3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Expression of envelope proteins by pre-s1/pre-s2 deletion mutants of HBV isolated from Southern African HIV-positive patients(2018) Simelane, DanielHepatitis B Virus (HBV) pre-S deletion mutants of genotypes B and C have been shown to predispose to hepatocellular carcinoma (HCC). Studies have reported that envelope protein expression can be affected by the deletion mutants, leading to endoplasmic reticulum (ER) stress, apoptosis and hepatic injury. In South Africa, genotype A/subgenotype A1 prevails and it also has a relatively high hepatocarcinogenic potential compared to non-A genotypes. The aim of the study was to determine whether strains of HBV subgenotype A1, isolated from HIV-infected patients, affect envelope protein expression in vitro. The strains included pre-S deletion mutants (also frequently found in HCC patients) and the strain isolated from HBsAg negative HIV positive individual. Different 1.28 mer replication-competent constructs, generated previously, including two wild type subgenotype A1 strains from an HBsAg-positive infection (A12C15; A12C15 ALT9.3), a strain from an occult infection (SHH193A) and 3 different deletion mutants: SHH011A (with deletions in the pre-S1/pre-S2 regions), SHH045A and SHH167A (with deletions in the pre-S2 region) were used. The plasmids were transfected into Huh7.5 cells and envelope protein expression followed by immunofluorescence using anti-HBs antibody and confocal microscopy on days 1, 3 and 5 post-transfection as well as quantification co-localization of the HBsAg using Zen software (Zeiss). Mean of fluorescence was quantified by Image J software. All the constructs expressed HBsAg and the HBsAg was located mainly in the cytoplasm for 100% of the transfected cells with a diffuse staining for wild-type (A12C15_OL and A12C15 ALT 9.3) and the 3 deletion mutants (SHH011A, SHH045A & SHH167A). The strain from an occult infection (SHH193A) showed aggregates of HBsAg in the cytoplasm with more extensive accumulation at the perinuclear region of the cells, a finding that is suggestive of envelope protein retention in the cellular compartments, which prevented secretion leading to the HBsAg-negative phenotype of the patient. In general, the deletion mutants (SHH011A, SHH045A & SHH167A), expressed envelope proteins at comparable amounts to that shown by the wild-type constructs (A12C15_OL and A12C15 ALT 9.3). Although other groups have shown the expression of the envelope proteins by pre-S deletion mutants of different HBV genotypes, this study is the first to show the expression of the envelope proteins for subgenotype A1 constructs. Immunocytochemistry and high resolution confocal microscopy was successfully used to follow the expression of HBsAg in the secretory pathway. The accumulation of HBsAg in the perinuclear region following transfection with the strain from the occult infection could account for the HBsAg-negative phenotype seen in the patient.Item Molecular characterization and genotyping of hepatitis B virus (HBV) from sudanese end stage renal disease (ESRD) patients with overt and occult HBV infection(2018) Nekhwevha, KhudaniHepatitis B virus (HBV), which causes infection globally, is endemic in sub-Saharan Africa. Currently, nine genotypes (A to I), distributed geographically, are recognised. In Sudan, located in north-east Africa, HBV prevalence ranges from 4% to 26%, and genotypes D and E predominate. Patients suffering from end-stage renal disease (ESRD) exhibit poor kidney function and require regular haemodialysis. HBV exposure in haemodialysis patients in Sudan is high, as the immunosuppressive nature of renal disease increases risk from HBV infection. The aim of this study was to molecularly characterise HBV from ESRD patients in Sudan undergoing haemodialysis and also to use phylogenetic analysis to determine if HBV infection was occurring in the four haemodialysis centres from which HBV DNA was successfully amplified. We undertook polymerase chain reaction (PCR) amplification and DNA sequencing of the basic core promoter/pre-core (BCP/PC) and the complete S (preS1/preS2/S) regions of 68 HBV DNA positive ESRD patients undergoing haemodialysis in four different treatment centres (Salma, Ibn Sena, Elturki and Alnaw) in Khartoum. In Hepatitis B surface antigen (HBsAg) positive (overt) HBV-mono-infected samples, PCR amplicons from the BCP/PC region were obtained from 28/46 (60.9%) of samples, with DNA sequences obtained from 24/46 (52.2%). Results for the S region were 23/46 (50%) and 15/46 (32.6%), for PCR amplification and sequencing, respectively. BCP/PC PCR amplification from 12 occult HBV-mono-infected samples resulted in 2/12(16.7%) amplifications of low density bands. Hence no DNA was obtained for sequencing. However, 4/12 (33.3%) of these samples yielded S region amplicons, with DNA obtained from 3/12 (25%). Ten HBV/HCV co-infected samples yielded 4/10 (40%) positive samples in the BCP/PC, with 3/10 sequenced (30%) in the complete S, 2/10 (20%) amplified with low DNA quality. Neighbour-joining phylogenetic analysis using 1000 bootstrap indicated that genotype D predominated at 11/15 (73.3%), followed by genotypes E and A, at 3/15 (20%) and 1/15 vi (6.7%), respectively. Genotype D was found in all centres and mostly detected at Salma 6/15 (40%), whereas genotype A was only detected at Elturki. Moreover, Genotype E was only detected in Salma and Ibn Sena. We identified a six nucleotide insertion occurring in the core region of HBV from four individuals from Salma centre. This was a novel finding in genotype D isolates as this insertion was previously found in genotype A. The preS1 mutation, ps1I48V, which occurs mostly in genotype A isolates, was found in one genotype A isolate from Sudan. We further identified a preS2 deletion in one genotype E isolate. The deletions in the pre S contribute to the development of hepatocellular carcinoma (HCC). The HBV reactivation marker, S174N was found in one HBsAg negative (occult) individual. We further identified HBV drug resistance mutations, rtS213 and rtQ215H occurring in two different individuals from Ibn Sena centre. Both of these individuals were not vaccinated. In conclusion, in Sudanese ESRD patients, genotype distribution varies between treatment centres, although genotype D predominates. In addition, drug resistance and vaccine escape similar to the ones found in South Africa exist in Sudan. The small sample size did not allow us to compare the mutations found in HBV isolates from individuals with overt or occult infection. Hence, further studies to identify distinguishing mutations between occult and overt samples, as well as in HBV/HCV co-infected individuals in Sudan are still needed.Item Molecular characterization of the hepatitis b virus in South Africa(2001) Bowyer, Sheila. M.The hepatitis B virus (HBV) partitions into 6 genotypes (A-F) and a seventh putative genotype, genotype G, which has recently been described. As southern Africa is highly endemic for HBV infection and little is known of the molecular epidemiology of HBV in this part of the world, we determined the genotype of viral strains circulating in both rural and urban South Africa and rural Namibia. Two screening assays were used. The first was based on size and restriction fragment length polymorphism (RFLP) analysis of specific polymerase chain reaction (PCR) fragments amplified from the preS 1 and preS2 regions of the surface gene. The second method was an enzyme immunoassay (EIA) based serological determination of genotype which used monoclonal antibodies raised against genotype-specific epitopes in the preS2-region. Only genotypes A and D were found in significant numbers in South Africa. The urban specimens partitioned as follows: 69.6 percent genotype A, 3.6 percent genotype B, 7.1 percent genotype C, 16.1 percent genotype D and 3.6 percent genotype E. Rural specimens were all either genotype A (89.2 percent) or genotype D (10.8 percent).Item Molecular methods to determine the presence of hepatitis B virus ( HBV ) genotypes in the serum of a hbv-infected chacma baboon(2004-03-17) Naicker., Tholsi. Jocelyn.In the course of a previous study (Kedda, et al., 2000), it became evident that the baboons had been inoculated with a mixture of HBV genotypes, namely, genotypes A and non-A. Therefore, a follow up study was undertaken to determine whether either A or non-A HBV genotypes predominated over the other during the time course of 52 weeks post-inoculation of HBV into baboons. HBV DNA extracted from sera obtained from one of the four HBV positive baboons, at two time points during a 52 week post-HBV inoculation period, was amplified using primers specific for the HBV core region that is well conserved in all genotypes.Item Hepatitis B virus serology : eight years of laboratory data in retrospect(1993-10-26) Sim, John. Gray. MalcolmAn analysis of 39,774 specimen records of diagnostic screening for hepatitis B virus (HBV), consisting of HBV surface antigen (HBsAg), antibody to surface antigen (anti- HBs) and antibody to HBV core antigen (anti-HBc) was performed. Specimens representative of all possible result combinations for these primary markers were found in the dataset and were investigated further by studying the distribution of extended marker frequencies within the groups defined by the primary marker combinations in an attempt to reconcile the patterns observed with standard profiles of HBV serology. The extended markers consisted of HBV e antigen (HBeAg), antibody to HBeAg (anti- HBe) and anti-HBc IgM. Unusual patterns of serological results which were identified were investigated further for the presence of HBsAg/anti-HBs immune complexing by acid dissociation techniques, and for the low level HBV chronic carrier state by polymerase chain reaction (PCR) amplification of HBV DNA. Results obtained indicated that immune complexing was not a common occurrence in the groups investigated, while preliminary findings in patients with isolated anti-HBc positivity demonstrated a 6.5% low level HBV carrier rate in this groupItem The association of HLA class II genetic and expression level variation with response to the hepatitis B vaccine in South Africa laboratory workers(2017) Goldfein, HadassaThe hepatitis B virus (HBV) vaccine has contributed greatly to decreasing the HBV epidemic. However, it remains unclear why 5-10% of individuals do not mount an adequate antibody response. Previous studies have shown that genetic variation influences HBV vaccine response. Since such studies are lacking in South African individuals, we examined the associations between HBV vaccine response and genetic variation in HLA-DPB1, additional candidate genes and HLA-DPBl expression levels in a South African cohort. HLA-DPA1 and -DPB1 allele typing was performed using Luminex technology, twenty-four candidate SNPs were typed by MassArray Analysis and HLA-DPBl mRNA expression levels were measured by qPCR. HLA-DPBl *01:01, *04:01:01G and *09:01 and SNPs and haplotypes in IL1B, 1L4, IL12B, 1FNG and the HLA region were significantly associated with HBV vaccine response. A trend of lower HLA-DPBl expression associating with better anti-HBs response was observed, although this was not significant. Response to the HBV vaccine is multi-genic but HLA-DP plays an important roleItem Generating obligate heterodimeric transcription activator-like effector nucleases to inactive hepatitis B viral replication(2018) Smith, Tiffany ShenayThe hepatitis B virus (HBV) remains a global health concern as chronic infection can lead to cirrhosis and hepatocellular carcinoma. Current therapies are only partially effective and do not target the HBV covalently closed circular DNA (cccDNA). cccDNA serves as a replication intermediate for the transcription of viral RNA, resulting in the persistence of infection. Several designer nucleases have therefore been developed to target cccDNA. Their mutagenic effect is mediated by causing double-strand breaks at target sites, consequently disrupting translation of viral proteins. Previous studies observed successful inhibition of HBV, with the use of homodimeric transcription activator-like effector nucleases (TALENs). TALENs are designed in pairs (left and right monomers) to cleave complementary strands of targeted DNA. They consist of a DNA-binding domain which is attached to the wildtype nuclease domain of FokI. TALENs have exhibited considerable predictable interaction with their target sites and have shown diminished toxicity in vitro and in vivo. Despite this success, TALENs containing wildtype FokI nuclease domains may be prone to off-target effects as two of the same domains (two left or two right) in close proximity on opposite strands of DNA can result in cleavage at unintended sites. To establish a more clinically applicable therapy, obligate heterodimeric TALENs targeted against the core (C) and surface (S) open reading frames (ORFs) of cccDNA were generated and referred to as C or S Variant TALENs. Variant TALENs contain mutant FokI nuclease domains. This allows for cleavage only when two different domains (one left and one right) are in close proximity on opposite strands of DNA thus reducing off-target effects. Overall the variant TALENs demonstrated significant knockdown of the HBV replication marker, hepatitis B surface antigen (HBsAg). Additionally variant TALEN-mediated targeted disruption occurred in ≥5.7% of total HBV DNA in vitro and ≥13% of total HBV DNA in vivo. Finally variant TALEN-treated mice also demonstrated significant knockdown of pregenomic RNA and surface mRNA levels as well as markedly reduced circulating viral particle equivalents. Together these results show that obligate heterodimeric TALENs are capable of HBV knockdown and serve as an advanced gene editing tool against chronic HBV infection.Item Inhibition of Hepatitis B virus using helper-dependent adenoviral vectors expressing pri-miRNAs from liver-specific MTTR promoter(2017) Mdunyelwa, AneleApproximately 240 million people are estimated to be chronic carriers of hepatitis B virus (HBV), placing them at high risk for complications like hepatocellular carcinoma (HCC) and cirrhosis. Infection with the virus is predominantly high in sub Saharan Africa, East and Southeast Asia. Current treatments are limited by the emergence of viral resistance and adverse side effects. These challenges prompted development of new therapy for HBV. The application of RNA interference (RNAi) as a form of treatment has shown successful HBV silencing. However, safe and efficient delivery of anti-HBV sequences remains a challenge. Recombinant adenoviruses (Ads) have a natural tropism for the liver, making them suitable for anti-HBV sequence delivery. Their use in gene therapy is limited by their ability to induce the innate and adaptive immune response, thus leading to diminished transgene expression. To overcome immune stimulation, third generation helper-dependent adenoviral vectors (HDAds), which are devoid of all their viral coding sequences, were developed. This study investigated the use of HDAds expressing primary micro RNA (pri-miRNA) sequences from the liver-specific MTTR promoter with the aim of inhibiting HBV replication. The anti-HBV pri-miRNA expression cassettes were successfully cloned into the adenoviral bearing genome and used for HDAd production. Infection of liver derived cell line with these HDAds resulted in efficient pri-miRNA expression and processing into expected guide sequences. This was accompanied by a significant inhibition of HBV replication. Injection of the HDAds into mice resulted in efficient liver transduction and no significant induction of the inflammatory response or liver toxicity. However, as a result of high levels of HBV replication markers produced in the transgenic mice used, this did not translate into significant HBV replication inhibition. This study therefore highlights the safety and therapeutic potential of HDAds against HBV infection. To demonstrate HBV gene silencing in vivo, transgenic mice expressing HBV replication markers will be used.Item Liver-targeted transcription activator-like effector repressors that inactivate HBV cccDNA(2017) Kaldine, HaajiraThe hepatitis B virus (HBV) continues to be a global health threat as chronic infection may lead to cirrhosis and hepatocellular carcinoma (HCC). Current treatments are limited in efficacy and do not target the stable HBV covalently closed circular DNA (cccDNA) minichromosome which forms the template for viral replication and is responsible for persistence of the infection. Using gene editing technologies to disable cccDNA presents a potential approach for treating HBV infection. Transcription activator-like effector (TALE) proteins provide specific and adaptable DNA binding modules, which can be used to generate engineered proteins capable of modifying DNA. Transcription activator-like effector nuclease (TALEN) mediated cleavage of cccDNA has been shown to effectively inhibit HBV replication. However, the approach to transcriptionally silence cccDNA, instead of cleaving it, may overcome the risk of unwanted host DNA cleavage. Repressor transcription activator-like effectors (rTALEs), which target and transcriptionally silence genes, have shown potential as antiviral agents. Here we generated Krüppel-associated box (KRAB)-based rTALEs targeted to the surface open reading frame (ORF) and HBx promoter region of HBV cccDNA to inhibit transcription. The rTALEs were placed under the transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, to restrict activity to hepatocytes thereby reducing the potential for off-target activity. In vitro the mTTR-driven rTALEs were shown to tissue specifically decrease secreted HBV surface antigen (HBsAg) levels by 63 - 92 %. Additionally, the mTTR-driven rTALEs were shown to tissue specifically decrease surface mRNA levels by 65 – 81 % and pregenomic RNA levels by 60 - 76 %. These results indicate that the KRAB domain was able to effectively suppress transcription from the basic core, Pre-S1 and/or vi Pre-S2 promoters which otherwise regulates HBV transcription. Furthermore, the observed inhibition was not associated with cytotoxicity or off-target effects. The work presented here is a proof-of-concept study demonstrating that highly specific transcriptional repressors designed to target and inhibit HBV viral replication without altering the genetic sequence or causing mutations in the host genome may be a promising antiviral approach. The capabilities of this technology to directly target cccDNA and inhibit its transcription, could contribute to addressing a global health problem.Item INHIBITING HEPATITIS B VIRUS GENE EXPRESSION WITH HAMMERHEAD RIBOZYMES THAT TARGET THE HBx OPEN READING FRAME(2002-10-28) Weinberg, Marc SaulHepatitis B virus (HBV) infection is endemic to several populous regions and is often complicated by cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches are an important medical objective. The X open reading frame (ORF) of HBV, HBx, is a conserved sequence that overlaps with the polymerase ORF and viral c/'s-elements, and is present within all viral transcripts. In addition, the HBx ORF encodes a 17 kDa transactivator protein, HBx, which is required for the establishment of viral infection and has been implicated in HBV-associated hepatocarcinogenesis. The HBx sequence thus represents a compelling target for applying nucleic acid hybridisation-based therapeutic agents for the inhibition of HBV gene expression and replication.