Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (MSc (Veterinary Tropical Diseases, Veterinary Science))--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

Thesis (M.S.)--University of Wisconsin--Madison, 1967. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The aim of this study was to initiate a structural analysis of the capsid polypeptides from several serotypes of bluetongue virus in order to provide insight into the relatedness and possible origins of the different serotypes. Tryptic peptide mapping of ¹²⁵I-labelled group antigen by ion exchange chromatography was used to assess the structural relatedness of seven BTV serotypes from Southern Africa, North America and Australia. Each serotype had several tyrosine containing tryptic peptides which were unique, but approximately 35% of the peptides analyzed were found to be highly conserved between all 7 serotypes. BTV-20 appeared to be closely related to BTV-B and these two serotypes with BTV-4 and BTV-17 appeared to form a closely knit central cluster. / KMBT_363 / Adobe Acrobat 9.53 Paper Capture Plug-in

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Bluetongue virus (BTV) is a pathogenic member of the Reoviridae family. BTV does not cause disease in humans, but is capable of selectively infecting and killing certain transformed human cell lines. Understanding BTV's oncotrophism may lead to new therapeutics for treating cancer. This study focused on the underlying mechanisms of BTV-induced cell death in carcinoma cell lines. It was our hypothesis that BTV infects human carcinoma transformed cells, produces mRNA and protein, induces a strong inflammatory response, induces mitogen activated protein kinase (MAPK)-based pro-apoptotic signaling, inhibits PKB-based signaling, and eventually kills the cell by inducing apoptosis. Three carcinoma cell lines (A498, HEP-G2, and A549) were independently infected with BTV. In each cell line we determined: (1) cell viability over the course of infection; (2) BTV induced cytokine expression profile and magnitude of expression; (3) BTV viral RNA expression profile and magnitude of expression; (4) BTV viral protein expression profile and magnitude of expression; (5) changes in BTV induced cell death and cytokine expression in cells with protein kinase B (PKB), p38-MAPK, extracellular receptor kinase (ERK-1/2), stress-activated protein kinase (SAPK-JNK), Src kinase, platelet-derived growth factor receptor (PDGFR) kinase, epidermal growth factor receptor (EDGFR) kinase, or Janus kinase (JAK) activity inhibited; (6) intracellular changes in PKB, p38-MAPK, ERK-1/2, and SAPK-JNK phosphorylation as a result of BTV infection; and (7) BTV-induced changes in tyrosine phosphorylation. We determined that BTV infects and kills all three cell lines in a cell line dependent manner. Relative cell death between cell lines was proportional to cytokine expression, but inversely proportional to viral protein expression. Only tyrosine kinase inhibitors influenced BTV-induced cell death and cytokine expression. Both A498 and A549 cells constitutively expressed phosphorylated PKB and p38 MAPK, of which both were de-phosphorylated during BTV infection. Tyrosine phosphorylation remained active, with elevated tyrosine phosphorylation exclusively in infected cells. We conclude that BTV-induced cell death and cytokine expression are a function of the cell's response to infection and are directly related through intracellular signaling. These pathways are only partially poly I:C inducible, but include PKB and tyrosine kinase signaling.

This study was undertaken to investigate the structure-function relationship of VP6 protein of bluetongue virus (BTV) using molecular cloning techniques. VP6 is present in small quantities in BTV and its enzymatic activity and role in the viral replication cycle have not been studied. Since the availability of large amounts of purified VP6 is essential for the analysis of VP6, a BTV -11 S3 gene was cloned into a prokaryotic protein expression system. VP6 protein was expressed in large amounts and purified to near homogeneity. A series of C-terminal and internal deletion mutants of S3 gene was constructed and the truncated VP6 proteins were expressed and purified. The nucleic acid binding activities of the VP6 protein towards dsRNA, dsDNA, and ssRNA were confirmed and a new ssDNA binding activity was also determined. The binding activities of VP6 were concentration-dependent. The sites responsible for the binding activities were mapped using the truncated proteins and synthetic sequence-specific oligopeptides. Two domains of VP6 were responsible for the nucleic acid binding activities and have been mapped within 28 amino acids near the middle and 11 residues near the carboxyl terminus of VP6. The binding affinities of the middle domain of VP6 towards single-stranded and double-stranded nucleic acid were slightly different. Three synthetic oligopeptides corresponding to these domains exhibited concentration-dependent nucleic acid binding activities. Based on these results I suggest that synthetic oligopeptides might be useful to screen nucleic acid binding activities and domains responsible for these activities. Expressed VP6 was used to produce polyclonal and monoclonal antibodies. Oligoclonal antibodies were raised by synthetic oligopeptides. Ten epitopes of VP6 were mapped and characterized. The amino acid sequences and sizes of six linear epitopes identified by oligoclonal antibodies were determined, and their locations were mapped and confirmed by deletion mutant analyses. These linear epitopes were surface-accessible except one. Based on these results I suggest that synthetic sequence-specific oligopeptides could mimic major components of antigenic determinants. Four epitopes recognized by four monoclonal antibodies were mapped and characterized. Three determinants were surface-accessible and three were conformational epitopes. These four determinants were distinct and different from the six linear epitopes determined using oligoclonal antibodies.

Bluetongue virus (BTV) is transmitted by arthropod vectors and causes bluetongue disease with serious economic loss in many regions of the world. The replication mechanism of bluetongue virus is still not clear. To have a better understanding regarding the viral replication, the function of each individual protein has to be identified. This study used molecular biology techniques to investigate the function of the inner core protein VP4. The M1 genes of United States bluetongue virus serotypes-2, -10, -11, -13, and -17 were cloned and sequenced. The length of each of the five M1 genes is 1981 nucleotides. The coding region of the M1 gene, which encodes the VP4 protein, possesses an open reading frame with an initiation codon (ATG) at nucleotides #9-11 and a stop codon (TAA) at nucleotides #1941-1943. This open reading frame encodes a protein of 644 amino acid residues with a predicted molecular weight of about 75 kDa. A potential leucine zipper motif was detected near the carboxyl terminus of the deduced VP4 amino acid sequence. The phylogenetic analysis of bluetongue viruses using the sequences of these five cognate M1 genes is consistent with the results of previous phylogenetic studies. Serotypes-10, -11, -13, and -17 are closely related and serotype-2 is the most distantly related among the five US BTV serotypes. Heterologously expressed bluetongue virus VP4 protein was purified to near homogeneity. Six linear epitopes of VP4 were mapped at both termini and in the middle of the protein. By using enzyme-linked immunosorbent assay and peptide competition assay, six linear epitopes were found to be surface accessible. The VP4 protein was shown to be an oligomer by chemical cross-linking. VP4 protein was identified as a ssRNA-binding protein. The VP4 protein has binding activity towards both capped and non-capped ssRNA. RNA-binding activity was not specific to BTV ssRNA. A leucine-zipper motif of VP4 is not required for RNA-binding activity. One RNA-binding domain was mapped between amino acid residues #112-158 by a Northwestern assay and by deletion mutant analysis. Using sequence-specific synthetic peptides corresponding to VP4 in the arginine-and lysine-rich regions, four potential ssRNA-binding domains of VP4 protein were mapped.

The bluetongue (BT) live attenuated virus vaccine has been used successfully in the control of BT in southern Africa and Europe. However, concerns about the safety, possible development of viraemia and clinical signs post vaccination (p.v.) presented an opportunity to investigate the possibility of reducing the current bluetongue virus (BTV) vaccine titre to below 104PFU/ml. A total of 83 merino sheep were used and vaccinated with BTV monovalent vaccines containing either serotypes 2, 4 or 8 with the following titres: 102, 103 and 104 PFU/ml. Positive and negative control sheep were also included. Animals were bled from Day 0, 3, 6, 9, 12, 15, 18, 21, 25 and 28 p.v and tested for viraemia. Seroconversion was determined on Day 0, 3, 9, 15, 21, 6 weeks, 3 and 4 months p.v. Vaccinated sheep were then challenged at 6 weeks p.v. using BTV infected blood and at 4 months using cell cultured material and evaluated for 14 days using the clinical reaction index. Seroconversion was demonstrated p.v. in more than 70% of sheep vaccinated with a low titre 102 PFU/ml of BTV serotypes 2, 4 and 8 from day 9 and at 4 months. All three serotypes did not demonstrate any viraemia p.v. at the three different titres (102, 103&104PFU/ml). Viraemia was demonstrated p.c. with cell culture material in sheep vaccinated with low titres (102&103 PFU/ml) of BTV serotypes 2 and 4. Viraemia could not be detected in sheep p.v. and p.c. with BTV serotype 8 in all different titres. Sheep challenged with cell culture material of BTV 2 and 4 showed mild clinical signs compare to those challenged with blood culture material that did not respond as expected as positive controls did not demonstrate any clinical signs of BT. It was demonstrated in this study that BTV monovalent vaccines containing serotypes 2, 4 and 8 with titres below 104 PFU/ml can protect more than 90% of vaccinated animals against clinical disease. Although certain serotypes failed to protect against viraemia, all serotypes protected against the development of clinical disease when challenged with either BTV-infected blood or cell cultured material. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

The spread of bluetongue virus (BTV) to previously disease-free regions which prohibit the use of the current BTV live-attenuated vaccine has highlighted the need for a new generation of vaccines (Ferrari, De Liberato et al. 2005; Veronesi, Hamblin et al. 2005). Subunit vaccines are one of the attractive alternative strategies. Subunit vaccines against BTV would target the outercapsid protein VP2, the main neutralization-specific antigen (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). A subunit vaccine based on the use of BTV-VP2 may be achieved by either using VP2 by itself or by means of virus-like particles (VLPs) on which VP2 proteins are exposed. In VLPs, the VP2 is co-expressed with other capsid and core proteins to form a particle that resembles the intact BTV. The BTV-VLP vaccine strategy is advantageous since it presents the neutralizing epitopes of more than one viral protein in a more authentic manner as found on the virus itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However there are difficulties associated with large scale production and a decrease in the stability of the particles over time (Berg, Difatta et al. 2005; Wang, Zhao et al. 2006). Studies have already demonstrated the vaccine potential of BTVVP2 by itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However if BTV-VP2 is to be used by itself as a single subunit vaccine, it is important that the protein is expressed under conditions where it is correctly folded and soluble. Solubility refers to the capacity of the expressed antigen to fold into an ordered tertiary structure that authentically exposes the neutralizing epitopes to the immune system (Dinner, Sali et al. 2000; Dobson 2003). However non-native interactions within and between in vitro synthesized viral proteins such as BTV-VP2 often leads to protein aggregation or insolubility. The immune response against aggregated or insoluble proteins is generally very poor. This problem of aggregation and insolubility may be alleviated to an extent by generating truncated versions of the protein from which hydrophobic regions that promote aggregation have been deleted leaving only the major neutralizing epitopes of the antigen (Fukumoto, Xuan et al. 2003; Bonafe, Rininger et al. 2009; Liu, Zeng et al. 2009; Seo, Pyo et al. 2009). The focus of the research presented in this dissertation was to evaluate the solubility of full-length BTV(10)-VP2 and truncated versions thereof after expression in a prokaryotic and baculovirus-Sf9 expression system. The full-length BTV(10)-VP2 (956 amino acids) gene and genes encoding truncated versions of BTV(10)-VP2 i.e. BTV(10)-VP2(aa450) (amino acid 1 to 450) and BTV(10)- VP2(aa650) (amino acid 1 to 650) were cloned into the bacterial expression vector pET160-DEST and the baculovirus expression vector pDEST™8. The C-terminal hydrophobic regions which might contribute to aggregation or insolubility of the protein when expressed in vitro were deleted from these truncated BTV(10)-VP2 proteins. The truncated proteins however still contained BTV neutralizing epitopes that were predicted from literature. The prokaryotic expression of the full-length BTV(10)-VP2 and the other truncated recombinant BTV(10)-VP2 proteins was carried out in E. coli BL21 Star DE3 expression strain. The initial pilot expression study confirmed high level expression of the recombinant proteins. The study also revealed that these proteins were insoluble. The optimization of the prokaryotic expression in order to increase the yield of soluble proteins by means of differential inducer concentrations, fermentation temperature and harvesting times did not produce soluble BTV(10)-VP2 and truncated BTV(10)-VP2 proteins. Previous studies have demonstrated the role of L-arginine in the recovery of soluble proteins from aggregation by reversing aggregation (Tsumoto, Umetsu et al. 2003). However in the current study, arginine treatment of the inclusion body and bacterial lysate containing the BTV(10)- VP2 and truncated recombinant proteins did not release soluble proteins. No soluble recombinant BTV(10)-VP2 proteins were detected when the recombinant proteins were expressed in BL21 host cells over-expressing heat-shock proteins (hsps) and chemical chaperones. However when the different recombinant proteins were co-expressed with the molecular chaperones dnaK-dnaJ-GrpE, it resulted in a fraction of soluble recombinant BTV(10)-VP2 proteins. In particular, approximately 50% of the total expressed BTV(10)-VP2(aa450) protein was soluble while approximately 20% of the total expressed BTV(10)-VP2(aa650) and full-length BTV(10)-VP2 were found soluble when coexpressed with dnaK-dnaJ-GrpE chaperones. These recombinant proteins could be eluted from a nickel affinity column further confirming that these proteins are in fact soluble. Interestingly the coexpression of the BTV(10)-VP2(aa450) protein with the above chaperones in combination with chaperones groEL-groES or only groEL-groES did not produce any soluble proteins. Baculovirus-insect expression of the aforementioned BTV(10)-VP2 recombinant proteins was carried out in Spodoptera frugiperda 9 (Sf9) cells. High level expression of the recombinant proteins was confirmed by an initial pilot expression study conducted at 42 hours post infection (p.i.). The pilot study also revealed that the recombinant proteins were insoluble. Arginine treatment of the lysate released a small fraction of soluble BTV(10)-VP2(aa450) and BTV(10)-VP2(ORF) proteins only detectable with immunoblot analysis using the anti-BTV(10) IgY antibodies. The amount of solubilized proteins was however too small to justify the cost associated with this expression system. / Dissertation (MSc)--University of Pretoria, 2010. / Genetics / unrestricted