Bibliography: leaves 155-183. Also available online.

Flaviviruses are a genus of positive-sense, single-stranded ribonucleic acid (RNA) viruses. This genus includes viruses which have caused significant public health and economic concerns worldwide, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and yellow fever virus (YFV). They are spread by arthropod hosts, mosquitos and ticks in particular, and generally cause illnesses that result in symptoms which range from mild influenza-like infection to hemorrhagic fever and encephalitis (Laureti et al., 2018). Many members of the flavivirus genus have re-emerged into the spotlight due to urban growth and increasingly hospitable environments for mosquitos due to climate change. Though effective vaccines are available against a few flaviviruses, there are still no effective vaccines available against DENV, ZIKV or WNV. Additionally, there are currently no antiviral therapeutics available for flavivirus infection; management of infection is generally supportive in nature. These challenges have caused flaviviruses to be of considerable interest to the infectious diseases research community (Collins & Metz, 2017). Virus entry into a host cell is a critical step of the virus life cycle. However, the mechanism that flaviviruses employ to enter host cells is still poorly understood. Identifying the viral and cellular factors which mediate this process could certainly allow for new inroads in the development of effective flavivirus therapeutics. Using YFV as a model flavivirus, this thesis project aims to characterize the role of specific cellular factors which govern flavivirus entry. Recent genetic screens from the Douam Lab have identified a cytoskeletal protein of interest as a potential candidate protein that regulates the YFV life cycle. As such, we hypothesized that this cytoskeletal protein is a mediator of flavivirus entry into host cells. For the first portion of our study, we sought to determine if chemical inhibition of this cytoskeletal protein could reduce the susceptibility of hepatoma cell lines to flavivirus infection, using the live attenuated yellow fever virus vaccine strain 17D (YFV-17D) as a prototypical flavivirus. We subsequently found that chemical inhibition of the activation of this cytoskeletal protein restricted YFV-17D infection of hepatoma cell lines in a dose dependent manner. In addition, we confirmed that the inhibition of infection was not due to cytotoxic effects imposed by our chemical inhibitor. For the second portion of our study, we sought to determine if this cytoskeletal protein specifically regulates YFV-17D entry. To assess this, we developed a novel YFV-17D reporter system, ΔNS1-17D, which is capable of entering cells, but is unable to produce viral particles de novo. As a whole, our study provides evidence that this cytoskeletal protein plays a significant role in regulating flavivirus infection. Future experiments will employ our new ΔNS1-17D reporter system to provide conclusive evidence that this cytoskeletal protein specifically promotes YFV-17D entry into host cells, and intend to identify the cell surface molecules bound to this cytoskeletal protein which mediate this process. By developing the foundation of a larger body of work which aims to identify host cell factors that regulate flavivirus entry, our findings could contribute to the development of innovative therapeutics against flaviviruses. / 2023-11-18T00:00:00Z

Bibliography: leaves 161-177.

Introduction: Cytomegalovirus (CMV) drug resistance mutations, because of the widespread use of ganciclovir, have been widely reported in international literature, particularly in the post-transplant setting. However, a genotypic assay to detect CMV drug resistance is not available in South Africa and the prevalence of these mutations is therefore unknown. We aimed to document the prevalence and types of CMV UL97 mutations following exposure to ganciclovir in adult and paediatric oncology patients, transplant recipients and HIV-infected patients in the local tertiary level hospitals: Red Cross War Memorial Children's Hospital, Groote Schuur Hospital and Tygerberg Hospital. Methods: The study had two components, the first component being a retrospective cross-sectional study using stored extracted DNA from patients with serially elevated CMV viral load levels. Thirty-three samples were tested for this component. The second component was a prospective case series on patients who were referred by clinicians for genotypic testing in whom CMV drug resistance was suspected. Eight samples were tested for this component. The CMV UL97 gene was amplified by conventional nested polymerase chain reaction (PCR) and Sanger sequencing performed. Results: CMV UL97 mutations were identified in five of thirty-three (15%) retrospectively screened samples while the prospective testing of eight patient samples identified drug resistance mutations in three patients (38%). Overall 8/41 (20%) patients had CMV UL97 mutations. A trend of higher risk for development of drug resistance mutations among haematological oncology patients 7/23 (30%) compared to solid organ transplant recipients 1/10 (10%) was observed, however, this difference was not statistically significant (P=0.306). Conclusion: This study, the first of its nature in South Africa, identified the presence of CMV UL97 mutations conferring resistance to ganciclovir in the haematological oncology, primary immunodeficiency and solid organ transplant patients in the Western Cape. The assay successfully detected CMV UL97 drug resistance mutations in whole blood and cerebrospinal fluid clinical samples. Ongoing viral replication in the background of intensive immunosuppression and prolonged antiviral therapy selects for the emergence of CMV UL97 drug resistance mutations.

The prevalence of HIV-1 is highest in Sub-Saharan Africa. Protective immune responses directed against HIV are complex and involve both cellular and humoral immunity. Based on the recent finding that the best correlate of protection against the first protective prophylactic RV144 vaccine were HIV-specific antibody responses, including those mediating natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC), there has been considerable interest in measuring alternative roles for HIV-specific binding antibodies. The aim of this MSc dissertation was to optimise and compare two high-throughput flow cytometry based approaches - the GranToxilux and PanToxilux assays - to measure HIV-specific ADCC responses. To do this, NK cells from a panel of healthy HIV-negative individuals were screened for their ability to directly kill the tumour cell line K562, as a measure of direct NK cell cytotoxicity. The individual with the highest granzyme B and caspase activity against K562 cells was chosen as the universal NK cell donor for this study.

Includes bibliographical references. / Immune system pressure on HIV-1 replication drives the antigenic changes seen over time. The monitoring of changes in viral sequences can provide important information on the nature of the immune response and the correlates of protection. Viral diversification may also occur due to other selective pressures such as cell availability and differences in viral fitness. Information on the genetic characteristics of HIV-1 variants present in the mother and her infected infant are useful data for establishing whether any common features exist between source infection and transmitted genotypes. This helps in the understanding of the mechanism of transmission and the selective pressures occurring during and following transmission. The overall aim of this study was to explore alternative methods other than DNA sequencing for the monitoring of genetic diversity in the third variable region (V3) of the HIV-1 env gene of integrated HIV-I variants in peripheral blood mononuclear cells (PBMC's) derived from infected mother-child pairs. Two methods for displaying DNA differences were compared: I-leteroduplex Mobility Assay (I-IMA) and Base Excision Sequence Scanning (BESS). These methods were validated using sequence data. Extracted PBMC DNA from infected mother-child pairs were used to amplify the V3 region by nested PCR. DNA fragments were cloned into plasmid vectors and analyzed by HMA and BESS to establish subtype and intrasample genetic diversity. In addition, a PCR-ELISA quantitation system was developed to measure copy numbers of integrated HIV-1 genomes in order to confirm whether a sufficient number of template molecules were present to be representative of the total viral quasispecies. In conclusion, this study compared two methods (HMA and BESS) as cost-effective alternatives to DNA sequencing for HIV-1 diversity studies. in addition, a novel application of the BESS assay was demonstrated. Diversity studies are reliant on estimation of adequate input of amplifiable copies. The PCR-ELISA quantitation system developed provided an efficient and specific method for determining DNA copy number.

The United States Environmental Protection Agency (USEPA) has established the Total Coliform Rule as an indication of health risks associated with microbial contamination of drinking and ground water. In addition, the fecal coliform test is used as an indicator to reflect the suitability of use by consumers of class A biosolids. However, numerous studies have shown that bacterial indicators are not predictive of enteric viruses, such as human adenovirus (HAdV), which are much more resistant to treatment methods than bacteria. Enteric viral contamination of estuarine waters and locally-harvested shellfish as a result of receiving effluent from wastewater treatment plants, as well as run-off from agricultural land treated with biosolids, can have serious implications for human health. Preliminary results suggest that HAdV is present in biosolids, wastewater effluent, estuarine waters receiving effluent and shellfish harvested from these receiving waters. The density, persistence and infectivity of human adenovirus in these environmental matrices are not known. The focus of this research was to address the presence, persistence and viability of HAdV in all four matrices. Presence and density of the virus was established through the use of a nested polymerase chain reaction (PCR) and quantitative PCR (qPCR). HAdV DNA was detected in 21 of the 26 biosolid samples and 21 of the 24 effluent samples assayed. The treatment method employed in the processing of the samples appeared to have an effect on the detection and concentration of HAdV DNA. Persistence of HAdV DNA in estuarine water was addressed in an in situ study using seeded microcosms containing either sterile river water or unfiltered river water under various environmental conditions during the spring, summer and fall. Unfiltered river water collected during the summer had the greatest deleterious effect on HAdV DNA persistence. HAdV DNA was most persistent, under all environmental treatments, during the fall. An in vitro study of sterile river water confirmed that temperature, not salinity, had a greater effect on HAdV DNA degradation. Laboratory tank studies revealed that oysters are capable of filtering and retaining HAdV from contaminated water. In each of the three tank studies conducted, HAdV DNA was detected in tissue samples from oysters exposed to seeded river water for 18 hours. It was also established that the oysters could depurate the virus, in an open system, in as little as three days. Integrated cell culture (ICC) - qPCR was used to determine the viability of detected viral particles. No direct correlation between the detection of HAdV DNA and the presence of viable viruses was found. Frequently, samples that contained HAdV DNA failed to produce viable virions. Current research corroborates these results, suggesting the detection and persistence of viral DNA is not sufficient evidence to support the assumption of viability.

No description available.

EBV is a human gammaherpesvirus that infects approximately 95% of the population worldwide and is associated with 1% of human cancer incidence. The EBV-genome-encoded latency membrane protein 1 (LMP1) is expressed in nearly all of EBV-associated malignancies and is the major EBV oncogene. Because LMP1 is essential for both establishing a latent EBV infection and promoting tumorigenesis, defining its role in these processes is critical in order to understand EBV-associated cancer development. Due to the importance of epigenetics in regulating EBV gene expression during latency, the goals of this dissertation were two-fold: to define the mechanisms of host epigenetic regulation of LMP1 expression and to establish how LMP1 alters the host epigenome. The host uses its epigenetic machinery to regulate EBV during latency. One mechanism is through the host protein CTCF, which acts as an insulator to prevent the intrusion of heterochromatin into euchromatic regions and prevent DNA methylation within the EBV genome. CTCF also participates in the three-dimensional organization of the EBV episome through the formation of a network of long-range interactions that, in turn, further regulate EBV gene expression. CTCF binds at several key regulatory regions in the EBV genome, including upstream latent gene promoters, with the strongest CTCF binding site in the EBV genome positioned at the LMP1 locus. However, the functional role of CTCF binding in regulating LMP1 has yet to be established. To define the role of CTCF binding at the LMP1 locus we utilized a genetically modified EBV bacmid that contained a site-directed deletion of the CTCF binding site at the LMP1 region. Using EBV-positive B cells we found that CTCF binding at the LMP1 locus is a key regulator of LMP1 expression. Loss of CTCF binding changed the epigenetic profile at the LMP1 locus resulting in the loss of euchromatic and a concomitant gain of heterochromatic histone modifications, as well as an associated increase in DNA methylation near the LMP1 promoter. These epigenetic changes mediated decreased LMP1 expression. Additionally, through chromosome conformation capture (3C) assays, we established that DNA loop formation between the LMP1 loci and the viral enhancer OriP is strictly dependent upon CTCF binding at LMP1. Taken together, these observations suggest an epigenetic mechanism by which the host, through CTCF, contributes to the regulation of LMP1 expression. LMP1 expression is also regulated by structural elements within the EBV genome called terminal repeats (TRs). The number of TRs within an EBV genome varies based upon the circularization of the viral genome, and LMP1 expression is inversely correlated with the number of TRs in epithelial cells. However, the mechanism by which TR number regulates LMP1 expression has yet to be elucidated. We hypothesized that TR number differentially regulates the epigenetic state of the LMP1 promoters to either promote or suppress their activity. By examining the epigenetic state at both LMP1 promoters in isogenic cell lines with different numbers of TRs, we identified differences in histone modifications and DNA methylation at the LMP1 promoters. Furthermore, decreasing the number of TRs eliminated a chromatin loop formed between the LMP1 loci and the viral enhancer OriP. This data suggests that the number of TRs regulates the epigenetic state of LMP1 and ultimately determines promoter usage, which is necessary for LMP1 expression. While the previous work focused on the host mechanisms that regulate LMP1 expression, we also explored if the reverse relationship existed, that is, if EBV hijacks the host epigenetic machinery as a means to alter host gene expression. LMP1 contributes to host cell proliferation and survival through the aberrant activation of biochemical signaling pathways that also modulate epigenetic regulation. Because the PARP1-mediated post-translational modification poly(ADP-ribosyl)ation (PARylation) regulates EBV latency, and LMP1 activates a PARP1 regulator MAPK/ERK, we hypothesized that LMP1 drives the induction of PARP1-mediated PARylation. PARP1 facilitates gene transcription by maintaining a euchromatic state, and as a result, the disruption in epigenetic regulation mediated by LMP1 through PARP1 may also induce changes in host gene expression, including genes that contribute to EBV-mediated tumorigenesis. A panel of EBV-positive cell lines were found to have higher PAR levels than EBV-negative B cells, establishing a relationship between latent EBV infection and cellular PARylation. Because expression of the EBV oncoprotein LMP1 was sufficient to induce cellular PARylation, we explored the model that disruption in cellular PARylation driven by LMP1 expression subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes through induction of EZH2 expression. Inhibition of PARP also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP activity in LMP1-induced gene expression and cellular transformation associated with LMP1. This dissertation reveals for the first time the importance of the host protein CTCF and the effect of the number of viral TRs in epigenetically regulating the expression of the oncogenic protein LMP1. This dissertation further establishes a mechanism by which EBV hijacks the host epigenetic machinery and identifies a novel role for LMP1 in driving the expression of tumor-promoting host genes by blocking the incorporation of an inhibitory epigenetic modification through the activation of PARP1, suggesting that PARP1 may be a target for treatment of EBV-associated malignancies. / Microbiology and Immunology

No description available.