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

This PhD thesis aimed at evaluating the contribution of participatory epidemiology (PE) to improve the foot-and-mouth disease (FMD) surveillance and control activities, especially the involvement of farmers at local level. The first objective aimed at assessing the effectiveness of the FMD surveillance and vaccination strategy at local level by using PE approach. The second objective aimed at assessing the feasibility of applying PE tools to improve the involvement of farmers in the FMD surveillance in Vietnam. PE methods performed in our study included informal interviews (focus group and individual), scoring tools (pairwise ranking, proportional pilling, disease impact matrix scoring and disease signs matrix scoring), visualization tools (mapping, timeline, flow chart) and sociological tools called Q methodology. 122 focus groups, 467 individual interviews, 339 questionnaire surveys were performed during two field studies in 2014 and 2015. 409 sera and 152 probang samples were taken. Conventional questionnaire surveys, Bayesian modelling and laboratory test (ELISA and rtRT-PCR) was used to validate the performance of PE in FMD surveillance. Disease was considered as the most important issues in animal production. FMD was the most important disease for dairy cattle production, followed by haemorrhagic septicaemia. For beef cattle production, it was recorded in reverse order. The most important disease for pig production was porcine reproductive and respiratory syndrome while FMD was ranked fourth. Farmers showed their abilities in differential diagnostic of important diseases based on its clinical symptoms. Sero-prevalence of FMD were estimated at 23% for population 1 (bordering with Cambodia) and 31% for population 2 (locating far from the border), respectively. Sensitivity and Specificity of PE were found to be 59% and 81%, respectively. The positive and negative predictive value were found to be 48% and 86% for population 1 and 58% and 81% for population 2, respectively. The presence of serotype A, lineage A/Asia/Sea-97 and serotype O with two separate lineages, O/ME-SA/PanAsia and O/SEA/Mya-98 supported virus circulation through trans-boundary animal movement activities. Dairy farms frequently applied quarantine, disinfection and vaccination as prevention methods. Beef farms preferred cleanliness and good husbandry management practices. Pig farms considered that all prevention methods had the same importance. Three distinct discourses “Believe”, “Confidence”, “Challenge”, representing common perceptions among farmers and accounting for 57.3 % of the variance, were identified based on Q methodology. Farmers take vaccination decisions themselves without being influenced by other stakeholders and feel more secure after FMD vaccination campaigns. However, part of the studied population did not consider vaccination to be the first choice of prevention strategy. The benefitcost ratio of FMD vaccination for dairy cow production in large-scale and in small-scale and meat cattle production were 37.2, 30.0 and 7.3, respectively. The sensibility analysis showed that FMD vaccination was profitable for all of production types even through the increase of vaccine cost and decrease of market price of milk and slaughter cattle. From the focus groups organized at sentinel villages, 18 new villages were identified as potentially infected by FMD. 77 suspected animals were confirmed positive for FMD, with viral serotypes O and A. Sensitivity and specificity of participatory surveillance were recorded at 0.75 and 0.65, respectively. The effectiveness of PE in FMD surveillance system to detect outbreak in Vietnam was demonstrated. It was demonstrated that vaccination was the most effective and economic method to prevent FMD. Through the application of simple, adaptive tools which facilitate direct and active participation of farmers, PE allowed to reach a better acceptability of surveillance and to obtain qualified information.

The thermostability of vaccines is of crucial importance in Africa, where the logistical process to get the vaccine from the manufacturer to the animal may take months, and in many remote regions transport and storage is in the absence of a cold-chain. Vaccines with improved stability and less reliance on a cold-chain are needed and could improve the longevity of immune responses elicited in vaccinated animals. In South Africa, cattle in the vaccination zone neighbouring the Kruger National Park have to be vaccinated thrice annually because of declining antibody responses at three months postvaccination. FMDV is known to be unstable, especially for O and SAT2 serotypes in mildly acidic pH conditions or at elevated temperatures, leading to dissociation of the capsid (146S particle) and loss of immunogenicity. The link between rapidly declining antibody responses and capsid stability have been reported by Doel and Baccarini, 1981. We hypothesized that more stable viruses, especially thermostability, will not only improve the protective immune response in animals but also require less frequent booster vaccinations. / The residues at the capsid inter-pentamer interfaces, and their interactions, are important for the infectivity and stability of the virion and mutations adjacent to these interfaces have an effect on the conformational stability of FMDV. However, experimental studies on the relative importance of residues and molecular interactions in viral capsid assembly, disassembly, and/or stability are still very limited, especially for the SAT serotypes of FMDV. This study investigated the effects of potential residues at the pentameric interfaces that are responsible for increased thermostability and potentially improved stability candidates were tested in small (guinea pigs) and large (cattle) animal vaccination trials to understand the role of stabilised antigens on immune responses. The biological variation in biophysical stability in SAT2 viruses in the southern Africa region was investigated to determine if any naturally occurring viruses have greater capsid thermostability. Naturally occurring stable viruses could be used as prospective candidates in vaccine production and therefore potentially result in increased duration of immune responses. / Our first aim was to investigate the role of different amino acid changes at the interface and their effect on capsid stability using models derived by Oxford University. These changes were introduced by mutating the SAT2 ZIM7/83 infectious genome-length clone (pSAT2) to derive mutated chimeric SAT2 viruses. We quantified the stabilizing effects of these mutations by using various stability assays. We established the novel thermofluor shift assay that is able to quantify the capsid stability of viruses. The growth kinetics, antigenicity, genetic stability, pH and salt sensitivity were investigated for each of the genetically engineered viruses (Chapters 2 and 3). / The second aim was to further our understanding on the correlation between improved stability and immune responses by performing small animal (Chapter 2) and large animals trials in cattle (Chapter 4) and comparing stabilised and wild-type antigens. This study for the first time for SAT vaccines, determined differences in IgG1 and IgG2 profiles, interferon gamma (IFN-γ) responses and differences in total and neutralising antibodies of stabilised and wild-type antigens over a six month period in cattle (Chapter 4). Animals were intra-dermolingually challenged with live virus to determine levels of protection the antigens have afforded. / In addition, a third aim will be to better understand the inherent thermostability variation of SAT2 viruses in the Southern African region (Chapter 5) by establishing a protocol for screening field isolates as potential vaccine strains and correlating their stability to amino acid residues at the interface of the 146S particles. / Thesis (PhD)--University of Pretoria, 2016. / Agricultural Research Council / Veterinary Tropical Diseases / PhD / Unrestricted

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

No description available.

Food safety scares affect consumption behavior, and food safety and animal health issues are increasingly impacting international agricultural trade. Foot-and-mouth disease (FMD) is a highly contagious viral-type disease, and has raised not only the concerns of animal health issue but also food safety issue. Over 58 countries in the world have experienced FMD outbreaks, and pork exports and imports among these countries are largely impacted. This dissertation focuses on how global pork trade is affected by FMD. This dissertation consists of three parts: first, this study specifically focuses on the market of U.S. pork exports. Results show that disease-affected pork importers are potential traders with the U.S., and only importing countries with a vaccination policy are more likely to increase pork imports from the U.S. rather than those importers with a slaughter policy. Second, a further investigation focuses basic hypothesis on import demand of FMD-affected importers by using a gravity model with fixed-effects to show how pork trade is affected by FMD among 186 countries. Results confirm that pork export falls when an exporting country develops FMD. Exporters with a vaccination policy have larger negative impacts than those with a slaughter policy. Further, pork importers that develop FMD and institute a slaughter policy will import more pork, but importers with a vaccination policy import the same level of pork. Third, the findings of part one and two reveal that FMD-free pork exporters face different market opportunities when pork importers have FMD outbreaks. Hence, four major FMD-free pork exporters, such as Canada, U.S., Germany, and Spain, are further investigated. Results confirm that the impacts of foreign FMD have altered pork exporters differently. Germany has gained the most exports during foreign FMD outbreaks in pork importers; the U.S. is second; Spain is third; and Canada is fourth. In sum, this dissertation contributes to the literature of gravity model when endogeneity and heteroskedasticity may coexist, when an extremely large number of zero observations are included, when single commodity for one specific exporter is analyzed, when a spatial econometric approach is compared, and when pork export market has been altered by foreign FMD outbreaks.

During 2007 the UK experienced outbreaks of three notifiable exotic livestock diseases; Foot and Mouth Disease (FMD), Highly Pathogenic Avian Influenza (HPAI) and bluetongue. Large epidemics of any of these diseases would have a serious impact on animal welfare, farming, food production and the economy. In light of this, understanding holdings which are most likely to acquire and spread infection and being able to identify areas at higher risk of an epidemic is valuable when preparing for and managing an epidemic. This thesis uses a spatial epidemiological framework and the detailed disease and demographic data from the 2001 Great Britain (GB) FMD epidemic to develop static models of the risk of FMD susceptibility and transmission. These models are used to develop maps of FMD risk. These methods are then applied to the outbreak of FMD in 2007. The inputs for this analysis comprised a set of data relating to the farms diagnosed with FMD and farms culled as part of the disease control measures. The cleaning of these data is described and data which were estimated relating to dates of infection and putative sources of infection are evaluated. The distribution of farm holdings and animals is taken from the June 2000 GB agricultural census, off-fields of farms in the agricultural census are recorded in other datasets and these have been identified and linked to census holdings. A model of holding level susceptibility is developed using both farm level variables and measures of animal numbers in the locality of the holding as well as the distance to the nearest farm infected before the ban on animal movements (seeds). The overall fit of the model was very good with an area under the Receiver Operator Characteristic (ROC) curve of 0.91. A further model was developed to describe the risk of FMD transmission. However, due to incompleteness of transmission data, this was a model of the risk of finding a subsequent Infected Premises (IP) within 3km of an IP. Risk factors were a combination of holding level variables and locality measures as well as data relevant to the infection, such as infectious period and the species initially infected. The area under the ROC curve for this model was 0.71, which is regarded as an acceptable fit. Geographical barriers to FMD transmission were investigated using a case-control methodology, linear barriers comprising rivers and railways had a significant protective effect with respect to disease transmission (odds ratio = 0.54, 95% CIs = 0.30,0.96, p=0.038). Modelled values for the transmission and susceptibility models were transformed to a raster surface in ESRI ArcMap for both the disease as it was seeded in the 2001 epidemic and a non-specific background risk surface independent of the distribution of seeds. A risk map generated for the outbreak of FMD in Surrey in August 2007 suggested that there was little risk of a large outbreak in Surrey. Potential disease introductions through livestock movements from Surrey into Scotland were identified and these suggested that if the disease were introduced into Scotland there was great danger of substantial local spread. These methods described in this thesis have been used to map risk of FMD and subsequently applied to inform the risk presented by a different outbreak of FMD. The study underlines the value of detailed data both disease and demographic, for epidemic management. Similar methods could and should be applied to other infectious diseases threats of livestock such as HPAI and bluetongue.

Foot-and-mouth disease virus (FMDV) is an economically devastating and highly contagious livestock pathogen. It exists as seven serotypes, comprising numerous antigenically distinct subtypes. The large amount of antigenic heterogeneity has confounded attempts at developing broadly reactive vaccines. In order to overcome this issue the fundamentals of the interactions between the virus and the host humoral immune response must first be understood. Previous work in this area using monoclonal antibody (mAb) escape mutants has identified five antigenic sites for the O serotype and efforts have been made to quantify their relative importance. However, this does not represent a complete picture of serotype O antigenicity. The work conducted in this thesis demonstrates the role of a limited number of dominant substitutions in mediating the antigenic diversity of serotype O Foot-and-Mouth disease virus. Two alternative but complementary methods for identifying epitopes were developed. The first used a mathematical model to analyse newly generated serological and sequence data from 105 viruses, cultured for this purpose (and cross-reacted to 5 reference antisera), in the context of an existing crystallographic structure to identify and quantify the antigenic importance of sites on the surface of the virus. The second approach was purely structural, using existing B cell epitope prediction tools to develop a method for predicting FMDV epitopes using existing crystallographic structures of FMDV. These techniques were validated by the use of reverse genetics, which confirmed the impact on cross reactivity of two predicted novel serotype O antigenic residues, with a further four novel residues identified by looking in depth at the interactions between two genetically close, but antigenically distant viruses. This increased knowledge of the antigenic composition of serotype O FMDV contributes to our understanding of the nature of vaccine efficacy and the breadth of protection, which, in the longer term, will aid in the goal of developing vaccines to better protect livestock from such a highly antigenically variable disease.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Michael W. Sanderson / The last outbreak for Foot and Mouth Disease (FMD) in the United States (U.S.) was in 1929. Since that time the U.S. has not had any exposure to the disease or vaccination, creating a very susceptible livestock population. The central U.S. has a large susceptible livestock population including cattle, swine, sheep, and goats. The impact of FMD in the U.S. would be devastating. Simulation modeling is the only avenue available to study the potential impacts of an introduction in the U.S. Simulation models are dependent on accurate estimates of the frequency and distance distribution of contacts between livestock operations to provide valid model results for planning and decision making including the relative importance of different control strategies. Due to limited data on livestock movement rates and distance distribution for contacts a survey was conducted of livestock producers in Colorado and Kansas. These data fill a need for region specific contact rates to provide parameters for modeling a foreign animal disease. FMD outbreaks often require quarantine, depopulation and disposal of whole herds in order to prevent the continued spread of the disease. Experts were included in a Delphi survey and round table discussion to critically evaluate the feasibility of depopulating a large feedlot. No clearly acceptable method of rapidly depopulating a large feedlot was identified. Participants agreed that regardless of the method used for depopulation of cattle in a large feedlot, it would be very difficult to complete the task quickly, humanely, and be able to dispose of the carcasses in a timely fashion. Simulation models were developed to assess the impact of livestock herd types and vaccination on FMD outbreaks in the central U.S. using the North American Animal Disease Spread Model (NAADSM), a spatially explicit, stochastic state-transition simulation model. Simulation scenarios with large vaccination zones had decreased outbreak length and number of herds destroyed. Vaccination did not provide additional benefit to control compared to depopulation alone when biosecurity and movement controls were high, however the ability to achieve high levels of biosecurity and movement control may be limited by labor and animal welfare concerns.