Thesis (Ph.D. (Biochemistry)) -- University of Limpopo, 2021 / Introduction: Rift Valley fever virus (RVFV) is an arthropod-born RNA zoonotic virus causing Rift Valley fever (RVF) disease. RVFV is prevalent across sub-Saharan Africa and the Arabian Peninsula with no existing effective and approved antiviral remedies for humans or animals. RVFV has developed mechanisms to hide from immune recognition and induce anti-apoptosis processes to keep the infected host cells viable in an attempt to advance their viral progeny. RVFV is a single-stranded enveloped RNA genome virus composed of 3 segments; the L, M and S segments. The S segment is known to encode a non-structural protein (NSs) identified to be the main virulence factor promoting viral replication through immune suppression. RVFV elicits a set of diverse symptoms ranging from a febrile illness to more severe symptoms that usually culminate in life-threatening haemorrhagic fever with high fatality rates. Thus, this study was designed to investigate the efficacy of lithium as a potential drug for reduction of RVFV load and amelioration of imbalanced and dysregulated inflammatory responses observed in Huvec and Raw 264.7 macrophages infected with this virus. Methods and results: The MTT and Cyquant viability assays were used to demonstrate that lithium exerts no cytotoxic effects on non-infected Raw 264.7 macrophage cells but rather promotes cell growth and proliferation. Conversely, lithium was shown to significantly induce cell death in RVFV-infected Raw 264.7 macrophages. The Annexin-V/PI apoptosis assay was employed to demonstrate that RVFV induces apoptosis as a mode of cell death on Raw 264.7 cells. RVFV-induced apoptosis was accompanied by antagonistic Bax/Bcl-2 protein expression ratios. RVFV-infected cells treated with lithium resulted in higher levels of apoptosis signals compared to untreated RVFV-infected cells. Analysis of apoptosis stages using the real-time cell analyser (RTCA) also revealed that lithium induced early forms of apoptosis in RVFV-infected cells. Interestingly, induction of early apoptosis in these cells corresponds with lower viral load, probably as a result of early inhibition of viral progeny replication, as determined using viral titration assay. Immune response profiles elicited in Raw 264.7 macrophages infected with RVFV and treated with lithium were monitored. An ELISA assay was used to determine the effect of lithium on cytokines and chemokine production in this cell model. The results obtained showed that lithium significantly stimulated production of IFN-γ as RVFV-infected lithium-treated cells produced high levels of IFN-γ compared to lithium-free RVFV-infected control cells. Furthermore, in the same setting, the secondary pro-inflammatory cytokine, IL-6, and chemokine, RANTES, were stimulated by lithium 12 hrs post-infection (pi). Lithium was shown to significantly stimulate TNF-α production as early as 3 hrs pi. In addition to TNF-α expression, the expression of the regulatory cytokine, IL-10, was significantly stimulated by lithium with the highest expression peak at 12 hrs pi. As determined using the H2DCF-DA and DAF-2 DA florigenic assays, reduced production of the ROS and RNS was observed in RVFV-infected lithium-treated cells as opposed to untreated RVFV-infected controls. This was further supported by the Western blot assay results that showed low expression of the iNOS while upregulating expression of heme oxygenase and IκB in RVFV-infected lithium-treated cells. Results from immunocytochemistry and Western blot assays revealed that lithium inhibits NF-κB nuclear translocation in RVFV-infected cells compared to lithium-free RVFV-infected cells and 5 mg/ml LPS controls. This study hypothesises persistent and deregulated inflammation as the central phenomenon responsible for endothelial damage and haemorrhagic fever in RVFV pathogenesis. Supernatants were collected from RVFV-infected macrophage cells treated with lithium and their effects on the integrity of endothelial cells were evaluated. The xcelligence real-time cell analyser system (RTCA) and transwell assay that measure endothelial monolayer integrity were used to demonstrate that lithium protects endothelial cells from RVFV-induced cellular damage. Moreover, lithium was shown to upregulate expression of cytoplasmic molecules such as α and β-catenins involved in attaching the cadherin molecules to the actin cytoskeleton on the endothelial cell. Expression of α-catenins, talins, zyxins and vinculins that attach integrins to the extracellular matrix and to other cells were observed to be upregulated by supernatants from RVFV-infected Raw 264.7 macrophage cells treated with lithium. Endothelial cell monolayer exposed to supernatants from RVFV-infected lithium-treated Raw 264.7 cells displayed upregulated expression of transmembrane molecules such as E-cadherins and N-cadherins. However, expression of VE-cadherins was observed to be lower compared to those treated with supernatants from lithium-free RVFV-infected Raw 264.7 control cells. Conclusion: These findings propose that lithium limits viral replication and viral load in macrophages by inducing early apoptosis in RVFV-infected cells. Since lithium was shown to promote Raw 264.7 macrophage proliferation, it is thus suggested that the use of lithium as an RVFV antiviral drug is less likely to elicit leukocytopenia. Lithium seems to regulate excessive inflammation in RVFV-infected Raw 264.7 macrophages by modulating the NF-kB signalling pathway. The endothelial integrity observed in the permeability assays has been supported by the expression of the molecules involved in keeping the cell to cell adhesion intact. This study links endothelial integrity patterns exerted by lithium with lowered production of inflammatory mediators such as ROS and RNS as these molecules are involved in destabilisation of cell junctions. Results from this study point towards the use of lithium as a potential treatment for RVFV infections by limiting viral replication, restricting viral spread and restoring the inflammation-regulating machinery. Key words. Lithium, Rift Valley fever virus, NF-kB, endothelial integrity, inflammation and apoptosis / Poliomyelitis Research foundation and National Research Foundation

Rift Valley fever virus (RVFV) is a mosquito-borne virus which has the ability to infect a large variety of animals including humans in Africa and Arabian Peninsula. The abortion rate among these animals are close to 100%, and young animals develop severe disease which often are lethal. In humans, Rift Valley fever (RVF) presents in most cases as a mild illness with influenza-like symptoms. However, in about 8% of the cases it progresses into a more severe disease with a high case fatality rate. Since there is such a high abortion rate among infected animals, a link between human miscarriage and RVFV has been suggested, but never proven. We could in paper I for the first time show an association between acute RVFV infection and miscarriage in humans. We observed an increase in pregnant women arriving at the Port Sudan Hospital with fever of unknown origin, and several of the patients experienced miscarriage. When we analysed their blood samples for several viral diseases we found that many had an acute RVFV infection and of these, 54% experienced a miscarriage. The odds of having a miscarriage was 7 times higher for RVFV patients compared to the RVFV negative women of which only 12% miscarried. These results indicated that RVFV infection could be a contributing factor to miscarriage. RVFV is an enveloped virus containing the viral glycoproteins n and c (Gn and Gc respectively), where Gn most likely is responsible for the initial cellular contact. The protein DC-SIGN on dendritic cells and the glycosaminoglycan heparan sulfate has been suggested as cellular receptors for RVFV, however other mechanisms are probably also involved in binding and entry. Charge is a driving force for molecular interaction and has been shown to be important for cellular attachment of several viruses, and in paper II we could show that when the charge around the cells was altered, the infection was affected. We also showed that Gn most likely has a positive charge at a physiological pH. When we added negatively charged molecules to the viral particles before infection, we observed a decreased infection efficiency, which we also observed after removal of carbohydrate structures from the cell surface. Our results suggested that the cellular interaction partner for initial attachment is a negatively charged carbohydrate. Further investigations into the mechanisms of RVFV cellular interactions has to be undertaken in order to understand, and ultimately prevent, infection and disease. There is currently no vaccine approved for human use and no specific treatments for RVF, so there is a great need for developing safe effective drugs targeting this virus. We designed a whole-cell based high-throughput screen (HTS) assay which we used to screen libraries of small molecular compounds for anti-RVFV properties. After dose-response and toxicity analysis of the initial hits, we identified six safe and effective inhibitors of RVFV infection that with further testing could become drug candidates for treatment of RVF. This study demonstrated the application of HTS using a whole-cell virus replication reporter gene assay as an effective method to identify novel compounds with potential antiviral activity against RVFV.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a virus of the family Bunyaviridae, genus Phlebovirus. It is responsible for extensive outbreaks of disease in livestock in Africa with significant mortality and economic impact. Virus neutralization is considered the gold standard for confirming Rift Valley fever virus (RVFV) infection but the procedure is time consuming and expensive. Real-time reverse transcription-polymerase chain reaction (rRT-PCR), histopathology, and immunohistochemistry (IHC) are the diagnostic methods most often used in South Africa to confirm or exclude a diagnosis of RVF in necropsied animals. Validated estimates of diagnostic accuracy of these tests, in naturally infected livestock, however, have not been published. The objective of this study was to estimate the diagnostic sensitivity and specificity of rRT-PCR, histopathology, and IHC using Bayesian latent class methods in the absence of a gold standard. A secondary objective was to estimate stratum-specific values based on species, age, degree of specimen autolysis, and the presence/absence of tissue pigments. The Sensitivity (Se) and Specificity (Sp) of qRT-PCR were 97.4% (95% credibility interval (CI): 95.2% - 98.8%) and 71.7% (95% CI: 65% - 77.9%) respectively. The extraordinary analytical sensitivity of PCR makes this test very susceptible to false positive reactions, and thus reduced specificity. This is more likely during large-scale epidemics due to crosscontamination of specimens at necropsy facilities or testing laboratories. The Se and Sp of histopathology were 94.6% (95% CI: 91% - 97.2%) and 92.3% (95% CI: 87.6% - 95.8%) respectively. Single cases of RVF could be confused with acute poisoning with plants, bacterial septicaemias, and viral diseases such as infectious bovine rhinotracheitis and Wesselsbron disease. Most of these conditions, however, can be excluded using histological examination of the liver, special stains, bacterial culture, and toxicological or serological investigations. The Se and Sp of IHC were 97.6% (95% CI: 93.9% - 99.8%) and 99.4% (95% CI: 96.9% - 100%) respectively. Immunohistochemistry is highly specific because characteristic positive immunolabelling of the cytoplasm of hepatocytes can be correlated with the presence of hepatocellular injury typical for RVFV infection. False negative results are sometimes obtained with IHC because of reader error or loss of the antigenic epitopes due to advanced autolysis. Scant positive immunolabelling might be missed or viral proteins might be absent from sections of liver with advanced hepatocellular damage. The stratified analysis suggested differences in test accuracy in foetuses and severely autolysed specimens. The Sp of histopathology in foetuses (83.0%) was 9.3% lower than the value obtained for the sample population (92.3%). Lesions in some foetuses are more subtle and the typical eosinophilic intranuclear inclusions are often difficult to detect. In severely autolysed specimens, the Se of IHC decreased by 16.1% and the Sp of rRT-PCR by 17.4%. There is no plausible biological explanation for this decrease in the Sp of rRTPCR since the RNA of RVFV is resistant to degradation in autolysed tissues. Conversely, the antibody used to detect RVFV using IHC detects epitopes raised against nucleoproteins of the virus and it is possible that viral proteins become too widely dispersed and/or degraded in autolysed tissues to detect by light microscopy. It is possible that the marked decrease in Se of histopathology and IHC in severely autolysed specimens caused an apparent decrease in Sp of rRT-PCR, due to the latent class method. In conclusion, the high estimated Sp (99.4%) of IHC and the low Sp of rRT-PCR (71.3%) suggests that the definitive diagnosis or exclusion of RVF should not rely on a single PCR test and that IHC would be an effective confirmatory test for rRT-PCR positive field cases necropsied during an epidemic. Immunohistochemistry results from severely autolysed specimens, however, should be interpreted with caution and aborted foetuses in areas endemic for RVF should be screened using a variety of tests. The diagnostic Se and Sp of histopathology was much higher than expected confirming the value of routine post mortem examinations and histopathology of liver specimens. The most feasible RVF testing option in areas that do not have suitably equipped PCR laboratories, and where disease is often not detected in livestock until after human cases have been diagnosed, would be routine histopathology screening with IHC confirmation. Key Words: Rift Valley fever; Rift Valley fever virus; Bayesian; latent-class model; real-time reverse transcription-polymerase chain reaction; immunohistochemistry; histopathology; diagnosis; sensitivity; specificity. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Paraclinical Sciences / unrestricted

From 1930 to 2000 Rift Valley Fever (RVF) was limited to the African continent. It is vector borne disease caused by a virus of the genus Phlebovirus, member of the Bunyaviridae family. The main vectors for transmission are Aedes and Culex. In September 2000 it was reported for the first time out of Africa, affecting Yemen and Saudi Arabia. This epidemic opened a new era in the history of RVF. It proved that the virus had the capaciity to affect new zones, different eco-systems and to spread to any area in the world. The outbreak lifted many hypothesis related to its introduction and to the factors associated with the outbreak. Although difficult to evaluate precisely its socio-economic impact was considered to be the heaviest in the modern history of Yemen animal diseases even when compared to the rinderpest outbreak that the country encountered in the seventies because of its zoonotic characteristic. To answer to these hypotheses it was of great importance to study and investigate all the factors associated with the outbreak. Thus after estimating the socio-economic impact of the disease in the world and more specifically in Yemen we studied the descriptive epidemiology of RVF in the first and most affected zone of the outbreak of 2000-2001 (Tihama Wadi Mawr) and then we analysed the socio-economic and environmental factors associated to the outbreak, to finish with the risk assessment of the re-introduction of RVFV from the Horn of Africa through legal animals trade. The descriptive study showed that at the national level 90% of the RVF cases were in the plain of Tihama coast, Hodiedah, Hajjah and Sadah governorates, the majority of the villages being located around the main canals of Wadi Mawr at an altitude < 300 m. Environmental as well as socio-economic factors likely to play a role in RVF transmission in Yemen were highlighted with the study of the period 1997-2007 in the country. As in previous RVF outbreaks in neighbouring countries in the Horn of Africa, the year 2000 presented above-normal vegetation index values, which reflected important precipitations, for both rainy seasons (the first occurring between March and May; the second between July and October). These environmental conditions favourable to the vectors populations were found concomitant with a late starting date of Eid-al Kabeer celebration (March) in 2000, related to high hosts (cattle, sheep and goats) densities. According to these criteria, 2000 was considered as an atypical year. Yemeni Veterinary Services did not declare any RVF outbreak since 2000. Thus, we assumed Yemen free of the disease when assessing the risk of introduction of RVF into Yemen via the legal trade of small ruminants from the Horn of Africa (Kenya, Somalia, Djibouti, and Ethiopia). After precisely describing the routes and volume of trade from the Horn of Africa to Yemen, the pathway and different scenarios for introduction were developed following the OIE risk assessment method. A matrix of likelihood combinations including four possible levels (very low, low, medium, high) was built and used to combine likelihood of events. The overall probability of introduction was assessed very low to medium depending on the period of the year and most likely to occur via ovine males exported during festival periods that change depending of the year considered. The uncertainty was considered to be low. The socio-economic impact although difficult to estimate was shown to be dramatic as RVF affects all the chain of life in particular of those associated to livestock and animal products trade. Despite the dramatic impact of the outbreak of RVF in 2000 but it had the advantage to draw the attention of decision makers, of international organizations and of local veterinary services on the importance of livestock diseases and their possible effects on human health and national economies. Veterinary education also improved significantly in Yemen. It enhanced the epidemiologists skills, disease surveillance in general and the cooperation between human health care people and veterinary services. A national P3 laboratory should also soon open with the help of the IAEA and the FAO and Al-Mukkah quarantine could be modernized and extended in the near future. Regional collaboration and improvement of knowledge on animal trade but also field studies related to the disease and its entomological features are seen as compulsory to hope improving the prevention and control of RVF.But the best way and strategy for prevention of Rift Valley Fever in Yemen as well as in the world is to develop more efficient surveillance and control tools to implement coordinated regional monitoring and control programmes. - Résumé - De 1930 à 2000, la Fièvre de la Vallée du Rift (RVF) était une maladie essentiellement africaine. Cest une maladie vectorielle causée par un virus du genre Phlebovirus de la famille des Bunyaviridae. Les principaux vecteurs sont les Aedes et les Culex. En Septembre 2000, des cas furent rapportés pour la première fois hors du continent africain touchant le Yémen et lArabie Saoudite. Cette épidémie ouvrit une nouvelle ére dans lhistoire de la RVF. Elle prouva la capacité du virus à infecter des zones nouvelles, des écosystèmes différents et sa capacité à se propager à nimporte quelle région du monde. Plusieurs hypothèses furent avancées concernant son introduction et les facteurs socio-économiques associés aux foyers. Sil est difficile dévaluer précisement son impact socio-economique, il fut considéré comme le plus lourd de lhistoire moderne des maladies animales au Yémen même comparé à celui de la peste bovine dans les années 70 à cause de son caractère zoonotique. Pour répondre à ses hypothèses, il était important détudier et de rechercher tous les facteurs associés aux foyers. Ainsi après avoir estimé limpact soci-èconomique de la maladie dans le monde et plus particulièrement au Yemen, lon sintéréssa à lépidémiologie descriptive de la RVF dans les premières zones touchées mais aussi les plus affectées lors des foyers de 2000-2001 (Tihama Wadi Mawr) Puis lon analysa les facteurs socio-économiques et environmentaux associés aux foyers pour finir avec lévaluation de risque de la réintroduction du RVFV à partir du commerce légal danimaux de la Corne dAfrique. Létude descriptive montre quau niveau national, en 2000-2001 environ 90 % des cas étaient dans la plaine de la côte de Tihama, Hodiedah, Hajjah et Sadah, la plupart des villages étant localisées autour des canaux principaux de Wadi Mawar à une altitude infereure à 300 Mètres (m). Les facteurs environmentaux et socioèconomiques susceptibles davoir un rôle dans la transmission de la RVF au Yémen furent soulignés avec létude de la période 1997-2007 dans le pays. Comme dans les foyers précédents de RVF dans les pays voisins de la Corne de lAfrique, lannée 2000 présentait des valeurs anormalement élevées dindex normal de végétation refletant dimportantes précipitations lors des deux saisons des pluies (la première entre mars et mai, la seconde entre juillet et octobre). Ces conditions environmentales favorables aux populations de vecteurs apparurent concommitantes avec une date tardive de début des festivités de lEid-al Kabeer en (mars) 2000 entraînant à une densité importante de populations hôtes (bovins, moutons, chèvres). Considérant ces facteurs lannée 2000 fut considérée comme une année atypique. Les services vétérinaires yéménites ne déclarèrent pas de foyers de RVF depuis 2000. Ainsi on fit lhypothèse que le Yémen nétait plus infecté pusqon lon sintéressa au risque de ré-introduction de la RVF au Yémen via le commerce légal de petits ruminants depuis la Corne de lAfrique (Kenya, Somalie, Djibouti et Ethiopie). Après avoir précisemment décrit les routes et volumes déchanges depuis la Corne de lAfrique jusquau Yémen, les chemins événementiels et différents scénarios dintroduction furent développé en suivant la méthode de lOIE. Une matrice de combinaison des probabilités incluant quatre niveaux (très bas, bas, modéré, élevé) fut construite et utilisée pour combiner les probabilité de réalisation des évènements. La probabilité globale dintroduction fut trouvée très basse à modérée en fonction de la période de survenue dans lannée et plus probablement via lintroduction dovins males exportés pendant les périodes de fêtes dont loccurrence change en fonction de lannée considérée. Lincertitude fut considérée basse. Si l'impact socio-économique de la RVF est difficile à évaluer, il est pourtant dramatique en atteignant tous les maillons du réseau de ceux dont la vie sorganise autour du bétail et de son commerce ou du commerce de ses produits. Malgré limpact dramatique des foyers de RVF en 2000, ceci a eu lavantage de dattirer l'attention de décideurs, dorganisations internationales et des services vétérinaires locaux sur l'importance des maladies de bétail et leurs effets possibles sur la santé humaine et les économies nationales. L'éducation vétérinaire s'est aussi améliorée de façon significative au Yémen. La qualification des épidémiologistes et la surveillance des maladies en général sen sont trouvées meilleures, ainsi que la coopération entre les services médicaux humains et les services vétérinaires. Un laboratoire national P3 devrait voir le jour prochainement avec laide de lIAEA (International Atomic Energy Agency) et de la FAO (Food and Agriculture Organization). La modernisation et l'extension de la quarantaine Al-Mukkah, est aussi prévue dans un avenir proche. La collaboration régionale, lamélioration de la connaissance du commerce du bétail mais aussi des études de terrain relatives à la maladie et à ses caractéristiques épidémiologiques sont indispensables pour espérer améloirer la prévention et le contrôle de la RVF. Mais la meilleures stragtègie pour la prevèntion de la fièvre de la vallée de Rift au Yemen comme dans le monde est de developper une surveillance plus efficace et controler des outils pour mettre en place un suivi regional coordonnée et des programmes de contrôle.

<p><font face="Times New Roman" size="3"><font face="Times New Roman" size="3"> <p align="left">The Social Capital approach to development is based on the premise that both cognitive and structural aspects of a given society determine the level of development performance. It is argued that norms of trust and reciprocity, networks, and mutual aid govern interaction among people, hold them together, facilitate opportunities to manage common property and pursue common goals, and ultimately contribute to development by facilitating their cooperation and collective action. In this thesis I explore the relationship between social capital and development by analysing the performance of household wellbeing in a given community. Two dominant views on social capital and the outcomes of development performance are currently in prominence in the development discourse. This debate centres on whether social capital is a sufficient cause on its own or whether it needs intermediary actors. The social capital theory argues that the development performance of a particular community can be explained directly by the prevailing level of social capital, and that the underlying levels of trust, social norms and networks are sufficient to explain development. On the other hand, the school of thought that supports the need for intermediary actors argues that social capital is unable to influence development performance at higher levels. If a larger impact and a more precise outcome are expected, intermediary actors who facilitate interest formation, aggregation and representation are necessary. Without them, social capital remains largely inactive and dormant. In exploring the possible link between the two variables, this thesis supports the second premises, introducing the notion of intermediary actors that might activate the stock of social capital and its performance on household wellbeing. The need to explore the relationship between the two variables demanded empirical research. The research was conducted in the rural villages of Oromia regional State of Ethiopia. Based on the empirical evidence, the relationship between the stock of social capital and performance in household wellbeing is generally positive. A large stock of social capital is generally accompanied by a higher level of performance in household wellbeing. However, I argue that social capital is only truly social when activated by an intermediary development actor. Failing this, though it contributes significantly to village solidarity and unity, social capital remains inactive and dormant. Therefore, the general conclusion of this thesis is that social capital matters, but its utilisation by intermediary agencies matters more.</p> </font></font></p>

<p><font face="Times New Roman" size="3"><font face="Times New Roman" size="3"> <p align="left">The Social Capital approach to development is based on the premise that both cognitive and structural aspects of a given society determine the level of development performance. It is argued that norms of trust and reciprocity, networks, and mutual aid govern interaction among people, hold them together, facilitate opportunities to manage common property and pursue common goals, and ultimately contribute to development by facilitating their cooperation and collective action. In this thesis I explore the relationship between social capital and development by analysing the performance of household wellbeing in a given community. Two dominant views on social capital and the outcomes of development performance are currently in prominence in the development discourse. This debate centres on whether social capital is a sufficient cause on its own or whether it needs intermediary actors.</p> </font></font></p>

Rift Valley Fever virus (RVFV) causes an infection with severe impact on animal and human health. The disease is endemic throughout almost the entire African continent and large regions of the Arabian Peninsula. During epidemics, high mortality is observed in animals, especially among cattle, goats, and sheep. In humans, the symptoms vary from a benign influenza-like disease to a life-threatening hemorrhagic fever. Due to the devastating effect on communities in endemic regions and the possibility of further spread of this virus, there is an imperative need to improve and develop control measurements against this emerging disease. Therefore, this thesis focuses on diagnostics and vaccines against RVFV. RVFV infection kinetics was studied in a mouse model system by detection and quantification of viral genomes, using a developed quantitative real-time PCR (QRT-PCR) method. This novel QRT-PCR method proved to be reliable and serves as a supplement to standard diagnostics, direct virus isolation and serological methods. High levels of viral RNA were found in blood and liver samples from experimentally infected mice during the first days post infection. Thereafter the levels declined rapidly and dropped below detection limit approximately seven days post infection. The QRT-PCR technique was also used in a study aimed to improve diagnosis of RVFV from field samples collected on filter strips. Today, the available RVFV vaccines are only approved for animal use and these vaccines have several shortcomings. Since RVFV is a highly pathogenic organism requiring bio-safety level 3 laboratories, two different none-replicating vaccine approaches have been applied and evaluated using a mouse model. A DNA based vaccine, administered via gene-gun, and the use of virus-like particles (VLP), by the intra-peritoneal route. RVFV specific and neutralising antibodies were raised with both vaccine approaches. However, VLP vaccination against Rift valley Fever proved to be more promising as a future vaccine, since higher titres of neutralising antibodies and improved survival rate were found upon a lethal RVFV challenge in mice. In conclusion, a sensitive and specific method for quantifying RVFV infection and a promising vaccine candidate against RVFV were developed.

A detailed study was made of the composition and distribution of modern and fossil animal and plant traces around saline, alkaline lakes in tectonically active, closed lake-basins. Modern and Pleistocene traces that were examined in lake basins of the Kenya Rift Valley (Lakes Bogoria, Magadi, and Nasikie Engida) were compared directly with fossil traces from the Eocene Lake Gosiute in the Green River Formation of Wyoming, U.S.A., which had a similar hydrochemistry. Analysis of lithofacies and the stratigraphic packaging of the sediments hosting biogenic structures was undertaken so that their vertical and lateral distribution could be used to interpret lake histories and to help to develop depositional models of enigmatic sedimentary successions. A focus was given to the application of the results for paleoecology and stratigraphy, and a model for predicting the position of different trace associations in vertical successions and in different parts of saline, alkaline lake basins has been developed. Evidence from the Kenyan lakes and Eocene Lake Gosiute shows that (1) sedimentary environments are diverse in underfilled basins, and frequent lake-level fluctuations strongly impact the distribution of sedimentary environments suitable for the production and preservation of biogenic structures; (2) the distribution of biogenic structures in underfilled basins is related to the geomorphological and structural setting, tectonic activity, catchment lithology, the basin margin or basin centre location, climate, and salinity and alkalinity, together with other finer-scale environmental and biological controls; (3) because saline environments are restrictive, sites of relatively dilute inflow (springs, rivers and deltas, ephemeral streams) provide oasis-like habitats for animals and plants, and contribute to the increased diversity and laterally variable distribution of saline-lake trace assemblages; and (4) the vertical distribution of trace fossils in a stratigraphic succession reflects changing environments through time; important stratigraphic surfaces, usually formed during periods of lake-level fall, can be recognized from the overprinting patterns of traces produced under different conditions.

A detailed study was made of the composition and distribution of modern and fossil animal and plant traces around saline, alkaline lakes in tectonically active, closed lake-basins. Modern and Pleistocene traces that were examined in lake basins of the Kenya Rift Valley (Lakes Bogoria, Magadi, and Nasikie Engida) were compared directly with fossil traces from the Eocene Lake Gosiute in the Green River Formation of Wyoming, U.S.A., which had a similar hydrochemistry. Analysis of lithofacies and the stratigraphic packaging of the sediments hosting biogenic structures was undertaken so that their vertical and lateral distribution could be used to interpret lake histories and to help to develop depositional models of enigmatic sedimentary successions. A focus was given to the application of the results for paleoecology and stratigraphy, and a model for predicting the position of different trace associations in vertical successions and in different parts of saline, alkaline lake basins has been developed. Evidence from the Kenyan lakes and Eocene Lake Gosiute shows that (1) sedimentary environments are diverse in underfilled basins, and frequent lake-level fluctuations strongly impact the distribution of sedimentary environments suitable for the production and preservation of biogenic structures; (2) the distribution of biogenic structures in underfilled basins is related to the geomorphological and structural setting, tectonic activity, catchment lithology, the basin margin or basin centre location, climate, and salinity and alkalinity, together with other finer-scale environmental and biological controls; (3) because saline environments are restrictive, sites of relatively dilute inflow (springs, rivers and deltas, ephemeral streams) provide oasis-like habitats for animals and plants, and contribute to the increased diversity and laterally variable distribution of saline-lake trace assemblages; and (4) the vertical distribution of trace fossils in a stratigraphic succession reflects changing environments through time; important stratigraphic surfaces, usually formed during periods of lake-level fall, can be recognized from the overprinting patterns of traces produced under different conditions.

Rift Valley Fever Virus is a plebovirus that causes epidemics and epizootics in sub-Saharan African countries but has expanded to Egypt and the Arabian Peninsula. The laboratory rat (Rattus norvegicus) is susceptible to RVFV and has been shown to manifest the characteristic responses of humans and livestock. The rat has frequently been used as a model to study RVFV pathogenesis. Several strains have been infected and some found to be resistant to hepatic disease while others were not. This resistance was found to be associated with a dominant gene inherited in Mendelian fashion. The congenic rat strain WF.LEW and several substrains of the parental strains were used to try and locate the resistance gene. Microsatellites and single nucleotide polymorphisms were used to characterize the genomes of various rat substrains in an attempt to map the gene. Breeding and viral challenge experiments were used to further characterize the strains and assign a location to the resistance gene. The LEW/SsNHsd rats showed approximately 37% genomic difference as compared with LEW/MolTac rats, and 8% difference as compared with LEW/Crl rats. WF/NHsd rats demonstrated a difference of approximately 8% as compared with WF/CrCrl rats. Genotyping of the congenic WF.LEW revealed Lewis markers on RNO3 and RNO9. Subsequent backcross experiments and viral challenge experiments assigned the resistance gene to the distal end of RNO3.