Thesis (M.S.)--University of Wisconsin--Madison, 1982. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 47-52).

Thesis (Ph.D.) - University of St Andrews, June 2009.

Thesis (M. Sc. (Biochemistry, Microbiology & Biotechnology))--Rhodes University, 2005.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves 168-176.

The technique of in situ hybridisation was optimised for the detection of viral RNA using radioactively-labelled single-stranded DNA and RNA probes, and applied to three areas of interest. Optimum hybridisation conditions were determined in vitro using cells infected with the single-stranded negative sense RNA paramyxoviruses. Transcription of RNA probes was the most rapid and efficient method of probe labelling, since electrophoretic purification was not required and large amounts of RNA were produced. However, their use for in situ hybridisation was problematic due to RNase contamination and low sensitivity. In contrast, DNA probes produced from M13 clones and oligonucleotide probes gave consistent hybridisation results and were preferred in subsequent studies for their ease of use, stability and sensitivity. The effect of virus-host interactions on the clearance of the paramyxovirus, SV5, in a mouse model was investigated by detection of viral RNA and protein in lung sections. Immunisation with purified SV5 proteins prior to infection provided protection against infection, indicated by a reduction in the level of viral RNA and protein, due to enhanced clearance of virus by primed T cells. X-irradiation of the host prior to infection resulted in prolonged or persistent infection in which RNA was detected up to 19 days post-infection. The potential of in situ hybridisation for detection of aetiological agents was demonstrated by investigation of the presence of measles virus in two chronic human diseases. Thus, measles virus RNA was detected in brain sections from a patient with subacute sclerosing panencephalitis and in the osteoclasts of bone sections from a patient with Paget's disease of bone. In situ hybridisation was used to analyse expression of the two immediate-early genes of herpesvirus saimiri, the 52K gene and the hinG gene. Differential expression was detected by hybridisation to mRNA using oligonucleotide probes, in productively-infected cells. The 52K gene was expressed asynchronously throughout the population in agreement with immunocytochemical detection of the 52K protein. In contrast, the hinG gene was expressed synchronously, with all cells showing similar levels of hybridisation, indicating a specific control mechanism for expression of the 52K gene, which differs from that of the hinG gene in requiring or being inhibited by additional factors. This may have relevance to the mechanism of establishment of latency in this virus.

Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.

No description available.

Conditional lethal mutants of poliovirus type 1 (Mahoney) were generated by treatment with the mutagen hydroxylamine. Temperature-sensitive mutants were selected by the replica plating technique at temperatures of 33°C (permissive) and 39°C (restrictive). New mutants were generated to achieve a larger population of mutants and also to generate additional RNA- mutants in this population. These mutants were characterized by two criteria: RNA synthesis and thermal stability. RNA synthesis is measured by the accumulation of labeled uridine incorporation into trichloroacetic acid (TCA) insoluble material. The thermal stability is determined by the difference in plaque forming units before and after treatment of the virion at 45°C. Complementation co-infections (5 MOI for each virus stock) were analyzed for the presence of the 150S virion particle of poliovirus after sedimentation through a linear sucrose gradient. Complementation is observed between RNA(+) mutants v.s. RNA(-) mutants, and between two RNA(-) mutants, but not between two RNA(+) mutants. Although reciprocal complementation has not been documented in this study some speculation on complementation is presented in this thesis.

The European honeybee (Apis mellifera) is a managed insect pollinator of global economic importance. Over the last few decades honeybees have been undergoing a major health crisis, with one of the biggest causes the parasitic mite, Varroa destructor and its role in changing the viral landscape of deformed wing virus (DWV), which consists of two major variants: DWV-A and DWV-B. Prior to the start of this project there was limited information known about the mechanisms behind the relationships between Varroa, DWV and honeybees. The overarching aim of this project was to further enhance our understanding of these complex relationships, focusing on the impact of Varroa DWV transmission and differences between the main DWV variants. One of the initial obstacles to understanding these complex interactions was the inability to accurately quantify DWV variants. Prior to the start of this project, there was a need for an accurate assay for the quantification of DWV-A, DWV-B and total DWV, allowing the role of both variants in viral transmission and establishment to be investigated. While primers did exist for DWV quantification, the majority did not distinguish between variants, or provide accurate levels of DWV. Given these challenges in variant detection, a new assay for the quantification of DWV-A, DWV-B and total DWV was designed and validated. The assay consists of an external plasmid standard with distinct sections, for the detection of variants and total DWV. This DWV variant plasmid assay was essential for further transmission studies in this project. DWV variant transmission was explored using a variety of different methods. A new in vitro feeding system was used, to allow investigations into Varroa DWV variant transmission in isolation. The feeding system utilises locust haemolymph, allowing changes in DWV transmission to be detected. In multiple feeding experiments significant changes in DWV transmission were detected. Significant changes in DWV composition within feeding Varroa were detected with decreased levels of DWV, and changing variant levels. Switches in variant composition within mites and transmission rates occurred during Varroa in vitro feeding. These variant switches occurred in both directions from DWV-A to DWV-B, and DWV-B to DWV-A dominance. These changes in mite variant composition corresponded to changes in levels of replicating strands. iv These changes in DWV transmission, composition and replicating strand detection were only seen due to the use of this in vitro feeding system. The in vitro work provided valuable information about Varroa variant transmission and composition changes during feeding but this is not a natural system. Honeybee pupae from a Varroa-free area with extremely low DWV titres provided the opportunity to investigate Varroa variant transmission and pupal DWV establishment. Over 96 hours total DWV levels underwent a 1339408X fold increase, within pupae following Varroa feeding, with a sharp increase after 12 hours, followed by a plateau after 60 hours. Within this time period, DWV-A underwent a similar increase, while DWV-B increased at a much slower rate (33X fold change). In contrast to the in vitro work, mite DWV levels did not decrease during feeding. The impact of natural Varroa cell infestation on L5 larvae was investigated, showing no significant effects between pupal total DWV levels and mite density, and DWV levels between infested and none-infested larvae. However, this lack of significance could be attributed to the use of L5 larvae, which had only undergone a maximum of 24 hours Varroa feeding within the cell. Additionally, the use of two drug treatments (ribavirin and hydroxyurea) to reduce DWV levels was explored. Both drug treatments were tested against Varroa and honeybees, using a variety of methods: immersion (Varroa), injections (honeybees) and feeding (both). While neither drug treatment resulted in consistent DWV decreases, some reduction in DWV levels were seen following Varroa soaking in drug solutions. A significant decrease in DWV was seen in honeybees following bolus and ad libitum feeding of drug treatments. Overall, information and insights have been gained regarding the complex relationship between Varroa, honeybees and DWV. A new DWV variant qPCR assay was developed and utilised in subsequent studies. DWV variant switches in both transmission rates and mite composition were found to occur in in vitro studies. Differences in DWV variant establishment within honeybees were detected following Varroa in vivo feeding, in low DWV pupae. Though the tested drug treatments did not affect DWV levels, this highlights the difficultly facing the establishment of any DWV treatment.