Thesis (Ph. D. in Chemistry)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2004. / Document formatted into pages; contains xiii, 73 p. Includes bibliographical references (p. 69-73). Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Mar. 24.

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.

No description available.

Thesis (M.Sc.(Zoology and Entomology))--University of Pretoria, 2005. / Abstract in English. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Jun 1.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / African swine fever is a hemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV), a double-stranded DNA virus and the only member of the family Asfarviridae. The structure of this multilayer virion contains more than 34 proteins including the protein p72 which is the major capsid protein. A single conformational neutralizing epitope has been identified on p72, but information on the other antigenic regions (epitopes) is lacking. The objective of this study was to identify p72 epitopes using polyclonal swine sera and a panel of monoclonal antibodies with the ultimate goal being the development of a blocking ELISA assay for the detection of anti-ASFV antibodies. The segment of the p72 protein from amino acids 1 to 345 was divided into five overlapping fragments which were then commercially synthesized. These fragments were cloned into the pHUE expression vector and transformed into Escherichia coli competent cells. The recombinant proteins were expressed in vitro, purified, and used as antigens in indirect ELISAs and western blots to test monoclonal antibodies and polyclonal swine sera. The monoclonal antibodies were produced against the p72 protein based on the ASFV Georgia/07 strain. The polyclonal sera were obtained from pigs immunized with a defective alphavirus replicon particle, RP-sHA-p72, expressing a recombinant protein composed of the extracellular domain of the ASFV HA protein together with the whole p72 protein. The polyclonal sera reacted to p72 in two distinct regions: between amino acids 1 and 83 and between amino acids 250 and 280. The anti-p72 reactive monoclonal antibodies reacted with p72 in three regions: between amino acids 100 and 171, amino acids 180 and 250, and amino acids 280 and 345. Fine mapping with oligopeptides allowed for the identification of six different linear epitopes. Among the monoclonal antibodies selected for blocking assay development, two have been shown to be promising candidates for further evaluation using sera from ASFV-infected pigs.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Manuel Borca / Stephen Higgs / African swine fever (ASF) is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus. ASFV is endemic in Sardinia and Saharan Africa and has been recently expanded from the Caucasus to Eastern Europe. There is no vaccine to prevent the disease and current control measures are limited to culling and restricted animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pret4 virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus) is completely attenuated. Swine infected with Pret4 Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 dpi showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi). This animal model was used to associate the presence of host immune response and protection against the challenge. Anti-ASFV antibodies and cytokines in serum, as well as ASFV-specific IFN-γ production in PBMCs, were assessed in each group. Interestingly, with the exception of ASFV-specific antibodies in the surviving swine challenged at 21 and 28 dpi, no solid association between any of the parameters assessed and the extent of protection could be established. These results were corroborated using a similar model based on the use of a rationally attenuated derivative of the highly virulent strain Georgia 2007. These results, encompassing data from 114 immunized swine, underscore the complexity of the system under study where it is very plausible that protection against disease or infection relies heavily on the concurrence and or interaction of different host immune mechanisms.

No description available.

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