Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Stephen Higgs / The genetic composition of arboviruses is a critical determinant of viral infectivity and the capacity for virus dissemination in arthropod vectors. Due to concerns related to a hypothetical potential for loss of attenuation, the supression of vector infection and dissemination is a critical component for the rationale-based design of live-attenuated flavivirus vaccine candidates. The yellow fever virus (YFV) 17D vaccine virus is not only attenuated in vertebrates, but also has low infectivity for Aedes agypti mosquitoes and since it does not disseminate, it is not transmissible. Using a reverse genetics system, the mutations present in the envelope protein YFV 17D virus were characterized in Ae. aegypti to determine the role of mutations in limiting the viral infectivity and dissemination capacity. This knowledge would contribute to the rational design of live attenuated vaccines with the desirable phenotype of being nontransmissible by arthropod vectors. The upper lateral portion of the YFV 17D envelope (E) protein domain III (EDIII) habors the T380R mutation in the FG loop. Experiments demonstrated that the T380R mutation was associated with the viral infectivity phenotype for mosquitoes, but did not influence dissemination into the secondary tissues. The G52R mutation in the molecular hinge region that is located between E protein domains I (EDI) and II, significantly reduced viral infectivity for mosquitoes. In contrast, when cloned into the Asibi wildtype virus genetic backbone, the T173I mutation in the loop structure between the G0 and H0 β- strands did not attenuate viral infection and dissemination. The double mutant virus containing both the G52R and T173I mutations in the E protein, showed a similar attenuated reduced infectivity to the single G52R mutant. The M299I mutation in the linker region between EDI and EDIII resulted in a significantly lower viral infectivity at the initial phase of viral infection at 7 days post-infection in Ae. aegypti. In conclusion, the characterization on four mutations in the YFV 17D vaccine E protein have demonstrated three genetic loci, that can influence the process of YFV infection in Ae. aegypti. These results provide new knowledge and understanding which may have broad applications for the rationale design of safe flavivirus vaccines, via targeting genetic loci and introducing specific mutations that preclude infection of, and transmission by arthropod vectors.

Call number: LD2668 .T4 1958 W47

African swine fever virus (ASFV) is a large double-stranded DNA virus which causes a lethal haemorrhagic fever in domestic pigs. This virus primarily infects cells from the monocyte/macrophage lineage and its ability to manipulate the function of these cells is key to the pathogenesis of this disease. ASFV encodes several proteins involved in immune evasion. One of these proteins, A238L, has been shown to inhibit host macrophage gene transcription. This protein has been shown to interact with several cellular proteins involved in signal transduction: a serine/threonine protein phosphatase, calcinerurin (CaN), the transcription factor NF-кB, and most recently the transcriptional co-activator CREB binding protein (CBP/P300). However its exact mechanism of action is not fully understood. Previous work has been limited to the investigation of individual signaling pathways and/or the expression of individual host genes. The aim of this study was to investigate the global effect of A238L on host macrophage gene transcription and also to carry out further investigation into the mechanism by which this protein functions. To determine the global effect of A238L on host macrophage gene transcription differential gene expression between porcine cells expressing A238L and control cells was examined using a porcine oligonucleotide microarray. These results demonstrated that A238L was a potent inhibitor of host macrophage gene expression. Functional characterisation of the annotated genes showed that a large proportion of A238L down-regulated genes are typically induced in response to cell stress. Significantly, genes regulated by the I kappa B kinase (IKK), mitogen-activated protein kinase (MAPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways were all shown to be down regulated by A238L. Genes associated with the MAPK pathways were particularly enriched. The transcription of A238L-regulated genes is controlled by numerous different transcription factors, including NF-кB. All of the transcription factors identified interact with the transcription co-activator CBP/P300. This provides a common link between these factors, and indicates that A238L may target CBP/P300 to inhibit gene transcription. This observation supports recent work demonstrating that A238L interacts with and inhibits CBP/P300 function. To explore the potential mechanisms involved in the nuclear localisation of A238L, ASFV-infected Vero cells, expressing A238L under the control of its own promoter, were examined under a range of conditions using confocal microscopy. The results demonstrated that A238L was actively imported into the nucleus and exported by a CRM 1 mediated pathway, although a pool of A238L protein remained in the cytoplasm. Sequence analysis of A238L identified the presence of two putative nuclear localisation signals (NLS-1 and NLS-2). NLS-2 was located within A238L’s CaN docking motif. Mutation of these motifs indicated that both NLS-1 and NLS-2 are active and exhibit functional redundancy. Mutation of the CaN docking motif alone, in the presence of intact NLS-2, resulted in a dramatic increase in the nuclear localisation of A238L. These results are consistent with a model in which A238L functions within both the nucleus and the cytoplasm and suggest that binding of CaN to A238L masks NLS-2, contributing to the cytoplasmic retention of A238L.

A transient three-dimensional simulation program was developed to study the effects of scanning speed, scanning pattern, blood perfusion, transducer choice and multiple tilted transducers with overlapping foci during scanned focussed ultrasound hyperthermia. The results showed that (1) the temperature fluctuations increase linearly with decreasing scanning speed, (2) the temperature fluctuations are a weak, increasingly exponential function of the blood perfusion rate, and (3) that the largest temperature fluctuation is always located at the acoustical focal depth on the scan path independently of focal plane depth. Simulations using multiple scan paths showed that relatively uniform average temperature distributions can be achieved at the focal zone as long as the spacing between the concentric scans was not greater than the diameter of the focus of the power field. Finally, the results showed that using multiple tilted transducers with overlapping foci, increased focussing can be obtained at the focal depth.

No description available.

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (MCF) in susceptible ruminants. Through an unknown mechanism, presence of the virus leads to proliferation of NK-like T cells that are not targetrestricted by the MHC class molecules. These host cells cause the symptoms found in MCF; fever, swollen lymph nodes, and necrotic lesions of the nasal, conjunctival, and oral mucosa, which usually leads to death of the host. MicroRNAs (miRNAs) are ~22 nt RNA molecules expressed by eukaryotes and viruses that regulate genes post-transcriptionally. Viral miRNAs have been found to regulate cellular genes to control the cell cycle and have a role in pathogenesis. It was hypothesised that OvHV-2 expresses miRNAs and these play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 encodes miRNAs. Bioinformatic analysis was conducted on deep sequencing data from RNA of OvHV-2- immortalised T cells. Candidate miRNAs were selected if they adhered to miRNA secondary structure. 46 candidate miRNAs were found, with three clusters on the minus strand; one at the 5’ end and the other two in a 9.3 kb region that contains no predicted open reading frames. The 8 most abundant candidates were successfully validated by northern hybridisation for small RNAs. The majority of the predicted targets for the 8 validated OvHV-2 miRNAs were from the OvHV-2 genome. This study adds OvHV-2 to the list of herpesviruses that encode miRNAs and provides another tool for studying the pathogenesis of MCF.

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.

Throughout the nineteenth century, New Orleans was repeatedly plagued by yellow fever epidemics. In this paper, cultural representations of yellow fever are considered in three novels: Baron Ludwig Von Reizenstein’s The Mysteries of New Orleans (1854-1855), George Washington Cable’s The Grandissimes (1880), and Mollie Evelyn Moore Davis’ The Queen’s Garden (1900). Because the etiology was unknown during the nineteenth century, yellow fever becomes a floating signifier on which to project the ills they observed in New Orleans society. Yellow fever thus becomes a representation of loose sexual mores, as well as a divinely retributive punishment for slavery, or a sign of adherence to an unequal, antiquated, aristocratic and un-American social system. Yellow fever, in these texts, exposes the struggles with race and racial superiority and illuminates tensions between groups of whites as New Orleans became an American city.

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.

MSc (Med) (Bioethics and Health Law), Faculty of Health Sciences, University of the Witwatersrand