Tesi sul tema "Rift Valley fever virus (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

Rift Valley fever phlebovirus (RVFV) is an arbovirus of medical and veterinary importance causing severe disease and mortality in humans and ruminants in endemic regions in Africa and the Arabian Peninsula. Understanding the capability, and limiting factors, of temperate British mosquitoes to support replication and transmission of RVFV is critical in order to understand the potential for RVFV establishment were it introduced to the UK. Using in vitro cell culture the effect of temperature on viral replication kinetics independently of the mosquito was investigated; demonstrating temperatures below 20 ̊C negatively affect RVFV replication. The full replication cycle was supported at 20 ̊C in vitro, and this was confirmed within in vivo mosquito experiments with wild-caught Aedes detritus demonstrating a transmission potential for RVFV at 20 ̊C and 25 ̊C. Experiments with two colonised lines of Cx. pipiens further demonstrated the transmission potential for RVFV by mosquitoes present in the UK. A novel RNA in situ hybridisation technique substantiated this result showing widespread dissemination of virus from the primary site of infection and evidence of secondary sites of replication within a single mosquito. Characterisation of the consensus sequence of RVFV propagated within these British mosquitoes in comparison to an in vivo mouse model showed potential for virus adaptation when switching between disparate hosts. Reproducible changes at the consensus level within each host had not previously been shown in early passages of RVFV in studies utilising in vitro models of replication. This suggests that RVFV replication generates genomic variation that may lead to adaptations that could promote potential survival in temperate regions. Taken together these findings indicate that transmission of RVFV within the UK by indigenous mosquitoes is possible. However, factors affecting mosquito survival including temperatures greater than 20 ̊C and ingestion of the higher virus dose (10^7 PFU/mL) will limit the likelihood of such events occurring.

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.

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 an emerging infectious disease found in both livestock and humans. RVF is associated with high abortion and mortality rates in livestock and can be fatal in humans. As such, RVF is economically and socially significant to affected smallholder and subsistence farmers, those infected, and national livestock industries. However, Rift Valley Fever virus (RVFV) vaccines are not commercially available outside of endemic areas or for humans, and current vaccines are limited in their safety and efficacy. A plant-based, viral nanoparticle vaccine offers a more affordable alternative to conventional vaccines that is safe, rapidly producible, and easily scalable, better meeting the needs of impacted communities. This project focuses on assessing the potential of using a Nicotiana benthamiana plant expression system to generate recombinant tobacco mosaic virus (TMV) nanoparticles displaying RVFV glycoprotein epitopes. Eight TMV-RVFV glycoprotein constructs were designed. Five TMV-RVFV constructs were successfully cloned, and four recombinant TMV constructs were successfully expressed in planta. The antigenicity of these constructs was examined for their possible use in RVFV vaccine development.

Les malalties transmeses per vectors representen un alt percentatge de les malalties infeccioses en el món. Concretament, les malalties causades per arbovirus (arthropod-born viruses), que circulen en la naturalesa entre artròpodes (els seus vectors), i els hostes vertebrats (els seus reservorios), poden causar malalties greus en els hostes vertebrats, però no causen una patologia significativa en els vectors. Durant dècades les malalties causades per arbovirus van ser oblidades, ja que en la seva gran majoria estaven localitzades en zones en vies de desenvolupament. En l'actualitat, factors ambientals, ecològics i socioeconòmics, com el canvi climàtic i la globalització, han contribuït a l'emergència i reemergencia de les malalties arbovirales. El constant moviment de persones i mercaderies ha donat lloc a la colonització i establiment d'espècies d'exòtiques al nostre país, com el mosquit tigre (Aedes albopictus), el qual és transmissor de molts arbovirus (e.g. el virus del dengue, el virus Zika (ZIKV) o el virus chikungunya). El desenvolupament d'aquesta tesi es va centrar en realitzar estudis de competència vectorial per al ZIKV i en un estudi del transcriptoma de Culex pipiens després de ser exposat al phlebovirus de la febre de la Vall del Rift (RVFV) per a comprendre les interaccions el virus i els mosquits locals. Els capítols I i II es van focalitzar a estimar la competència vectorial per a ZIKV de diferents espècies de mosquits de camp presents al nostre país: Aedes albopictus, Aedes caspius i Culex pipiens. A més, es van desenvolupar experiments de transmissió vertical per a determinar si la generació de mosquits provinents de femelles infectades amb el ZIKV és capaç de disseminar-ho. Durant el desenvolupament d'aquests estudis, s'ha demostrat que els mosquits locals de l'espècie Ae. albopictus són vectors competents per al ZIKV. No obstant això, les espècies Cx. pipiens i Ae. caspius són refractàries per a aquest arbovirus. Respecte a l'experiment de transmissió vertical, es va demostrar que la progènie de les femelles inoculades amb el virus de manera intratoràcica va ser susceptible a la infecció del virus, però no van ser capaços de disseminar-lo. D'altra banda, el capítol III es va centrar en l'estudi de les interaccions a nivell molecular entre l'espècie de mosquit Cx. pipiens i RVFV, amb l'objectiu caracteritzar les alteracions a nivell molecular de l'expressió dels gens corresponents al sistema immune del mosquit durant la infecció per RVFV mitjançant una anàlisi del transcriptoma de novo. Com a resultat, es van obtenir 48 gens diferencialment expressats en els mosquits davant la presència del virus que servir de diana per a controlar la infecció, ja sigui per a desequilibrar la tolerància dels mosquits al virus com per a inhibir la infecció en els mosquits. Els resultats obtinguts de l'estudi de les alteracions del transcriptoma de mosquits de l'espècie Cx. pipiens exposats a RVFV estableixen les bases per a la realització de futurs estudis funcionals dels gens involucrats a controlar/permetre la infecció per RVFV. En el seu conjunt, el desenvolupament d'aquesta tesi incrementa el coneixement per a millorar el disseny d'estratègies eficients per a la vigilància de vectors transmissors del ZIKV i del RVFV.
Las enfermedades transmitidas por vectores representan un alto porcentaje de las enfermedades infecciosas en el mundo. Concretamente, las enfermedades causadas por arbovirus (arthropod-borne viruses), que circulan en la naturaleza entre artrópodos (sus vectores), y los hospedadores vertebrados (sus reservorios), pueden causar enfermedades graves en los hospedadores vertebrados, pero no causan una patología significativa en los vectores. Durante décadas las enfermedades causadas por arbovirus fueron olvidadas, ya que en su gran mayoría estaban localizadas en zonas en vías de desarrollo. En la actualidad, factores ambientales, ecológicos y socioeconómicos, como el cambio climático y la globalización, han contribuido a la emergencia y reemergencia de las enfermedades arbovirales. El constante movimiento de personas y mercancías ha dado lugar a la colonización y establecimiento de especies de exóticas en nuestro país, como el mosquito tigre (Aedes albopictus), el cual es transmisor de muchos arbovirus (e.g. el virus del dengue, el virus Zika (ZIKV) o el virus chikungunya). El desarrollo de esta tesis se centró en realizar estudios de competencia vectorial para el ZIKV y en un estudio del transcriptoma de Culex pipiens después de ser expuesto al phlebovirus de la fiebre del Valle del Rift (RVFV) para comprender las interacciones el virus y los mosquitos locales. Los capítulos I y II se focalizaron en estimar la competencia vectorial para ZIKV de diferentes especies de mosquitos de campo presentes en nuestro país: Aedes albopictus, Aedes caspius y Culex pipiens. Además, se desarrollaron experimentos de transmisión vertical para determinar si la generación de mosquitos provenientes de hembras infectadas con el ZIKV es capaz de diseminarlo. Durante el desarrollo de estos estudios, se ha demostrado que los mosquitos locales de la especie Ae. albopictus son vectores competentes para el ZIKV. Sin embargo, las especies Cx. pipiens y Ae. caspius son refractarias para este arbovirus. Con respecto al experimento de transmisión vertical, se demostró que la progenie de las hembras inoculadas con el virus de forma intratorácica fue susceptible a la infección del virus, pero no fueron capaces de diseminarlo. Por otro lado, el capítulo III se centró en el estudio de las interacciones a nivel molecular entre la especie de mosquito Cx. pipiens y RVFV, con el objetivo caracterizar las alteraciones a nivel molecular de la expresión de los genes correspondientes al sistema inmune del mosquito durante la infección por RVFV mediante un análisis del transcriptoma de novo. Como resultado, se obtuvieron 48 genes diferencialmente expresados en los mosquitos ante la presencia del virus que servir de diana para controlar la infección, ya sea para desequilibrar la tolerancia de los mosquitos al virus como para inhibir la infección en los mosquitos. Los resultados obtenidos del estudio de las alteraciones del transcriptoma de mosquitos de la especie Cx. pipiens expuestos a RVFV sientan las bases para la realización de futuros estudios funcionales de los genes involucrados en controlar/permitir la infección por RVFV. En conjunto, el desarrollo de esta tesis incrementa el conocimiento para mejorar el diseño de estrategias eficientes para la vigilancia de vectores transmisores del ZIKV y del RVFV.
Vector-borne diseases represent a 17 % of infectious diseases in the world. Among them, those diseases caused by arboviruses (arthropod-borne viruses), which circulate in the nature between arthropods (their vectors) and vertebrate hosts (their reservoirs), are currently provoking serious diseases in humans and animals. For decades, the arboviral diseases were neglected, since most of them were located in developing areas. Nowadays, environmental, ecological and socioeconomic factors (e.g., globalization and climate change) have contributed to the emergence and re-emergence of arboviral diseases. The constant movement of people and merchandise has allowed the colonization and establishment of exotic mosquito species in our country such as the tiger mosquito (Aedes albopictus), which is a potential vector of many arboviruses (e.g., dengue virus, Zika virus or chikungunya virus). This thesis focused on conducting vector competence and transmission studies in local mosquito species for Zika virus (ZIKV) and on the study of the Culex pipiens transcriptome alteration after being exposed to the Rift Valley fever phlebovirus (RVFV) in order to better understand how virus-vector interaction influences on ZIKV and RVFV transmission. Chapters I and II focused on estimating the vector competence for ZIKV of different field-collected mosquito species present in our country: Aedes albopictus, Aedes caspius and Culex pipiens. In addition, vertical transmission studies were performed to determine if the progeny of females infected with ZIKV were able to disseminate the virus. The results of these studies showed that local populations of Ae. albopictus were competent vectors for ZIKV and Cx. pipiens and Ae. caspius species were refractory for this arbovirus. Moreover, it was demonstrated that ZIKV was able to be transmitted to the progeny but the later could not disseminate the virus. Chapter III focused on the study of interactions between the Cx. pipiens mosquito species and RVFV at molecular level, with the aim to characterize the alterations in the expression of the mosquito genes related to the immune system during RVFV infection by analyzing de novo transcriptome. As a result, 48 immune differentially expressed genes in mosquitoes exposed to RVFV were altered, which could serve as potential targets to control the infection, either by unbalancing the mosquito tolerance to RVFV or by inhibiting the infection in mosquitoes. The results obtained on the Cx. pipiens transcriptome alterations due to exposure to RVFV pave the way for future functional studies about genes involved in the control/tolerance of RVFV infection. Overall, this thesis increased the knowledge to better design efficient strategies for ZIKV and RVFV surveillance and control.

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.

Rift Valley fever (RVF) is a haemorrhagic fever agent caused by an infection with an enveloped negative-stranded RNA Rift Valley fever virus (RVFV). It belongs to the genus Phlebovirus in the family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing high numbers of neonatal fatalities in animals and occasional fatalities in humans. It is endemic to parts of Africa and the Arabian Peninsula, but is described as an emerging virus due to the wide range of mosquitoes that could spread the disease into non-endemic areas, posing serious health and agricultural problems. The disease can be prevented by vaccination, but there is currently no Food and Drug Administration-approved RVFV vaccine that can be used outside endemic areas, while there are two live attenuated vaccines available for use in endemic areas. These vaccines have the potential for reversion, and are therefore not recommended for use in countries where RVFV is not endemic. This indicates the need for more RVFV vaccine research and development. This work focused on the development of a RVFV vaccine candidate that would allow for differentiation between infected and vaccinated animals as well as humans.

Serosurveillance is a powerful tool fundamental to understanding infectious disease dynamics. The presence of virus neutralising antibody (VNAb) in sera is considered the best evidence of infection, or indeed vaccination, and the gold standard serological assay for their detection is the virus neutralisation test (VNT). However, VNTs are labour intensive, costly and time consuming. In addition, VNTs for the detection of antibodies to highly pathogenic viruses require the use of high containment facilities, restricting the application of these assays to the few laboratories with adequate facilities. As a result, robust serological data on such viruses are limited. In this thesis I develop novel VNTs for the detection of VNAb to two important, highly pathogenic, zoonotic viruses; rabies and Rift Valley fever virus (RVFV). The pseudotype-based neutralisation test developed in this study allows for the detection of rabies VNAb without the requirement for high containment facilities. This assay was utilised to investigate the presence of rabies VNAb in animals from a variety of ecological settings. In this thesis I present evidence of natural rabies infection in both domestic dogs and lions from rabies endemic settings. The assay was further used to investigate the kinetics of VNAb response to rabies vaccination in a cohort of free-roaming dogs. The RVFV neutralisation assay developed herein utilises a recombinant luciferase expressing RVFV, which allows for rapid, high-throughput serosurveillance of this important neglected pathogen. In this thesis I present evidence of RVFV infection in a variety of domestic and wildlife species from Northern Tanzania, in addition to the detection of low-level transmission of RVFV during interepidemic periods. Additionally, the investigation of a longitudinal cohort of domestic livestock also provided evidence of rapid waning of RVF VNAb following natural infection. Collectively, the serological data presented in this thesis are consistent with existing data in the literature generated using the gold standard VNTs. Increasing the availability of serological assays will allow the generation of robust serological data, which are imperative to enhancing our understanding of the complex, multi-host ecology of these two viruses.

Rift Valley Fever is a zoonotic, arthropod-borne disease that adversely affects ungulates and people. The etiologic agent, Rift Valley fever virus (RVFV; Bunyaviridae, Phlebovirus), is primarily transmitted through mosquito bites, yet can be transmitted by exposure to infectious aerosols. Presently, there are no licensed vaccines or therapeutics to prevent or treat severe RVFV infection in humans. We have previously reported on the activity of favipiravir (T-705) against the MP-12 vaccine strain of RVFV and other bunyaviruses in cell culture. Additionally, efficacy has been documented in mouse and hamster models of infection with the related Punta Toro virus. Here, we characterize a hamster RVFV challenge model and use it to evaluate the activity of favipiravir against the highly pathogenic ZH501 strain of the virus. Subcutaneous RVFV challenge resulted in substantial serum and tissue viral loads and caused severe disease and mortality within 2-3 days after infection. Oral favipiravir (200 mg/kg/day) prevented mortality in 60% or greater in hamsters challenged with RVFV when administered within 6 h post-exposure and reduced RVFV titers in serum and tissues relative to the time of treatment initiation. In contrast, although ribavirin (75 mg/kg/day) was effective at protecting animals from the peracute RVFV disease, most ultimately succumbed from a delayed-onset neurologic disease associated with high RVFV burden in the brain observed in moribund animals. When combined, T-705 and ribavirin treatment started 24 h post-infection significantly improved survival outcome and reduced serum and tissue virus titers compared to monotherapy. Our findings demonstrate significant post-RVFV exposure efficacy with favipiravir against both peracute disease and delayed-onset neuroinvasion, and suggest added benefit when combined with ribavirin.

Rift Valley fever (RVF) is an arthropod-borne viral disease of significant importance in both livestock and humans. Epidemics occur periodically in domestic ruminants, typically after heavy rains, which encourage rapid multiplication of mosquito vectors. Clinical symptoms in livestock vary from inapparent infection to abortions and peracute deaths. The disease has significant zoonotic potential. People in contact with infected livestock may develop disease that varies from mild flu-like symptoms to severe neurological and haemorrhagic disorders and death. An important way of controlling the disease is through vaccination of susceptible livestock. Rift Valley fever virus (RVFV) clone 13 is a relatively new livestock vaccine against RVF that is derived from an avirulent natural mutant strain of RVFV. This vaccine has been shown in previous studies to confer protective immunity against infection with live virus. The effect of this vaccine on semen quality in male animals has never been tested. The purpose of the current trial was to determine whether RVFV clone 13 vaccine had any effect on semen quality in rams. The hypothesis tested was that animals vaccinated with RVFV clone 13 vaccine would not experience a reduction in semen quality (measured by evaluating the percentage progressively motile and percentage morphologically normal spermatozoa in successive ejaculates) relative to unvaccinated control animals. A test/control model was used to evaluate the effect of this vaccine on semen quality. A group of peripubertal ram lambs were tested for antibodies to RVFV using a serum neutralisation test (SNT). Animals without detectable antibodies (n=23) were then randomly allocated to either a test group (n=12) or a control group (n=11). Daily rectal temperature measurements were taken and weekly semen evaluations were conducted. Blood samples were drawn weekly to assess serum antibody titres. Seven animals were subsequently eliminated from the statistical analysis because of potential confounding factors. Of these seven, five animals had extremely poor semen quality at the start of the trial, one animal was found to have a persistent febrile response commencing at the start of the trial, and one animal had seroconverted to Rift Valley fever virus in the period between the initial screening and onset of the trial. Logistic regression analysis was performed on data gathered from the remaining animals to determine whether an association existed between animal group, rectal temperature and semen quality parameters. It was found that no correlation existed between treatment group and values obtained for the semen quality parameters measured. There was no statistically significant post-vaccination temporal decline in the percentage of live morphologically normal spermatozoa, or the percentage of progressively motile spermatozoa, either when assessed amongst all animals or when assessed within individual groups. Based on the data from this trial, the hypothesis was not rejected. Despite this finding, it should be stated that the elimination of animals from the analysis had some effect on the statistical power of the study. A repeat of the trial with a larger sample size and a more comprehensive pre-screening process to avoid the inclusion of animals with poor semen quality may be indicated.
Dissertation (MMedVet)--University of Pretoria, 2014.
tm2015
Production Animal Studies
MMedVet
Unrestricted

Master of Science
Department of Diagnostic Medicine/Pathobiology
Juergen A. Richt
Rift Valley fever virus (RVFV) is a vector-borne zoonotic pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that causes severe disease in ruminants and humans. RVFV is a significant threat to US livestock and public health due to a lack of licensed, efficacious vaccines and its ability to become established in non-endemic areas. Subunit vaccine candidates based on RVFV N- and C-terminal glycoproteins (Gn and Gc) are a viable option for use in ruminants due to their ease of production, safety, and ability to induce immune responses that offer differentiation between infected and vaccinated animals (DIVA). Importantly, subunit Gn+Gc vaccine candidates have demonstrated efficacy in sheep. However, despite the efficacy of a dual glycoprotein vaccine, no studies have directly compared protective efficacies of the individual glycoproteins. Furthermore, although RVFV demonstrates 2.1% maximum pairwise amino acid strain divergence within Gn/Gc ectodomains, it remains unclear how this may affect cross-protective vaccine efficacy. In this study, we used a BALB/c mouse model to determine the median lethal dose (LD₅₀) of 3 wildtype RVFV strains and used this information to standardize challenge doses in subsequent vaccine efficacy studies using baculovirus-expressed Gn/Gc antigens derived from RVFV strain Zagazig Hostpital 1977 (ZH548). Strains Kenya 2006 (Ken06) and Saudi Arabia 2001 (SA01) demonstrated equally high virulence (LD₅₀= 7.9pfu), while recombinant strain South Africa 1951 (rSA51) was less virulent (LD₅₀=150pfu). Following prime-boost vaccination, 100% (10/10) of the Gn+Gc vaccinated mice survived challenge with x1000 LD₅₀ Ken06 and SA01, while only 50% (5/10) of Gn+Gc vaccinated mice survived challenge with rSA51. Additionally, 90% (9/10) of Gn-only vaccinated and 40% (4/10) of Gc-only vaccinated mice survived challenge with Ken06. These data suggest that a Gn-only subunit vaccine is an efficacious alternative to dual glycoprotein vaccine candidates and that our ZH548-derived Gn+Gc vaccine has the potential to cross-protect against divergent RVFV strains. Results from this study can be used to optimize current vaccine formulations and inform future vaccine efficacy and licensure studies in ruminants.

El virus de la Fiebre del Valle del Rift (RVFV) es un flebovirus zoonótico transmitido por mosquitos que afecta a los rumiantes causando abortos y hepatitis aguda con necrosis multifocal. Los síntomas de la RVF varían desde el síndrome gripal hasta la retinitis y la encefalitis. En esta tesis, mostramos la susceptibilidad leve-subaguda de las ovejas Ripollesas de 9-10 semanas de edad a la infección por RVFV. Cuatro aislamientos virales diferentes de brotes de campo se replicaron de forma eficiente in vivo tras la inoculación subcutánea experimental, detectándose viremia y diseminacion viral dependientes del aislado de RVFV. Se demostró la transmisión horizontal a un cordero centinela no infectado. La infección en corderos únicamente causó pirexia transitoria, aunque se observó opacidad corneal ("ojo azul") en 3 de 16 ovejas inoculadas subcutáneamente. En una aproximación preliminar, se investigó el tejido formolado y parafinado procedente de estos animales afectados por la opacidad corneal mediante histopatología y qRT-PCR, para su caracterización. En cuatro corderos infectados se diagnosticó uveítis anterior con endotelitis linfoplasmocítica. Para evaluar la protección conferida por una única dosis subcutánea de una vacuna de virus Ankara modificado (MVA) que codifica las glicoproteínas Gn y Gc de RVFV en corderos, se inmunizaron 3 grupos de 6 a 7 corderos de 5-7 semanas de edad de la siguiente manera: Un grupo vacunal (denominado rMVA-GnGc), un segundo grupo control vectorial (vector MVA) y un tercer grupo control no vacunado (solución salina). Catorce días más tarde, todos los animales se sometieron a un desafío subcutáneo con 105 TCID50 del aislamiento virulento RVFV 56/74 y se evaluó la eficacia de la vacuna usando puntos finales estándar. Dos corderos (uno del grupo vacunado y uno del control vectorial) sucumbieron al desafío, mostrando lesiones hepáticas características. Los corderos de los grupos control vectorial y control no vacunado fueron febriles de los días 2 a 5 después del desafío (pc) mientras que los del grupo rMVA-GnGc mostraron un único pico de pirexia al día 3 pc. Se detectó ARN de RVFV en ambos hisopados nasal y oral de los días 3 a 7 pc en algunos corderos de los grupos control vectorial y no vacunado, pero no se pudo detectar diseminación viral en los corderos supervivientes vacunados. Caracterizamos patológicamente la nueva lesión ocular detectada en un abordaje secundario. Se seleccionaron 2 grupos de 5 corderos por grupo (n = 10) de la base de datos histórica de infecciones experimentales de RVFV realizadas en el Centro de Investigación en Sanidad Animal (España, NBS3), en base a sus datos clínicos, viremia y diagnóstico de lesiones ocular y hepática. Se caracterizó la uveítis anterior diagnosticada (8 de 10) con endotelitis linfoplasmocítica (2 de 10). CD3, CD20 e infiltrados inflamatorios mononucleares positivos a la lisozima se observaron en ojos parafinados RVFV-positivos. El marcaje positivo de CD20 sólo se observó en los infiltrados en uvea anterior. También se diagnosticó una nueva retinitis T dependiente (CD3 positivo) en 5 de 10 corderos infectados con RVFV. Se desarrolló un protocolo de inmunoquímica contra RVFV basado en un anticuerpo monoclonal murino. En conclusión, la raza Ripollesa se infecta fácilmente con RVFV sin manifestaciones clínicas evidentes. Un modelo de desafío con corderos de 5 a 10 semanas de edad ha demostrado ser eficaz para testar vacunas. Se sugiere que una sola dosis de la vacuna rMVA-GnGc puede ser suficiente para reducir la diseminación de RVFV y la duración de la viremia, pero no proporciona inmunidad estéril ni protección contra la enfermedad. Hasta donde sabemos, esta es la primera descripción patológica de uveítis anterior con lesión retiniana en un modelo ovino, muy parecida a las lesiones oculares humanas.
Rift Valley Fever Virus (RVFV) is a mosquito-borne zoonotic phlebovirus that primarily affects ruminants by causing abortions and acute hepatitis with multifocal necrosis as major findings. Human RVF symptoms range from flu-like syndrome to retinitis and encephalitis. The increasing interest in RVFV deserve revisiting experimental sheep infection. In this thesis, we show the susceptibility of 9–10 weeks old European sheep (Ripollesa breed) to RVFV infection, showing a mild, subacute form of disease. Four different viral isolates from field outbreaks efficiently replicated in vivo after subcutaneous experimental inoculation, and consistent viral loads in blood and RVFV-isolate dependent virus shedding were detected, showing horizontal transmission to a noninfected, sentinel lamb. RVFV infection caused transient pyrexia in old lambs and no other clinical symptoms were observed, although corneal opacity (“blue eye”) was found in 3 out of 16 subcutaneously inoculated sheep. To better characterize this corneal opacity, in a preliminary approach, formalin-fixed paraffin wax-embedded tissue from these ocular condition-affected animals was investigated by histopathology and quantitative real time reverse transcriptase polymerase chain reaction. Anterior uveitis with lymphoplasmacytic endotheliitis was diagnosed in these four RVFV-infected lambs. To evaluate the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the RVFV glycoproteins Gn and Gc in lambs, 3 groups of 6 to 7 Ripollesa lambs of 5–7 weeks old were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 105 TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. We characterize pathologically the new ocular detected condition in a secondary approach. Two groups of five lambs per group were selected (n=10) from the historical database of RVFV experimental infections performed in Center of Research in Animal Health (Spain, NBS3 facility) in the basis of their clinical data, viremia and diagnosed ocular and hepatic lesions (two previous experiments). The previously diagnosed anterior uveitis (8 out of 10) with lymphoplasmacytic endotheliitis (2 out of 10) was characterized. CD3, CD20 and lysozyme-positive mononuclear inflammatory infiltrates were observed in RVFV-positive paraffin-embedded eyes. CD20 labelling was only observed in infiltrates in anterior uvea. A novel T-cell dependent retinitis was also diagnosed in 5 out of 10 RVFV-infected lambs based on CD3-positive labelling. An immunochemistry protocol based on a murine monoclonal antibody was developed at CReSA BLS2 facility. In conclusion, Ripollesa sheep are readily infected with RVFV without apparent clinical manifestations. A 5-10 weeks old Ripollesa breed challenge model has proven to be effective in vaccine testing because of its susceptibility to virus. It is suggested that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. To our knowledge, this is the first pathological description of RVFV-related anterior uveitis with retinal injury in a RVFV-challenge sheep model, resembling ocular human lesions.

There are two broad aims to this project. The first aim is to compare and characterise two lumpy skin disease virus (LSDV) vaccines namely the vaccine based on attenuated Neethling LSDV (nLSDV) and Herbivac®LS (Herbivac). The second aim is to construct a recombinant LSDV expressing Rift Valley fever virus (RVFV) genes. An LSDV vaccine is critical for sustainable control of lumpy skin disease (LSD). There are four commercially available live attenuated vaccines for LSDV, nLSDV, Herbivac, Lumpyvax and the Kenyan strain sheeppox virus (KS-1). In this study Herbivac was characterised by comparing it to its parent, nLSDV. Growth curves of the two viral strains were conducted in cell culture as well as in embryonated hens’ eggs. No notable difference in the growth rate of the two strains could be detected when the viruses were grown in cell culture, however a notable difference was detected when the viruses were grown on the chick allantoic membranes (CAMs) of embryonated hens’ eggs. When grown on CAMs a faster growth rate was observed for nLSDV compared to Herbivac. nLSDV also killed the embryos at 4 d.p.i where Herbivac did not. The two strains were then further characterised through histological analysis of CAMs after infection with each of the viruses. Overall, higher levels of hyperplasia and hypertrophy were observed in CAMs infected with either nLSDV or Herbivac compared to uninfected CAMs. Herbivac-infected CAMs resulted in thicker chorionic membranes and larger pocks compared to nLSDV. RVFV and LSDV both contribute to the disease burden among cattle in Africa and the Arabian Peninsula. The main aim of this study was to construct a recombinant Herbivac which expresses immunogenic proteins of Rift Valley fever virus (Herbivac-RVFV). Herbivac-RVFV was designed to express specific RVFV genes selected for their antigenic properties. The genes selected are also representative of the genes from recent viral outbreaks in the horn of Africa. The selection of outbreak relevant RVFV genes involved phylogenetic analysis of all full length M-segment and NC gene sequences available on Genbank. Phylogenetic trees were constructed for M-segments and NC genes and groups identified which were highly representative of sequences from recent outbreaks of the virus. Consensus sequences were derived from these groups and included in the transfer vector. The phylogenetic analysis also revealed that the sequences of current RVFV vaccines are phylogenetically distant from viruses isolated from current outbreaks, although high levels of sequence conservation was maintained across all viral strains. This is the first study in which the RVFV genes coding for proteins that will induce a protective immune response (Gn and Gc, as well as the nucleocapsid (NC) gene) were selected so as to be representative of current outbreak strains of the virus. These genes were inserted between LSDV ORFs 49 and 50, a novel insertion site. The transfer vector also contained an eGFP marker gene and an ECO-GPT selection gene, located outside of the LSDV flanking sequences. This meant a two-step isolation procedure, first to isolate the recombinant containing the entire transfer vector with eGFP and ECO-GPT, and then to isolate a recombinant with only the RVFV genes and not eGFP and ECO-GPT. Transient expression of RVFV proteins in cells infected with Herbivac and then transfected with the transfer vector was confirmed via western blotting and immunofluorescence. Here the proteins Gn, Gc and NC were shown to be expressed. In the present study, a single crossover Herbivac-RVFV recombinant was isolated through multiple passaging of cell lysates, originally obtained from Herbivac-infected FBT cells transfected with the transfer vector, in the presence of mycophenolic-acid selection medium.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa that presents a significant health threat to both humans and animals. Outbreaks have serious economical implications. Two facts cause concern. Firstly, recent outbreaks in the Arabian Peninsula and effects of global warming have the implication that the virus might spread further into non-endemic RVF regions since it utilizes a wide range of mosquito vetors. Secondly, RVFV is a potential bio-terrorism agent. Therefore, quick, safe, robust and cost-effective methods of detection and appropriate diagnostic reagents are needed for early diagnosis, disease surveillance and bio-defence field monitoring. The development of several effective ELlSAs for the detection of antibodies to RVFV have been reported (Paweska et al., 2003a, 2003b and 2005a; 2005b). A serious drawback of these ELlSAs is that they use an inactivated virus as antigen. The availability of a recombinant antigen would lower the safety risks of antigen preparation and the costs of diagnosis. The purpose of this study was to clone and express a recombinant RVFV nucleoprotein (N) from a southern African RVFV strain (Zim 688/78), and to evaluate its suitability as a diagnostic antigen in an ELISA for the detection of antibodies to RVFV in humans and animals. The gene encoding the N protein was amplified by RT-PCR using primers specifically designed to contain restriction enzyme sites for cloning. The nucleic acid sequence of the gene was determined and compared to that of the 7 other published N gene sequences of different strains. The N gene of the Zim688178 strain had 5 unique nucleic acid differences, all in wobble positions which did not cause amino acid changes. The nucleic acid sequence of the Zim688I78 strain was the closest related to the Clone1 3 strain (98.6%), and its amino acid sequence was identical to Clone 13. A fragment of the gene encoding the glycoprotein G2 was also amplified, its nucleic acid sequence was determined and compared to the published G2 sequences of other strains. The G2 fragment was closest related to the BEgy93 (98.7%), HEgy93 (98.7%) and ZH548 (98.7%) strains. The Zim688/78 N gene was cloned into two bacterial expression system vectors, pGEX4T-1 and pET32(a)+ and three baculovirus expression vectors, pFastBac-1, pFastBacHT-B and pBACgus-1. Low amounts of completely insoluble recombinant N protein (rN) were expressed by baculovirus recombinants generated using pFastBacHT-B. The expression level of rN using pFastBac-I was higher and rN was more soluble. These constructs, including the pBACgus-1 construct, was sent to Biovac (Pinelands, South Africa) for further optimization of baculovirus expression. Bacterial expression using the pGEX4T-1 vector resulted in a high yield of rN, but it was mostly insoluble. However, IPTG induced bacterial expression with the pET32(a)+ construct resulted in a high yield of soluble rN. The recombinant nucleoprotein was purified with Ni-affinity column purification, using the pET32(a)+ fusion tag with 6x His residues. The purified rN was of high quality and therefore it could be used for direct coating of immunoplates in indirect ELISA (I-ELI%). Various coating procedures were tested. Overnight coating in carbonatelbicarbonate buffer (pH 9.6) on a MaxiSorp (Nunc) plate gave the best results. The recombinant 5 nucleoprotein antigen based I-ELISA was used to measure IgG and IgM antibodies in titrated sera from vaccinated personnel and naturally infected humans respectively. The test was able to distinguish between sera with high and low concentrations of antibodies. The rN-based I-ELISA was also used to measure IgG and IgM antibody responses in vaccinated and experimentally infected sheep. The test was able to show seroconversion in both vaccinated and infected sheep, track the rise and decline of IgM antibodies and detect the expected lower levels of the immune response in sheep vaccinated with liveattenuated virus compared to that of sheep infected with wild type virus. The rN also proved to be suitable as an antigen in IgG sandwich and IgM capture ELlSA formats. The production of the RVFV rN under experimental procedures used was quick, easy, relatively inexpensive and safe. The findings of this project demonstrated that the recombinant is a high quality diagnostic antigen for use in I-ELISAs for the detection of antibodies in humans and animals.
Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Rift Valley fever (RVF) is a disease of ruminants in Africa, Madagascar and the Middle East, affecting primarily domestic, but also many wild species. The disease can be peracute to acute and is characterised by a necrotic hepatitis and a generalised haemorrhagic syndrome. The disease is caused by a mosquito-borne virus of the Phenuiviridae family. Outbreaks occur after heavy rains, which favour the breeding of the mosquito vectors. The disease is a zoonosis and humans become infected by bites from infected mosquitoes or contact with tissue from infected animals. The first records of RVF in southern Africa date back to 1950 when a large epidemic occurred in South Africa and there have been many outbreaks since, some major, with the most recent major outbreak in SA in 2010. During the 2010 outbreak multiple indigenous wildlife species were affected, including springbok (Antidorcas marsupialis), blesbok (Damaliscus pygargus phillipsi), bontebok (D. pygargus pygargus), waterbuck (Kobus ellipsiprymnus), African buffalo (Syncerus caffer), sable (Hippotragus niger), kudu (Tragelaphus strepsiceros), nyala (Tragelaphus angasii) and gemsbok (Oryx gazella). Even though no cases have been recorded in roan (Hippotragus equinus) antelope to date, the fact that such a wide array of wildlife was affected, and taking into account the close phylogenetic relationship between sable and roan antelope, it is reasonable to assume that roan will also be susceptible to RVF. Many control methods are aimed at vector control, but since the epidemiology of the disease is still poorly understood, this has limited value. Vaccination thus provides the best means of disease prevention and RVFV Clone 13 vaccine is a new vaccine proven to be effective and safe in domestic animals. It has not yet been scientifically tested in wildlife. To date there is no published research on the use of RVF vaccine in wildlife, nor are there any recommended vaccine protocols for wildlife. Many of these species are valuable in conservation and financial terms, and hence the need for this research. The purpose of the research was: To determine whether Clone 13 RVFV vaccine is effective in creating a humoral immune response in the African savannah buffalo and roan antelope. To determine if there are any obvious clinical side effects, such as general malaise or abortion due to vaccination. The World Organisation for Animal Health (OIE) lists both the enzyme linked immunosorbent assay (ELISA) and serum neutralization test (SNT) as recommended methods for detecting an immune response in populations considered free from disease, as well as post-vaccination to detect an immune response. As reported previously, the ELISA and SNT have been used to detect antibodies against RVFV in serum of a variety of animal species.
Dissertation (MMEDVET)--University of Pretoria, 2017.
Production Animal Studies
MMEDVET
Unrestricted

Le Virus du Nil Occidental (VNO) et le Virus de la Fièvre de la Vallée du Rift (VFVR) sont deux arbovirus transmis par le moustique Culex pipiens comprenant deux biotypes: pipiens et molestus. Au cours de ce projet, nous avons évalué la circulation du VNO au Liban dans des populations de moustiques, des humains, des chevaux et des poulets. Nous avons aussi évalué la compétence vectorielle des populations locales de Cx. pipiens à transmettre le VNO et le VFVR.Des moustiques ont été récoltés et testés pour la présence d’un gène spécifique du VNO. En plus, des sérums humains, de chevaux et de poulets ont été analysés pour rechercher des anticorps spécifiques par ELISA puis confirmés par neutralisation. En outre, des spécimens de Cx. pipiens ont été infectés avec la lignée 1 du VNO ou la souche de VFVR Clone 13. Ensuite, les taux d’infection, de dissémination et de transmission ont été déterminés à différents jours après infection des moustiques. La compétence vectorielle a été comparée entre les différents biotypes.Les résultats entomologiques ont révélé que Cx. pipiens est dominant (87.2%). Tous les moustiques analysés étaient négatifs pour le VNO. Les taux de séroprévalence étaient de 1.01% et 1.98% parmi les humains et les chevaux respectivement. De plus, Cx. pipiens s’est révélé bien plus compétent pour transmettre le VNO que le VFVR. Le biotype molestus est capable de transmettre le VNO plus tôt que celui de pipiens. Cette étude présente des preuves sur une faible circulation du VNO au Liban. Cx. pipiens s’est révélé compétent pour assurer cette transmission. Ainsi, il est essentiel d'établir des programmes de surveillance pour prévenir les éventuelles épidémies
West Nile virus (WNV) and Rift Valley Fever virus (RVFV) are two emerging arboviruses that have never been reported in Lebanon. They can be transmitted by Culex pipiens mosquito species including two biotypes: pipiens and molestus. During this project, we assessed the circulation of WNV among mosquitoes, human, horse and chicken populations in Lebanon. Moreover, we evaluated, under experimental conditions, the capacity of local Cx. pipiens biotypes to transmit both viruses.Adult mosquitoes were collected, identified and tested to detect WNV RNA. Besides, human, horse and chicken blood samples were collected and screened for WNV antibodies using an in-house ELISA and then confirmed by neutralization assay. Moreover, local Cx. pipiens specimens were experimentally infected with WNV lineage 1 or RVFV Clone 13 strain. The viral infection, dissemination and transmission were then estimated at different days post infection.The vector competence was compared between Cx. pipiens biotypes.Entomological results revealed that 87.2% of collected adult mosquitoes were Cx. pipiens. Screened mosquitoes were negative for WNV. Seroprevalence rates were 1.01% and 1.98% among humans and horses respectively. Besides, local Cx. pipiens were highly competent for WNV transmission and to a lesser extent to RVFV. The molestus biotype was able to transmit WNV earlier than pipiens biotype.The present study provides new evidence of a low circulation of WNV among human and horses in Lebanon. Cx. pipiens is the suspected vector and is experimentally competent to ensure transmission. Therefore, there is a need to establish surveillance program to predict and prevent potential outbreaks

La Fièvre de la Vallée du Rift (VFVR) est une arbovirose zoonotique affectant principalement les ruminants et les humains. Son éco-épidémiologie complexe implique de nombreuses espèces de vecteurs, d'hôtes et de voies de transmission. Ainsi, différents mécanismes de transmission et d'émergence sont impliqués dans la circulation du virus de la FVR (VFVR) et ceux-ci dans des écosystèmes contrastés d'Afrique, de la Péninsule Arabique et des îles du sud-ouest de l'Océan Indien, dont l'île de Madagascar.Par sa superficie, sa grande diversité éco-climatique et sa faune et flore endémique, Madagascar est considérée comme une île continent. On y retrouve, en effet, des écosystèmes variés plus ou moins favorables aux moustiques : semi-arides dans le sud-ouest, humides et froids sur les hautes terres centrales, per-humide dans l'est et humides et chaud dans le nord-ouest. Madagascar a été affectée par deux épidémies de FVR en 1990-91 puis 2008-09. Une étude menée lors de la dernière épidémie a montré que le virus avait largement diffusé dans l'île de façon hétérogène.Compte tenu de la complexité des mécanismes de transmission de la FVR et de la diversité des écosystèmes de Madagascar, nous avons supposé que cette hétérogénéité spatiale était due à des mécanismes de transmission et d'émergence qui variaient en fonction des écosystèmes de l'île. Ainsi, le premier objectif de ce travail de thèse étaient de déterminer les mécanismes et les dynamiques de transmission de la FVR inhérents aux différents écosystèmes de Madagascar. Le second objectif a été d'identifier les mécanismes d'émergence de la FVR à Madagascar et de déterminer s'il sera possible, et nécessaire, de prédire cette émergence à l'échelle des écosystèmes.Dans le cadre de ce travail de thèse deux enquêtes sérologiques nationales, l'une bovine (2008) et l'autre humaine (2011-13) ont, premièrement, été analysées par un modèle linéaire mixte généralisé afin d'identifier les facteurs environnementaux et comportementaux favorables à la circulation du virus chez les bovins et les humains. Deuxièmement, deux enquêtes sérologiques bovines, l'une réalisée en 2008 et l'autre en 2014, ont été analysées pour reconstruire la dynamique de transmission de la FVR dans les différents écosystèmes de l'île. Cette reconstruction a été réalisée à partir de données de séroprévalence et d'âge inclues dans un modèle Bayésien hiérarchique pour estimer la force d'infection annuelle de 1992 à 2014. Enfin, afin de faire le lien biologique avec les résultats des travaux menés à une échelle nationale et de décrire les mécanismes de transmission à une échelle fine, des enquêtes longitudinales entomologiques et sérologiques ont été réalisées entre 2015 et 2016 dans un écosystème à risque. Et ceci, afin de décrire la transmission saisonnière du VFVR chez les ruminants associée à la dynamique de transmission des vecteurs potentiels.Nos résultats ont montré que la région du nord-ouest de l'île est une région à risque de transmission. D'une part, elle est constituée d'environnements associant une forte densité de bovins à des zones humides, inondables et irriguées, favorables aux espèces d'Anopheles et Culex. D'autre part, le VFVR semble avoir circulé de façon relativement intense lors de la période inter-épizootique de 1992 à 2007, puis sa transmission a soudainement augmenté en 2007-2008, ce qui est concomitant avec l'apparition des foyers de FVR en 2008. Pour finir, 6 ans après l'épidémie de FVR à Madagascar, le virus semble toujours circuler à bas bruit dans la région. Cette circulation étant probablement due à une transmission vectorielle favorisée par l'abondance de vecteurs potentiels dans la région.Les résultats de ces différents travaux nous ont permis de présenter des hypothèses de transmission dans les différents écosystèmes de l'île et ainsi de proposer des stratégies de surveillance, de prévention et de lutte contre la FVR adaptées au contexte de Madagascar
Rift Valley fever (RVF) is a zoonotic vector-borne disease affecting ruminants and humans. Its complex eco-epidemiology involves several species of vectors, hosts and transmission routes. These particularities allowed the circulation of RVF virus (RVFV) in a variety of ecosystems involving different transmission and emergence mechanisms. Indeed, the RVFV has affected contrasted eco-regions in Africa, Arabian Peninsula and South-West Indian Ocean islands, including Madagascar.Madagascar is considered as a continent island due to its ecological diversity and its endemicity level of the flora and the fauna. In particular, the variation of the Malagasy ecosystems (semi-arid in the south, humid and cold in the highlands, humid and warm in the north-west and per-humid in the east) has an impact in their presence and /or the relative abundance of some mosquito species. Madagascar was heavily affected by RVF in 1990-91 and 2008-2009, with evidence of a large and heterogeneous spread of the disease.Thus considering the diversity of RVF eco-epidemiological cycles and the variety of Malagasy ecosystems, we hypothesized that, in Madagascar, the mechanisms of transmission would be different according to these ecosystems. Therefore, the first objective of this thesis was to understand the mechanisms and the dynamics of transmission of RVFV in the different ecosystems. The second objective was to determine the mechanisms of emergence of RVFV and if it would be necessary and possible to predict the emergence of RVFV outbreaks according to the ecosystems.Firstly, we analyzed both cattle and human serological data performed at the national level using generalized linear mixed models to identify the environmental and behavioral factors associated with RVF transmission in both cattle and human. Secondly, we reconstructed the dynamic of transmission of RVF in the different Malagasy ecosystems. Seroprevalence data of cattle of known age were fitted using Bayesian hierarchical models to estimate the annual force of infection from 1992 to 2014. Thirdly, to understand the biological process link to the mechanisms of transmission at the national scale, we investigated the fine scale mechanisms of transmission of RVFV in pilot area of an at-risk region. We, thus, performed both longitudinal entomological and serological surveys between 2015 and 2016, in order to describe the seasonal transmission of RVFV among ruminants and its association with the dynamics of RVFV potential vectors.Our results showed that the northwestern part of Madagascar is an at-risk region for RVFV transmission. On one hand, it is characterized by high cattle densities associated with humid, floodplain and irrigated areas suitable for RVFV potential vector like Anopheles and Culex species. On the other hand, RVFV had probably circulated intensively in the region during the 1992-2007 inter-epizootic period and its transmission increased suddenly in 2007-08, almost concomitantly with the first outbreaks recorded in 2008. Finally, RVFV was still circulated in the northwestern region at low level, 6 years after the last epidemic. This circulation is likely due to vectorial transmission favoring by the abundance of several potential vectors of RVFV in this pilot region.Finally, our better understanding of the mechanisms of transmission of RVFV throughout Madagascar allowed us to propose hypothesis of transmission in different ecosystems of Madagascar and consequently refine strategies for RVF surveillance and prevention

Le virus de la fièvre de la vallée du Rift (VFVR) est un arbovirus endémique en Afrique qui a un impact sur le plan économique et en santé publique en affectant les ruminants et les humains. Le génome viral est composé de trois segments L, M et S de polarité négative ou ambisens. Le segment M code deux glycoprotéines structurales majeures, GN et Gc, et deux protéines auxiliaires, P78/NSm-GN et P14/NSm. Nous avons également identifié une protéine de 13 kDa, nommée NSm', qui correspond à une forme tronquée de P14/NSm. Dans cette étude, nous avons analysé l'impact de ces trois protéines sur la propagation du VFVR in vitro, et in vivo chez la souris ou le moustique. Seule l'absence d'expression des protéines P14/NSm et P13/NSm' mène à une réduction drastique de la mortalité chez la souris, démontrant le rôle majeur de P14/NSm et de son substitut P13/NSm' dans la virulence virale. Ces observations corrèlent avec une production fortement réduite du virus mutant dans des macrophages murins. De façon surprenante, P78/NSm-GN n'a pas d'effet sur la virulence du VFVR chez la souris. En revanche, elle apparaît comme un déterminant majeur de la dissémination virale chez les moustiques infectés et un virus mutant n'exprimant pas P78/NSm-GN présente une production virale altérée lors de l'infection aigüe et persistante de cellules de moustique. Cette étude a permis de mettre en évidence la contribution des protéines auxiliaires codées par le segment M du VFVR dans la propagation du virus chez les hôtes mammifères et arthropodes, la protéine NSm/NSm' étant essentielle pour la virulence chez la souris et la glycoprotéine P78/NSm-GN conditionnant la dissémination virale chez le moustique
Rift Valley fever virus (RVFV) is an arbovirus endemic in Africa, which has an economic and; public health impact by affecting ruminants and humans. The viral genome is divided into three segments, L, M and S, in negative or ambisense polarity. The M segment encodes the two major structural glycoproteins, GN and Gc, and at least two additional proteins, P78/NSm-GN and P14/NSm which are non-essential for virus growth in vitro. We identified an additional 13 kDa protein, referred to as NSm', which corresponds to a N-terminally truncated form of P14/NSm. We further assessed the role of the NSm-related proteins on 1 RVFV propagation by comparing mutant viruses expressing various sets of the NSm related proteins to a rescued wild-type virus in experimentally infected mice and mosquitoes. Only the lack of P14/NSm and P13/NSm' proteins affected virulence drastically in the mouse model, identifying these proteins as the major virulence factor encoded by the M segment. These observations correlated with reduced growth of a P14/NSm and P13/NSm' KO mutant in murine macrophages. Additionally, P78/NSm-GN appeared as a major determinant of virus dissemination in infected mosquitoes and a mutant virus lacking P78/NSm-GN was found to show altered virus production during acute and persistent infection of mosquito cells. This study demonstrates the differential contribution of the NSm- related proteins to virus propagation in mammalian and arthropod hosts, the NSm/NSm' protes being essential for viral virulence in mice and the P78/NSm-GN glycoprotein conditioning virus spread in the mosquito vector

Transmis par les moustiques, le virus de la fièvre de la vallée du Rift (vFVR) est un virus zoonotique qui affecte principalement les ruminants en Afrique et conduit à des pertes économiques importantes. Il n’existe actuellement pas de traitements et les seuls vaccins disponibles sont à usage vétérinaire. Le développement de nouveaux vaccins plus sûrs contre le vFVR est une priorité de l’OMS en raison du risque d’émergence de cet arbovirus dans d’autres continents. Dans cette étude, nous avons développé une vaccination à ADN optimisée contre le vFVR qui consiste à administrer par voie cutanée un plasmide codant pour l’ectodomaine de la glycoprotéine de surface Gn du vFVR (eGn) en présence d’un plasmide adjuvant codant le GM-CSF et combinée avec une électroporation. De plus, nous avons également optimisé la vaccination à ADN en l’associant à la stratégie de ciblage des cellules dendritiques (DCs) via un plasmide qui code des fragments d’anticorps scFv fusionnés avec l’eGn dirigés contre les récepteurs DEC205 et CD11c exprimés à la surface des DCs. Les vaccins ont été testés chez le mouton, hôte naturel du virus et dans le modèle murin pour étudier les mécanismes de protection. Dans nos deux modèles d’études, l’immunisation par le plasmide codant l’eGn confère une meilleure protection après une épreuve virale ainsi qu’une forte production d’anticorps non neutralisants par rapport au ciblage des DCs. En revanche, le ciblage d’eGn vers des récepteurs de DCs protège partiellement contre une épreuve virale et induit une immunogénicité différente dans les deux espèces. Nous avons confirmé le rôle protecteur de ces anticorps anti-eGn par un transfert passif dans le modèle murin et le mécanisme d’action de ces anticorps protecteurs reste encore à être déterminé. Notre étude montre pour la première fois la protection par un vaccin à ADN contre le vFVR chez le mouton
The Rift valley fever virus (RVFV) is a mosquito-borne virus that mainly affect ruminants in Africa, resulting in economic burden. There is currently no treatment and only vaccine for veterinary use against the RVFV are available. The development of new and safer vaccine is urgently needed due to the risk of introduction of this arbovirus to other continents. In the present work, we developed an optimized DNA vaccination against RVFV using a plasmid encoding the ectodomain of surface glycoprotein Gn (eGn) of RVFV into the skin with plasmid adjuvant encoding GM-CSF and electroporation in sheep. We further optimized the DNA vaccination using dendritic cell targeting strategy with a plasmid encoding a single chain fragment variable (scFv) fused with eGn directed to two DC receptors, DEC205 and CD11c. The efficacy of the vaccines were tested in the sheep, the natural host and in the mouse model to investigate the mechanism of protection. In both models non-targeted eGn vaccine confer a better clinical protection and higher non-neutralizing antibody production than DC-targeted vaccine. However, in both models eGn targeting to DEC205 differentially affected the immune response and induced a partial protection after a challenge. We further demonstrated that non-neutralizing antibodies induced by native eGn protect mice by passive transfer. The mechanism mediated by these antibodies remains to be investigated. Overall, this work indicates the proof of concept that DNA vaccine can confer protection against the RVFV in the sheep

Bunyaviridae is one of the biggest known viral families, and includes many viruses of clinical and economic importance. The major virulence factor of most bunyaviruses is the non-structural protein (NSs). NSs is expressed early in infection and inhibits the innate immune response of the host by blocking several steps in the interferon induction and signalling pathways. Hence, NSs significantly contributes to the establishment of a successful viral infection and replication, persistent infection and the zoonotic capacity of bunyaviruses. Although functions and structures of many viral interferon antagonists are known, no structure of a bunyavirus NSs protein has been solved to date. This strongly limits our understanding of the role and the mechanism of interferon antagonism in this large virus family. In this work the first structure for a bunyavirus interferon antagonist, the core domain crystal structure of NSs from the Rift Valley fever virus (RVFV) is presented. RVFV is one of the most clinically significant members of the Bunyaviridae family, causing recurrent epidemics in Africa and Arabia, often featuring high-mortality haemorrhagic fevers. The structure shows a novel all-helical fold. The unique molecular packing of NSs in the crystal creates stable fibrillar networks, which could correspond to the characteristic fibrillation of NSs observed in vivo in the nuclei of RVFV infected cells. This first NSs structure might be a useful template for future structure-aided design of drugs that target the RVFV interferon antagonism. Attempts at characterising other bunyavirus NSs proteins of other genera were made, but were hampered by problems with obtaining sufficient amounts of soluble and folded protein. The approaches that proved unsuccessful for the solubilisation of these NSs proteins, however, should inform future experiments aimed at obtaining recombinant NSs for structural studies.

La fièvre de la Vallée du Rift (FVR) est une arbovirose zoonotique décrite pour la première fois en 1930 au Kenya. Transmise principalement entre ruminants par des moustiques des genres Aedes, Culex et Anopheles, elle peut aussi se transmettre à l'homme par contact direct avec des produits d'avortement ou des fluides corporels d'animaux virémiques. Cette maladie a été décrite dans de nombreux pays d'Afrique ainsi que sur la péninsule arabique et dans l'Océan Indien. La circulation du virus de la FVR (VFVR) a été décrite dans 3 écosystèmes distincts : (i) les zones semi-arides sud- et est-africaines, (ii) les mares temporaires des zones arides, (iii) les zones irriguées adjacentes à de grands fleuves. Au sein de chacun de ces écosystèmes, le rôle des mouvements d'animaux dans l'introduction du VFVR et des moustiques vecteurs dans sa transmission ont fortement été mis en avant. Malgré l'existence de modèles statistiques pouvant prédire l'émergence du VFVR en Afrique de l'est notamment grâce au niveau de pluviométrie, les mécanismes de transmissions en jeu dans les autres régions sont encore incertains. L'apparition de foyers dans un écosystème tempéré et montagneux de Madagascar, où les espèces et abondances vectorielles ne sont pas favorable à la persistance du VFVR, suscite des interrogations quant à ces mécanismes. L'objectif de cette thèse est d'identifier les facteurs et les processus épidémiologiques permettant la circulation récurrente du VFVR dans un écosystème tempéré de Madagascar. L'étude a pris en considération les pratiques socio-économiques ainsi que les principaux moustiques vecteurs du virus de la zone tempérée des hautes terres malgaches. Les principaux modes de diffusion du virus entre bovins ont été étudiés au cours de 3 années de suivi sérologique. Un modèle mathématique calibré sur la base de nombreuses données empiriques collectées dans cette zone est proposé. Deux pratiques commerciales ont pu être distinguées : le commerce classique et une pratique traditionnelle de troc. Les résultats de l'analyse suggèrent une implication différente de ces deux pratiques dans la circulation du VFVR. Alors que le commerce pourrait permettre l'introduction du virus, la pratique du troc serait quant à elle le support de la circulation au sein de la zone. Un modèle déterministe est construit afin de comparer quatre scénarios pouvant expliquer la circulation virale dans cet écosystème peu favorable. Les résultats suggèrent que, si la transmission vectorielle reste la principale voie de transmission dans cet écosystème inhabituel, la transmission directe lors du vêlage de vaches virémiques pourrait également jouer un rôle. La circulation du VFVR est de plus favorisée par les pratiques socio-économiques de la zone qui permettent, malgré la saison sèche, l'exposition des bovins introduits et potentiellement virémiques aux moustiques vecteurs
Rift Valley fever (RVF) is a zoonotic disease first described in 1930 in Kenya. Primarily transmitted between ruminant by mosquitoes of Aedes, Culex and Anopheles genus, it can also be transmitted to humans by direct contact with abortion products or body fluids of viraemic animals. This disease has been described in many African countries, in the Arabian Peninsula and the Indian Ocean. The circulation of RVF virus (RVFV) has been reported in three ecosystems: (i) semi-arid areas of South and East Africa, (ii) temporary ponds in arid areas, (iii) irrigated areas near large rivers. Within each of these ecosystems, the role of animal movements in the introduction of RVFV and that of mosquitoes in virus transmission has been strongly emphasized. Despite the existence of statistical models that predict the emergence of RVFV in East Africa based on rainfall level, the transmission mechanisms involved in other areas are still uncertain. The occurrence of an outbreak in a temperate and mountainous ecosystem of Madagascar, where the species and vector abundances are unfavorable to the persistence of RVFV, raises questions about these mechanisms.The objective of this thesis is to identify the factors and the epidemiological processes that support the RVFV recurrent circulation in a temperate ecosystem of Madagascar. The study took into account the socio-economic practices as well as major mosquito vectors of the area. The main modes of virus spread between cattle were studied during a three years serological follow-up. A mathematical model was elaborated and calibrated using empirical and field data collected in the area. Two cattle exchange practices could be distinguished: the usual trade and a traditional practice of barter. The results of the analysis suggest a different impact of these two practices in RVFV circulation. While trade may allow virus introduction, the barter practice would support its spread within the area. A deterministic model was built to compare four scenarios that could explain the recurrent virus circulation in this unfavorable ecosystem. Results suggest that, if the vector-based transmission remains the main transmission mode, direct transmission from viremic cows at calving could also play a role. RVFV circulation is favored by socio-economic practices of the area that led, despite the dry season, to the exposure of introduced, and potentially viremic, cattle to vectors

La Fièvre de la Vallée du Rift (FVR) est une zoonose observée pour la première fois au Kenya en 1930 qui s'est peu à peu propagée à la plupart des pays d'Afrique. La FVR est une maladie à transmission vectorielle dont le virus appartient au genre Phlebovirus de la famille des Bunyaviridæ. En Afrique de l'Est, l'émergence de foyers est prédite par des modèles statistiques, ce qui n'est pas le cas en Afrique de l'Ouest où les facteurs et les mécanismes en jeu sont encore mal définis. L'objectif de cette thèse est d'identifier les facteurs et les processus épidémiologiques expliquant l'émergence de foyers de FVR au Sénégal, en mettant en œuvre une approche éco-épidémiologique centrée sur les principaux moustiques vecteurs du virus. Par l'étude de variables environnementales et climatiques et par leur exploitation dans des modèles mathématiques, nous avons tenté de répondre à deux questions épidémiologiques majeures : (1) quelles sont les zones potentiellement à risque, et (2) quelles sont les périodes favorables à l'apparition de foyers. L'étude a été menée à l'échelle locale, dans une zone d'environ 10 km2 autour du village de Barkedji situé dans la région sylvo-pastorale du Ferlo.Pour localiser les zones à risque de transmission du virus, nous utilisons la télédétection et l'analyse paysagère afin de caractériser l'environnement favorable aux deux principaux candidats vecteurs du virus, Aedes vexans et Culex poicilipes. Pour identifier les périodes à risque, nous avons développé un modèle d'abondance de populations de moustique des deux espèces vectrices prenant en compte la dynamique des gîtes larvaires (les mares), et dont les simulations ont été validées avec des données de terrain de capture de moustiques. Pour se faire, nous avons dû préalablement développé un modèle dynamique de hauteur d'eau des mares temporaires, modèle calibré et validé à partir de données de terrain et de données d'observation de la Terre.Les résultats de l'analyse paysagère ont confirmé que les milieux favorables aux vecteurs de la maladie pouvaient être caractérisés par télédétection. Ils ont aussi mis en évidence l'importance des mares et de la densité de végétation environnante, et ont abouti à une cartographie de l'hétérogénéité spatiale du risque de circulation de la FVR. Les résultats de l'analyse temporelle ont montré que les années de circulation active du virus coïncidaient avec les années pour lesquelles les deux espèces de moustiques étaient présentes en forte quantité. On observe ainsi deux années à très forte densité des deux moustiques vecteurs, en 1987 et en 2003, correspondant aux années d'épidémie/épizootie les plus importantes dans la région.

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

Foreign animal disease outbreaks can cause substantial economic losses. Policy makers need information on both the vulnerability of the food supply to disease epidemics and the impacts of alternative protection actions. This research focused on the assessment of the U.S. agricultural sector and human vulnerability to a Rift Valley Fever (RVF) outbreak and the value of a select set of alternative disease control strategies. RVF is a vector-borne, zoonotic disease that affects both livestock and humans; thus both animal and human consequences of an outbreak were examined. This research was conducted in two parts. Livestock impact assessment used an integrated epidemic/economic model to examine the extent of RVF spread in the animal population and its consequences plus the outcome of implementing two different control strategies: emergency vaccination and larvicide vector control. The number of infected, aborted, and dead animals is best controlled by coupling vaccination along with larvicide, but results in the second highest median national welfare loss. Therefore, careful decisions must be made as to what actions should be taken. Total national producer welfare is reduced with each scenario, and is more severe than the total national welfare loss (producer, consumer, and processor together). Consumer welfare is increased with each scenario due to a drop in prices of some commodities, and in some instances, an increase in supply as well. The majority of the national welfare loss can be attributed to the producers' and processors' loss in welfare. The highest damages are seen in the regions of the outbreak such as the South Central (SC). Other regions such as the Corn Belt, Lake States, and South East regions also see high damages due to price changes. The outbreak did not have substantial price effect on dairy products, but did have noticeable price changes for live cattle such as heifer calves, stocked yearling, and dairy calves. Prices for substitutes such as pork, chicken, and turkey experienced a price reduction, which can also be a factor resulting in consumer welfare gains. Human impact assessment utilized an inferential procedure for estimating the human consequences which comprise of a cost of illness calculation to assess the dollar cost of human illnesses and deaths, as well as a Disability Adjusted Life Year calculation to give an estimate of the burden of disease on public health as a whole. With potential costs above $2 billion for human illness, and with this number not accounting for loss or damages to other sectors of the economy, it can be highly probable that investing in a human vaccination campaign can be cost-effective and possibly cost-reducing. This cost along with the economic loss of the agriculture sector suggests substantial potential losses to the U.S. if this hypothetical situation were to become reality. Combining total loss estimates from the cost of illness and ASM models, potential damage of a RVF outbreak could range from 121 million to 2.3 billion US 2010$. The results of this study show the economic damages of an outbreak in the livestock population being much greater relative to the outbreak in the human population (roughly 16 times greater). It should be pointed out that both cost estimates are most likely under estimated. The animal outbreak is not incorporating all susceptible livestock (e.g. hogs and goats), and the human illness is not incorporating other damages to society (e.g. damages due to loss of tourism). By providing estimates on the potential economic outcomes, policy makers can better choose where, when, and how to invest their resources.

Master of Science
Department of Diagnostic Medicine/Pathobiology
A. Sally Davis
Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic Phlebovirus that is a significant threat to ruminants and humans. RVFV is categorized as an overlap Select Agent by the Department of Health and Human Services and US Department of Agriculture. Therefore, the study of RVFV’s pathogenesis and the development of novel diagnostic tools for the prevention and control of outbreaks and virus spread is crucial. RVF is endemic to sub-Saharan Africa but has spread beyond the continent to the Arabian Peninsula indicating the competence of the virus to emerge in new areas. Thus, the high likelihood of RVF’s spread to other non- endemic countries also spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In the US, RVFV is a Select Agent, requiring BSL-3 enhanced containment practices for research work. First, we developed a method for the detection of RVFV RNA by reverse transcriptase real-time PCR (RT-qPCR) using non-infectious, formalin- fixed, paraffin-embedded tissues (FFPET). The results from FFPET RT-qPCR were compared to prior results for fresh-frozen tissues (FFT) RT-qPCR, as well as immunohistochemistry and histopathology completed on the same FFPET blocks. We developed a novel technique using a rapid and low cost magnetic bead extraction method for recovery of amplifiable RVFV RNA from FFPET. FFPET RT-qPCR can serve as an alternative tissue-based diagnostic test, which does not require a BSL-3 research facility. Second, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies. The cELISA results were compared to the virus neutralization test, the gold standard serological assay for RVFV. This prototype cELISA is easy to implement, sensitive, specific, and safe test for the detection of antibodies to RVFV in diagnostic and surveillance applications. RVF is an important transboundary disease that should be monitored on a regular basis. The diagnostic tests developed and validated in this thesis could be used in endemic or non-endemic countries for the early detection of RVF and assist with the implementation of countermeasures against RVFV.

Rift Valley fever (RVF) is a disease endemic to Africa, which has recently spread outside of Africa to the Arabian Peninsula. Rift Valley fever virus (RVFV) is the causative agent of RVF and manifests as severe hepatitis, encephalitis and haemorrhagic fever, resulting in mortality in approximately 1% of human cases. RVFV also affects agriculture as it causes high mortality rates in young ruminants (>90% in new-born lambs) and is associated with high levels of abortions, which results in devastating economic losses. RVFV is a single-stranded RNA virus with a genome comprising of three separate genetic elements referred to as the Large (L), Medium (M) and Small (S) segments. The negative sense L segment encodes an RNA-dependent RNA polymerase (RdRp) while the M segment encodes two glycoproteins, Gn and Gc, and two non-structural proteins, NSm1 and NSm2. The glycoproteins are important for viral entry, genome packaging and mature virion formation as well as being the main antigen for the elicitation of neutralising antibodies by humoral immunity. The NSm proteins are required for mosquito vector transmission and preventing viral-induced apoptosis in host cells. The ambisense S segment encodes in the positive orientation a non-structural (NSs) gene, and in the negative orientation the nucleocapsid (N) gene. NSs is an important virulence factor involved in subverting host defences and the loss of NSs results in a highly attenuated RVFV infection. N is required for RNA synthesis and encapsidation of viral genomes. There are currently very few treatments in the early stages of development and vaccines for RVFV are not readily available. The overall lack of therapeutic strategies for RVFV urges novel therapeutic development such as RNA interference (RNAi). Endogenous RNAi is triggered by dsRNA and is involved in gene regulation through sequence specific suppression of target mRNA. Therapeutic RNAi exploits the RNAi pathway to facilitate targeted degradation of viral genes and has been applied effectively to the inhibition of a number of viruses that cause chronic and acute infections. There are fewer studies that have used RNAi to inhibit highly pathogenic viruses. Efficacy has been demonstrated against Ebola virus, Lassa virus and Dengue fever virus, which suggests applicability to the inhibition of RVFV. In this thesis, short hairpin RNAs (shRNAs) were generated to target the NSs, N and M genes of RVFV, which are important proteins in the viral life cycle. To determine the knockdown efficacy of the shRNAs, HEK293 cells were transiently transfected with the shRNAs and a vector expressing the respective shRNA gene target fused to a luciferase reporter. The reporter levels were assessed using a dual-luciferase assay and several shRNAs were selected for further characterisation as a result of effective target knockdown. Consequently, the shRNAs reduced the levels of expressed FLAG-tagged NSs, N and M encoded proteins, which were detected using western blot analysis. ShRNAs directed against NSs were shown to disrupt this protein’s function to result in alleviation of pathogenic properties. Specifically, NSs was shown to suppress the transcription levels of a luciferase reporter as well as prevent the activation of an IFN-β promoter. When the shRNAs were transiently transfected into HEK293 cells, they were able to reverse NSs-induced suppression in the reporter assays. Furthermore, NSs is cytotoxic as determined by observing cell morphology under transmitted light microscopy, which was quantified using a MTT viability assay and cells that subsequently received anti-NSs shRNAs had improved viability. This class of anti-pathogenic shRNAs should be able to down-regulate NSs in vivo and attenuate RVFV virulence. However, NSs is not essential for viral replication and as a result of the aggressive pathology of haemorrhagic RVF, essential structural genes were targeted to investigate shRNAs with anti-replicative properties. ShRNAs directed against N were transfected 24 hrs prior to infection with RVFV. The inhibition of viral replication was determined by collecting supernatant over 3 days and measuring the levels of N antigen using an ELISA. The shRNAs demonstrated effective suppression of RVFV but N antigen was detected at 72 hrs post-infection, which suggested that the shRNAs were overwhelmed by the virus. A series of shRNAs against M were subsequently tested and the anti-M shRNAs effectively suppressed viral replication in cultured cells over an extended 96 hr experiment, demonstrating that M is a good target for RNAi-mediated inhibition of RVFV. In this thesis, the potential of RNAi-based therapeutics against RVFV was demonstrated and these data contribute to the growing knowledge that RNAi should be developed further as a potential treatment for haemorrhagic fever viruses. Finally, some DNA viruses such as HBV form cellular reservoirs from which new virus can be produced and the DNA is resistant to RNAi-mediated inhibition. RVFV is an RNA virus with an acute infection, which makes it more susceptible to RNAi and an excellent target for this particular therapeutic modality.

Doctor of Philosophy
Department of Diagnostic Medicine/Pathobiology
A. Sally Davis
William Wilson
Rift Valley fever virus (RVFV) is a zoonotic arbovirus that is a significant threat to livestock and humans. It is listed as #3 for most dangerous animal threats and is in the top 10 pathogens needing urgent research in preventative and control measures. Although RVFV has never been reported in the US or Europe, outbreaks outside the African continent have sparked renewed interest in developing diagnostics and vaccines to protect both agriculture and public health. Having specific and versatile diagnostics is critical for vaccine development and application. For example, diagnostic tools that aid in identifying key immunogens and understanding the virus-host interaction directly contribute to developing protective vaccines. Additionally, vaccines that are used prophylactically or in response to an outbreak require diagnostic tests to differentiate infected from vaccinated animals (DIVA). This is critical for assessing the return to ‘disease free’ status after an outbreak. Unfortunately, there are limited RVFV diagnostic tests that are versatile and DIVA compatible with the newest RVFV vaccines. We describe the development of several diagnostic tools that are DIVA compatible for detecting RVFV nucleic acid, antibodies, and antigens. First, we evaluate a fluorescence microsphere immunoassay (FMIA) for the detection of antibodies against a RVFV surface glycoprotein and the nucleocapsid protein. The targets developed in this assay provide the basis for a DIVA-compatible serological assay with a candidate RVFV Gn/Gc subunit vaccine, as well as, offer a multiplexing platform that can simultaneously screen for several ruminant diseases. Second, we describe a novel chromogenic in situ hybridization (ISH) assay to detect RVFV in formalin-fixed, paraffin-embedded (FFPE) tissues. This molecular assay offers a highly sensitive, multiplexing platform that detects RVFV RNA on the cellular level of diagnostic tissue samples. Moreover, we demonstrate the first application of ISH as a DIVA-compatible assay for candidate RVFV gene-deletion vaccines. Third, we provide working protocols for western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) that use monoclonal or polyclonal antibodies against key RVFV antigens. These tools can be applied to pathogenesis research and used in the development of vaccine and therapeutic countermeasures against RVFV. The RVFV diagnostic methods developed and evaluated in this dissertation can serve as a model for developing diagnostic strategies for other transboundary animal diseases.

A thesis submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg for the De +ee of Doctor of Philosophy Johannesburg, 1992
The structural, functional and antigenic properties of the envelope glycoproteins of Rift Valley fever virus (RVFV) were analyzed using a panel of monoclonal antibodies (MAbs). In order to gain a better understanding of the role of the RVFV surface proteins in infection and pathogenesis, the mechanisms ofanti:~odymediated neutralization of the virus were examined, as well as the fl}action of the glycoproteins in viral attachment and penetration. Of the twenty three MAbs which were generated, fourteen were directed against the G1 and nine against the G2 protein of RVFV. The topological relationship of the antigenic determinants to each other on the viral glycoproteins was achieved using competitive binding assays with enzyme-labelled MAbs. For the RVFV 01 protein, four antigenic domains which may be interlinked were identified. The domains G1 I, II and IV were involved in virus neutralization and haemagglutination, while G1 III was associated with low level C'-dependent neutralization. With regard to the G2 protein, four antigenic domains which appear to be spatially distinct were identified. Domain G2 I exhibited significant neutralizing and haemagglutination activity, while G2 II was involved in haernagglutination and weak C(-dependent neutralization. The remaining 02 regions neutralized to a low level only in the presence of C'. The majority of the epitopes on bath viral glycoproteins were highly conformational, indicating that the native protein structure is necessary for the recognition and expression of the functional activities of these particular antibodies. Protective determinants were shown to occur on both Gland 02, demonstrating that both RVFV envelope proteins are important in viral pathogenesis. The neutralization studies, in turn, revealed that the inhibition of virus attachment is not the principal means of antibody-mediated neutralization of RVFV. Instead, such ..ieutralization appears to be the result of several different processes, including synergistic neutralization by combinations of different antibodies, prevention of virus binding, virus internalization and the blocking of the viral life cycle at an intracellular stage. Further insight into RVFV infectivity was obtained by showing that both glycoproteins are involved in virus entry into the host cell. Finally, the present. findings strongly support an endosomal route of entry and penetration for RVFV, associated with concomitant allosteric changes in the 01 protein.

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.

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.

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.

Rift Valley fever virus is a zoonotic pathogen that is transmitted between mosquitoes and mammals such as sheep, cattle and humans. It is an enveloped negative-sense single-stranded RNA virus, which is a member of the family Bunyaviridae and the genus Phlebovirus. During replication, RVFV produces a 78 kDa glycoprotein NSm1 of unknown function, believed to be nonstructural. Here I show that NSm1 is incorporated into RVFV virions assembled in C6/36 mosquito cells, but not in virions assembled in Vero E6 mammalian cells. The presence of NSm1 in insect-amplified virions was demonstrated through repeated immunoblots of purified virions and further supported by mass spectrophotometric confirmation of the identity of immunoblot-positive protein bands. This research appears to be the first evidence that distinct viral protein profiles are correlated to the host cell in which replication occurred.