Infectious disease is an important issue for animal breeders, farmers and governments. Solutions to control infectious disease are needed and research focused on the genetic loci determining variation in immune-related traits has the potential to deliver solutions. The primary aim of this thesis is to discover regions of the bovine genome which influence the immune response post immunisation. To accomplish this two types of immunising agents, a Foot-and-Mouth Disease Virus (FMDV) peptide (FMDV15) and a commercial vaccine for Bovine Respiratory Syncytial Virus (BRSV), were used to immunise the second generation (F2 and backcrosses) of the Roslin Bovine Genome (RoBoGen) herd, a Charolais Holstein cross population. The FMDV15 peptide consisted of two sections of the VP1 protein located on the FMDV capsid, together encompassing the major neutralising antibody sites that are known to be immunogenic. Protection against FMDV is generally believed to relate to the levels of neutralising antibody and has been correlated with IgG1 and IgG2 levels as well as interferon- . In addition it has been shown that T cell responses also play a role in protection against FMDV. Thus all of these were used as phenotypic measurements post immunisation to the FMDV15 peptide. The BRSV vaccine used was an attenuated live vaccine. Protective mechanisms against BRSV infection include IgA, IgG1, IgG2 and IgM BRSV-specific antibodies and antibody titres particularly those of the IgG isotypes are considered to be correlates of protection. Thus, IgG1 and IgG2 antibody levels were measured post vaccination with the BRSV vaccine. All phenotypes were measured across time, and allowed analysis of the primary and secondary adaptive immune responses. Both agents caused considerable variation in the phenotypes measured post immunisation, with significant responses detected two weeks post immunisation. REstricted Maximum Likelihood (REML) analysis attributed much of this variation to sire, highlighting the heritable component, and environmental effects. Significant positive correlations were detected across time within each trait for both the FMDV and BRSV responses. The FMDV and BRSV antibody levels also correlated with each other at later time points, suggesting that there may be animals which are genetically predisposed to be high or low responders in general. Initially a linkage mappingapproach was followed using 165 microsatellite markers, which detected 77 QTL in response to the FMDV peptide and 27 QTL in response to the BRSV vaccine. There were some overlapping QTL, for example QTL which spanned the Major Histocompatibility Complex. Further analysis was conducted by developing a Perl scripted program which genotyped the RoBoGen herd in two ways; 1) Single Nucleotide Polymorphism(s) (SNP) were genotyped within the confidence intervals of the previously discovered QTL and 2) SNP were genotyped via a candidate gene approach. Association study methodology, accounting for relationship stratification via principal components of the genetic relationship matrix, was used to detect significant SNP, in response to both the FMDV peptide and the BRSV vaccine. Twenty significant SNP associations were discovered across 19 traits, with some SNP located in genes with known biological relevance to an immune response, such as the Toll-Like Receptors (TLR), TLR4 and TLR8. This thesis has detected regions of the genome which are significantly associated with the immune responses elicited by two different agents, suggesting similar pathway(s)/gene(s) may be used in defence of multiple pathogens. Once regions of significance were detected, further analysis using SNP markers identified significant, non-synonymous SNP that were associated with the immunising agents. The novel markers discovered in this study may aid breeding for resistance to disease via marker assisted selection. In addition, they may also have highlighted new targets for vaccinologists to develop ‘next generation’ vaccines.

Foot-and-mouth disease (FMD) is a highly infectious disease of wild and domestic cloven–hoofed animals such as cattle, swine and deer. It is caused by one of the most contagious animal diseases known; FMD virus (FMDV). Since the disease is endemic in many countries, transmission by international travel/trade presents an on-going potential threat to the UK. Very little is known at the molecular level about how FMDV replicates within host cells. In this study, FMDV replicons have been used to investigate FMDV RNA replication and to improve our understanding of the viral life cycle: a process which will aid in the production of a new generation of live-attenuated vaccine candidate strains. Sequences encoding the capsid coding region of the genome have been replaced with green fluorescent protein (GFP) such that replication can be monitored in live cells via GFP fluorescence. This provides a rapid, non-invasive screen for replicative fitness that can be used outwith high disease security facilities. Differences between replicating and non-replicating forms could easily be distinguished, highlighting the potential of this system to screen for attenuated genomes. The FMDV replicon system was improved through a series of construct modifications until an optimal system was produced. A range of different methods were used to attenuate the replication of these genomes. Of major significance is the finding that increasing dinucleotide frequencies were shown to decrease growth kinetics of Echovirus 7 – as opposed to altering the codon-pair bias - and the application of this finding to construction of further replicon systems (and RNA viruses in general) is described.

Large animal species, such as cattle, sheep and pigs, have great potential value to scientific research. This is due to their physiological similarity to humans, meaning they make excellent disease models in addition to their inherent agricultural value. However, the efficiency with which such animals can be created has been a critical barrier to their use in bioscience. Research into creating genetically modified large animals has not progressed as rapidly as research on smaller mammals, such as mice, for two main reasons. Firstly, technologies such as pluripotent stem cells, which are well established in rodents, are lacking for large animals. Secondly, large animals cannot produce as many offspring within a given time frame as mice or rats. This, combined with the low efficiencies and lack of precision of current transgenic methods, severely reduces the likelihood of obtaining an animal with a desired genotype within a viable amount of time. Recently, new tools known as ’genome editors’ have been developed to facilitate genetic modification of animals. The vastly enhanced efficiency of these editors in comparison to previous gene targeting methods, combined with the fact that genome editors do not require marker genes to be used, mean that creating genetically modified livestock is now far more feasible. This thesis investigates whether two types of genome editor, TALENs and CRISPR/Cas9, can be used to produce genetically modified large animals for a range of applications. Genome editors were combined with interspecific blastocyst complementation techniques to produce chimeric rodents where the haematopoietic system is partially or fully derived from the donor cells. This work was carried out with a long-term aim of producing chimeric animals which could produce human organs suitable for transplantation. Initial blastocyst complementation experiments were carried out by injecting murine ESCs into wildtype rat blastocysts. One animal resulting from these injections showed chimerism in several tissues. Further experiments were carried out using rat ESCs and mouse blastocysts which were either Runx1-/- or Rag1-/-, however no additional chimeras were identified. In addition to these experiments, TALENs and sgRNAs were designed against Runx1 and Rag1 in sheep and pigs in order to create a large animal model for future blastocyst complementation experiments. Increasing animal productivity is a key step in meeting the demands of an increasing global population and tackling future food insecurities. TALENs and sgRNAs for use in the CRISPR/ Cas9 system were created to target the myostatin gene in sheep. Myostatin is a negative regulator of muscle growth and animals which acquire natural inactivating mutations in both myostatin alleles exhibit a well-characterised double-muscled phenotype, where total muscle mass is about 20% greater than that of a wildtype animal. Embryo microinjections were carried out using both types of genome editor and two edited lambs were produced, one from each editor. The TALEN-edited lamb was mosaic for a deletion of arginine 283 which, upon further analysis of the muscle, did not appear to cause a significant phenotype. The CRISPR-edited lamb was heterozygous for a 20bp deletion, causing the formation of a premature stop codon and severe truncation of the mature myostatin protein. Based on data from other myostatin-knockout animals, including the Belgian Blue cattle breed, this truncated protein is not thought to be functional. To determine if this is indeed the case, the CRISPR-edited lamb is now part of a breeding programme to amplify the edited allele. To discover if genome editors could be applied to create disease-resistant animals, the project focused on foot and mouth disease. Through a literature search and bioinformatic analysis of the bovine and porcine proteomes, three host genes which are cleaved by the virus were identified; eIF4A1, eIF4G1 and IKBKG. TALENs were designed to bind and cut at the FMDV protease cleavage sites in all three genes in order to disrupt protease cleavage and reduce viral replication by slowing viral disruption of the host translation and innate immune response pathways. Although none of the TALENs showed any signs of activity, this thesis sets out some potential directions for future work. In conclusion, this thesis shows that, despite some technical issues, genome editors are a promising technology for the creation of genetically modified livestock.

The structure, kinetics, and interactions of the conserved 16mer and 15mer RNA motifs of the internal ribosome entry site (IRES) of the Foot-and-Mouth Disease virus (FMDV), have been investigated by homonuclear and heteronuclear NMR techniques. The 16mer RNA is endowed with a classic GNRA tetraloop motif, which is essential for IRES activity and the 15mer RNA motif is a potential tetraloop receptor. We have determined three high resolution NMR solution structures of the 16mer apo-RNA, the 16mer Mg2+ RNA complex and the 15mer apo-RNA with RMSDs of 0.17Å, 0.16Å and 0.35Å, respectively. The high precision of these NMR structures was achieved by including a large number of NMR experimental restraints, derived from NOEs and coupling constants, and validating them using the MolProbity program. The 16mer RNA structure comprised of six base pairs with a GUAA tetraloop and the 15mer RNA structure comprised of four base pairs and a large heptaloop; this is the first heptaloop to be studied by NMR.Addition of Mg2+ to the 16mer apo-RNA caused selective chemical shift changes to the stem G177 and loop G178 imino proton resonances, suggesting Mg2+-induced conformational change to the GUAA tetraloop. This was supported by a significant chemical shift change to the selectively 19F-labelled loop U179 in the 5-FU 16mer RNA. Furthermore, variable temperature experiments revealed retarded imino proton exchange for the stem and loop imino protons, demonstrating the enhanced thermodynamic stability conferred by Mg2+. This enhancement in stability was confirmed by measuring the imino proton exchange rates for the 16mer apo-RNA and the 16mer Mg2+ RNA complex. Analysis of the 16mer apo-RNA and its Mg2+ RNA complex NMR solution structures revealed that Mg2+-induced structural changes to the GUAA tetraloop act to stabilise the loop via stronger base stacking and intramolecular interactions. Fascinatingly, we discovered that Mg2+ ions provide increased stability required for the formation of a G.A sheared base pair in the GUAA tetraloop. RNA-RNA interactions between the 16mer and 15mer RNAs and their fluorinated analogues were studied by NMR spectroscopy. Small changes to chemical shift and linewidth of proton peaks in the non-fluorinated RNA-RNA complex provided evidence for a weak interaction between the loop of the 16mer RNA and the stem of the 15mer RNA. 19F-NMR experiments revealed additional peaks for the 19F-labelled U179 of the fluorinated 16mer/15mer RNA complex providing further good evidence of RNA-RNA interaction. The NMR structures of the conserved RNA motifs and their interactions have yielded important information in understanding the properties and behaviour of RNA. This will provide the first stepping stone in understanding the IRES mechanism and its use in antiviral therapy and biotechnology.

Within East African countries many of the known infectious diseases of animals occur commonly and are poorly controlled. Foot-and-mouth disease (FMD) is one of the contagious viral diseases that has great impact on economic development both in terms of direct and indirect losses. The epidemiology of the disease is complex due to the presence of six of the seven serotypes and the presence of large numbers of both wild and domestic susceptible animals in the region. Decision-making to determine the importance of FMD control relative to the economic consequences and what FMD control strategies should be applied based on the epidemiological information is required. In this regard the first step is to investigate the genetic relationships/variability of East African isolates and their phylogeographic distribution. These can provide base-line information for designing control strategies by vaccination as well as the determination of the sources of infection. Sufficient genetic information on the FMO serotypes O, SAT-1 and SAT-2 are lacking and therefore the number of viral Iineages and genotypes or topotypes from East African countries could not be determined. Published studies on the relative occurrence and genotype distribution of FMO are largely confined to the southern and western part of the continent. In this study, the genetic profile of the 3 most prevalent serotypes (0, SAT-2 and SAT-1) recovered from outbreaks in East Africa between 1957 and 2003 was addressed. Phylogenetic analysis of partial and complete sequences of the 10 gene revealed the presence of distinct lineages and genotypes for East Africa as well as historical relationships of some of the genotypes with isolates from other regions. A great variation in the occurrence and distribution of these serotypes were found. All the African and the Middle East/South East Asian isolates of serotype O included in this study clustered into one lineage having 8 distinct topotypes. These results indicated that between countries as well as inter-regional (east and west Africa) spread of viruses occurred in the past. Inter-regional spread of the virus between eastern Africa and western Africa was also confirmed for SAT-1 viruses. The fact that phylogenetic links are found with both serotypes implies that the spread of viruses was possibly associated with unrestricted animal movement due to nomadic movement in Africa. The phylogenetic relationships of SAT-1 viruses are more diversified in Africa. Eight lineages and 11 genotypes were identified when the optimal nucleotide sequence differences of ≥ 23% for lineages and ≥ 6% for genotypes were used as a cut-off values. It was observed that viruses from Uganda are evolving independently from viruses elsewhere on the continent and clustered into 3 discrete lineages. In contrast, viruses from countries neighbouring Uganda, Kenya and Tanzania, clustered into one lineage. Uganda also harboured 3 topotypes of SAT-2 virus isolates, one is distinct for Uganda and the other are shared with Kenya and Zaire (DRC). This study highlighted distinct lineages found in Uganda and needs further investigation. Within SAT-2, 67 isolates from 22 African countries and Saudi Arabia clustered into 5 lineages which consisted of 15 genotypes. Clustering of viruses into distinct genotypes (topotypes) according to year of isolation and geographical origin was observed showing countries with common boundaries shared common epizootics in the past. These results also showed a link between eastern and southern African countries. Attempts were also made to investigate the incidence of FMD in Ethiopia using sera collected from cattle, small ruminants and wildlife. The results obtained from the liquid phase blocking ELISA and the 3ABC ELISA indicated the presence of SAT-1 and SAT-2 in buffalo populations in the southern part of Ethiopia while results from small ruminants and other wildlife were not indicative of any significant role in the epidemiology of FMD. Serological results also indicated that SAT-1 is present in cattle, although this serotype has not been previously identified. The cumulative molecular epidemiological results from this and previous studies indicated that genetic variability of FMD viruses can be independently maintained within country/countries or regions as well as inter-regions of Africa. The serological results from buffaloes in East Africa are also suggestive of a possible reservoir of the SAT types FMD in the region which has a great impact on the control of the disease. Furthermore, the numerous lineages and genotypes of FMD virus isolates in Africa having distinct or overlapping distributions as well as the genetic linkage between regions will complicate the epidemiology of the disease. Therefore, it is strategically important to consider a regional approach and the use of a vaccine which contains a cocktails of antigens of FMD virus strains. / Thesis (PhD)--University of Pretoria, 2004. / Veterinary Tropical Diseases / unrestricted

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of this study is to evaluate the adjuvant effect of interferon alpha (IFNα) in swine vaccinated with a recombinant replication-defective adenovirus containing foot-and-mouth disease virus (FMDV) protein coding regions, as well as to understand the molecular mechanisms involved in the interaction of FMDV with its host. In the first part of this thesis, the adjuvant effect of pIFNα was evaluated in swine vaccinated with a recombinant vaccine delivered by a human adenovirus type 5 (Ad5) vector containing FMDV capsid (P1-2A) and 3Cpro proteinase coding regions (Ad5-A24). Swine were separated into 5 groups and inoculated with low (5x108 PFU) or high (5x109 PFU) doses of Ad5-A24 in the presence or absence of pIFNα (Ad5-pIFN, 109 PFU). Control animals received 6x109 PFU of an Ad5 vector containing the glycoprotein gene of vesicular stomatitis virus (Ad5-VSNJV-G). All swine were challenged at 42 days post vaccination (dpv) with FMDV-A24. Prior to challenge, blood samples were examined for IFN production, induction of IFN-induced genes (ISG s), FMDV-specific neutralizing antibodies and FMDV-specific antibody isotypes. After challenge, a number of parameters were analyzed including clinical score, viremia, lymphopenia and antibodies against FMDV structural (S) and non-structural (NS) proteins. The results indicate that both groups that received high-dose Ad5-A24 developed an FMDV-specific neutralizing antibody response by 14-21 dpv, which was maintained until the day of challenge. Both high-dose groups developed high levels of IgG1 and IgG2, however the IgG1 response was higher. The high-dose Ad5-A24 with IFN group developed higher levels of IgG1 than the group administered only high-dose Ad5-A24 and this difference was statistically significant. Antiviral activity and IFNα were detected in the groups that received IFN. The three ISG s examined, PKR, OAS and Mx1, were detected by real time RT-PCR in leukocytes from Ad5-pIFNα-vaccinated swine. After challenge, all animals in the control group developed early viremia, vesicular lesions, considerable lymphopenia and antibodies to FMDV NS proteins. The animals that received low-dose Ad5-A24 without IFN had similar clinical signs, except that fewer animals had viremia. In contrast, pigs inoculated with the low-dose Ad5-A24 and IFNα had a delayed onset of vesicular lesions and only one animal had detectable viremia. Animals vaccinated with high-dose Ad5-A24 without IFNα had no viremia, showed fewer lesions, one animal had no lesions, and delayed onset of disease compared to the low-dose Ad5-A24 groups. Four of five pigs vaccinated with high-dose Ad5-A24 and IFNα were completely protected from disease and only one animal in this group had a vesicular lesion restricted to the site of challenge virus inoculation. The results indicate that IFNα enhances the level of protection induced by the Ad5-FMD vaccine against homologous FMDV, supporting the use of IFNα as a potential adjuvant in FMD vaccination strategies. To investigate the effect of FMDV infection on the induction of the host IFN-α/β response, swine cells were infected with wild-type (WT) FMDV and a mutant FMDV lacking the L proteinase (Lpro) coding region (A12-LLV) at different multiplicities of infection. The synthesis of IFN-α and IFN-β mRNAs and three well characterized ISG s, PKR, OAS, and Mx1 mRNA, were evaluated by real time RT-PCR. A12-LLV infection resulted in significantly higher levels of induction of IFN-β mRNA as compared to WT virus infected cells, while IFN-α mRNA was not induced after either infection. The increased levels of IFN-β mRNA in A12-LLV-infected cells correlated with higher levels of induction of PKR, OAS and Mx1 mRNAs and antiviral activity. By using RNA interference (RNAi) technology to knock-down PKR mRNA expression, it was possible to demonstrate that the yield of A12-LLV was increased up to 200-fold, supporting the role of PKR as an inhibitor of FMDV replication. These results confirm that Lpro down regulates the innate immune response to FMDV infection at multiple levels. Previous studies indicated that control was at the translation initiation level by Lpro cleavage of translation initiation factor eIF-4G. The present data demonstrates that regulation also occurs at the level of transcription by inhibition of IFN-β mRNA induction through an unknown mechanism. / Esse trabalho tem como objetivo avaliar o efeito adjuvante do interferon alfa suíno em animais imunizados com uma vacina recombinante de um adenovírus defectivo contendo as regiões codificadoras das proteínas do FMDV, bem como investigar alguns dos aspectos moleculares envolvidos na interação FMDV e a célula hospedeira em uma espécie susceptível. Na primeira fase do trabalho, o efeito adjuvante do interferon alfa suíno (pIFNα) foi avaliado em suínos imunizados com uma vacina recombinante, tendo como vetor o adenovirus humano tipo 5 (Ad5), contendo regiões do capsídeo do FMDV A24 e da proteinase 3Cpro do FMDV A12 (Ad5-A24). Os suínos foram separados em 5 grupos e inoculados com baixa (5x108 PFU) e alta (5x109 PFU) dosagem de Ad5-A24 na presença ou na ausência de pIFNα (Ad5pIFNα, 109 PFU). O grupo controle foi inoculado com 6x109 PFU da glicoproteína do vírus da estomatite vesicular (VSV) cepa New Jersey (Ad5VSNJV-G). Todos os suínos foram desafiados aos 42 dias pós-vacinação (dpv) com FMDV-A24. Após a inoculação, as amostras de sangue foram examinadas para a produção de IFN, a indução de genes induzidos pelo IFN e os anticorpos neutralizantes e resposta de imunoglobulinas específicos para o FMDV. Depois do desafio um número de parâmetros foram analisados incluindo a avaliação clínica, viremia, linfopenia; além dos anticorpos contra as proteínas estruturais e não estruturais do FMDV. Os resultados obtidos indicam que ambos os grupos que receberam Ad5-A24 em alta dosagem desenvolveram níveis de anticorpos neutralizantes pelos 14-21 dpv, que foram mantidos até o dia do desafio. Os níveis de IgG1 foram maiores que de IgG2 nesses dois grupos, sendo que a IgG1 é considerada a mais relevante para conferir proteção ao FMDV. Dentre esses grupos, o que recebeu o IFN apresentou níveis significativamente mais altos desta imunoglobulina. Atividade antiviral e o IFNα foram detectados nos animais que receberam o IFN. A respeito da presença dos genes induzidos pelo IFN nos leucócitos dos suínos vacinados com Ad5-pIFNα, todos os três genes incluídos neste estudo, PKR, OAS e Mx1, foram detectados pelo real time RT-PCR. Após o desafio todos os animais do controle desenvolveram viremia, linfopenia, lesões vesiculares e os anticorpos contra as proteínas não estruturais do FMDV. Os animais que receberam baixa dose de Ad5-A24 sem IFN tiveram sinais clínicos similares, exceto que poucos animais desenvolveram viremia. Porém, os suínos inoculados com a mesma dose da vacina de Ad5-A24 com o IFN apresentaram as lesões vesiculares com início tardio e somente um animal teve detectável viremia. Os animais vacinados com a alta dose de Ad5-A24 sem IFN não tiveram nenhuma viremia e poucas lesões foram detectadas tardiamente após a inoculação do FMDV. Quatro dos cinco suínos, que receberam a alta dose da vacina com o IFN, foram protegidos da doença e somente um animal neste grupo teve uma lesão vesicular, restrita ao local do inoculação do vírus por ocasião do desafio. Esses resultados indicam que IFNα realça o nível da proteção induzido pela vacina do adenovírus-FMD contra o FMDV homólogo, suportando o uso do IFNα como um adjuvante potencial em estratégias de vacinação de FMD. Para avaliar os efeitos da infecção pelo vírus da FMD na indução da resposta de IFNα⁄β do hospedeiro, células de origem suína foram previamente infectadas em diferentes multiplicidades de infecção com o FMDV e com um vírus mutante que teve o gene que codifica a protease L ou Lpro deletado, o FMDLLV. A síntese de mRNA do IFNα e β bem como de três dos genes induzidos pelo IFN, PKR, OAS e Mx1 foram avaliados por real time RT-PCR. A infecção das células pelo FMDLLV induziu altos níveis de mRNA do IFNβ quando comparados com os do FMDV original. Contudo, não foi possível detectar os níveis de mRNA do IFNα na presença de ambos os vírus. O aumento nos níveis de mRNA do IFNβ foi relacionado ao aumento nos níveis de indução dos mRNAs de PKR, OAS e Mx1, assim como dos altos níveis de atividade antiviral. Pelo uso da tecnologia de interferência do RNA, usando siRNA (silencing RNA) para bloquear a expressão do mRNA da PKR, foi possível demonstrar que o título do FMDLLV aumentou cerca de 200 vezes. Desta forma foi possível confirmar o papel desta proteína como um inibidor da replicação do FMDV. Os resultados obtidos demonstram que a Lpro tem um importante papel na regulação da resposta imunológica inata do hospedeiro quando da infecção pelo FMDV em vários níveis. Estudos anteriormente realizados indicaram que o controle era efetuado ao nível da tradução pela clivagem do eIF4G. Os dados obtidos neste trabalho indicam que a regulação também ocorre ao nível da transcrição e pela inibição da indução do IFNβ através de um mecanismo ainda não conhecido.

Foot-and-mouth disease (FMD) is a highly contagious disease caused by the FMD virus (FMDV) belonging to the Picornaviridae family. The virus affects cloven-hoofed animals and occurs as seven immunologically distinct serotypes where six of the seven serotypes occur in Africa. This fact, as well as the role of wildlife in virus maintenance, makes eradication and control of FMDV in Africa difficult. Thus, it is imperative to attain more information regarding the genetic diversity of FMD viruses prevalent on the African continent to further our knowledge of the virus as well as to enable better control strategies and the development of improved vaccines. Sufficient genetic information regarding the Leader (L) and complete capsid-coding, P1, region of serotype A and O viruses prevalent on the African continent is lacking, although the SAT isolates have been extensively characterised in the past. In this study the sequence of the L/P1-coding region was successfully determined using a genome-walking approach for a small number of A and O viruses recovered from outbreaks isolated from various species in East and West Africa over the last 33 years. Phylogenetic analysis of the P1 and capsid-coding regions 1A, 1B, 1C and 1D revealed that the African isolates grouped strictly according to serotype and geographic region which indicated the possibility of transboundary spread of the virus within East and West African countries respectively. In contrast, phylogenetic analysis of the non-structural, Lpro-coding region revealed a different tree topology compared to the capsid-coding regions for the A and O isolates with sub-grouping according to serotype and geographic regions was less apparent. The relatedness between the serotype A and O L region might be the result of genetic recombination. The inter and intratypic nucleotide and amino acid variation of the A and O isolates revealed that the most variable capsid-coding region was the externally located VP1 whilst the internally located VP4 capsid protein was the most conserved. The observed variation is in agreement with other studies and reflects the selective pressures on these proteins which either allow or prevent the occurrence of genetic changes for structural constraints or immune escape. Surprisingly, the L protease-encoding region also displayed a high degree of variation. A detailed analysis of the L/P1 amino acid alignment of the A and O isolates revealed that although the extent of variation is high in these regions, the amino acids identified in previous studies as important for FMDV structure (for the capsid-coding regions) and function were found to be conserved, indicating that the virus has adapted itself to elude the host immune response without affecting its vital functional and structural abilities. Additionally, it was observed that the amino acid residues identified as being important for FMDV attaching to the host cell receptors e.g. the RGD amino acid motif of VP1 was highly conserved for all isolates. To further investigate the FMDV-receptor interaction, RT-PCRs were developed to examine the mRNA expression level of the known FMDV receptors. The â integrins that facilitate FMDV cell entry i.e. â1, â3, â6, â8 and heparan sulphate proteoglycans (HSPG) were investigated in susceptible cell lines used for FMDV vaccine production i.e. IB-RS-2 and BHK-21. The RT-PCRs were successfully developed and optimised. The results showed that the mRNA expression levels were variable for all receptor cDNAs tested across 36 passage levels of IB-RS-2 and BHK-21 cells. No distinct differences in virus susceptibility for three FMDV strains with continuous cell passage of IB-RS-2 and BHK-21 cells at passage levels 5, 21 and 36 could be found. The information gained from this study regarding the viral L and P1 region genetic diversity, and phylogenetic analysis has indeed impacted on our understanding of FMDV African viruses. Additionally, the investigation of the FMDV receptor mRNA expression levels and virus susceptibility on two cell lines with continuous cell passage has proved a vital starting point to determine the possible receptors expressed on the surface of cells used by the vaccine production division at the ARC-OVI-TADP and forms the basis for further investigations of the FMDV receptors on the protein level and the development of a real-time RT-PCR for FMDV receptor expression. / Dissertation (Msc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

No description available.

For the second study, a web-based survey was developed and distributed to all members of four major health education organizations. A total of 1,925 HEs’ completed the survey and 1,607 responses were utilized in the final analysis. This study indicated that participants had deficient knowledge and unfavorable attitudes toward the CDCproposed genomic competencies. In the third study, a theoretical model was developed to predict HEs’ likelihood to incorporate genomic competencies into their practice. Using techniques from Structural Equation Modeling (SEM), the model was tested with the same data of the second study. Findings supported the proposed theoretical model. While genomic knowledge, attitudes, and self-efficacy were significantly associated with HEs’ likelihood to incorporate genomic competencies into their practice, attitudes was the strongest predictor of likelihood. In summary, these studies indicated that participating HEs had deficient genomic knowledge, unfavorable attitudes toward a set of CDC-proposed genomic competencies, and low likelihood to adopt genomic competencies into health promotion. Relevant training should be developed and advocated. As the SEM analysis results indicated the survey findings supported the proposed theoretical model, which can be utilized to steer future training for HEs. statistics, 2) unadjusted inferential statistics, 3) stratified analysis, and 4) multivariable models. My investigation produced results in accord with generally accepted notions in addition to significant findings that interestingly counter current preconceptions. Intraspecies contact was more common than inter-species, with indirect contact occurring more frequently than direct. Direct contact between species occurred extremely rarely. The most important factors that influenced the rate of contact for both species were water, winter, and cultivated fields. Information regarding probability of infectious agent survival and transfer will be used in the future to advance current epidemiological models, including geographicautomata (Ward et al. 2007: In Press) and cellular automata models (Doran and Laffan 2005) to better understand and manage integrated domestic cattle and free-ranging wildlife populations. Such modeling provides essential and necessary knowledge for developing prevention, detection, response, and recovery strategies – employed in advance, during, and after a disease outbreak, respectively.

No description available.