Dogs in the Tucson area with a known history of clinical Valley Fever were tested with two skin test reagents to determine their ability to detect delayed type hypersensitivity (DTH) to the Coccidioides spp. The reagents used were Spherusol, from Nielsen Biologicals, and coccidioidin, which is no longer commercially available. Skin tests were read 48 hours after placement and evaluated for erythema and/or induration.

Histopathology of a murine lymph node, 9 days post infection with Coccidioides. Magnification 10X

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

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

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

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

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

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.

abstract: Valley Fever (VF), is a potentially lethal fungal pneumonia caused by Coccidioides spp., which is estimated to cause ~15-30% of all community-acquired pneumonias in the highly endemic Greater Phoenix and Tucson areas of Arizona. However, an accurate antigen-based diagnostic is still lacking. In order to identify protein and glycan antigen biomarkers of infection, I used a combination of genomics, proteomics and glycomics analyses to provide evidence of genus-specific proteins and glycosylations. The next goal was to determine if Coccidioides-specific glycans were present in biological samples from VF patients. Urine collected from 77 humans and 63 dogs were enriched for glycans and evaluated by mass spectrometry for Coccidioides-specific glycans and evaluated against a panel of normal donor urines, urines from patients infected with other fungi, and fungal cultures from closely related pneumonia-causing fungi. A combination of 6 glycan biomarkers was 100% sensitive and 100% specific in the diagnosis of human VF subjects, while only 3 glycan biomarkers were needed for 100% sensitivity and 100 specificity in the diagnosis of dog VF subject. Additionally, a blinded trial of 23 human urine samples was correctly able to classify urine samples with 93.3% sensitivity and 100% specificity. The results of this research provides evidence that Coccidioides genus-specific glycosylations have potential as antigens in diagnostic assays. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2019

Spatio-temporal systems with heterogeneity in their structure and behavior have two major problems. The first one is that such systems extend over very large spatial and temporal domains and consume a lot of resources to simulate that they are infeasible to study with current platforms. The second one is that the data available for understanding such systems is limited. This also makes it difficult to get the data for validation of their constituent processes while simultaneously considering their global behavior. For example, the valley fever fungus considered in this dissertation is spread over a large spatial grid in the arid Southwest and typically needs to be simulated over several decades of time to obtain useful information. It is also hard to get the temperature and moisture data at every grid point of the spatial domain over the region of study. In order to address the first problem, we develop a method based on the discrete event system specification which exploits the heterogeneity in the activity of the spatio-temporal system and which has been shown to be effective in solving relatively simple partial differential equation systems. The benefit of addressing the first problem is that it now makes it feasible to address the second problem.We address the second problem by making use of a multilevel methodology based on modeling and simulation and systems theory. This methodology helps us in the construction of models with different resolutions (base and lumped models). This allows us to refine an initially constructed lumped model with detailed physics-based process models and assess whether they improve on the original lumped models. For that assessment, we use the concept of experimental frame to delimit where the improvement is needed. This allows us to work with the available data, improve the component models in their own experimental frame and then move them to the overall frame. In this dissertation, we develop a multilevel methodology and apply it to a valley fever model. Moreover, we study the model's behavior in a particular experimental frame of interest, namely the formation of new sporing sites.