Thesis (M.Sc.(Microbiology)--University of Pretoria, 2006. / Summary in English. Includes bibliographical references.

Thesis (Ph. D.)(Genetics)--University of Pretoria, 2005. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, has a 10-segment double-stranded (ds)RNA genome enclosed within a double capsid. In addition to seven structural proteins (VP1-VP7), four non-structural proteins (NS1, NS2 and NS3/NS3A) are synthesized in infected cells and are involved in virus morphogenesis. Due to the lack of a reverse genetic system for orbiviruses, analyses regarding AHSV gene function have been limited to the characterization of individual virus proteins following their expression in heterologous expression systems. The phenomenon of RNA interference (RNAi), has, however, revolutionized approaches to study the function of individual genes. RNAi is an evolutionary conserved mechanism by which RNA duplexes, known as short interfering RNA (siRNA), can reduce gene expression through enzymatic cleavage of complementary mRNA. In addition to synthetic siRNA, RNAi can also be induced in mammalian cells by plasmid and viral vector systems that express short hairpin RNAs (shRNAs) that are subsequently processed to siRNAs by the cellular machinery. Consequently, the aim of this investigation was to establish a plasmid DNA vector-based RNAi assay whereby the expression of the AHSV-9 NS2 gene could be suppressed in BHK-21 cells. Complementary oligonucleotides corresponding to selected AHSV-9 NS2 gene sequences were chemically synthesized, annealed and cloned into the pSUPER shRNA delivery vector, downstream of the RNA polymerase III H1 promoter. The vector-expressed shRNAs targeted regions within the NS2 gene corresponding to nucleotides 211 to 230 (shRNA-211), 377 to 396 (shRNA-377) and 958 to 977 (shRNA-958), respectively. To determine whether the NS2-directed shRNAs was capable of silencing NS2 protein expression, BHK-21 cells were co-transfected with the respective pSUPER shRNA delivery vectors and a NS2 reporter plasmid, pCMV-NS2-eGFP. Fluorescence microscopy indicated that NS2-eGFP expression was makerdly reduced in these cells compared to cells transfected with the reporter plasmid only, and fluorometry analysis indicated that the level of inhibition mediated by the shRNAs were in excess of 80%. The potential of the NS2-directed shRNAs to reduce the level of NS2 transcripts in AHSV-9 infected BHK-21 cells was also investigated by transfection of the BHK-21 cells with the respective pSUPER shRNA delivery vectors, followed by virus infection. Results obtained by means of semi-quantitative real-time reverse transcriptase-polymerase chain reactions indicated that shRNA-377 interfered the most efficiently with NS2 mRNA expression, and the greatest silencing effect was observed at 24 h post-infection. During the course of this investigation it was also attempted to establish a BHK-21 cell line that stably expressed the NS2-directed shRNA-377. For this purpose, a recombinant pSUPER.Retro.Puro retroviral vector was constructed and following transfection of BHK-21 cells, stable transfectants were selected by growth in the presence of puromycin. Results indicated that although the derived cell line suppressed AHSV-9 NS2 mRNA expression, the plasmid DNA was maintained extrachromosomally. Overall, the results of this investigation have provided evidence that AHSV-9 NS2 gene expression can be suppressed in mammalian cells by vector-derived shRNAs. / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc Agric (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Thesis (M. Sc.(Natural and agricultural science))-University of Pretoria, 2006. / Includes bibliographical references. Available on the Internet via the World Wide Web.

African horsesickness virus (AHSV) is a member of the Orbivirus genus and part of the family Reoviridae. It is a double stranded RNA virus with ten genome segments that encode seven structural and four nonstructural proteins. AHSV nonstructural protein NS2, encoded by segment eight, is a single stranded RNA (ssRNA) binding protein. The formation of viral inclusion bodies (VIBs) as well as the selection and condensation of the ten different virus genome segments during encapsidation, are proposed functions of NS2. This study investigates the binding ability of NS2 to ssRNA by looking at the protein structure, as well as its affinity for virus specific mRNAs. NS2 has an α-helix rich C-terminal region and α-helixes are known to be involved in ssRNA binding. BTV NS2 has an α-helix content of 69% and AHSV NS2 an α-helix content of 47%. AHSV NS2 has a lower binding affinity for ssRNA than bluetongue virus (BTV) NS2. There thus seems to be a correlation between the α-helix content of different NS2 proteins and their ssRNA affinity. In order to determine if the difference between the ability of AHSV NS2 and BTV NS2 to bind nonspecifically to poly(U)Agarose can be ascribed to differences in the α-helix rich C-terminal of NS2, a chimeric NS2 protein was constructed that contained the α-helix rich C-terminal of BTV NS2 and the N-terminal of AHSV NS2. The binding affinity of the chimeric NS2 to poly(U)Agarose was compared to that of AHSV NS2 and BTV NS2 and it was found that it correlated well with that of AHSV NS2. The α-helixes therefore do not seem to play a major role in ssRNA binding. The binding affinity of NS2 rather seems to be determined by the N-terminal of the protein. In order to investigate the specific affinity of AHSV NS2 for AHSV mRNAs, it was necessary to obtain mRNA transcripts with terminal ends identical to AHSV mRNAs. The AHSV genes were cloned into a transcription vector with an internal ribozyme. The 3' ends of the transcripts were generated by autolytic cleavage mediated by the ribozyme immediately downstream of the viral insert. The vector's promoter was constructed in such a way that the 5' ends of the genes were inserted at the plus one position for transcription. Full-length mRNA transcripts of four AHSV genes were obtained, the genes encoding NS2, NS3, VP6 and VP7. mRNA transcripts of NS3 and VP7 genes, lacking three to five terminal nucleotides at either the 5' or 3' ends, were also obtained. A nonspecific mRNA control was derived from an AHSV gene cloned in the wrong transcriptional orientation. Preliminary binding assays showed that NS2 has at least a nonspecific affinity for all these mRNAs. The fluctuation in the results also suggests that mRNA concentration may playa role in protein/mRNA interactions. We suggest that NS2 may play a role in encapsidation by binding nonspecifically to all the mRNAs in the infected cell and thus presenting it at the VIBs for selection and encapsidation. / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

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

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