stained positive and all the positive cases were correctly identified. Therefore, there was 100 % concordance between immunohisto chemistry (when applied to formalin-fixed, paraffin-embedded heart and/or lung and/or spleen tissues from positive horse cases that had been archived for less than 10 years) and virus isolation results. Heart and lung had consistently more positive signal than spleen. The Hamblin antiserum did not cross-react with closely-related orbiviruses (specifically equine encephalosis virus and bluetongue virus) in selected horse and sheep tissues, respectively. Characteristic positive staining was observed in lung, heart and spleen samples from two dogs that died of African horsesickness. Positive signal was not affected by long-term storage in formaldehyde (up to 365 days). Also, specific positive staining could be detected in heart and/or lung and/or spleen samples in more than 95 % of positive horses where tissue blocks had been stored for between 10 and 83 years. The principal target cells in the horse and dog cases were microvascular endothelial cells, intravascular monocyte-macrophages and, to a lesser extent, interstitial macrophages in lung, spleen and liver, in particular. Positive staining is intracytoplasmic with a bead/dot and/or granular character. Beads, dots or granules may occur singly or in clusters. Occasionally, linear deposits of positive signal delineate segments of capillary vessels. The veterinary pathologist must look for characteristic positive signal in target cells, because, occasionally, certain bacteria (Rhodococcus equi and Helicobacter sp.) cross-react with the Hamblin antiserum. Clearly, the test is highly sensitive, specific and robust, sufficiently so for the routine diagnosis of African horsesickness virus. / Dissertation (MSc)--University of Pretoria, 2008. / Paraclinical Sciences / unrestricted

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 horsesickness is an infectious but non-contagious disease caused by an orbivirus belonging to the Reoviridae family. The disease is classified as notifiable by the OlE because of the potential severe economic consequences that can result from outbreaks. Bluetongue, an arthropod-transmitted disease of wild and domestic ruminants, is caused by the bluetongue virus, which is the prototype species of the genus Orbivirus. Bluetongue is also a notifiable disease because it can spread very rapidly in naive populations of susceptible livestock. Strict restrictions have been issued for the trade in animals and animal products from infected areas. In the present study, a duplex one-step real-time RT-PCR using the fluorogenic dye SYBR® Green I was developed for the specific detection and identification of AHSV and BTV in one reaction, using melting temperatures (Tm) to discriminate between the viruses. Two primers pairs were designed to bind to areas of homology within genome segment 7 (VP7) of AHSV and genome segment 5 (NS1) of BTV respectively. The duplex real-time RT -PCR based test utilizes single tube RT -PCR amplification in which AHSV and BTV primers were used simultaneously. The RT-PCR primers amplified 232 bp of genome segment 7 from all nine serotypes of AHSV and 79 bp of genome segment 5 from all 22 BTV serotypes that were tested. When both viruses were present, two melting peaks were simultaneously generated at 76.30°C and 80.04°C representative of BTV and AHSV amplification products respectively. Serogroup-specific products were amplified from dsRNA of field isolates of AHSV and BTV. dsRNA from EHDV and EEV failed to demonstrate either the 232 bp specific AHSV PCR product or the 79 bp specific BTV product. These results indicate that the duplex real-time RT-PCR could be a useful technique for detection of AHSV and BTV from isolated viral dsRNA. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / 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