La fièvre aphteuse (FMD pour Foot-and-mouth disease en anglais) est une maladie très contagieuse touchant les animaux biongulés. Elle provoque des dégâts économiques considérables sur toute la surface du globe. La fièvre aphteuse est provoquée par un virus, le FMDV. Il s'agit d'un virus à ARN simple brin, de polarité positive appartenant au genre Aphtovirus dans la famille Picornaviridae. Ce virus se réplique et se propage dans l'hôte très rapidement. Dans les zones infectées, les deux principales stratégies de contrôle utilisées sont l'abattage systématique des animaux infectés et la vaccination. A l'inverse, les vaccins ne sont pas utilisés dans les zones sans FMDV, mais l'apparition d'une épidémie nécessite des stratégies pour arrêter ou au moins limiter la diffusion du virus. Actuellement, les vaccins inactivés sont les vaccins les plus utilisés pour prévenir la maladie. Cependant, ils requièrent une production à grande échelle du virus, et malgré les mesures mises en place (laboratoire sécurisé, etc), des épidémies ont été provoquées par le passé du fait de la fuite de virus FMDV. De plus, il est difficile de distinguer les animaux infectés des vaccinés (DIVA). Il est donc nécessaire de développer de nouveaux vaccins. Au cours de l'infection, la polyprotéine du virus est clivée par des protéases virales en précurseurs structural (P1) et non structuraux (P2 et P3). La protéase 3C est responsable de la majorité des clivages ; ainsi, le précurseur P1 est clivé par la 3C en trois protéines structurales, VP1, VP3 et VP0, formant le protomère de FMDV, l'unité de base de la capside virale. La protéine VP1 joue des rôles importants dans l'attachement, la protection et le sérotypage. Du fait de la présence d'un site antigénique linéaire suffisant à la protection par production d'anticorps neutralisants, VP1 est considérée comme la protéine la plus immunogénique du virus. Dans cette étude, nous avons développé un nouveau vaccin contre la FMD, basé sur l'adénovirus canin de type 2 (Cav2). L'évaluation du transfert de gène médié par Cav2 chez le porc et le bétail in vitro montre des résultats prometteurs pour le développement de vaccins pour ces espèces, notamment l'expression des antigènes de FMDV par les candidats vaccins Cav2-FMDV. L'immunogénicité de ces candidats vaccins a été montrée chez les modèles murins et cobayes. De plus, des résultats encourageants ont été observés chez le cobaye, suggérant que la réponse immunitaire élicitée par les vecteurs recombinants pouvait conduire à une protection partielle des animaux après épreuve. Cependant, une optimisation de l'immunisation doit être faite dans le but de confirmer ces résultats. Ce type de vaccin peut de plus être utilisé comme vaccin marqueur, car il ne contient aucune protéine non structurale. / Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease affecting cloven-hoofed livestock worldwide. Foot-and-mouth disease virus (FMDV) is the causative agent of FMD and one of the most infectious known animal viruses. FMDV is a positive-sense, single-stranded RNA virus belonging to the Aphthovirus genus in the Picornaviridae family. FMDV replicates and spreads in the host extremely rapidly. Slaughter and vaccination are the two major strategies used to control FMD in infected countries. In FMDV-free countries, vaccines are not used, and once the disease breaks out in these areas, strategies are required to stop or at least slow the spread of the virus. Currently, inactivated vaccines are by far the most commonly used vaccines to prevent FMD. Such vaccines, however, require large-scale production of virus, and despite the use of bio-safety facilities, vaccine production has led to inadvertent virus release and FMDV outbreak. Another limitation of inactivated vaccines is the difficulty in distinguishing between infected and vaccinated animals (DIVA). Therefore, improved vaccines need to be developed.During infection, the FMDV polyprotein is cleaved into structural (P1) and non-structural (P2 and P3) precursors by a viral protease. The non-structural 3C protein is the protease that is responsible for most of the maturation events. The P1 precursor is processed by 3C protease into three structural proteins, VP1, VP3 and VP0, forming the FMDV protomer. The VP1 protein plays important roles in attachment, protective immunity and serotype specificity. VP1 is considered to be the major immunogenic protein, as it contains a linear antigenic site that is able to induce neutralizing antibodies that suffice to protect animals against the disease.In this project, we developed a novel vaccine against FMD, based on canine adenovirus type 2 (Cav2). In vitro evaluation of Cav2 mediated gene transfer in pigs and cattle showed that the Cav2 vector holds promise for the development of vaccines for pigs and cattle. Study of these recombinant viruses indicated that Cav2-FMDV supported expression of FMDV capsid proteins in vitro. The immunogenicity of these recombinant viruses was evidenced in mouse and guinea pig models, and encouraging results in guinea pigs suggested that the immune response elicited against FMD by recombinant virus could afford partial protection against FMDV challenge. In the future, immunization with Cav2-derived vector should be optimized to confirm these preliminary results. This type of vaccine, when designed to express capsid but not non-structural proteins of FMDV, can serve as a marker vaccine against FMD.

Foot-and-mouth disease (FMD) is an economically devastating picornaviral disease affecting over 40 species of cloven-hoofed animals. The virus occurs as seven immunologically distinct serotypes which are characterized by high levels of intra- and intertypic variation. The three South African Territories (SAT) serotypes 1-3 are endemic to sub-Saharan Africa, a region where the epidemiology of the disease is particularly complex due to the presence of six of the seven serotypes, the role of wildlife in virus maintenance and the apparently higher levels of variation in the endemic serotypes. These factors make it imperative to establish methods suited to elucidating the regional epidemiology. One of the integral parts of this process is the genetic characterization of regionally representative viruses in order to assess the variation in the field and to clarify the role of wildlife. Nucleotide sequence data and methods suited to studying the SAT-types are however limited. A first priority was therefore to establish a PCR-based nucleotide sequencing technique targeting the highly immunogenic and phylogenetically informative 1D genome region encoding the VP1 protein. The screening of multiple serotypes and subtypes prevalent on the African continent confirmed that this method was robust and well-suited to molecular epidemiological studies in the southern Africa region. The method was first applied in the characterization of FMD virus recovered from the reproductive tract of free-living African buffalo in the Kruger National Park. Nucleotide sequencing assisted in authentication of the results and indicated that carrier status was likely, but it was not possible to unequivocally demonstrate persistent infection of FMDV. In a separate study, the role of impala antelope (Aepyceros melampus) in the epidemiology of the disease in South Africa was assessed. Genetic characterization of impala and African buffalo (Syncerus caffer) viruses collected over an eleven year period confirmed that inter-species transmission occurred on several occasions and that virus can persist in impala populations for more than 12 months. Inter-species transmission and investigation of the possible mechanisms facilitating virus transmission from persistently infected buffalo focussed on the Kruger National Park in South Africa. In order to ensure regional relevance the study was broadened to incorporate buffalo populations throughout southern Africa. Viruses of the three SAT-types recovered from diverse African buffalo populations were therefore characterized. The results reveal that independently evolving viral lineages occur in distinct geographical regions for each of the SAT-types examined and that the levels of intratypic variation are in the order of 52 - 55 % on nucleotide level across the genome region characterized. Given the strict locality-specific grouping of buffalo viruses the likely usefulness of this database for tracing the origin and course of contemporary and historical SAT-type outbreaks was investigated. Molecular epidemiological studies conclusively show that buffalo are indeed the ultimate source of infection for susceptible cloven-hoofed animals occurring in close proximity, that interspecies transmission occurs between cattle and antelope and that trans-boundary transmission of virus remains a threat to disease security in southern African countries. / Thesis (PhD (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Development of transfrontier conservation areas (TFCAs) in southern Africa depends, among other, on the ability of stakeholders to find practical and sustainable solutions for wildlife‐livestock integration in the conservation landscape. Due to the presence of buffalo Syncerus caffer in most of the TFCAs in southern Africa, foot‐and‐mouth disease (FMD) has to be controlled in susceptible livestock species sharing the rangelands with wildlife. Conventional FMD control measures act as an additional burden on communal livestock producers and may hamper rural development and wildlife‐livestock integration even further. However, commodity‐based trade in the form of an integrated approach to the control of both food safety and disease risk along the entire beef value chain has been proposed as a more favourable alternative for ensuring market access for beef produced at the wildlife‐livestock interface. Such a non‐geographic based approach could allow for trade to continue despite high risk of FMD if appropriate disease risk and food safety measures are implemented by farmers and subsequent role players along the value chain and hence, could promote greater wildlife‐livestock compatibility. / The objective of the present study was to analyse beef production, health and trade systems of farmers at the wildlife‐livestock interface within foot‐and‐mouth disease (FMD) protection zones in order to identify challenges, risks and limitations that may limit compliance with proposed commodity‐based trade prerequisites as well as value chain participation. Based on the findings of this study a holistic, integrated approach is proposed at the village level that could be implemented to serve as an incentive for equitable participation by farmers whilst 1) addressing the risks and limitations of a farming system, 2) ensuring greater wildlife‐livestock compatibility, and 3) promote consistent market access by fulfilling the requirements of an integrated value chain approach based on commodity‐based trade standards. / A farming systems approach was used to investigate beef production, health and trade systems in FMD protection zones mainly within the Zambezi Region (ZR) of Namibia, which is situated within the KAZA TFCA (Kavango‐Zambezi Transfrontier Conservation Area), but also the Mnisi study area (MSA) in South Africa adjacent to the Great Limpopo Transfrontier Conservation Area (GLTFCA). A combined qualitative and quantitative approach was used to assess and describe farmers’ perceptions in selected study areas about beef production, trade, and wildlife conservation. Secondary data obtained from state veterinary services, the Meatco abattoir in Katima Mulilo, as well as previous studies were analysed and modelled to describe spatial‐temporal trends in trade as well as cattle distribution in relation to resource availability. / The results indicate that beef production systems in some of the most remote areas of the ZR as well as in the MSA resemble a typical low‐input low‐output production system, mainly due to the high level of risk farmers had to cope with and the limited opportunity to offset losses. The major challenges within livestock farming in all the areas studied were animal diseases, grazing competition, predation, stock theft and contact with wildlife, although the importance of each varied between study areas. Herd size effect in the MSA significantly explained the variation in attitude towards trade, production and management of cattle between farmers with below average and farmers with above average herd sizes. In the MSA, home slaughter contributed significantly more to direct household food security in households with larger herd sizes than in households with smaller herd sizes, and in the ZR farmers with smaller herd sizes were discouraged from participation in formal trade. / The attitudes and perceptions of farmers In the ZR towards wildlife and conservation often varied between survey areas as a consequence of the variation in the geophysical properties of the landscape, proximity to conservation areas, as well as the form of the interface with conservation areas. The perceived spatial‐temporal movement of buffalo varied between survey areas in the ZR. However, the frequency and nature of buffalo‐cattle interaction was generally high and intimate. Most farmers associated buffalo with risk of disease, especially FMD, but some were more concerned about grazing competition and the negative effect on husbandry practises. Farmers readily deployed traditional risk mitigation tactics in the form of kraaling at night and herding at day to control the movement of their animals and to reduce risks. Herding was found to be a potential strategy to specifically mitigate cattlebuffalo contact despite the lack of evidence that an overall strategic approach to herding exist. Although the majority of farmers in the ZR were in favour of conservation and its benefits, the negative impact of increasing wildlife numbers on farmers’ attitudes was an indication that the generally positive sentiment was changing and may in future deter conservation efforts. / Indications are that the cattle population in the ZR at its estimated density and distribution had reached the ecological capacity of the natural resource base in the ZR and animal performance and survival was therefore subjected to increased variability in resource availability linked to climate change. The cattle population’s existence at ecological capacity and the inability of farmers to offset the loss of condition in the dry season with supplementary feed were reflected in the changes in carcass quality and grades across seasons. However, there was sufficient forage produced in the ZR to sustain animal performance to some extent throughout the year, but those areas with surplus forage existed beyond the assumed grazing range around villages and perennial rivers where most cattle and wildlife concentrate. The future ability of farmers to access such underutilised grazing resources in order to strategically counter the negative consequences of climate change and growing wildlife numbers could be an important coping and risk management mechanism linked to commodity‐based trade and sustained animal quality. / Regular FMD outbreaks had a significant impact on the consistency with which the Meatco abattoir in the ZR operated between the years 2007‐2011, with negative consequences to both farmers and the abattoir itself. It was found that the formal trade system in the ZR discriminated against farmers with below average herd sizes, and that the disposition held by farmers with smaller herd sizes are most significant in areas further than approximately 55km away from quarantine camps. Vegetation type and possible contact with buffalo or previous FMD outbreaks in the area did not significantly affect market participation nor off‐take rates at a crush‐pen level in the ZR. The negative effect that distance from a quarantine station had on formal off‐take rate and the level of sales to Meatco at crush‐pen level, was the most significant in the winter months and crush‐pens situated beyond 55km from a quarantine station. The results indicate that the trade range of the Meatco abattoir was less than its trade threshold which contributed to its struggle to sustain throughput and profitability. / Finally the loss of income farmers experienced in both the ZR and the MSA during simultaneous FMD outbreaks in the year 2012 was quantified, as well as the impact it had on livelihoods in the ZR. A commodity‐based trade approach may have reduced the impact on farmers’ income significantly. However, we farmers are unable to comply with the proposed requirements for mitigating risk and ensuring food safety and quality in such communal systems in the absence of interventions to build the necessary capacity and awareness. It is recommended that at the wildlife‐livestock interface such as those investigated in this study, an integrated value chain approach to trade could serve as a catalyst to incentivise and enable farmer participation in holistic, integrated rangeland and livestock management practises that will promote conservation and rural development. / Thesis (PhD)--University of Pretoria, 2016. / The Institute for Tropical Medicine, Antwerp, Belgium / University of Pretoria / National Research Foundation of South Africa / Veterinary Tropical Diseases / PhD / Unrestricted

This dissertation reports on the technology of cell-free protein synthesis (CFPS) including 1) stabilized lyophilized cell-free systems and 2) enhanced heterogeneous cell extracts. This work further considers applications of CFPS systems in 1) rapid vaccine development, 2) functional virus-based nanoparticles, 3) site-specific protein immobilization, and 4) expanding the language of biology using unnatural amino acids. CFPS technology is a versatile protein production platform that has many features unavailable in in vivo expression systems. The primary benefit cell-free systems provide is the direct access to the reaction environment, which is no longer hindered by the presence of a cell-wall. The “openness" of the system makes it a compelling candidate for many technologies. One limitation of CFPS is the necessity of freezing for long-term viable storage. We demonstrate that a lyophilized CFPS system is more stable against nonideal storage than traditional CFPS reagents. The Escherichia coli-based CFPS system in this work is limited by the biocatalytic machinery found natively in E. coli. To combat these limitations, exogenous biocatalysts can be expressed during fermentation of cells prepared into extract. We demonstrate that simple adjustments in the fermentation conditions can significantly increase the activity of the heterogeneous extract. Towards virus-based particles and vaccines, we demonstrate that the open nature of CFPS can be utilized for coexpression of virus proteins and self-assembly of virus particles. This technique allows for the rapid production of potential vaccines and novel functional virus-based nanoparticles. Unnatural amino acids expand the effective language of protein biology. Utilizing CFPS as an expression system, we demonstrated that the incorporation of a single specific unnatural amino acid allows for site-specific immobilization, thus stabilizing the protein against elevated temperatures and chemical denaturants. Current unnatural amino acid incorporation technologies are limited to one or few simultaneous incorporations and suffer from low efficiency. This work proposes a system that could potentially allow for upwards of 40 unnatural amino acids to be simultaneously incorporated, effectively tripling the protein code. These projects demonstrate the power and versatility of CFPS technologies while laying the foundation for promising technologies in the field of biotechnology.

No description available.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease that affects even-toed hoofed mammals. The FMD virus (FMDV) is the causative agent of FMD, of which there are seven clinically indistinguishable serotypes. Three serotypes, namely, South African Territories (SAT)1, SAT2 and SAT3 are endemic to southern Africa and are the most antigenically diverse among the FMDV serotypes. A negative consequence of this antigenic variation is that infection or vaccination with one virus may not provide immune protection from other strains or it may only confer partial protection. The identification of B-cell epitopes is therefore key to rationally designing cross-reactive vaccines that recognize the immunologically distinct serotypes present within the population. Computational epitope prediction methods that exploit the inherent physicochemical properties of epitopes in their algorithms have been proposed as a cost and time-effective alternative to the classical experimental methods. The aim of this project is to employ in silico epitope prediction programmes to predict B-cell epitopes on the capsids of the SAT serotypes. Sequence data for 18 immunologically distinct SAT1, SAT2 and SAT3 strains from across southern Africa were collated. Since, only one SAT1 virus has had its structure elucidated by X-ray crystallography (PDB ID: 2WZR), homology models of the 18 virus capsids were built computationally using Modeller v9.12. They were then subjected to energy minimizations using the AMBER force field. The quality of the models was evaluated and validated stereochemically and energetically using the PROMOTIF and ANOLEA servers respectively. The homology models were subsequently used as input to two different epitope prediction servers, namely Discotope1.0 and Ellipro. Only those epitopes predicted by both programmes were defined as epitopes. Both previously characterised and novel epitopes were predicted on the SAT strains. Some of the novel epitopes are located on the same loops as experimentally derived epitopes, while others are located on a putative novel antigenic site, which is located close to the five-fold axis of symmetry. A consensus set of 11 epitopes that are common on at least 15 out of 18 SAT strains was collated. In future work, the epitopes predicted in this study will be experimentally validated using mutagenesis studies. Those found to be true epitopes may be used in the rational design of broadly reactive SAT vaccines / Life and Consumer Sciences / M. Sc. (Life Sciences)

Foot-and-mouth disease virus (FMDV) is a highly infectious virus affecting cloven-hoofed animals. The most prominent of its clinical signs is the development of vesicular lesions on the feet and in or around the mouth, which are a consequence of extensive FMDV-induced epithelial cell death. Currently, there is no certain biological knowledge on why extensive epithelial cell death occurs in some FMDV-infected tissues, but not in others. Using the epithelial tissues of tongue and dorsal soft palate as examples of a tissue where lesions occur and one that does not visibly exhibit FMDV-induced cell death, this work aims to identify the potential drivers of epithelial cell death and survival. A partial differential equation (PDE) model informed by experimental data on epithelial structure, is used to test epithelium thickness and cell layer structure as potential determinants. A second PDE model investigates FMDV-interferon (IFN) dynamics and their impact on the levels of cell death and survival, while an experimental study is undertaken to provide data for model validation. The work carried out casts light on the important role of a variety of factors including FMDV replication, IFN production and release, and IFN antiviral action.

L’augmentation depuis une trentaine d’années des maladies infectieuses dites émergentes ou ré-émergentes chez l’homme, causées à plus de 70% par des pathogènes issus d’espèces hôtes animales (i.e. Ebola, SIDA), stimule l’étude de systèmes éco-épidémiologiques à l’interface entres populations humaines et animales (i.e. sauvages et/ou domestiques).Le contact entre hôtes est un phénomène important dans l’étude de ces systèmes car il permet la transmission des pathogènes entre individus et la diffusion de maladie au sein et entre populations. Nous avons choisi la maladie de la fièvre aphteuse comme modèle d’étude de la transmission de pathogènes des populations sauvages vers les populations domestiques. Le buffle africain (Syncerus caffer) étant le réservoir présumé de cette maladie fortement contagieuse, nous nous sommes interrogés sur les conditions de transfert au bétail (Bos taurus et Bos indicus) du virus aphteux aux frontières de trois parcs nationaux africains qui constituent des interfaces entre espaces anthropiques et protégés perméables aux mouvements d’animaux. Dans le cadre de ce doctorat 4 protocoles ont été mis en place entre 2010 et 2011 au Zimbabwe. Premièrement, des colliers GPS (Global Positionning System) ont été déployés sur des bovins sauvages/domestiques pour décrire leurs déplacements dans le paysage et quantifier les contacts interspécifiques. Des colliers furent également posés sur l’une des espèces prédatrices de ces ongulés: le lion (Panthera leo). L’intégration de la guilde des prédateurs nous a permis d’estimer les modifications de l’utilisation de l’espace par les herbivores en réponse à la présence de carnivores et les conséquences en termes de contacts et de transmission interspécifique de pathogènes. Deuxièmement, un suivi longitudinal sérologique sur le bétail a complété le protocole télémétrique avec des prélèvements répétés sur des individus marqués selon le cycle saisonnier. Troisièmement, les contacts au sein des populations de bovins domestiques ont été caractérisés par des enquêtes auprès des éleveurs. Quatrièmement, le rôle potentiel de la diversité des hôtes sur le risque infectieux d’un écosystème a été exploré par l’estimation de densité de macro-parasites dans le paysage selon une variation de la gamme d’hôtes potentiels (i.e. (i) sauvages, (ii) sauvages et domestiques et (iii) uniquement domestiques).Nos résultats montrent que (1) les taux d’interaction interspécifiques, estimés par télémétrie, varient entre sites et présentent une saisonnalité prononcée (i.e. pic saison sèche chaude). (2) La distribution des ressources conditionne la périodicité et la distribution de ces contacts dans les différents compartiments du paysage. (3) La fréquence des incursions du bétail dans un espace protégé ainsi que les taux de contacts avec les buffles influencent positivement la probabilité d’acquisition d’anticorps anti-aphteux chez le bétail. La probabilité de perte d’anticorps est également fonction du niveau d’interaction avec les buffles mais selon une relation négative. (4) La densité du réseau d’interaction intra-spécifique domestique influence positivement l’incidence sérologique de la fièvre aphteuse. (5) La présence de prédateurs supérieurs dans le paysage permettrait de limiter les incursions du bétail dans les espaces protégés et diminuerait la probabilité d’infection par les populations d’hôtes sauvages. (6) Enfin les densités de macro-parasites dans la végétation sont supérieures dans des espaces communaux sans interaction avec les populations sauvages et où la richesse spécifique des hôtes est plus faible. Les résultats de cette étude sur la transmission interspécifique de pathogènes entre populations sauvages et domestiques dans les écosystèmes tropicaux ouvrent des champs de réflexion encore largement inexplorés, notamment sur l’évolution de la virulence et des modes de transmission des pathogènes ayant comme hôtes des populations sympatriques sauvages et domestiques. / Emerging or re-emerging diseases in human populations have increased over the last thirty years. Since 70% of such diseases are caused by pathogens originating from animal hosts (i.e. Ebola, AIDS, and avian influenza), this increase has prompted the study of eco-epidemiological systems that occur at the interface between human and animal populations (i.e. wild and/or domestic). Contacts between hosts are particularly important factors in these systems since they result in pathogen transmission among individuals and, therefore, disease diffusion within and among populations. We used foot-and-mouth disease (FMD) as a model to study pathogen transmission from wild to domestic populations. As the African buffalo (Syncerus caffer) is the presumed reservoir of this highly contagious disease, we examined the conditions in which the virus was transmitted to cattle sensitive to the disease (Bos taurus and Bos indicus) at the borders of African national parks; these areas are interfaces between anthropogenic and protected areas in which animals can move freely.In the context, 4 protocols were implemented between 2010 and 2011 in Zimbabwe. First, GPS (Global Positioning System) collars were placed on cattle and buffalo in order to describe and analyze their movements across the landscape as well as to quantify interspecific contacts. In one of the study sites, collars were attached to one of the predators of these ungulates: the lion (Panthera leo). By integrating the predator guild into our telemetry protocols, we could examine the potential changes in spatial use by cattle and buffalo in response to predator presence and their consequences for contact dynamics and interspecific pathogen transmission. Second, a longitudinal serological survey was conducted in which tagged individuals were sampled repeatedly over the course of different seasons. Third, to characterize contacts within the domestic host population, interviews were conducted with cattle owners regarding their husbandry practices across seasons. Fourth, to describe the potential role of host diversity in ecosystem infection risks, macroparasite density (i.e. ticks) was estimated for landscape compartments that contrasted in terms of potential hosts present (i.e. (i) wild, (ii) domestic and wild, and (iii) domestic only).Our study primarily shows the following results. (1) Interspecific interaction rates, as estimated by telemetry, vary between sites and have a pronounced seasonality (peak occurs during the hot dry season). (2) Resource distribution (i.e. water and grazing areas) seems to condition the frequency and distribution of these contacts in the different landscape compartments. (3) Cattle incursion frequencies into protected areas and the frequency and intensity of contact with buffalo significantly positively affect the probability of foot-and-mouth antibody acquisition in cattle. The probability of antibody loss in cattle is also a function of diminished rates of interaction with buffalo. (4) Intraspecific interaction densities positively influence FMD serological incidence in cattle. (5) Top predator presence in the landscape could limit cattle incursion into protected areas and reduce the likelihood of their being infected by wild host populations. (6) Finally, the estimated densities of macroparasites in the vegetation were higher in communal spaces where there was no interaction with wild hosts and where host species richness was weak.The results of this study on the interspecific transmission of pathogens between wild and domestic populations in tropical ecosystems encourage the exploration of research topics that are still largely unexplored, including the evolution of virulence transmission modes of pathogens hosted by sympatric wild and domestic populations.

A Dengue e a Febre Aftosa são infecções virais que ocorrem tanto no Brasil como no mundo, apresentando enorme impacto socioeconômico. As proteases virais são reconhecidas como alvos de grande interesse para o planejamento de antivirais, devido ao seu papel fundamental no ciclo de vida de muitos vírus, incluindo-se os flavivirus (vírus da Dengue DENV) e os picornavirus (vírus da Febre Aftosa FA). Com o objetivo de buscar e identificar novos inibidores de proteases virais (da NS2B/NS3pro do DENV ou da Lbpro do vírus da FA) propusemos modelos de busca virtual, incluindo diferentes filtros de seleção (ex. farmacofórico, drug-like, similaridade e ancoramento) aplicados, sequencialmente, a bancos de dados de compostos comerciais (280x103 a 23x106 compostos). A seleção final dos compostos foi sempre feita por inspeção visual. Para NS2B/NS3pro do DENV, construíram-se quatro modelos de busca virtual (Modelo I-DENV a Modelo IV-DENV). O primeiro foi construído, baseando-se na estrutura cristalográfica ligada a um inibidor peptidomimético, e aplicado ao banco ZINC. Ao final, dez compostos foram comprados e submetidos a testes de inibição enzimática, frente à NS2B/NS3pro, para validação experimental deste modelo. Dois compostos mostraram alguma atividade inibitória (IC50 150 - 300 µM). Visando-se melhorar estes resultados, a flexibilidade da NS2B/NS3pro foi incluída, usando simulações de dinâmica molecular (DM), e um novo banco de dados construído (ZINC-Curated). Através de uma análise extensiva do banco ZINC-Curated, usando ferramentas estatísticas/quimiométricas, confirmou-se que este foi enriquecido com compostos com características de fármacos. Outros três modelos de busca virtual foram construídos incluindo-se diferentes informações obtidas nas simulações de DM. O modelo II-DENV foi feito usando ancoramento e aplicado ao banco ZINC-Curated, selecionando dezesseis compostos. Nenhum deles apresentou atividade inibitória significativa frente à NS2B/NS3pro do DENV. Os modelos III-DENV e IV-DENV utilizaram modelos farmacofóricos, que tiveram seus desempenhos previamente avaliados usando dados de literatura, e foram aplicados aos bancos NCI e ZINC-Curated, respectivamente. O modelo III-DENV selecionou quinze compostos, tendo quatro deles apresentado atividade inibitória (IC50 30 - 100 µM). O modelo IV-DENV selecionou dezoito compostos, sendo quatro ativos frente a esta protease (IC50 4 - 90 µM), representando uma taxa de acerto de ~22 %. Ainda, uma série de treze análogos estruturais do composto mais ativo foi construída, sendo três deles também ativos. Portanto, as modificações incluídas na busca virtual permitiram melhorar, significativamente, os resultados obtidos. Para Lbpro do vírus da FA, construíram-se dois modelos de busca virtual (Modelos I-FA e II-FA). O primeiro foi construído usando sua estrutura cristalográfica, sem ligantes, e uma série in house de potenciais inibidores covalentes. Seis compostos foram selecionados e testados frente à Lbpro, tendo dois deles baixa atividade inibitória (IC50 300 - 600 µM). A partir da disponibilidade da estrutura da Lbpro com ligante, o modelo IIFA foi construído e aplicado ao banco ZINC-Curated, selecionando quinze compostos. Estes foram adquiridos e testados frente à Lbpro, não apresentando atividade inibitória significativa. Assim, as modificações incluídas ainda não foram suficientes para selecionar inibidores mais potentes. No entanto, estes modelos/resultados contribuíram para o entendimento da(s) interação(ões) no sítio ativo da Lbpro. / Dengue and Food-and-mouth disease are viral infections that occur in Brazil and in the world, causing a huge socioeconomic impact. Viral proteases are recognized as targets for antiviral design, because they are crucial for the life cycle of many viruses, such as flavivirus (Dengue virus DENV) and picornavirus (Food-and-mouth disease virus FMDV). In order to discovery novel inhibitors of viral proteases (of NS2B/NS3pro of DENV or of Lbpro of FMDV) virtual screening models were proposed comprising a sequence of different filters (e.g. pharmacophore, drug-like, similarity and docking) applied to databases of commercial compounds (280x103 to 23x106 compounds). In all models, the final selection of compounds was always done by visual inspection. For DENV NS2B/NS3pro, four virtual screening models were proposed (Model I-DENV to Model IV-DENV). Model I-DENV was built, based on the crystal structure bound to a peptidemimetic inhibitor, and applied to ZINC database. Finally, ten compounds were purchased and submitted to enzymatic assays against this protease to the experimental validation of this model. Two compounds showed some inhibitory activity (IC50 150 - 300 µM). In order to improve these results, NS2B/NS3pro flexibility was included, using molecular dynamics (MD) simulations, and a novel database was built (ZINC-Curated). Throughout an exhaustively analysis of ZINC-Curated, using statistical/chemometrics tools, we confirmed that this new database was enriched with drug like compounds. Other three virtual screening models were built including different information from MD simulations. Model II-DENV was built using docking and applied to the ZINC-Curated database, selecting sixteen compounds. None of them showed a significant inhibitory activity against DENV NS2B/NS3pro. Models III-DENV and IV-DENV were built using pharmacophore models, which have their performance previously evaluated using literature data, and applied to NCI and ZINC-Curated databases, respectively. Model III-DENV selected fifteen compounds, showing four of them inhibitory activity (IC50 30 - 100 µM). Model IV-DENV selected eighteen compounds. Four of them were active against this protease (IC50 4 - 90 µM), representing a hit rate of ~22 %. Moreover, a set of thirteen structural analogues of the most active compound were built, being three of them also active. Thus, the modifications done in the virtual screening procedure really improved our results. For FMDV Lbpro, two virtual screening models were built (Models I-FMDV and II-FMDV). The first one was based on the crystal structure, without ligands, and used a set of in house potential covalent inhibitors. Six of the in house compounds were selected and tested against this protease. Two of them showed a weak inhibitory activity (IC50 300 - 600 µM). Later on, the Lbpro bound with ligands was available being therefore used to build another model. Model II FMDV was applied to the ZINC-Curated database, selecting fifteen compounds that were purchased and also tested against the target protease. But none of them showed a significant inhibition. Thus, the incorporated changes were not enough to retrieve active compounds. However, these models/results contributed to better understand Lbpro binding site interactions

O presente trabalho busca analisar a dinâmica econômica dos fluxos da pecuária bovina no Mato Grosso do Sul (MS) e identificar sua distribuição geográfica dentro do estado. Dado que as barreiras sanitárias e técnicas constituem, atualmente, alguns dos principais obstáculos ao comércio internacional, o conhecimento sobre as características dessa dinâmica e a identificação dos municípios centrais na pecuária do estado permitem analisar o processo de difusão da febre aftosa no MS em um eventual de surto da doença. Dessa forma, foram identificadas as áreas de maior risco de ocorrência e de potenciais maiores impactos econômicos. A metodologia aplicada consiste na análise de redes socioeconômicas, construídas a partir das Guias de Trânsito Animal registradas no estado em 2014 e 2015. Verificou-se que a movimentação dentro do MS se traduz em redes fortemente conectadas, o que poderia resultar em uma alta velocidade de difusão da doença no território. Os municípios de Campo Grande, Corumbá e Ribas do Rio Pardo mostraram-se os mais centrais nesse processo, pois recebem e enviam muitos animais para outros municípios, além de atuarem como intermediadores de fluxos animais dentro das redes de movimentação. Por conta do fornecimento de animais para outros estados, a difusão pode ocorrer a nível nacional, elevando os impactos econômicos da doença. Nesse sentido, foram estimadas as perdas de exportação e custos de controle do último surto de febre aftosa ocorrido no MS, em 2005/2006. As exportações do estado recuaram 81% em 2006, com relação ao ano anterior. A partir do modelo teórico de simulação e das estimativas de custos do último surto, conclui-se que, diante de uma potencial difusão de febre aftosa, com origem no MS, os impactos diretos na pecuária bovina seriam significativos, o que torna indispensável a promoção de estudos sobre impactos e riscos da entrada e difusão da doença no país, de modo a otimizar a alocação de recursos em termos de sua prevenção e, em caso de crise, no Plano de Emergência. / This work seeks to analyze the economic dynamics of cattle flows in Mato Grosso do Sul (MS) state and to identify their geographic distribution within the state. Given that sanitary and technical barriers are currently some of the main obstacles to international trade, knowledge about the characteristics of this dynamics and the identification of central municipalities in terms of livestock flows allow analyzing the diffusion process of the foot-and-mouth disease in MS in a possible outbreak. In this way, the municipalities with the greatest risk of occurrence and potential economic impacts were identified. The applied methodology consists on socioeconomic network analysis, constructed from the Animal Transit Guides registered in MS in 2014 and 2015. The cattle movement within MS produces heavily connected networks, which could result in a high-speed dissemination of the disease in the territory. The municipalities of Campo Grande, Corumbá and Ribas do Rio Pardo were the most central in this process, since they receive and send many animals to other municipalities, besides acting as intermediaries of animal flows within the movement networks. Due to the supply of animals to other states, the diffusion can occur at a national level, raising the economic impacts of an outbreak. In this sense, the export losses and the costs of emergence to control last FMD outubreak in MS, in 2005/2006, were estimated. The State exports declined 81% in 2006, compared to the previous year. Considering the theoretical model of simulation and the costs and losses estimated for the last outbreak, we conclude that a potential diffusion of the FMD, originating in MS, might generate significant direct impacts on livestock. This conclusion highlights how essential it is to promote studies about the impacts and risks of the FMD entry and diffusion in Brazil, in order to optimize the allocation of resources to prevention strategies and, in case of an outbreak, to implement the Emergency Plan.