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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Studies into host macrophage transcriptional control by the African Swine Fever Virus protein A238L

Silk, Rhiannon Nicola January 2010 (has links)
African swine fever virus (ASFV) is a large double-stranded DNA virus which causes a lethal haemorrhagic fever in domestic pigs. This virus primarily infects cells from the monocyte/macrophage lineage and its ability to manipulate the function of these cells is key to the pathogenesis of this disease. ASFV encodes several proteins involved in immune evasion. One of these proteins, A238L, has been shown to inhibit host macrophage gene transcription. This protein has been shown to interact with several cellular proteins involved in signal transduction: a serine/threonine protein phosphatase, calcinerurin (CaN), the transcription factor NF-кB, and most recently the transcriptional co-activator CREB binding protein (CBP/P300). However its exact mechanism of action is not fully understood. Previous work has been limited to the investigation of individual signaling pathways and/or the expression of individual host genes. The aim of this study was to investigate the global effect of A238L on host macrophage gene transcription and also to carry out further investigation into the mechanism by which this protein functions. To determine the global effect of A238L on host macrophage gene transcription differential gene expression between porcine cells expressing A238L and control cells was examined using a porcine oligonucleotide microarray. These results demonstrated that A238L was a potent inhibitor of host macrophage gene expression. Functional characterisation of the annotated genes showed that a large proportion of A238L down-regulated genes are typically induced in response to cell stress. Significantly, genes regulated by the I kappa B kinase (IKK), mitogen-activated protein kinase (MAPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways were all shown to be down regulated by A238L. Genes associated with the MAPK pathways were particularly enriched. The transcription of A238L-regulated genes is controlled by numerous different transcription factors, including NF-кB. All of the transcription factors identified interact with the transcription co-activator CBP/P300. This provides a common link between these factors, and indicates that A238L may target CBP/P300 to inhibit gene transcription. This observation supports recent work demonstrating that A238L interacts with and inhibits CBP/P300 function. To explore the potential mechanisms involved in the nuclear localisation of A238L, ASFV-infected Vero cells, expressing A238L under the control of its own promoter, were examined under a range of conditions using confocal microscopy. The results demonstrated that A238L was actively imported into the nucleus and exported by a CRM 1 mediated pathway, although a pool of A238L protein remained in the cytoplasm. Sequence analysis of A238L identified the presence of two putative nuclear localisation signals (NLS-1 and NLS-2). NLS-2 was located within A238L’s CaN docking motif. Mutation of these motifs indicated that both NLS-1 and NLS-2 are active and exhibit functional redundancy. Mutation of the CaN docking motif alone, in the presence of intact NLS-2, resulted in a dramatic increase in the nuclear localisation of A238L. These results are consistent with a model in which A238L functions within both the nucleus and the cytoplasm and suggest that binding of CaN to A238L masks NLS-2, contributing to the cytoplasmic retention of A238L.
2

Use of Random Peptide Reactivities to Analyze Host Immune Responses of African Swine Fever Virus Infection and Immunization

January 2011 (has links)
abstract: African Swine Fever (ASF), endemic in many African countries, is now spreading to other continents. Though ASF is capable of incurring serious economic losses in affected countries, no vaccine exists to provide immunity to animals. Disease control relies largely on rapid diagnosis and the implementation of movement restrictions and strict eradication programs. Developing a scalable, accurate and low cost diagnostic for ASF will be of great help for the current situation. CIM's 10K random peptide microarray is a new high-throughput platform that allows systematic investigations of immune responses associated with disease and shows promise as a diagnostic tool. In this study, this new technology was applied to characterize the immune responses of ASF virus (ASFV) infections and immunizations. Six sets of sera from ASFV antigen immunized pigs, 6 sera from infected pigs and 20 sera samples from unexposed pigs were tested and analyzed statistically. Results show that both ASFV antigen immunized pigs and ASFV viral infected pigs can be distinguished from unexposed pigs. Since it appears that immune responses to other viral infections are also distinguishable on this platform, it holds the potential of being useful in developing a new ASF diagnostic. The ability of this platform to identify specific ASFV antibody epitopes was also explored. A subtle motif was found to be shared among a set of peptides displaying the highest reactivity for an antigen specific antibody. However, this motif does not seem to match with any antibody epitopes predicted by a linear antibody epitope prediction. / Dissertation/Thesis / M.S. Biological Design 2011
3

KINETIC STUDIES OF TWO ERROR-PRONE DNA REPAIR ENZYMES: POSSIBLE MECHANISMS FOR VIRAL MUTAGENESIS

Showalter, Alexander Keith 28 March 2002 (has links)
No description available.
4

Evaluation of cross protection by an attenuated African swine fever virus isolate against heterologous challenge

Souto, Ricardo Gomes January 2012 (has links)
African Swine Fever Virus (ASFV) is an Asfivirus and is the only member of the family Asfarviridae. It manifests as a disease that varies from acute to sub-acute or chronic forms. A true carrier state in domestic pigs is unknown but chronically affected individuals may carry and spread the virus for extended periods. African Swine Fever (ASF) is a socio-economically important disease characterized by high morbidity and mortality affecting the livelihood of many small to big scale farmers and seriously compromising international trade. Strategic measures to control this disease are by physical containment and culling in outbreak situations. There is no vaccine available. Nevertheless, every pork producer should ideally be actively involved in having biosecurity measures in place to avoid contamination and contacting their veterinary services in case of suspicion of ASF to have appropriate samples analysed. Official veterinary services must be equipped with proper diagnostic tools in order to provide a quick response. The sensitivity of currently available diagnostic tests at the Transboundary Animal Diseases Programme, Onderstepoort Veterinary Institute was analysed in order to report the best technique available. Sensitivity to ASF virus infection and therefore diagnostic potential of cell primary cultures as bone marrow macrophages, blood macrophages and alveolar macrophages was done via comparison of titre results from inoculations of ASFV SPEC 257 as control, and ASFV MOZ 1/98. In addition, molecular detection of specific DNA fragments within the viral genome were compared using five different PCRs. Bone marrow macrophage cultures and blood macrophage cultures were the most reliable cells whereas alveolar macrophages more often showed contamination. Results show that PPA PCR and real time PCR detected the highest diluted samples, thus the lowest concentration of virus, in both trials done with ASFV MOZ 1/98 and ASFV SPEC 257. In addition, animal trials were performed by inoculating domestic pigs with four different ASFV isolates of varying pathogenicity. These viruses were all from distinct geographic origins. Non-virulent ASFV OURT 3/88 and high virulent ASFV BENIN 1/97 were previously described and used as reference viruses. ASFV MOZ 1/98, suspected of having high virulence and ASFV MKUZE, which was thought to be of low virulence were included in this study to provide further information on the pathological and clinical outcome of the disease as well as measuring viral replication in various organs and blood. The study showed that ASFV MKUZE was of intermediate virulence, whilst ASFV MOZ 1/98 was highly virulent with a high mortality rate. Results confirmed the inadequacy of ASFV MKUZE to act as vaccine opposed to ASFV OURT 3/88. Following this, a potential vaccine by use of attenuated Portuguese ASFV OURT 3/88 tested against virulent heterologous challenge with a strain now known with certainty to cause acute ASF, the isolate ASFV MOZ 1/98 collected from a diseased pig in Mozambique. Domestic commercial pigs where submitted to either one or two vaccinations before challenge. Viral load in blood and tissue samples was higher in unvaccinated animals and higher in single vaccinated than in pigs vaccinated twice. However, acute ASF afflicted all groups with severe clinical signs and post-mortem lesions. Although it did not confer total immunity it was determined that pigs vaccinated with European attenuated ASFV OURT 3/88 acquired partial protection against challenge with virulent southern Africa ASFV MOZ 1/98. / Dissertation (MSc)--University of Pretoria, 2012. / gm2014 / ab2015 / Veterinary Tropical Diseases / unrestricted

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