The highly hydrophobic viral protein (VP) 7 of African horse sickness virus (AHSV) folds into a trimeric structure that aggregates to form flat, hexagonal crystals (Chuma et al., 1992). These crystals are composed of flat sheets of hexameric rings, similar to the rings of trimers seen in the outer core surface layer. The crystals have been shown to be highly immunogenic when used as a subunit vaccine and are able to elicit a strong immune response against subsequent viral infections (Wade-Evans et al., 1997). The aim of this study is to investigate the structural constraints of using these structures as a particulate, multiple peptide vaccine delivery system. Three hydrophilic regions at amino acid position 144, 177°and 200 on the VP7 surface of this trimeric structure were targeted for insertion of peptides and a new vector was constructed in this study with a multiple cloning site at each one of the three top domain sites. The newly constructed three-site VP7 mutant gene was expressed in the Bac- To-Bac expression system and the recombinant proteins were investigated for its solubility and crystal formation by sucrose density gradient centrifugation. The structure and stability of the modified, trimeric VP7 was confirmed and further analyzed. Scanning electron microscopy showed the formation of large structures by the trimeric modified VP7 protein units. These' structures differed from the hexagonal crystals formed by unmodified VP7, resulting in rough-looking, flat circular structures attached by protein cables. The high yield of protein expression and the ease, with which these particles can be purified, makes this vector ideal for vaccine use. These protein structures also seemed to remain stable after being stored under different conditions. Studies were also conducted on the stability of these structures after sonication, enabling a range of different size particles to be presented to the immune system. The purpose for the creation of multiple cloning sites was for the vaccine to be able to accommodate and efficiently present multiple epitopes to the immune system. An investigation was launched into the effect of peptide insertion at one or more of the multiple cloning sites. The initial study included the insertion of two small peptides from AHSV VP2 at amino acid sites 144 and 177 respectively. The size of the peptides that can be inserted is also very important in the use of virus-like particles as antigen carriers. In order to utilize the full potential of the VP7 particles as an antigen presentation system, it must be possible to accommodate large epitope-containing insertions. At the extreme, a stretch of 250 amino acids from AHSV VP2 was inserted into the 177 amino acid multiple cloning site of the three-site VP7. Structural evaluation of all these expressed proteins indicated that the structure of the VP7 subunit vaccine is stable and still retains the ability to form large aggregated structures from the trimeric units. Scanning electron microscope revealed that all these peptide-containing constructs retain approximately the same structural shape as the structures formed by the three-site VP7 mutant. / Dissertation (MSc(Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

A biosystematic study of seven Afrotropical and two Oriental species of the Imicola species-group was undertaken; this group of Culicoides of the subgenus Avaritia includes C. imicola the most important vector of the viruses of bluetongue (BT) and African horsesickness (AHS) known in the Old World. Five African species are redescribed i.e. C. imicola, C. pseudopallidipennis, C. bolitinos, C. miombo and C. loxodontis. Two new species are described, and the extralimital C. brevitarsis and C. nudipalpis are discussed where relevant. These nine species comprise the Imicola group, one of 10 groups constituting the subgenus worldwide. Due to confusion in the literature, the Imicola group is redefined and distinguished from the Orientalis group (also redefined); 21 species of approximately 70 world species of Avaritia are reassigned to either of the two groups. A key to all nine known species of the lmicola group is given; shortcomings in the taxonomy of the Orientalis group are discussed. The adult morphology of both sexes of the nine Imicola group species was studied; this revealed deficiencies in the descriptive format currently used in taxonomic studies of world Culicoides. Accordingly, greater detail has been introduced into descriptions and includes the use of new character states. Methods for mounting Culicoides on glass slides are also improved to ensure more accurate observation and measurement of diagnostic taxonomic features; furthermore, the descriptions are based on long series of each sex. Illustrations were made from specimens mounted symmetrically, and no feature omitted from any bodypart illustrated. Certain aspects of the life-cycle of most species were investigated but especially those of C. bolitinos, C. loxodontis and C. kwagga. The immatures of these develop exclusively in the dung of the elephant, the buffalo, the blue wildebeest, both species of rhinoceros and Burchell's zebra. Two of these species, C. bolitinos and C. kwagga, have broadened their resource range as they can invade and mature in the dung of cattle and horses. This has obvious implications for the transmission of viruses, especially where indigenous herbivores are run with domesticated livestock. In an intensive two-year survey a comparison was made between the Culicoides fauna of a natural area, the Kruger National Park (KNP), and that found in areas changed by Man, namely livestock farms adjoining the KNP. This part of the eastern Transvaal lowveld is a main focus of AHS in South Africa. Light-trapping, rearing from dung, and pootering off live hosts, revealed that some species of the Imicola group are exclusively associated with certain herbivores; these include the elephant and the zebra which are suspected or proven reservoir hosts for AHS. The results thus throw further light on the epidemiology of this disease, and also show that Man plays a decisive role in determining the numbers, and distribution, of particular Imicola group species under certain conditions. In the case of C. imicola, the commonest and most widespread of all species, this range expansion, or establishment of foci, is due to man's maintenance of domesticated livestock in confined species, and especially where these are kept on irrigated pastures. However, the serendipitous discovery of a large imicola-free zone in South Africa indicates that edaphic conditions likely play an even more important role than Man and climate in determining the prevalence and abundance of C. imicola. This area is the sandy dune field west of Port Elizabeth and holds promise as a natural quarantine zone for the import and export of livestock. AFRIKAANS : 'n Biosistematiese ondersoek van sewe Afrotropiese en twee Orientale spesies van die Imicola groep is gedoen; by hierdie groep Culicoides van die subgenus Avaritia word C. imicola wat as die mees belangrikste vektor van bloutong-(BT) en perdesiekte (AHS) virus in die Ou Wereld beskou word, ingesluit. Vyf Afrika spesies, nl. C. imicola, C. pseudopallidipennis, C. bolitinos, C. miombo en C. loxodontis, is herbeskryf. Twee nuwe spesies is beskryf asook die suid-oos Asiese spesies C. brevitarsis en C. nudipalpis word bespreek waar van toepassing. Hierdie nege spesies vorm die Imicola groep, een van die 10 groepe waaruit die subgenus Avaritia wêreldwyd bestaan. As gevolg van verwarring in die literatuur is die Imicola groep hergedefinieer en geskei van die Orientalis groep ( ook hergedefinieer); 21 spesies van ongeveer 70 wereld spesies van Avaritia is heringedeel in die twee groepe. 'n Sleutel vir al nege wereld spesies van die Imicola groep, asook 'n verspreidingskaart vir elke spesie, word gegee. Tekortkominge in die taksonomie van die Orientalis groep word ook bespreek. Die volwasse morfologie van beide geslagte van die nege Imicola groep spesies is bestudeer; dit het gebreke in die formaat wat tans vir die taksonomiese beskrywing van wêreld Culicoides gebruik word aan die lig gebring. Gevolglik is daar meer data in die beskrywings, wat nuwe karakterkenmerke insluit. Die metode van die montering van Culicoides op glasplaatjies is ook verbeter om meer akkurate ondersoek en meting van die diagnostiese kenmerke te verseker; verder, is beskrywings gebaseer op lang reekse van elke geslag. Illustrasies is gemaak van voorbeelde wat simmetries gemonteer is en geen kenmerk is uitgelaat van enige gelllustreerde liggaamsdeel. Aspekte van die lewensiklus van die meeste van die spesies, veral C. bolitinos, C. loxodontis en C. kwagga is ondersoek. Die onvolwassenes van hierdie spesies ontwikkel slegs in die mis van olifante, buffels, wildebeeste, renosters en zebras. Twee van die spesies, C. bolitinos en C. kwagga, het hulle broeimediums van voorkeur vergroot en kan eiers lê en tot volwassenheid ontwikkel in die mis van beeste en perde. Dit het vanselfsprekende gevolge vir virusoordrag tussen inheemse herbivore en vee, veral in gebiede waar die twee groepe saamloop. In 'n intensiewe twee-jaar studie, is 'n vergelyking gemaak tussen die Culicoides fauna soos aangetref in 'n ongerepte deel van Afrika, die Kruger Nasionale Park (KNP), en in gebiede aangrensend aan die KNP wat deur die mens in veeplase omskep is. Die deel van die Oos Transvaalse laeveld is bekend as 'n perdesiekte "hotspot" in Suid-Afrika. Ligvalvangste, uitbroei van Culicoides uit mis en versameling vanaf lewendige gash ere het aangedui dat sekere spesies van die Imicola groep eksklusief met sekere herbivore geassosieeris; hierby ingesluit is die olifant en zebra wat onderskeidelik verdagte en bevestigde gashere van AHS is. Die resultate dra by tot die verklaring van die epidemiologie van hierdie siekte en wys ook dat die mens 'n beslissende rol speel in die vasstelling van die getalle sowel as die verspreiding van spesifieke spesies van die Imicola groep. In die geval van C. imicola, die mees algemeenste en wydverspreidste spesie, is die uitbreiding, of daarstelling van fokuspunte, te wyte aan die mens se instandhouding van vaste bloedbanke op besproeide weiding. Die toevallige ontdekking van 'n groot imicola-vry sone elders in Suid-Afrika dui daarop dat grondtipe moontlik 'n bepalende rol kan speel in die aanwesigheid en volopheid van C. imicola. Hierdie "skoon" area is die sandduine-veld wes van Port Elizabeth en lyk belowend as 'n natuurlike kwarantyn gebied vir die invoer en uitvoer van lewende hawe. / Dissertation (MSc)--University of Pretoria, 2013. / Animal and Wildlife Sciences / unrestricted

African horsesickness virus (AHSV) , a member of the genus Orbivirus in the family Reo viridae, is the aetiological agent of African horsesickness, a highly infectious non-contagious disease of equines. The AHSV virion is composed of seven structural proteins organised into a double layered capsid, which encloses ten double-stranded RNA segments. The double stranded (ds) RNA genome of AHSV encodes, in addition to the seven structural proteins, at least three non-structural proteins (NS1 to NS3). The assembly of viral proteins in AHSV-infected cells results in at least three characteristic particulate structures. The first of these structures are the complete virions and viral cores. Empty virions or particles that simulate the virion surface can be produced synthetically by the co-expression of various combinations of AHSV structural genes in insect cells. Apart from the core particles and complete virions, there are two additional structures observed in AHSV-infected cells. Unique virus-specified tubular structures, composed of NS1, are observed in the cytoplasm of all orbivirus-infected cells. The second structure, distinctive hexagonal crystals, is unique to AHSV and is composed entirely of VP7, the major core protein. The assembly of all these particles can be produced synthetically when expressed individually in an insect cell expression system. The aim of this investigation was first of all to investigate the structure and assembly of these structures and secondly to evaluate their use as vehicles for foreign immunogens. The NS1 gene of AHSV-6 was cloned as a complete and full-length cDNA fragment from purified dsRNA genome segment 5 and the complete nucleotide sequence determined. The gene was found to be 1749 bp in length with one major open reading frame (ORF) of 1645 bp, encoding a protein comprising 548 amino acids. The 5' and 3' termini of the gene were found to contain the conserved terminal hexanucleotide sequences of AHSV RNA fragments, followed by inverted heptanucleotide repeats. The deduced amino acid sequence was analysed and found to define a hydrophobic protein of 63 kDa. Antigenic profile analysis indicated a hydrophilic domain with relative high antigenicity in the C-terminus of the protein. This represents a possible insertion site for immunogenic epitopes. The cloned NS1 gene of AHSV-6 was modified at the 5' and 3' terminal ends to facilitate expression of the gene. In vitro expression yielded a protein corresponding to the predicted size of NS1. The gene was also expressed in insect cells, using a recombinant baculovirus and yields of approximately 1.0mg NS1 protein/106 cells were obtained. Expression of NS1 in insect cells resulted in the intracellular formation of tubular structures with diameters of 23 ±2 nm. Biophysical analysis of the AHSV tubules suggests that they are more fragile and unstable than BTV NS1 tubules. To gain more insight into the structure, assembly and the biochemical characteristics of AHSV cores and virions, a number of baculovirus multigene expression vectors have been developed and utilised to co¬express various combinations of AHSV genes. Cells infected with a dual-recombinant baculovirus, expressing AHSV-9 VP3 and VP7 genes, contained high levels of VP7 and low levels of VP3. The simultaneous expression of the two proteins resulted in the spontaneous intracellular assembly of empty multimeric core-like particles (CLPs) with a diameter of approximately 72 nm. These particles structurally resembled authentic AHSV cores in size and appearance. The yield of CLP production was low as a result of the insolubility of VP7, which aggregates preferably into large hexagonal crystal as well as the low yield of VP3. The interaction of CLPs with either VP2 or VP5 was investigated by co-infection of the VP3 and VP7 dual recombinant baculovirus with a VP2 or VP5 single recombinant baculovirus. Each of the outer capsid proteins interacted separately with CLPs. Co-expression of all four major structural proteins of AHSV, using two dual recombinant baculoviruses one expressillJg VP2 and VP3, the other VP5 and VP7, resulted in the spontaneous assembly of empty virus-like particles with a diameter of 82 nm. Although co¬expression of the different combinations of AHSV proteins was obtained, the levels of expression were low. This low levels of the AHSV capsid proteins and the aggregation of VP7 down regulated the assembly process. In order to investigate the possibility of the use of CLPs and VP7 crystals as particulate delivery systems, insertion analysis of VP7 was used to identify certain sequences in the VP7 protein that are not essential for the assembly of CLPs or trimer-trimer interactions in the crystals. Two insertion mutants of VP7 (mt177 and mt200) were constructed. In each case three unique restriction enzyme sites were introduced that coded for six amino acids. In mt177 these amino acids were added to the hydrophilic RGD loop at position 177-178 and for mt200 to amino acid 200 - 201. Both regions were located in the top domain of VP7. Insertion mt177 increased the solubility of VP7, but did not abrogate trimerisation and CLP formation with VP3. The yield of mutant CLPs was significantly higher than the normal CLPs, possibly due to the increased solubility and availability of VP7 trimers. Evidence about the size of an insert that can be accommodated by VP7 was provided by the insertion of a 101 amino acid region of VP2, containing a previously identified immunodominant region of VP2. The two chimeric VP7/TrVP2 proteins were investigated for their ability to form crystal structures and CLPs. The chimeric proteins did not produce the typical hexagonal crystal structure, but rather small ball-like structures. This investigation yielded valuable information regarding the structure and assembly of AHSV tubules, CLPs and VLPs. These findings also have practical value, since the multimeric structures can be utilised as delivery systems for immunogens, like the AHSV VP2 immunodominant epitopes. / Thesis (PhD (Genetics))--University of Pretoria, 2007. / Veterinary Tropical Diseases / unrestricted

Please read the abstract in the section 00front of this document / Thesis (PhD (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

The aims of this investigation were to compare the nonstructural proteins, NS3 and NS3A, of African horse sickness virus (AHSV) and to further characterize the cytotoxic properties of these proteins. NS3 and NS3A are encoded from two in-phase overlapping reading frames on the smallest double-stranded (ds) RNS genome segment, segment 10 (S10) of AHSV. The proteins differ only with respect to the presence of an additional 10 or 11 amino acids, depending on the serotype, at the N-terminal of NS3. All known orbiviruses have been shown to encode two closely related proteins from S10. Sequence analysis of the N-terminal region of the NS3 proteins of the different serotypes of AHSV and various orbiviruses revealed that this region is not highly conserver. Both AHSV NS3 and NS3A are membrane-associated and cytotoxic to insect cells causing membrane permeabilisation and eventual cell death when expressed individually (Van Staden et al., 1995; Van Staden et al., 1998; Van Niekerk et al., 2001a). AHSV infection of Vero cells results in the synthesis of both proteins in equimolar amounts (Van Staden, 1993). The effect on the cytotoxic properties of these proteins when expressed together in insect cells was therefore investigated here. Whether co-expressed or expressed individually NS3 and NS3A caused a dramatic decrease in the viability of insect cells. The NS3 protein is therefore representative of the NS3 and NS3A proteins together in terms of its cytotoxic effect. The effect of the exogenous addition of NS3 on the membrane permeability of Vero cells was also investigated. The NS3 protein was found to cause a rapid increase in the membrane permeability of Vero cells. The cytotoxic properties of NS3 appear therefore not to be limited to their endogenous effects on insect cells. The AHSV-3 NS3 and NS3A proteins were expressed as histidine tagged recombinants in the baculovirus expression system, to allow for the purification of large quantities of protein for functional and comparative studies. The resulting NS3 histidine fusion product, however, displayed a decrease in solubility, probably as a result of incorrect folding due to the presence of this histidine tag extension at the N-terminus of the protein. To produce antibodies that detect NS32, and not NS3A, in AHSV infected cells, the N-terminal region unique to AHSV-3 NS3 was displayed on the surface of the AHSV core protein, VPO7. The chimeric protein VP7-NS3 displayed the same structural characteristics as the wild type VP7 protein, aggregating into highly insoluble crystals. Antiserum was prepared against purified VP7-NS3 and analysed in terms of its ability to recognize denatured and non-denatured AHSV-3 NS3. Although the antiserum was shown to contain antibodies directed against VP7 epitopes no immune reaction with NS3 was observed. The use of alternate sites on the surface region of VP7 for the display of such a small peptide needs to be investigated. Although no functional differences between NS3 and NS3A were identified in this investigation, the finding that NS3 causes membrane permeability of damage to Vero cells represents the first indication that this AHSV protein causes extra cellular membrane damage in mammalian cells. Many viral membrane damaging proteins or viroporins are thought to contribute significantly to the severity of virus-induced pathogenesis. The mechanism of membrane damage and the contribution of the membrane damaging properties of NS3 to AHSV-induced pathogenesis needs to be investigated / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

African horsesickness is caused by the AHSV, a member of the genus Orbivirus, family Reoviridae. Nine serotypes have been identified. The viral genome consists of ten double stranded (ds) RNA segments encoding at least 7 structural and 4 non¬structural proteins. The major core proteins VP3 and VP7 together with the minor core proteins VP1, VP4 and VP6 form the core particle surrounding the 10 dsRNA segments. An outer capsid, consisting of two major structural proteins VP2 and VP5 surrounds the core. VP2 is the most variable of the proteins within the AHSV serogroup and carries serotype specific epitopes which induce a protective immune response against virulent homologous AHSV challenge. The VP2 protein is therefore the antigen of choice for the development of a subunit vaccine against AHSV. It has been shown that protection against AHSV-4 can be achieved by vaccination with AHSV VP2 protein. The AHSV-3 VP2 protein has previously been cloned and expressed as baculovirus recombinant protein in our laboratory. The recombinant protein induced only a weak neutralising immune response. It has been determined in this investigation that the majority of recombinant AHSV-3 VP2 proteins expressed in Sf-9 insect cells are in an insoluble, aggregated form. This is likely to be the cause of the poor neutralising immune response induced by this protein. In order to investigate this problem two strategies were adopted. First an attempt was made to chemically solubilise the particulate VP2 protein and refold the protein into a form that may present the neutralising epitopes more appropriately. The solubilisation of the protein with 6M Guanidinium HCI was successful, but the largest percentage of the protein was again rendered insoluble during the refolding process which involves the removal of Guanidinium HCI by column chromatography. The chemical solubilisation therefore proved to be too inefficient to provide a solution to the problem. The second method for increasing the solubility and immunogenicity of the VP2 protein was by co-expression of VP2 and VP5, the two outer capsid proteins of AHSV¬3. For the dual expression of the two proteins it was necessary to characterise the AHSV-3 VP5 gene and express it as a baculovirus recombinant first. The VP5 gene was therefore sequenced. A nucleotide sequence of 1566 bp was determined encoding a peptide of 505 amino acids with a predicted size of 56K. The VP5 was expressed as baculovirus recombinant using the baculovirus Bac-to-Bac™ expression system. The yield of VP5 was low but was nevertheless better than the expression levels of AHSV-9 VP5 gene using an alternative baculovirus expression system. AHSV-3 VP2 and VP5, were cloned respectively under the polyhedrin and p10 promoters of the pFastbac dual transfer vector of the Bac-to-Bac™ baculovirus expression system. mRNA transcription of both AHSV-3 VP2 and VP5 genes in Sf-9 cells was shown. The expression of VP2 was also demonstrated but VP5 was very poorly expressed by the dual recombinant. Further research to determine the effect co-expression of AHSV-3 VP5 in the AHSV-3 VP2 antigenicity is needed. / Dissertation (MSc Agric (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

Two particulate structures based on AHSV major structural protein VP7, are under investigation as possible vectors for epitope display. Due to the extreme insolubility of the VP7 protein it aggregates into large hexagonal crystalline particles when expressed in an insect cell expression system (Chuma et aI, 1992). To investigate its ability to present immunologically important epitopes to the immune system, VP7 mutants have been constructed that allow the presentation of foreign peptides on the surface these particles (Maree, 2000). In part of this study AHSV core-like particles resulting from the co-expression of the two major structural proteins VP3 and VP7 were under investigation. Due to the low solubility of VP7, low core-like particle yields are obtained during co-expression (Maree, 2000). As a result not enough of the particles can be produced to make its use viable. To increase the core-like particle yield it is necessary to increase the solubility of VP7. Several amino acids have been implicated in the observed low solubility of VP7 (Monastyrskaya et aI, 1997). One of these amino acids was targeted for site-specific mutation in an effort to increase protein solubility. Leucine 345 is located on the ninth C-terminal helix in the bottom domain of VP7 and was substituted to arginine, a polar positively charged residue. The substitution was made in wild type VP7 as well as in insertion mutants 177 and 200. The mutation was effected via PCR and the resulting mutant genes were expressed in the Bac-To-Bac expression system. The newly constructed mutant proteins were further investigated for an increase in solubility by differential and sucrose density centrifugation. The site specific mutation in wild type VP7 and insertion mutant 200 resulted in a slight increase in solubility whereas the mutation in mutant 177 had no effect on solubility. As a result of failing to significantly increase the solubility of these mutants, no increase in the core-like particle yield was achieved. In the second part of this study recombinant VP7 crystals were constructed which present a single and triple repeat of the HIV-1 transmembrane protein gp41 neutralizing antibody epitope, ELDKWA. To this end, oligonucleic acid adaptors coding for the epitope repeats were designed and cloned into VP7 insertion sites 144 and 177. The newly constructed VP7 mutant genes were expressed in the Bac-to-Bac expression system and the recombinant proteins were investigated for its solubility and crystal formation by sucrose density gradient centrifugation. All of the epitope presenting constructs were significantly less soluble than insertion mutants 144 and 177 and retained its ability to assemble into large hexagonal crystals. Gradient purified particles were injected into Balb/c mice and the epitope specific antibody response determined by dot and western blot analysis. Although a humoral immune response was induced against the VP7 constructs none were able to induce antibody responses specific to the ELDKWA epitope. / Dissertation (MSc (Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

Factors that determine the molecular basis of African horsesickness virus (AHSV) virulence are unclear. Several proteins and different events in the viral replication cycle together with different environmental factors are likely to contribute to the virulence phenotype. The aim of this investigation was to study the possible contribution of AHSV nonstructural protein NS3 to virus virulence. NS3 is a cytotoxic protein that localizes in areas of plasma membrane disruption and is assumed to be associated with the release of virus particles from infected cells. A conserved feature in all AHSV NS3 proteins is the presence of two hydrophobic domains. To investigate whether these hydrophobic domains interact with membranes, cell surface localization and membrane association studies were conducted on NS3 and the two hydrophobic domain mutant forms of NS3. Results indicated that mutations in either hydrophobic domain did not prevent membrane targeting but abolished membrane anchoring. This prevented cell surface localization and obviated the cytotoxic effect of NS3. AHSV NS3 is a highly variable protein. This level of variation may influence virulence properties. To compare the NS3 sequence variation level of South African AHSVs and bluetongue viruses (BTV/s) with those previously described, the NS3 protein sequences of field isolates, reference and vaccine strains were determined and analysed. The variation level of AHSV NS3 was found to be higher than previously reported. Comparison of the AHSV vaccine and field strain NS3 sequences revealed no obvious NS3 virulence marker. The level of AHSV NS3 sequence variation could distinguish between field isolates and vaccine strains and differentiate between sub-populations within a serotype. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters with exception of AHSV-8 and one AHSV-6 encoded NS3. BTV NS3 inferred phylogeny indicated that the three BTV NS3 clusters that occur as geographically defined entities are all simultaneously present in S.A. This would suggest that BTV originated in southern Africa. To investigate the impact of minor NS3 variation on AHSV virulence, the membrane permeabilization properties of AHSV-2 vaccine and virulent NS3 proteins were compared. The AHSV-2 S10 genes were cloned and NS3 was expressed using the BAC-TO-BAC system. The membrane permeabilization effect of NS3 expression was monitored by calcium influx into insect cells. Expression of either the vaccine or virulent associated AHSV-2 NS3 protein equally increased membrane permeability. The NS3 sequence variation therefore had a limited influence on membrane permeabilization properties and the virulence status of the vaccine and virulent AHSV-2 strains in this study. The possible effect of larger NS3 sequence variation levels on AHSV virulence was investigated by NS3 associated properties such as virus release and membrane permeabilization. AHSV infections increased the permeability of cell membranes and the different strains had different virus release properties. Virus release was not exclusively related to increased membrane permeability of infected cells or to the yield of the virus in the infected cells. The level of NS3 variation may influence AHSV release mechanisms and membrane permeabilization properties thereby contributing to AHSV virulence properties. / Thesis (PhD (Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

African horse sickness virus (AHSV) is a ten-segmented, dsRNA virus, classified as a distinct species within the genus Orbivirus, family Reoviridae. There are nine serotypes of AHSV, any of which can cause African horse sickness (AHS), an extremely severe ‘transboundary’ and notifiable disease of horses, listed by OIE. AHS is currently restricted to sub-Saharan Africa, but has occasionally emerged causing major outbreaks in other geographic regions. Complete genome nucleotide sequences were determined for nine reference-strains of AHSV (different serotypes). The selection-pressure on each genome-segment and its encoded proteins, in relation to protein function were analysed in phylogenetic comparisons. This initial AHSV sequence database also provided a basis for molecular-epidemiology studies. In particular, the role of the Seg-2 in determination of virus-serotype was used to identify viruses involved in a multi-serotype outbreak, identifying Ethiopia as an important area of AHSV circulation. Phylogenetic-relationships were also investigated between different isolates of AHSV serotypes 2, 4, 6, 8 and 9, from various locations in Africa. Two real-time RT-PCR assays were developed for detection of the highly conserved genome segments 1 and 3, and molecular diagnosis of AHSV. Real-time RT-PCR assays were also developed, targeting Seg-2, for detection and identification of the nine AHSV serotypes. These assays are suitable for ‘high throughput’ characterisation of AHSV outbreak-strains, from either endemic or AHSV-free zones. The results obtained would facilitate rapid design and implementation of appropriate virus control measures. Cross-contamination was detected in four of the original AHSV reference-strains. A new set of plaque-cloned and monotypic reference-strains was therefore identified and characterised, and is available through the Orbivirus Reference Collection (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/AHSV-Nos.htm). Serological relationships were analysed using antibodies against VP2 of AHSV-9 (expressed by recombinant MVA) and the nine monotypic reference-strains, showing neutralisation of both AHSV-9 and AHSV-6, in agreement with their closer phylogenetic relationship within Seg-2/VP2.

Thesis (MSc (Veterinary Science))--University of Pretoria, 2002. / Includes bibliographical references.