Thesis (MMedVet (Gyn))--University of Pretoria, 2005. / Includes bibliographical references.

The Foot-and-mouth disease virus (FMDV) affects cloven-hoofed animals and is endemic in most parts of Africa, South America and southern Asia. South Africa is considered a FMDV-free zone but the virus is maintained within the wildlife in the Kruger National Park (KNP), making mitigation of outbreaks a high priority. Diagnostic methods are usually costly due to the high production cost of the reagents used, meaning that regular monitoring and diagnosis of animals around the KNP for FMDV is expensive due to the large amounts of serum continuously being tested. I propose an alternative plant expression platform for the local production of more cost effective diagnostic reagents capable of distinguishing between infected and vaccinated animals (DIVA). I selected the non-structural 3ABC polyprotein of FMDV to express, as it is a suitable candidate as a coating antigen in a competitive enzyme linked immunosorbent assay (C-ELISA) for the detection of neutralizing antibodies in livestock sera. I also chose other variations of the full polyprotein (3AB, 3AB1 and 3B) for expression as they have previously been shown to be effective in FMDV diagnosis. I also selected a second reagent to be expressed: this was the CRAb-FM27 single chain variable fragment (scFv), which binds a 3B epitope on the 3ABC polyprotein and has previously shown to be effective as a competing antibody in a C-ELISA. The 3B antigen and the scFv were successfully expressed and purified from N. benthamiana, which to my knowledge is the first time either has been shown. The plant produced scFv successfully bound the 3B antigen in an I-ELISA. Separately, the plant produced 3B antigen could be used to successfully differentiate FMDV infected and vaccinated guinea pig serum in an I-ELISA. However, testing of these reagents in tandem within a C-ELISA to DIVA sera was inconclusive, and further research is required to optimise C-ELISA conditions.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

Foot-and-mouth disease virus (FMDV) is one of the most contagious viruses of animals and is recognised as the most important constraint to international trade in animals and animal products. Two fundamental problems remain to be understood before more effective control measures can be put in place. These problems are the FMDV „carrier state‟ and the short duration of immunity after vaccination which contrasts with prolonged immunity after natural infection. The aim of this thesis was to study the interaction between FDMV and cells in lymphoid tissue in the natural bovine host, in order to improve our understanding of the protective immune response. Using laser capture microdissection in combination with quantitative real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and corroborated by in situ hybridization, it is shown that FMDV locates rapidly to, and is maintained in, the light zone of germinal centres following primary infection of naïve cattle. Maintenance of non-replicating FMDV in these sites may represent a source of persisting infectious virus and also contribute to the generation of long-lasting antibody responses against neutralising epitopes of the virus. The role of T-lymphocyte subsets in recovery from FMDV infection in calves was investigated by administering subset-specific mouse monoclonal antibodies. Depletion of circulating CD4+ or WC1+ γδ T cells was achieved for a period extending from before challenge to after resolution of viraemia and peak clinical signs, whereas CD8+ cell depletion was only partial. Depletion of CD4+ cells was also confirmed by analysis of lymph node biopsies 5 days post-challenge. Depletion with anti-WC1 and anti-CD8 antibodies had no effect on the kinetics of infection, clinical signs and immune responses following FMDV infection. Three of the four CD4+ T-cell-depleted calves failed to generate an antibody response to the non-structural polyprotein 3ABC, but generated a neutralising antibody response similar to that in the controls, including rapid isotype switching to IgG antibody. These data suggest that antibody responses to sites on the surface of the virus capsid are T cell-independent whereas those directed against the non-structural proteins are T cell-dependent. CD4 depletion was found to substantially inhibit antibody responses to the G-H peptide loop VP1135-156 on the viral capsid, indicating that responses to this particular site, which has a more mobile structure than other neutralising sites on the virus capsid, are T cell-dependent. Depletion of CD4+ T cells had no adverse effect on the magnitude or duration of clinical signs or clearance of virus from the circulation. In conclusion, CD4+ T-cell-independent antibody responses play a major role in the resolution of primary infection with FMDV in cattle.

The goal of the work presented herein was to further our understanding of Bovine Leukocyte Antigen (BoLA) class I diversity of Holstein cattle and develop tools to measure class I restricted T cell responses to intracellular pathogens such as foot and mouth disease virus (FMDV) following vaccination. BoLA is a highly polymorphic gene region that allows the bovine immune system to differentiate pathogen-infected cells from healthy cells. Immune surveillance by CD8+ T cells plays an important role in clearing viral infections. These CD8+ T cells recognize BoLA class I molecules bearing epitopes (antigenic peptides) of intracellular origin in their peptide binding groove. Polymorphisms in the peptide binding region of class I molecules determine affinity of peptide binding and stability during antigen presentation. Different antigen peptide motifs are associated with specific genetic sequences of class I molecules. In order to better understand the adaptive immune response mediated by BoLA molecules, technologies from human medicine such as high-throughput sequencing, biochemical affinity and stability assays, tetramers and IFN-γ ELIspot assays could be applied. Therefore, it was hypothesized that we can translate these technologies from the study of human T cell responses to the study of cattle immunity. The first objective was to establish a comprehensive method for genotyping BoLA of Holstein cattle by using Illumina MiSeq, Sanger sequencing and polymerase chain reaction sequence-specific primers (PCR-SSP) (See Chapter 2). This is an important first step in order to study the BoLA restricted immune responses following FMDV vaccination. The second objective was to define the FMDV capsid protein peptide repertoire bound by BoLA class I molecules using bioinformatics and biochemical affinity and stability assays to facilitate the identification of T cell epitopes (See Chapter 3). The third objective was to demonstrate clonal T cell expansion for specific epitope polypeptides using ex-vivo multi-color flow cytometric MHC-epitope complexes (tetramers), followed by IFN-γ production measured by an ELIspot assay to quantify and define the antigen specific response of Holstein cattle to FMDV vaccination (see Chapter 4). In this, my dissertation studies aimed to improve our understanding of the BoLA class I restricted T-cell responses to candidate FMDV vaccines in Holstein cattle. In this manner, my research will improve animal health through the production of assays for characterizing the bovine immune response to intracellular pathogens and enhance vaccine design leading to improved biologicals to protect cattle from devastating infectious diseases.

published_or_final_version / Zoology / Master / Master of Philosophy

Thesis (MSc (Veterinary Tropical Diseases))--University of Pretoria, 2008. / Includes bibliographical references. Also available in print format.

Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Structural biology forms the basis of all functions in an organism from how enzymes work to how a cell is assembled. In silico structural biology has been a rather isolated domain due to the perceived difficulty of working with the tools. This work focused on constructing a web-based Functional Genomics Information Management System (FunGIMS) that will provide biologists access to the most commonly used structural biology tools without the need to learn program or operating specific syntax. The system was designed using a Model-View-Controller architecture which is easy to maintain and expand. It is Python-based with various other technologies incorporated. The specific focus of this work was the Structural module which allows a user to work with protein structures. The database behind the system is based on a modified version of the Macromolecular Structure Database from the EBI. The Structural module provides functionality to explore protein structures at each level of complexity through an easy-to-use interface. The module also provides some analysis tools which allows the user to identify features on a protein sequence as well as to identify unknown protein sequences. Another vital functionality allows the users to build protein models. The user can choose between building models online on downloading a generated script. Similar script generation utilities are provided for mutation modeling and molecular dynamics. A search functionality was also provided which allows the user to search for a keyword in the database. The system was used on three examples in Foot-and-Mouth Disease Virus (FMDV). In the first case, several FMDV proteomes were reannotated and compared to elucidate any functional differences between them. The second case involved the modeling of two FMDV proteins involved in replication, 3C and 3D. Variation between the several different strains were mapped to the structures to understand how variation affects enzymes structure. The last example involved capsid protein stability differences between two subtypes. Models were built and molecular dynamics simulations were run to determine at which protein structure level stability was influenced by the differences between the subtypes. This work provides an important introductory tool for biologists to structural biology. / Thesis (PhD)--University of Pretoria, 2009. / Biochemistry / unrestricted

Foot-and-mouth disease (FMD) virus causes a highly contagious, economically important disease of cloven-hoofed animals, including livestock animals such as cattle and swine. In South Africa, the disease is controlled primarily through prophylactic vaccination and strict animal movement control. To control effectively the spread of FMD, it has become increasingly more important to develop diagnostic tests that can differentiate FMDV-infected animals from those that have been vaccinated. Currently, the detection of antibodies to nonstructural proteins, especially the 3ABC nonstructural polypeptide, is considered to be the most reliable method to distinguish virus-infected from vaccinated animals. Towards the development of such a serological test, the primary aim of this investigation was to express the 3ABC nonstructural polypeptide of the SAT2 type FMD virus ZIM/7/83 in both a prokaryotic and eukaryotic expression system in order for the recombinant protein to be used as a diagnostic antigen. The nucleotide sequence of the 3ABC-encoding region of SAT2/ZIM/7/83 was determined, the amino acid sequence deduced and subsequently compared to corresponding sequences of other virus isolates representing all seven FMDV serotypes. Phylogenetic analysis revealed that the 3ABC polypeptide of the SAT serotypes, which are mostly restricted to sub-Saharan Africa, clustered separately from the euroasiatic FMDV serotypes (types A, O, C and Asia1). Amino acid sequence alignments also indicated considerable variation in the 3A, 3B and 3C proteins between the SAT and euroasiatic types located mainly in previously identified epitope-containing regions. These results suggest that the sensitivity and specificity of diagnostic tests based on the 3ABC nonstructural polypeptide of the European FMDV types may be compromised when applied to the African sub-continent. Therefore, a SAT-specific diagnostic assay is required to distinguish virus-infected from vaccinated animals. The 3ABC-encoding region of SAT2/ZIM/7/83 was subsequently expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein using the bacterial expression vector pGEX-2T, and in Spodoptera frugiperda insect cells using the BAC-to-BACTM baculovirus expression system. Although high-level expression of the recombinant GST-3ABC protein was obtained, the GST-3ABC protein was insoluble and could not be purified by glutathione affinity chromatography. Therefore, the recombinant GST-3ABC fusion protein was purified from reverse-stained SDS-polyacrylamide gels and shown to be immunoreactive in Western blot analysis using an FMDV-specific serum. Expression of the 3ABC polypeptide in insect cells infected with a recombinant bacmid yielded soluble recombinant protein, but the level of expression was lower compared to that obtained in E. coli. In addition, Western blot analysis of cell extracts prepared from recombinant bacmid-infected cells revealed the presence of three immunoreactive proteins of 47, 25 and 18 kDa. These correspond with the size of the FMDV proteins 3ABC, 3AB and 3A, respectively, suggesting that the 3C protease was responsible for proteolytic cleavage of the 3ABC polypeptide. Based on the results obtained, the bacmid expression system appears to be more suitable for the production of the 3ABC polypeptide. / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted