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

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

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 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.

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

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 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.

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.

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.

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