A Thesis submitted to the Faculty of Science in ful lment of the requirements for the degree of Doctor of Philosophy, School of Computer Science and Applied Mathematics. Johannesburg, 2016. / In this thesis in order to study the complex dynamics of Rift Valley fever (RVF) we combine two modelling approaches: equation-based and simulation-based modelling. In the first approach we first formulate a deterministic model that includes two vector populations, Aedes and Culex mosquitoes with one host population (livestock), while considering both horizontal and vertical transmissions. An easy applicable expression of the basic reproduction number, R0 is derived for both periodic and non-periodic environment. Both time invariant and time varying uncertainty and sensitivity analysis of the model is carried out for quantifying the attribution of model output variations to input parameters over time and novel relationships between R0 and vertical transmission are determined providing important information useful for improving disease management. Then, we analytically derive conditions for stability of both disease-free and endemic equilibria. Using techniques of numerical simulations we perform bifurcation and chaos analysis of the model under periodic environment for evaluating the effects of climatic conditions on the characteristic pattern of disease outbreaks. Moreover, extending this model including vectors other than mosquitoes (such as ticks) we evaluate the possible role of ticks in the spread and persistence of the disease pointing out relevant model parameters that require further attention from experimental ecologists to further determine the actual role of ticks and other biting insects on the dynamics of RVF. Additionally, a novel host-vector stochastic model with vertical transmission is used to analytically determine the dominant period of disease outbreaks with respect to vertical transmission efficiency. Then, novel relationships among vertical transmission, invasion and extinction probabilities and R0 are determined. In the second approach a novel individual-based model (IBM) of complete mosquito life cycle built under daily temperature and rainfall data sets is designed and simulated. The model is applied for determining correlation between abundance of mosquito populations and rainfall regimes and is then used for studying disease inter-epidemic activities. We find that indeed rainfall is responsible for creating intra- and inter-annual variations observed in the abundance of adult mosquitoes and the length of gonotrophic cycle, number of eggs laid per blood meal, adults age-dependent survival and fight behaviour are among the most important features of the mosquito life cycle with great epidemiological impacts in the dynamics of RVF transmission. These indicators could be of great epidemiological significance by allowing disease control program managers to focus their e orts on specific features of vector life cycle including vertical transmission ability and diapause. We argue that our IBM model is an ideal extendible framework useful for further investigations of other relevant host-vector ecological and epidemiological questions for providing additional knowledge important for improving the length and quality of life of humans and domestic animals. / LG2017

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 183-189).

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 4 uppsatser.

Rift Valley fever virus (RVFV) is a Phlebovirus member of the Bunyaviridae family and it is the causative agent of Rift Valley fever (RVF), a mosquito-borne viral zoonotic disease that poses a significant threat to domestic ruminants and human health in Africa. The RVFV is an encapsulated, negative-sense, single-stranded RNA virus with a tripartite segmented genome, containing L (large), M (medium) and S (small) segments. The S segment codes for two proteins, namely the nucleocapsid (N) protein and non-structural protein (NSs). There is evidence that the NSs protein is involved in virulence by blocking the expression of the interferon beta (IFN-β) promoter. It has been recently demonstrated that the SAP30-NSs-YY1 multiprotein complex represses the IFN-β promoter. Consequently, the interferon expression is blocked, allowing virus to replicate. A total of 45 isolates of RVFV recovered over a period of 53 years in 14 African countries, Madagascar and Saudi Arabia were characterized by full sequencing of the S segment of the virus. This data was added to another 27 strains of RVFV available on GenBank for phylogenetic analysis using MEGA4, giving a total of 72 strains analyzed. Alignments were made of the entire S segment, the NSs gene, the N gene, and their deduced amino acid sequences. The laboratory strains, clone 13, MP12 and Smithburn, were also included in the alignments. Two isolates were passaged ten times through two different amplification systems to asses the potential for sequence variation to occur in the original material through routine laboratory manipulations. Sequencing data was generated from the virus RNA present in the original clinical specimens and from the extracted RNA from the tenth passage of virus in each amplification system. The results showed 100% homology for each respective isolate, demonstrating that the RVFV S segment remained stable during ten serial passages in different propagation systems. Phylogenetic analysis was conducted on the naturally occurring RVFV strains (n = 72) and the findings indicate that circulating strains are compartmentalized and belong to one of three major lineages, namely Egyptian, western African, and central, eastern and southern African. The strains clustered in the Egyptian lineage had an average p-distance of 1.0%, the western African strains 0.9%, and the central, southern and eastern African strains 2.0%. The overall average p-distance was 2.5%, with a range from 0 to 4.1%. For the N gene, the range was from 0 to 4.2%, with an average of 2.2%. For the N protein, the range was from 0 to 2%, with an average of 0.2%. The NSs gene had a range of 0 to 4.6%, with an average of 2.4%. The NSs protein had a range of 0 to 3.8%, with an average of 1.7%. The intergenic region (IGR) had a range of 0 to 9.2%, with an average of 4.8%. Results of the study suggest that RVF outbreaks can result from either the rapid spread of a single strain over vast distances or from an increased activity of a strain circulating at an endemic level within an area/region during prolonged dry periods. Sequencing alignment showed that the length of the S segment ranged from 1690 to 1692 nucleotides. This difference in length was due to insertions and deletions found in the IGR, which is also the region with the most sequence divergence (4.8%). Both the NSs and N genes had neither insertions nor deletions, and were both found to be stable, though the NSs gene was slightly more variable than the N gene (2.5% versus 2.2%) The deduced amino acid sequences of the NSs protein were considerably more variable than that of the N protein (1.7% versus 0.2%). Alignment of the NSs protein demonstrated that the 5 cysteine residues at positions 39, 40, 150, 179 and 195, are highly conserved among the isolates analyzed. These residues are important for conservation of the three-dimensional structure of the protein and the formation of filamentous structures observed in cells infected with natural strains of RVFV. The NSs protein is now implicated as the major factor of virulence and that its pathogenicity is associated with the blocking of interferon production. Therefore, any amino acid changes that result in changes to the filamentous structure of the NSs protein might impact on the binding kinetics between the NSs protein, SAP30 (Sin3A Associated Protein 30) and YY1 (Yin Yang-1). There were 6 amino acid changes in the NSs-SAP30 binding domain, with one being unique to the live-attenuated Smithburn vaccine strain. Generated sequencing data contributes to global phylogenetic characterization of RVFV isolates and and molecular epidemiology of the virus. In addition, findings of this study will further aid investigation on reassortment events occurring between strains of RVFV and genetically related viruses, the role of the NSs protein in the replicative cycle of the virus, the pathogenic effects of the NSs protein within the RVFV-infected host cells, and might help to identify molecular basis of RVFV virulence.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Rift Valley fever virus (RVFV) is an enveloped, negative-sense, ssRNA virus with a tripartite genome that causes morbidity and mortality in both livestock and humans. Although RVFV is mainly circulating in mainland Africa, this arthropod-borne virus is a potential threat to the other parts of the world. No fully licensed vaccines for human or animal use in the U.S., and effective antiviral drugs have not been identified. As virulent RVFV strains are only handled in biosafety level (BSL) 3 or higher level facilities in the U.S., few laboratories have access to RVFV which limits antiviral development. However, it is crucial to develop effective antivirals to protect public and animal health. Animal models that reproduce Rift Valley fever are vital to identifying and developing antiviral compounds. The currently available attenuated RVFV strain, MP12, provides a BSL-2 challenge model virus for preliminary investigations of RVFV prior to using the virulent RVFV strains. All strains of RVFV have a highly conserved genome, indicating that antivirals or vaccines effective against any RVFV strain will most likely be effective for all RVFV strains. Therefore, we hypothesize that the MP12 is a suitable model virus that can be used for identification and evaluation of effective RVF antivirals. The first objective of this project was to establish a mouse model susceptible to MP12 infection. Based on the literature, we selected and screened six different strains of mice to test their susceptibilities to MP12. We found the STAT-1 knockout mice are the most susceptible to MP12 infection based on clinical symptoms, mortality, viremia, virus replication, histopathological, and immunochemical analyses. Importantly, these mice displayed acute-onset hepatitis and delayed-onset encephalitis similar to severe cases of human RVFV infection. Our second objective was to identify potential antiviral drugs in vitro. We developed and employed a cell-based assay using the recombinant MP12 virus expressing Renilla luciferase to screen a library of 727 small compounds purchased from National Institutes of Health. Of the compounds, 23 were identified and further tested for their inhibitory activities on the recombinant MP12 virus expressing green fluorescent protein. Further plaque reduction assays confirmed that two compounds inhibited replication of parental RVFV MP12 strain with limited cytotoxic effects. The 50% inhibitory concentrations using an MP12 multiplicity of infection (MOI) of 2 were 211.4 µM and 139.5 µM, respectively. Our third objective was to evaluate these two candidates, 6-azauridine and mitoxantrone, in vivo using our mouse model. After one-hour post MP12 infection via an intranasal route, treatment was given intranasally twice daily. Mice treated with placebo and 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurological signs, while mice treated with ribavirin (a known antiviral drug) or mitoxantrone showed delayed onset of disease. This result indicates that the mitoxantrone can improve the outcome of RVFV infection in our mouse model. The underlying mechanism of mitoxantrone to inhibit RVFV replication remains to be investigated. Our studies build the foundation for identification and development of antivirals against RVFV in a BSL-2 environment.

Rift Valley fever (RVF) is a peracute or acute disease of domestic ruminants and humans in sub-Saharan Africa, caused by a mosquito-borne virus. It is a high priority pathogen because of its potential to cause severe economic harm to the livestock industry and to cause life threatening haemorrhagic disease in humans. The disease was first recorded in southern Africa when a large epidemic occurred in the South Africa in 1950, and the first recorded outbreak in Namibia was in 1957. Since then, occasional large epidemics have occurred in southern Africa, with long interepidemic periods. The epidemiology of RVF is complex and many questions regarding the movements of the virus and its survival during the interepidemic period remain unanswered. The aim of this study was to compile a comprehensive description of the history of RVF in Namibia and to describe its epidemiological characteristics. This was accomplished using information available in the scientific literature, annual reports, disease reports and reports to the OIE. The geographical location and temporal occurrence of each outbreak was recorded as accurately as allowed by available records. Also recorded were suspected RVF outbreaks, defined as those outbreaks in which samples were not collected for laboratory analysis or RVF was not confirmed on submitted samples but where the clinical picture was suggestive of the disease. Serological surveys done in humans and animals were also included in the study. The collected data were analysed descriptively, by risk mapping and by cluster analysis. The relatively low number of recorded outbreaks and the poor spatial resolution of much of the data prevented more detailed multivariable analysis. Maps were produced to show the districts affected for the outbreaks with no coordinates and the exact location of the outbreaks which had coordinates. This was then followed by a detailed description of each outbreak showing the species affected and the mortalities caused. Risk mapping was done to identify areas of the country which are at high risk of having outbreaks. A quarter degree square grid was used to show the cumulative number of confirmed outbreaks occurring from 1957 to 2011. The accuracy of this was, however, limited due to the poor spatial resolution of data prior to 1986, which recorded only the district(s) affected. The risk map was visually compared with maps of sheep and cattle density and rainfall. A space-time permutation model, using case-only data, was used to detect space-time clusters with high rates, using SaTScan software on all the confirmed outbreaks with GPS coordinates. The objective was to detect areas of significantly high rates of RVF in Namibia, testing whether the outbreaks were randomly distributed over space and time. Space time permutation requires the use of precise geographic coordinates; therefore the only confirmed outbreaks that could be used for this analysis were those occurring during 2010 and the 2011. A total of six years had outbreaks of RVF in Namibia, the major outbreaks occurring in 1957, 1974, 1984, 2010 and 2011. Rift Valley fever was confirmed in the Karas, Hardap, Khomas, Erongo, Otjozondjupa, Omaheke and Oshikoto regions, with suspected outbreaks occurring in the Kavango and Caprivi regions. SaTScan analysis showed that there were two statistically significant outbreak clusters observed, one in the Hardap region in 2010 and the other in the Oshikoto region in 2011. The south-eastern part of the country was shown to be predisposed to RVF outbreaks; this correlated with sheep population density. The southern part of Namibia receives less rainfall and is hotter than the north, with colder winters, factors which may reduce vector and virus survival and therefore limit continuous viral circulation. This likely renders livestock highly susceptible to infection and if there is an introduction of the virus a severe epidemic may occur. In the Northern Communal Areas and adjacent Etosha National Park the positive serological results in humans and wildlife show that there is continuous or intermittent low level circulation of the virus. This could be leading to high levels of herd immunity and hence no confirmed outbreaks recorded in these areas to date. Nevertheless, all suspected cases should be tested for RVF to avoid misdiagnosis and under-reporting of cases. / Mini-dissertation (MSc)--University of Pretoria, 2015. / tm2016 / Veterinary Tropical Diseases / MSc / Unrestricted

The serodiagnosis of Rift Valley fever (RVF) relies on the use of inactivated whole virus based reagents which present biosafety, financial and operational constraints. There are no vaccines for humans, the availability of animal vaccines is limited and they have several drawbacks. The aim of this study was to evaluate a bacterially expressed recombinant RVF virus (RVFV) nucleocapsid protein (recNP) as a safe immunodiagnostic reagent, and an immunogen in a mouse and host animal model. Several enzyme-linked immunosorbent assays (ELISAs) were developed in this study, enabling sensitive and specific detection of antibodies and RVFV antigen in human and animal specimens. The recNP was combined with different adjuvants and used to immunize mice and sheep subsequently challenged with a virulent wild type RVFV strain. Depending on the recNP/adjuvant combination, protection against disease in mice ranged between 17 and 100%, with sterilizing immunity elicited in some experimental groups, compared to 100% morbidity/mortality and excessive viral replication in adjuvant and PBS control mice. Immunization with recNP combined with Alhydrogel, an adjuvant that biases immunity towards Th2 humoral immunity, that yielded 100% protection, induced an earlier and stronger type I interferon response in mice after challenge, compared to repression of the same gene in adjuvant and PBS control mice. There was massive activation of pro-inflammatory responses and genes with pro-apoptotic effects in the livers of control mice at the acute phase of infection, accompanied by high viral replication, possibly contributing to the pathology of the liver. There was also evidence of activation and repression of several genes involved in activation of B- and T-cell immunity in control mice, some indicating possible immune evasion by the challenge virus. Immunization of sheep with the same recNP/adjuvant combinations were, however, not able to decrease replication of challenge virus. The recNP based ELISAs are an important addition to and improvement of the currently available serodiagnostic tests for RVF. The mechanism by which recNP immunization protects mice from developing severe disease during the acute phase of infection is now better understood, but the mechanism for earlier clearance of the virus needs further investigation.

The Adames strain of Punta Toro virus (PTV-A) causes acute hepatic disease in hamsters and mice similar to that seen in natural Rift Valley fever virus (RVFV) infection, while the Balliet strain (PTV-B) is apathogenic. The ability of PTV-A to suppress the interferon (IFN) response has been demonstrated in hamsters and is thought to be a contributing factor to PTV-A's pathogenicity in hamsters. PTV-B is not assumed to exhibit this IFN-antagonistic activity, as it stimulates production of significantly higher IFN-β levels. To elucidate the role of IFN in resistance of mice to PTV-B infection, we utilized mice deficient in a critical IFN signaling protein, STAT-1. We found that these mice were drastically more susceptible to PTV-B, which caused 100% lethality compared to 0% in their wild-type counterparts. STAT-1 deficient mice were also more susceptible to PTV-A, as these mice succumbed to infection significantly earlier than wild-type mice (p=0.0058). We sought to determine whether PTV-A's IFN-antagonistic mechanism is functional in mice by examining expression of IFN-β in primary macrophages infected with either strain. We found that IFN-β protein concentration is higher in samples taken from PTV-B-infected cells. We employed quantitative PCR arrays specific to IFN signaling and response pathways to evaluate changes in gene expression throughout the course of infection with either virus strain. We found several genes with differentially regulated expression between PTV-A- and PTV-B-infected macrophages, including Ifnβ1 and multiple Ifnα subtypes. Also, several genes coding for inflammatory and chemotactic molecules, Cxcl11, Cxcl10, Cxcl9, Vcam1, and Il6, demonstrated increased expression in PTV-B samples compared to PTV-A. Of particular interest, Isg20, a 3'-5' exonuclease with specificity for single-stranded RNA, was stimulated ~2-fold higher by PTV-B, and Iigp1, from the family of GTPases associated with host defense against intracellular pathogens, was stimulated ~2.7-fold higher by PTV-B. The individual functions of each of these genes in mouse resistance to PTV-B could be a focus of future studies to better understand essential host defense mechanisms to phleboviral infection.

Please read the abstract in the 00front part of this document / Dissertation (MSc Agric (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Rift Valley fever (RVF) belongs to the group of viral haemorrhagic fevers (VHFs), most of which are zoonotic diseases causing outbreaks in animals and humans all over Africa. In the absence of haemorrhagic or specific organ manifestations, these diseases are clinically difficult to diagnose. Rapid laboratory confirmation of cases is therefore essential for timely execution of supportive treatment, appropriate case management, infection control, and tracing of contacts. Rift Valley fever virus (RVFV), a mosquito-borne pathogen, is responsible for high mortality rates and abortion in domestic ruminants, resulting in significant socio-economic losses. Furthermore, the virus is potentially infectious by aerosol, can replicate in a wide range of mosquito species and poses a bioweapon threat. The recent spread of the virus outside of the African continent, demonstrates its ability to move northwards to RVF free regions, e.g. to Europe and Northern America. Such fears fuel the international demand for reliable and validated diagnostic tools for rapid diagnosis of RVF. The aim of this study was to develop a rapid and accurate molecular tool for the detection of RVFV. A real-time loop-mediated isothermal amplification assay (LAMP) targeting the L segment of RVFV, was developed and evaluated. The assay proved to be highly specific and able to detect RVFV strains representing the genetic spectrum of the virus. Furthermore, the assay did not amplify the RNA of other genetically and antigenically related phleboviruses. The sensitivity of the assay was compared to that of a previously published TaqMan RTD-PCR protocol and found to be equal. Similarly, the assay demonstrated very high diagnostic sensitivity and specificity in various clinical human and animal specimens, collected during natural outbreaks of the disease in Africa. The detection of specific viral genome targets in positive clinical specimens was achieved in less than 30 minutes. As a highly accurate, rapid and very simple nucleic acid detection format, the RT-LAMP assay has the potential to be used in less well equipped laboratories in Africa. The assay format can be adapted to a portable device that can be utilized during RVF outbreaks in remote areas, and can be a valuable tool for differential diagnosis of VHFs. / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted