3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Targeted inactivation of hepatitis B virus genome using enhanced transcription activator-like effector nucleases containing a sharkey fokI nuclease domain(2018) Singh, PrashikaDespite the availability of an effective vaccine, hepatitis B virus (HBV) infections are a major public health problem with approximately two thirds of the world’s population having been exposed to this virus. This DNA virus infects hepatocytes causing inflammation and is the causative agent of hepatitis B. Upon infection the viral genome is transported to the nucleus and repaired to covalently closed circular DNA (cccDNA). The cccDNA intermediate is responsible for the persistence of HBV in chronically infected individuals and acts as a viral reservoir. Current treatments are not able to target the stable cccDNA and highlight the need for the development of new therapies. This project focused on the development of anti-HBV transcription activator-like effector nucleases (TALENs) with improved efficiency and safety. TALENs are designer nucleases that induce double-stranded breaks (DSBs) within DNA. This stimulates host repair pathways that results in insertions or deletions (indels) that cause frame-shift mutations and disrupt targeted genes. TALENs are highly promising as they are able to target cccDNA and could lead to a functional cure for HBV. Existing anti-HBV TALENs have been designed to target conserved sites in the S and C open reading frames of the HBV genome. The third generation obligate heterodimeric Sharkey variants are enhanced FokI nuclease domains that were generated by altering certain amino acid residues in the wild-type FokI nuclease domain. Sharkey TALENs were produced by substituting the wild-type FokI domain of existing anti-HBV TALENs with Sharkey variants. In vitro analysis of the anti-HBV Sharkey TALENs demonstrated expression of the nucleases in liver-derived cells. However, the wild-type anti-HBV TALENs exhibited superior inhibitory effects as compared to the Sharkey TALENs. Sequence-specific cleavage was achieved by both surface (S) and core (C) Sharkey TALENs however the latter achieved greater targeted cleavage. Mutagenesis analysis of HBV DNA from extracted HepG2.2.15 cells revealed the Sharkey C TALEN achieved 12.1% targeted cleavage greater compared to the Sharkey S TALEN. Efficacy was assessed in vivo in the mouse hydrodynamic injection model of HBV replication. In vivo viral markers of HBV replication were assessed and the Sharkey anti-HBV S and C TALENs were able to reduce viral surface antigen secretion by 57% and 78% respectively, whereas both Sharkey TALENs achieved a 96% reduction of circulating viral particle equivalents. The Sharkey TALENs were able to reduce viral mRNA production by 35% and 45% and pregenomic RNA levels by 73% and 80%. Minimal toxicity in vitro and in vivo was observed using the Sharkey TALENs indicating that reduction of markers of HBV replication were as a consequence of the action of the Sharkey anti-HBV TALENs. Results for the Sharkey C TALEN were comparable to the wild-type TALENs in vitro and in vivo. Despite the inhibition of HBV replication achieved with third generation Sharkey TALENs, a more comprehensive analysis of anti-HBV Sharkey TALEN activity in different in vitro and in vivo models of HBV replication is necessary. In addition, in depth analysis of potential off-target effects is required to comprehensively examine the safety of these variants. Nonetheless, in this study the Sharkey TALENs are able to inhibit HBV replication cccDNA and transcription, thereby further validating the use of TALENs as a potential therapy for chronic HBV infection.Item Expression of anti-HBV primary micro-RNA shuttles using an inducible promoter system.(2014-03-28) Mlambo, TafadzwaHepatitis B virus (HBV) infection is an important global health concern and chronic carriers of the virus are at high risk of developing hepatocellular carcinoma (HCC) and cirrhosis. Current therapies are only partially effective, which emphasises the need for improved treatment strategies. Harnessing the RNA interference (RNAi) pathway as a treatment strategy against HBV has shown great promise. However, there are obstacles that need to be overcome before RNAi-based treatment of HBV infection is realised. These include problems of liver tissue targeting and dose regulation. This study investigated the use of a liver specific and mifepristone-inducible RNA polymerase (Pol) II promoter system for the specific and precise regulation of anti-HBV sequence expression. The inducible system used consists of two expression cassettes; one containing the regulator/transactivator protein and another containing the transgene. Natural primary microRNA (pri-miR) mimics, pri-miR-31/5 and pri-miR-31/5/8/9, were used as anti-HBV sequences. Firefly luciferase gene expression was used to test modulation by the inducible system and to determine optimal induction conditions. The pri-miR-31/5, pri-miR-31/5/8/9 and luciferase encoding fragments were incorporated into the plasmid vector pRS17 that bears the inducible promoter, creating pRS-31/5, pRS-31/5/8/9 and pRS-Luc respectively. Firefly luciferase expression with this system was shown to be inducible and mifepristone dose-dependent. Effective knockdown of HBV gene expression was achieved with both pRS-31/5 and pRS-31/5/8/9 in vitro and in vivo. However, with high vector amounts, similar efficiency in silencing of HBV gene expression was observed in the presence and absence of the inducer mifepristone suggesting leaky expression of the pri-miRs. To confirm this, knockdown studies were carried out with the pri-miR-31/5/8/9-expressing cassette separated from the transactivator cassette. HBV gene expression knockdown was observed with the pri-miR-31/5/8/9 cassette alone confirming leaky expression from the inducible system. Leakiness appears to be as a result of the E1B promoter driving the expression of the pri-miRs in the absence of mifepristone. However, reducing the vector amounts decreased basal expression and improved the inducibility of the system in cell culture studies. Successful propagation of an inducible and liver-specific RNAi-activating expression system will address the difficulty of achieving dose control of RNAi effectors and contribute to advancing the use of RNAi for HBV treatment.Item Nucleic acid sequence analysis of the distal X gene region containing the basic core promoter of the hepatitis B virus in southern African asymptomatic carriers of the virus and hepatocellular carcinoma patients(2014-03-07) Baptista, Marina Da Conceicao Pinto AzevedoThe purpose of this study was to identify mutations in the basic core promoter and enhancer II region a* the hepatitis B virus (HBV) that might result in the hepatitis B virus e antigen (HBeAg)-negative phenotype and contribute to hepatocarcinogenesis in black African carriers of the virus. The basic core promoter/enhancer II overlaps the X gene. HBV DNAfrom serum of 47 asymptomatic carriers and 50 patients with hepatocellular carcinoma and from 29 tumorous and 10 nontumorous liver tissues was amplified and sequenced directly. That part of the enhancer II region not overlapping the basic core promoter was reasonably well conserved in all samples. Missense mutations at positions 1809 and 1812 were found in 80% of all sequences and may represent wiidtype sequence in southern African isolates. Nucleotide and amino acid divergences were higher in the basic core promoter of hepatocellular carcinoma patients than of asymptomatic carriers (p<0.0001). This applied particularly to the paired 1762 adenine to thymine (1762T) and 1764 guanine to adenine (1764*) missense mutations, the prevalence of which was 66% in patients with hepatocellular carcinoma compared with 11% in asymptomatic carriers (p<0.0001). There was no association between the presence of 1762T1764A and the rate of HBeAg negativity, although these mutations suppressed HBeAg titres in HBeAg-positive patients. Suppression of HBeAg expression as well as alteration of amino acid sequence of the X protein may be contributing factors in the development of hepatocarcinogenesis.Item Genotyping and molecular characterization of hepatitis B virus(HBV) from human immunodeficiency virus (HIV) infected individuals in southern Africa(2012-01-26) Makondo, EuphodiaHepatitis B virus (HBV) and human immunodeficiency virus (Thakur et al.) are endemic in South Africa. There no data on the HBV genotypes prevailing in HIV-infected South Africans. The aim this study was to determine the HBV genotypes in HIV-infected patients and to identify mutations occurring in the basic core promoter/preccore (BCP/PC) and complete S regions. Twenty six HBV isolates from HBV-HIV co-infected individuals from Helen Joseph urban hospital prior to and at six month after initiation of HAART were analyzed. Three hundred HBV isolates from the rural Shongwe Hospital were recruited prior to treatment. Restriction fragment length polymorphism (RFLP) together with sequencing of the BCP/PC and complete surface region amplicons were employed for genotyping and analysis. There was no significant difference in the HBV genotypes from both cohorts. Subgenotype A1 was the predominant genotype. HBV DNA was detected in 13/26 (50 %) Helen Joseph patients and 72/300 (24%) HBV DNA was detected in patients from Shongwe cohort: 28/300 (9.3%) HBsAg-positive and 44/300 (14.7%) HBsAg-negative. The BCP/PC region of HBV isolates from both cohorts showed mutations that could account for the HBeAg negativity, although in the case of the Helen Joseph cohort the HBeAg status was unknown because of depletion of serum. The HBeAg negativity in 44/49 Shongwe patients (89,7%) could be accounted for by the following mutations: 1) the basic core promoter mutations T1753C A1762T G1764A, which down regulate transcription of precore mRNA; 2) the Kozak sequence mutants that affect HBeAg translation, 3) the G1862T mutation, which interferes with post-translational modification of the HBeAg-precursor, and 4) the classical G1896A stop codon mutation with C1858T. The G1862T mutation occurred in HBV from 24.1% HBsAg-positive Shongwe patients but in none of the HBsAg-negative patients (p<0.05). PreS deletion mutants were found in isolates from both cohorts. These have previously been described in hepatocellular carcinoma patients. The Helen Joseph HBV isolates had the “a” determinant and immune/vaccine escape mutants. In addition to these, Shongwe isolates had HBV reactivation markers mutations V168A + S174N in 23.8% of the HBsAg-negative and in none of the HBsAg-positive infections. Drug resistant mutations were found in three Shongwe HBV isolates from ART naïve individuals and in none Helen Joseph HBV isolates. In conclusion, South African HIV-infected individuals were predominantly infected with subgenotype A1 of HBV. Mutations in the BCP and precore region of HBV isolates could account for the HBeAg negativity in the majority of patients. A number of HBV isolates displayed both immune escape and drug resistance mutations that were responsible of the HBsAg-negativity in patients from which they were isolated.Item The relationship between hepatitis b virus and apoptosis in humans and in a transgenic mouse model(2012-01-17) Viana, Raquel ValongoHepatitis B virus (HBV) has been found to be highly endemic in Africa and south east Asia. In southern Africa, subgenotype A1 and genotype D prevail while in south east Asia genotype B and C predominate. Infection with HBV can lead to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma (HCC). It has been shown that viral factors as well as a number of host and environmental factors can influence the course of HBV infection. Development and progression of various liver diseases are associated with either an increase or decrease in hepatocyte apoptosis. Dysregulated apoptosis itself may be a fundamental feature of most acute and chronic human liver diseases. The purpose of this study was to characterise the subgenotype A1 and genotype D HBV infection, prevailing in South Africa. To control for the influence of host factors on HBV infection as well as to avoid the use of in vitro cell lines, such as Huh-7, that have defective apoptotic pathways, the in vivo urokinase plasminogen activator severe combined immunodeficient (uPA-SCID) transgenic mouse model was utilised. The HBV infection of the transgenic mice infected with HBV positive sera containing either subgenotype A1 wildtype, subgenotype A1 with the G1862T mutation, subgenotype A2 or genotype D, was compared. For the first time, we were able to demonstrate the successful infection of the uPA-SCID transgenic mouse model with subgenotype A1 of HBV. The successful establishment of the in vivo HBV infection with different genotypes or subgenotypes in the uPA-SCID transgenic mice was demonstrated by the increase of HBV DNA levels, the presence of cccDNA and HBV transcripts as well as the detection of the core and/or surface HBV antigens in the liver tissue of the chimeric mice. Differences between the HBV infections with the various genotype/subgenotypes were observed. Subgenotype A1 with the G1862T mutation showed the earliest detection and therefore highest levels of cccDNA as well as the highest HBV DNA levels when compared to the other strains. The highest HBV DNA levels were recorded for the subgenotype A1 G1862T infected transgenic mouse followed by genotype D, subgenotype A2 and the lowest levels observed in the subgenotype A1 wild-type infected transgenic mouse. HBsAg was also only detected in the livers of mice infected with subgenotype A1 with the G1862T mutation. HBcAg staining in the chimeric liver was positive when the mice were infected with genotype D, which concurs with previous observations that genotype D is characterised by high HBcAg expression. Subgenotype A1 with the 1862 mutant showed the highest levels of apoptosis as a result of the abnormal precore precursor protein accumulation shown to be associated with this 1862 missense mutation. Thus different genotypes and subgenotypes as well as variations within genotypes can influence HBV infection. Moreover, the results of these experiments in the immunocompromised chimeric mice, grafted with liver cells from a single donor, suggests that even when host and environmental risk factors are controlled for, the subgenotype or genotype can influence the course of infection. The limitations of the uPA-SCID transgenic mouse model include the lack of an immune system and the short life-span of the animal; therefore a population based study was carried out to investigate the influence of host factors on HBV infection in various disease groups. The study cohort comprised 635 serum samples from South Africa, China and Japan. Of these samples, 564 were HBsAg-positive and the remaining 71 HBsAg-negative, HBV DNA negative controls. The study cohort included asymptomatic carriers; chronically infected HBV patients as well as patients with HBV associated HCC. Possible associations were determined between HBV genotype, HBV viral load, apoptosis levels, disease group and the age and gender of the patient where available. Apoptosis levels were quantified by the measurement of cleaved cytokeratin 18 (M30) in serum. Patients infected with genotype C or subgenotype A1 were shown to possess a higher odds ratios of developing HCC compared to subgenotype B2 or genotype D, respectively. Significantly higher HBV viral loads were observed in genotype C compared to subgenotype B2. Among the Asian cohort, it was also shown that the male gender was positively associated with high viral loads in HCC patients. Moreover, a positive association between higher HBV viral load levels and HCC in the South African cohort was observed. Male gender, older age, HBV viral load, subgenotype A1 and the presence of the G1862T mutation were shown to be positively and significantly associated with higher levels of apoptosis. In this study it was discovered that the levels of cleaved cytokeratin 18 could potentially be used as a biomarker for the severity of HBV infection because a significant difference was observed with the apoptosis levels between the asymptomatic and HCC patient disease groups. We conclude that even when the influence of host and environmental factors is controlled for, as is the case in the chimeric mouse model, the HBV genotype can affect the progression of infection. Moreover, it was shown in the population based study that the effect of HBV genotype on the outcome of HBV infection can be influenced by host factors. The subgenotype A1 G1862T mutation was shown in both studies to affect both HBV infection and apoptosis. This suggests that HBV variants should be investigated to ascertain their potential impact on the course of HBV infection as it may differ from the wild-type. Apoptosis was shown to be associated with HBV infection in both studies and could possibly be an ideal marker of the progression of HBV infection. These findings are important in helping us to understand factors influencing the course of HBV infection. We have therefore shown in both the studies that differences do exist between the South African subgenotype A1 and genotype D, and that these differences should be taken into consideration for the future evaluation of HBV infection and treatment of South African HBV infected patients. Moreover, cleaved cytokeratin 18 may provide an ideal surrogate marker for HBV disease progression and monitoring.Item Occult hepatits B virus (HBV) infection in the Chacma Baboon (Papio ursinus orientalis)(2011-11-23) Dickens, CarolineMembers of the family Hepadnaviridae have been detected in both avian and mammalian species. They have a very limited host range, and among the nonhuman primates, have been found to occur naturally in chimpanzees, gorillas, gibbons, orang-utans and woolly monkeys. The human hepatitis B virus (HBV) has been shown to infect chimpanzees, Barbary macaques and tree shrews. During the course of a previous study, to determine the susceptibility of baboons (Papio ursinus orientalis) to HBV infection, HBV DNA was detected in the serum of 2 baboons prior to their inoculation with HBV-positive human serum, raising the possibility that baboons are naturally infected with a hepadnavirus. Therefore the aim of this study was to determine the prevalence of HBV in wildcaught baboons and to molecularly and functionally characterise the virus isolated from these baboons. DNA was extracted from the sera of wild-caught baboons and four separate regions of the HBV genome amplified by nested polymerase chain reaction (PCR). Samples were only considered to be positive for HBV if at least three of these regions amplified. DNA was extracted from the liver tissue of one of the HBV DNA-positive baboons using a proteinase K digestion followed by a phenolchloroform extraction and ethanol precipitation. From this extract, the complete HBV genome was amplified by nested PCR of eight overlapping subgenomic fragments, and sequenced. This sequence was analysed phylogenetically using both the PHYLIP and Simmonic software packages. A selective real time PCR using SYBR®-green detection was used to detect covalently closed circular (ccc) DNA. RNA was extracted from the baboon liver tissue using a guanidinium-acidphenol extraction method, reverse transcribed and portions of the HBV genome amplified by nested PCR. Transmissibility of the virus was tested by injecting four experimentally naïve baboons individually with serum from four HBV DNApositive baboons and followed for 26 weeks. HBV was detected in the serum of 5/69 (7,2%) wild-caught baboons by Southern hybridization and in 11/49 (22,4%) adult and 4/20 (20,0%) juvenile wild caught baboons. This gave an overall prevalence of 21,7% in the baboon population surveyed. Serologically, the baboon sera were negative for all markers of HBV infection and alanine aminotransferse (ALT) levels were normal. In the one baboon liver tissue available, HBcAg was detected by immunohistochemical staining in some of the hepatocyte nuclei, but HBsAg was not detected. Phylogenetic analysis of the complete genome of the HBV isolate found it to cluster with subgenotype A2, a surprising result considering that subgenotype A1 predominates in South Africa. However, unlike other subgenotype A2 isolates, the basic core promoter had the G1809T / C1812T double mutation characteristic of subgenotypes A1 and A3 and the precore region had the G1888A mutation unique to subgenotype A1. These mutations in the basic core promoter and precore regions have previously been shown to reduce the expression of the precore and core proteins. Four additional mutations in the polymerase, surface, X and core open reading frames (ORFs) further differentiated the baboon HBV strain form the majority of previously sequenced subgenotype A2 isolates. cccDNA was detected at low levels in the baboon liver tissue. Regions of the precore/core and surface ORFs were amplified off reverse transcribed cDNA. These results demonstrate HBV replication in the baboon liver. Transmission of the virus was shown by the detection of HBV DNA in the sera of the four inoculated baboons at various times throughout the 26 week follow-up period. These baboons also showed transient seroconversion for HBsAg and HBeAg during this period with intermittent fluctuations in ALT levels. Moreover, using DNA extracted from liver tissue obtained at necropsy from one of the injected baboons, the sequence of the HBV surface gene amplified was found to be identical to the sequence of the isolate from inoculum. The finding of subgenotype A2 in the baboon is paradoxical because subgenotypes A1 and A3 as well as genotypes D and E predominate in Africa. The possibility exists that subgenotype A2 is an older strain that has been overtaken by these other strains. There is however a scarcity of subgenotype A2 sequencing data from Africa and a higher circulation of this subgenotype could be uncovered with more extensive molecular epidemiological studies in more remote areas. Alternatively, a recent discovery of alternative compartmentalization of subgenotype A2 infections in the peripheral blood lymphocyte population of individuals from India, where subgenotype A1 also predominates, could explain the lack of detection of this subgenotype in Africa. Occult hepatitis B infection is defined as the presence of HBV DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for HBsAg by currently available assays. The detection of HBV DNA in the baboon liver and serum in the absence of serological markers therefore classifies this infection as occult. To our knowledge, this is the first study to demonstrate a naturally occurring occult HBV infection in non-human primates.Item The effect of the accumulation of Hepatitus B virus e-antigen precursor on cell viability(2006-11-17T13:05:26Z) Viana, Raquel ValongoThe G1862T mutation in the bulge of the RNA encapsidation signal, in the precore region of hepatitis B virus, results in reduced expression of HBeAg and accumulation of the HBeAg precursor in the endoplasmic reticulum (ER)/Golgi apparatus of the cell. This accumulation can disturb the functioning of the ER and lead to the ER stress response that can affect various cellular pathways, in turn affecting cell viability. The aim of this study was to determine whether apoptosis or necrosis occurred when cultured Huh7 cells were transfected with a plasmid expressing the G1862T mutation. Plasmid constructs, with and without the G1862T mutation, were used to transfect cells. To differentiate between necrosis and apoptosis cells were stained with propidium iodide or YO-PRO-1®, respectively. These were analyzed quantitatively using flow cytometry and qualitatively using confocal microscopy. Confocal microscopy, using monoclonal anti- HBe and the Hoechst stain, was performed to ensure that apoptosis was present as a result of the accumulation of the G1862T mutant HBeAg precursor. Caspase profiling was carried out using a fluorogenic-based assay. When cells were transfected with wild-type plasmid, necrosis predominated over apoptosis. Apoptosis predominated when the cells were transfected with the G1862T mutant plasmid. The highest levels of apoptosis occurred at 72 hours post-transfection. Confocal microscopy revealed the co-localization of aggregates of mutant HBeAg precursor with apoptotic nuclei. Transfection with G1862T mutant plasmids resulted in significant differences in the expression of caspase 3, 8, and 9 relative to the wild-type, at 48 and 72 hours post-transfection. The accumulation of the G1862T mutant HBeAg precursor, in the ER/ Golgi compartment, leads to apoptosis and affects the levels of caspase expression.