PhD, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Chronic infection with the hepatitis B virus (HBV) is a major risk factor for cirrhosis and hepatocellular carcinoma, which is the sixth most common cancer worldwide. Available treatment for chronic HBV infection has limited efficacy in preventing associated complications. The compact and multifunctional nature of the viral genome limits its mutability making HBV an ideal candidate for therapy based on nucleic acid hybridisation. The potent and specific gene silencing that can be achieved with RNA interference (RNAi) has fueled interest in exploiting this pathway as a therapeutic modality. Synthetic and expressed RNA sequences have been used to activate RNAi. These engineered sequences mimic natural substrates of the RNAi pathway, which allows them to enter and reprogramme the pathway to effect silencing of intended targets. Tradionally expressed RNAi activators have been transcribed as short hairpin RNA (shRNA) sequences from RNA polymerase III (Pol III) promoters. These shRNA mimic precursor microRNA (pre-miRNA) and consequently enter the RNAi pathway at a relatively late stage. Overexpression of shRNA sequences from Pol III promoters, specifically the U6 promoter, has been associated with toxic side effects and has raised concerns about the use of expressed RNAi activators. Another concern of developing therapeutic RNAi expression cassettes is the emergence of HBV mutants that are resistant to silencing by a single expressed RNAi effecter. These points have highlighted the need for the development expressed RNAi activators that are effective at low concentrations and capable of combinatorial silencing. To address these issues the aim of this study was to assess the feasibility of anti HBV effecter sequences that mimic an early substrate (viz. primary miRNA or pri-miRNA) of the RNAi pathway. Pri-miRNA expression is typically under the transcriptional control of Pol II promoters. Consequently RNAi activators that Abstract - xi - mimic pri-miRNA, so-called pri-miR shuttles, may be expressed from Pol II promoters. Initially a panel of shRNA expression cassettes driven by a Pol III promoter was constructed and silencing of HBV replication assessed. Pri-miR shuttles were then designed by incorporating guide sequences of the most effective anti HBV U6 shRNA into naturally occurring pri-miR-122 and pri-miR-31. Potent inhibition of viral replication was observed with both Pol III and Pol II-driven pri-miR shuttle expression cassettes in vitro and in vivo. Subsequently liver-specific pri-miR-122 and multimeric pri-miR-31 shuttle expression cassettes were created. Pri-miR-122 shuttle sequences expressed from the alpha-1 antitrypsin promoter and HBV basic core promoter exhibited the best liver-specific silencing. Polycistronic pri-miR-31 shuttle sequences were shown to produce multiple RNAi activators capable of silencing multiple target sequences. Silencing by the pri-miR shuttle sequences was independent of toxic effects that arise from induction of the interferon response or saturation of the endogenous miRNA pathway. Pri-miR shuttles clearly represent an improved option for the use of expressed shRNA and brings therapeutic RNAi technology a step closer to clinical application.

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand / The hepatitis B virus remains a major public health problem even after decades of its discovery. Horizontal transmission during early childhood is the predominant mode of transmission in highly endemic regions such as sub-Saharan Africa. Infection exhibits a wide spectrum of clinical manifestations, from an asymptomatic stage to severe liver disease which may result in hepatocellular carcinoma (HCC). The HBV X protein (HBx) has been implicated in carcinogenesis, which often has a poor prognosis, consequently the use of highly specific monoclonal antibodies (mAbs) directed against HBx in an enzyme-linked immunosorbent assay (ELISA) could lead to early identification of HBV carriers at risk of developing liver cancer. A variety of mixed hybridoma cell cultures secreting anti-HBx antibodies were cloned and sub-cloned by “limiting dilution”. Clonal supernatants were assessed for anti-HBx antibody production by Indirect ELISA and Western/Immunoblotting. Monoclonal antibodies were then characterized according to their relative binding affinity (Indirect ELISA) and relative epitope specificity (Competitive ELISA). One of our monoclonal antibodies was found to bind to the same epitope on HBx as the commercial anti-HBx antibody and with the same high affinity. In the developed Sandwich ELISA, our monoclonal antibody proved effective as the „detecting‟ antibody when the commercial anti-HBx antibody was deployed as the „capture‟ antibody. This Sandwich ELISA will be further developed in our laboratory with the object of applying it to patient sera.

Degree awarded with distinction on 6 December l995. A dissertation submitted to the Faculty of Science, University the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. March. 1995 / Hepatitis B virus (HBV) infection resulting in integration of the viral DNA into host liver cell DNA is associated with the development of hepatocellular carcinoma (HCC). This is indicated by epidemiological trends, molecular studies and studies of animal models infected with viruses closely related to HBv. However, little is known about the mechanism by which the integrated HBV DNA includes HCC despite continuing analysis of the integrated HBV DNA and its surrounding cellular sequences. [Abbreviated Abstract. Open document to view full version]

Ph.D. thesis, Faculty of Health Sciences,University of the Witwatersrand, 2009 / Hepatitis B virus (HBV) infection is endemic in Africa. As many as 98% of black Africans are infected during their lives and about 10% (65 million) have chronic HBV infection, which is the cause of 70-80% of all hepatocellular carcinoma (HCC) cases. Despite this high prevalence of HBV and the high incidence of HCC in Africa, relatively few complete HBV genomes from African HCC cases have been deposited in international data bases. In order to gain a clearer understanding of the role of genetic variants and mutants in the development of HCC, the complete genomes of HBV isolated from southern African HCC patients were amplified and molecularly characterized. HBV DNA was extracted from the serum forty HBsAgpositive HCC patients. Twenty six complete genomes were successfully amplified, cloned and sequenced from nine HCC patients. Phylogenetic analyses of the complete genomes and the individual open reading frames of HBV isolates from the HCC patients, led to the classification of all the isolates within subgenotype A1. No isolates belonging to subgenotype A2 and genotype D were identified, even though these genotypes/subgenotypes have been shown to circulate in South Africa. Three patients contained the uncommon combination of serological subtype ayw1 in the subgenotype A1 strain. This combination has been found previously in South Africa and the Phillipines. Seventy-eight percent of the patients carried HBV strains with the double basic core promoter (BCP) mutation (1762T/1764A), previously shown to reduce HBeAg expression. Furthermore, complete genome sequence analysis has revealed a complex combination of mutations, which include at least three or five of these residues 1753C1762T1764A1766T1768A1809T1812T occurring as the dominant HBV strains isolated from 5/9 HCC patients. These mutations have previously been shown to regulate gene expression at various levels, to enhance viral replication and simultaneously decrease HBeAg expression. All five HBV genomes isolated from one patient contained novel complex BCP rearrangements, which introduced 2 HNF1 and 1 putative HNF3 transcription factor binding sites. These mutations can enhance viral replication and simultaneously abolish HBeAg expression at a transcriptional level. Furthermore, truncated core proteins would be expressed from 4/5 isolates and none would express wild-type HBx. Several mutations were identified in the pre-S/S genes of 2/5 isolates, which would result in the expression of novel 3’ truncated medium surface proteins (MHBst) and large surface proteins (LHBst). The majority of the mutations would contribute to hepatocyte pathogenesis and transformation by activating cell proliferating pathways. Two patients also contained rare HBV variants not previously identified in HBV strains from southern Africa. These included an HBV splice variant and a poly (dA) variant from patient 10 and patient 6, respectively. These variants occurred in combination with other isolates within the respective patients. The envelope genes were characterised in a total of 18 HCC patients, the pre-S gene of HBV contained deletions in 72% of the patients. Deletions across pre- S1/pre-S2, pre-S2 initiation codon mutations with internal deletions, and S gene nonsense mutations were prevalent. Mutated envelope proteins have been shown to accumulate within the hepatocyte endoplasmic reticulum (ER) and are a characteristic histopathological hallmark of HCC known as ground glass hepatocytes. HBV induced ER stress has been shown to dysregulate several cell cycle regulatory pathways, which contribute to HCC. In addition several novel LHBst and MHBst have been described. These potential transactivators require further investigation. The HBV mutations described in this study have been associated with increased risk for HCC. Despite the obvious heterogeneity HBV displays within and between patients, there are common characteristics shared between the HBV variants which emerge during the development of HCC. These include the BCP and pre-C (1753C1762T1764A1766T1768A1809T1812T) mutations and the pre-S/S mutations. These mutations are able to affect HBV replication and gene expression, and may work synergistically to promote liver dysfunction and HCC.

by Lau Tze Chin, Gene. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (Leaves 170-195). / Abstract --- p.1 / Acknowledgement --- p.3 / List of tables --- p.4 / List of figures --- p.6 / List of abbreviations --- p.7 / Chapter Chapter One - --- Introduction --- p.9 / Chapter 1.1. --- Historical Aspects --- p.9 / Chapter 1.2. --- Classification of hepatitis B virus --- p.12 / Chapter 1.2.1. --- Hepadnaviruses --- p.12 / Chapter 1.2.2. --- Comparative properties of hepadnaviruses --- p.13 / Chapter 1.2.2.1. --- Physical properties --- p.13 / Chapter 1.2.2.2. --- Genetic relatedness --- p.15 / Chapter 1.2.2.3. --- Pathogenesis --- p.16 / Chapter 1.3. --- Structural and morphological properties of HBV --- p.17 / Chapter 1.4. --- Molecular biology of HBV --- p.20 / Chapter 1.4.1. --- Molecular structure of HBV --- p.20 / Chapter 1.4.1.1. --- Biochemistry of the virion envelope --- p.20 / Chapter 1.4.1.2. --- The nucleocapsid --- p.21 / Chapter 1.4.1.3. --- Structural features of HBV genome --- p.23 / Chapter 1.4.2. --- Genetic organization of HBV --- p.24 / Chapter 1.4.3. --- Infection cycle of HBV --- p.29 / Chapter 1.4.3.1. --- Viral attachment and internalization --- p.29 / Chapter 1.4.3.2. --- Replication of HBV --- p.30 / Chapter 1.4.3.3. --- Gene expression and regulation --- p.31 / Chapter 1.4.3.4. --- Host-virus DNA interaction --- p.33 / Chapter 1.5. --- Epidemiology and transmission of HBV --- p.34 / Chapter 1.5.1. --- World wide prevalence --- p.35 / Chapter 1.5.1.1. --- HBsAg prevalence --- p.35 / Chapter 1.5.1.2. --- Cumulative rate of HBV infection --- p.35 / Chapter 1.5.1.3. --- Age specific pattern of HBV infection --- p.36 / Chapter 1.5.2. --- Epidemiological pattern of HBV in Hong Kong --- p.37 / Chapter 1.5.3. --- Mode of transmission --- p.38 / Chapter 1.6. --- Clinical outcomes of HBV infection --- p.38 / Chapter 1.6.1. --- Acute infection --- p.41 / Chapter 1.6.2. --- Chronic infection --- p.42 / Chapter 1.6.3. --- Primary hepatocellular carcinoma --- p.43 / Chapter 1.7. --- Laboratory diagnosis of hepatitis B --- p.44 / Chapter 1.7.1. --- The HBV markers --- p.47 / Chapter 1.7.1.1. --- HBsAg and anti-HBs --- p.47 / Chapter 1.7.1.2. --- HBcAg and Anti-HBc --- p.47 / Chapter 1.7.1.3. --- HBeAg and anti-HBe --- p.49 / Chapter 1.7.1.4. --- HBV-associated DM polymerase --- p.49 / Chapter 1.7.1.5. --- HBV-DNA --- p.49 / Chapter 1.7.2. --- Methodology in the detection of hepatitis B markers --- p.50 / Chapter 1.7.2.1. --- Direct detection of HBV and HBV antigens --- p.50 / Chapter 1.7.2.2. --- Serological detection of HBV markers --- p.51 / Chapter 1.7.2.3. --- HBV-associated DNA polymerase assay --- p.51 / Chapter 1.7.2.4. --- Molecular technique for the detection and quantitation of HBV-DNA --- p.52 / Chapter 1.8. --- Antiviral therapy in hepatitis B --- p.52 / Chapter 1.8.1. --- Therapeutic agents for treatment of HBV infection --- p.53 / Chapter 1.8.1.1. --- Steroids --- p.53 / Chapter 1.8.2.2. --- Nucleoside analogs --- p.54 / Chapter 1.8.1.3. --- Interferon --- p.55 / Chapter 1.8.2. --- Clinical trials of interferons --- p.55 / Chapter 1.9. --- Extrahepatic tissue tropism of HBV --- p.62 / Chapter 1.10. --- Objective and design of study --- p.65 / Chapter 1.10.1. --- Objectives of study --- p.65 / Chapter 1.10.2. --- Study design --- p.66 / Chapter 1.10.2.1. --- Cross-sectional study --- p.67 / Chapter 1.10.2.2. --- Longitudinal study --- p.67 / Chapter 2.1. --- Materials --- p.71 / Chapter 2.1.1. --- Patients recruitment and clinical materials --- p.71 / Chapter 2.1.1.1. --- Cross-sectional study --- p.71 / Chapter 2.1.1.2. --- Longitudinal study --- p.71 / Chapter 2.1.2. --- Bacteria] stock --- p.71 / Chapter 2.1.3. --- "Chemicals, equipments and consumables" --- p.72 / Chapter 2.1.4. --- Buffers and solutions --- p.72 / Chapter 2.1.4.1. --- Phosphate buffer saline (PBS) --- p.72 / Chapter 2.1.4.2. --- Leucocyte lysis buffer (X 5)(LLB) --- p.72 / Chapter 2.1.4.3. --- Buffer equilibrated phenol (BEP) --- p.76 / Chapter 2.1.4.4. --- Phenol-Chloroform mixture --- p.76 / Chapter 2.1.4.5. --- 3.0M sodium acetate (pH 5.2) --- p.76 / Chapter 2.1.4.6. --- Tris-EDTA buffer (pH 8.0) (TE) --- p.76 / Chapter 2.1.4.7. --- Stock salmom sperm DNA solution --- p.77 / Chapter 2.1.4.8. --- Tracking dye --- p.77 / Chapter 2.1.4.9. --- Tris-borate electrophoresis buffer (TBE) --- p.77 / Chapter 2.1.4.10. --- Luria-Bertani Broth (LB) --- p.77 / Chapter 2.1.4.11. --- Solution ] --- p.78 / Chapter 2.1.4.12. --- Solution ]] --- p.78 / Chapter 2.1.4.13. --- Potassium acetate buffer (pH 5.4) --- p.78 / Chapter 2.1.4.14. --- Column elution buffer (CEB) --- p.78 / Chapter 2.1.4.15. --- NPMEB solution --- p.79 / Chapter 2.1.4.16. --- Neutralizing solution --- p.79 / Chapter 2.1.4.17. --- Standard saline citrate (SSC) --- p.79 / Chapter 2.1.4.18. --- Denhardt solution --- p.79 / Chapter 2.1.4.19. --- Prehybridization solution (PS) --- p.80 / Chapter 2.1.4.20. --- NETFAP Solution --- p.80 / Chapter 2.1.4.21. --- Heparin solution --- p.81 / Chapter 2.1.4.22. --- Hybridization mix for oligo-nucleotide probe --- p.81 / Chapter 2.1.4.23. --- NEPS solution (pH 7.0) --- p.81 / Chapter 2.1.4.24. --- Restriction endonuclease and buffer --- p.82 / Chapter 2.2. --- Methods --- p.82 / Chapter 2.2.1. --- Sample preparations --- p.82 / Chapter 2.2.1.1. --- Isolation of plasma and peripheral blood leucocytes (PBL) --- p.82 / Chapter 2.2.1.2. --- Extraction of DNA from Peripheral blood leucocytes --- p.83 / Chapter 2.2.1.3. --- Quantitation of Peripheral blood leucocyte DNA --- p.83 / Chapter 2.2.2. --- Preparation of radio-labelled HBV-DNA probe --- p.84 / Chapter 2.2.2.1. --- Plating and selection of bacterial stock --- p.84 / Chapter 2.2.2.2. --- Growth of E. coli HB101 and amplification of pAM6 --- p.84 / Chapter 2.2.2.3. --- Harvesting of E. coli and extraction of plasmid pAM6 --- p.84 / Chapter 2.2.2.4. --- Purification of plasmid pAM6 --- p.86 / Chapter 2.2.2.5. --- Large scale isolation and purification of HBV genome from plasmid pAM6 --- p.86 / Chapter 2.2.2.6. --- Radio-labelling of HBV-DNA --- p.88 / Chapter 2.2.2.6.1. --- Nick-translation of total HBV-DNA genome --- p.88 / Chapter 2.2.2.6.2. --- Multi-primer labelling of total HBV- DNA genome --- p.88 / Chapter 2.2.2.6.3. --- End-labeling of 21-base HBV oligo- nucleotide --- p.88 / Chapter 2.2.2.6.4. --- Determination of labelling efficiency --- p.89 / Chapter 2.2.2.7. --- Purification of labelled HBV-DNA probe --- p.90 / Chapter 2.2.2.7.1. --- Total genomic HBV-DNA probe (pAM6 probe) --- p.90 / Chapter 2.2.2.7.2. --- Oligo-nucleotide HBV-DNA probe (oligo probe) --- p.90 / Chapter 2.2.3. --- Hybridization study of clinical samples --- p.91 / Chapter 2.2.3.1. --- Solution hybridization of sera samples --- p.91 / Chapter 2.2.3.2. --- Spot hybridization of sera samples --- p.91 / Chapter 2.2.3.2.1. --- "Pre-hybridization treatment of sera samples (adapted from Lin et al.,1987)" --- p.91 / Chapter 2.2.3.2.2. --- Pre-hybridization and hybridization of the membrane --- p.92 / Chapter 2.2.3.2.3. --- Washing of membrane --- p.92 / Chapter 2.2.3.2.4. --- Final treatment and autoradiography: --- p.92 / Chapter 2.2.3.3. --- Quantitation of HBV-DNA in the sera samples: --- p.93 / Chapter 2.2.4. --- Assay for serological Hepatitis B marker --- p.93 / Chapter Chapter Three - --- Results --- p.93 / Chapter 3.1. --- Preparation of HBV-DNA probes --- p.95 / Chapter 3.2. --- Radiolabelling of HBV-DNA --- p.95 / Chapter 3.3. --- Hybridization methodology --- p.98 / Chapter 3.4. --- Comparison of the performance of HBV-DNA probes --- p.100 / Chapter 3.4.1. --- Quantitation of serum HBV-DNA --- p.100 / Chapter 3.4.2. --- Comparative hybridization performance of different HBV-DNA probes --- p.105 / Chapter 3.5. --- Clinical application of HBV-DNA probe:Detection of HBV-DNAin serum and peripheral blood leucocytes (PBL) --- p.109 / Chapter 3.5.1. --- Cross-sectional study --- p.112 / Chapter 3.5.1.1. --- Frequency of HBV-DNA detection in relation to different clinical manifestations --- p.112 / Chapter 3.5.1.2. --- Frequency of HBV-DNA detection in relation to the serological status --- p.114 / Chapter 3.5.1.3. --- Distribution of serum and PBL HBV-DNA level in chronic hepatitis B patients in relation to the different HBV-related manifestations --- p.119 / Chapter 3.5.2. --- Longitudinal study of patients with chronic hepatitis B under interferon therapy with prednisolone pretreatment --- p.123 / Chapter 3.5.2.1. --- Features of patients under study --- p.123 / Chapter 3.5.2.2. --- Correlation between the occurrence of HBV- DNA and HBeAg in serum --- p.123 / Chapter 3.5.2.3. --- Outcome of clinical trial: --- p.126 / Chapter 3.5.2.3.1. --- Number of patients responding to therapy: --- p.126 / Chapter 3.5.2.3.2. --- Variation in serum HBV markers during the course of study --- p.128 / Chapter 3.5.2.3.3. --- Change of HBV-DNA statusin peripheral blood leucocytes --- p.134 / Chapter Chapter Four - --- Dicussion --- p.140 / Chapter 4.1. --- Preparation of HBV-DNA hybridization probes --- p.140 / Chapter 4.1.1. --- Source of HBV-DNA --- p.140 / Chapter 4.1.2. --- Raidolabelling of HBV-DNA --- p.141 / Chapter 4.2. --- Hybridization methodology --- p.141 / Chapter 4.2.1. --- Optimization of hybridization conditions --- p.141 / Chapter 4.2.2. --- Comparison of the performance among different HBV- DNA probes --- p.144 / Chapter 4.3. --- Detection of HBV-DNA in clinical serum samples --- p.148 / Chapter 4.3.1. --- Crossectional study of patients with various categories of HBV related diseases --- p.148 / Chapter 4.3.1.1. --- HBV-DNA detection in serum --- p.148 / Chapter 4.3.1.2. --- Detection of HBV-DNA in peripheral blood mononuclear cells --- p.153 / Chapter 4.3.2. --- Longitudinal studies of patients undergoing antiviral therapy --- p.159 / Chapter 4.3.2.1. --- Serum HBV-DNA and HBeAg --- p.159 / Chapter 4.3.2.2. --- HBV-DNA in peripheral blood leucocytes --- p.163 / Conclusion --- p.166 / Future perspectives --- p.168 / References --- p.170

ABSTRACT In sub-Saharan Africa a causal relationship has been established between hepatitis B virus (HBV) infection and membranous nephropathy (MN), especially in Black children. The most common method of treatment is interferon therapy, which is however, only effective in 30-40% of patients. The reason for this is unclear. The objective of this pilot study was to determine whether mutations in the complete surface gene of HBV isolated from Black children with HBV-associated MN before, during and after treatment with interferon, had any effect on treatment response and vice versa. HBV DNA was extracted from the serum of a responder, reverter and non-responder patient before, during (4 and 16 weeks) and after (40 weeks) IFN treatment. The preS1/preS2/S region was amplified and cloned, and the clones sequenced. Sequence analyses revealed the preS2 region to be the most variable in the reverter and non-responder and HBsAg was the most variable in the non-responder. Phylogenetic analysis showed that the viral population dynamics between the responder strains and the reverter/non-responder strains differed as a result of various mutations found within the surface gene. Thus the presence of mutations in preS2 and HBsAg of the non-responding patients may carry predictive markers for nonresponse but further investigation would be needed to conclusively prove this.

Thesis (D Phil. (Medical Virology))--2008. / Background and Objectives: South Africa is one of the countries highly affected by human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections. Some drugs (e.g. lamivudine) used as part of combination antiretroviral regimens for HIV treatment have dual activity against HBV and HIV. Despite high infection rate with both viruses, routine screening for HBV before initiation of treatment for HIV is not yet a standard practice. This study undertook to investigate: (1) the burden of HBV co-infection in HIV-positive patients enrolling for highly active antiretroviral therapy (HAART) at Dr George Mukhari hospital, (2) the impact of anti-HBV containing HAART regimens on HBV during the management of HBV/HIV co-infected patients, (3) the co-evolution of HBV and HIV drug-resistant strains, and (4) the correlation of HBV genotypes with response to anti-HBV containing HAART regimens. Study Population and Methods: To investigate the burden of HBV/HIV co-infections, a cohort of 192 HIV patients who were candidates for ARV treatment at Dr George Mukhari hospital were studied by screening for HBV serological markers (HBsAg, anti-HBs and anti- HBc) (Elecsys 2010, Roche Diagnostics) and HBV DNA with an in-house nested PCR assay targeting HBV polymerase gene. Quantitation of HBV DNA positive samples was performed with Roche Cobas Taqman HBV test 48 assay. To investigate the impact of lamivudine-containing HAART regimens on HBV during the management of HBV/HIV co-infected patients, as well as the coevolution of HBV and HIV drug-resistant strains, a total of 78 patients were studied. HBV virological response against lamivudine containing-HAART regimens [1a (lamivudine, stavudine and efaverenz); 1b (lamivudine, stavudine and neviripine)] was measured (Cobas Taqman HBV test 48, Roche diagnostics). HBV direct sequencing targeting HBV polymerase gene was performed on all baseline samples (n=78) and additional samples collected at various time points (n = 45). Direct sequencing was also performed on 30 HIV baseline samples targeting the HIV reverse transcriptase and protease genes (Spectru-Medix SCE 2410 Genetic Analysis System and ABI PRISM® 3100 Genetic Analyzer version 3.7). To explore the genetic diversity of HBV and HIV strains circulating in Pretoria and surrounding areas, as well as the correlation of HBV genotypes with response to lamivudine-containing-HAART regimens in co-infected patients, all baseline and follow-up HBV and HIV sequences were analysed, compared and correlated with treatment. Sequence alignments and phylogenetic studies for both HBV and vi HIV were conducted with MAFFT, Mega 4 and neighbour joining phylogenetic trees generated with the PHYLIP programme. Results: Three significant findings were observed in this study. Firstly, the majority of South African HIV patients enrolling for HAART were exposed to HBV infection and either had acute or chronic HBV infections. A total of 63.0% of patients were found to have one or more HBV markers, with 40.6% having detectable HBV DNA as an indication of replication. The study also detected 22.9% with positive HBsAg, and 23% of 77% HBsAg-negative patients having occult hepatitis B infection. Secondly, HBV/HIV co-infected patients do benefit during the management of HIV infections with lamivudine-containing HAART regimens. A total of 68.4% of patients responded to HAART, with undetectable HBV DNA during 18 to 24 months of follow-up. A total of 91.3% of HIV patients also responded to HAART with an undetectable HIV viral load during 6 to 12 months of follow-up. However, a total of 18% of patients had persistent HBV DNA, yielding various HBV virological responses against lamivudine containing-HAART regimens. This proportion of patients poses a question regarding the management of HBV and HIV coinfections, as guidelines on the use of HAART with anti-HBV activity from developed countries, may not necessarily be followed in developing countries. The results further showed that baseline drug-resistance was more frequent with HIV than HBV in this cohort of patients. The following HIV primary drug resistant mutants were observed: nine major NRT's primary mutants, M41L (1/30), E44A (1/30), V75M (1/30), F77L (1/30), V118I (1/30), M184V (1/30), L210S (1/30), T215Y (1/30) and V90I (1/30), and five major NNRT’s primary mutants were also detected, K103N (3/30), Y318CFSY (1/30), E138Q (1/30), P225H (1/30) and K238T. However, all followup samples had undetectable HIV viral load. In contrast to HIV, only one patient was detected with HBV mutant, M204I, at baseline. The mutant reversed to wild type during 6 months and other follow-up (12, 18 and 24 months). Finally, this study indicated that the HBV genotype A is still the most prevalent genotype circulating in South Africa. Of the 78 HBV sequences, 77 were genotype A and 1 sequence was genotype G. This is the first report from Africa of the detection of HBV genotype G. HIV subtype C remains the predominant prevailing subtype in South Africa. HBV genotype or HIV subtype C was not observed to influence any treatment outcome following treatment with vii lamivudine-containing HAART regimens. The study also indicated that patients on lamivudinecontaining HAART regimens do benefit not only by suppressing HIV and HBV viral load, but also improving immunity (i.e. CD4 cells count increases). Conclusion: Overall, the present study highlights the need for screening HBV before initiation of any HAART containing anti-HBV regimens in HBV/HIV co-infected patients. It necessitates the use of molecular assays for effective laboratory in diagnosis of occult HBV infections in HIVpositive patients, especially in developing countries where these assays are not widely available. While lamivudine-containing HAART regimens do benefit both HBV and HIV patients in co-infected individuals, however, whether HBV virological response is temporary or sustained is unknown at this stage. What is certain is that these patients require an effective monitoring programme as (1) a small percentage experience variable HBV virological responses (partial, reactivation, or no response), and (2) hepatitic flares are likely to develop if HAART is terminated (e.g. by patient), or the current HAART regimen is switched to another regimen without anti-HBV activity. HBV genotype A remains the dominant genotype in South Africa, but novel genotypes can be detected. HIV subtype C was found to be the prevalent subtype. HBV genotype or HIV subtype C were not seen to influence any treatment outcome following treatment with lamivudine-containing HAART regimens. Recommendations: HIV patients should be screened for HBV before initiation of anti-HBV containing HAART regimens. The screening of HBV in HIV patients is also important since some drugs included as part of HAART (e.g. nevirapine) may cause hepatotoxicity and exacerbate HBV infections leading to increased morbidity and mortality due to liver complications. Immunization and immune boosters of HIV patients with low (< 10IU/L) or no immunity against HBV should be done as this could be beneficial, although these patients may not respond optimally, or their immunity may wane faster due to immunocompromised status. Monitoring of both HBV and HIV resistant strains should be conducted for timely detection for the occurrence of multiple resistant mutations, which could limit future therapeutic option for both viruses.

The hepatitis delta virus (HDV) is the smallest human pathogenic RNA virus and relies heavily on host proteins for its replication. The objective of my research was to observe the effect of HDV replication on host gene expression, using a HEK-293-based cell system engineered to mimic HDV replication. A high-throughput sequencing was performed and allowed to establish a total of 3,561 genes differentially expressed by HDV RNA. Among those genes, 3,278 were upregulated by HDV RNA and 283 downregulated. A Gene Ontology (GO) enrichment analysis was performed on those dysregulated genes and revealed that upregulated genes were predominantly part of these four pathways: RNA processing, G-protein coupled receptor signaling pathway, protein transport, and organelle organization. On the other hand, downregulated genes were part of the nucleosome assembly pathway. The expression of several genes was confirmed by RT-qPCR. Moreover, protein complexes whose expression at the gene level was affected were identified. A total of 30 complexes were found to be significantly affected by HDV replication. Among them, we found many chromatin and histone related complexes. Lastly, a flow cytometry analysis revealed an increase in cell cycle arrest in G0/G1 and a reduction in the percentage of cell in S phase. Moreover, there was a difference in cell size for arrested cells in G0/G1 in HDV replicating cells. Overall, my results support the hypothesis that HDV replication induces cell cycle dysregulation.

The viral dynamics of the hepatitis C virus (HCV) in newly acquired infection are not well understood. HCV exists within an individual as a spectrum of minor variants termed quasispecies. The evolution of minor variants may contribute to viral escape of the host?s immune response, thereby facilitating development of chronic infection. The hypervariable 1 region (HVR1) is the most heterogeneous part of the HCV genome and contains a putative B-cell epitope. Thus, diversity in HVR1 could be a strategy used to evade neutralising antibodies. Acutely infected individuals (n=24) were examined with the aim of defining HVR1 quasispecies diversity in acute infection. The characterisation of the E1/HVR1 sequence and host specific evolution of HCV minor variants in treatment nonresponders was also investigated. HCV E1/HVR1 fragments were amplified from 48 sera using a combined reverse transcription-polymerase chain reaction (RT-PCR). Products were TA cloned into pCRIITOPO and approximately 10-20 clones were sequenced from each sample. HVR1 quasispecies diversity was examined longitudinally via sequence analysis. Quasispecies diversity was characterised primarily by mean nucleotide diversity. The mean HVR1 diversity of the acute cohort (n=48; 2.12% ?? 2.22) was lower than the diversity obtained for a cohort of chronically infected individuals (n=99; 4.5% ?? 5.1). There was no significant difference in mean HVR1 diversity between the HIV/HCV co-infected and HCV mono-infected groups (p=0.99) or between the clearer and non-clearer groups (p=0.85). Examination of amino acid usage and the hydropathic profile of each position in HVR1 revealed that sequence variation was confined to specific sites. The investigation of host specific evolution of HVR1 quasispecies demonstrated that minor variants (comprising 10- 20% of a population) became the dominant species over time in two treatment non-responders. These variants bore mutations that were not reflected in the consensus sequence of their respective populations at the initial timepoint analysed. Common infection was identified by 98% HVR1 sequence homology within two pairs of individuals. The evolution of common strains appeared to be different between individuals, suggesting host pressures may influence quasispecies evolution. This thesis provided an insight into the viral dynamics and host specific evolution of acute phase quasispecies.

Includes five copies of author's previously published articles in back pocket. / Bibliography: leaves 120-142. / xvi, 142, [59] leaves, [25] leaves of plates : ill. (some col.) ; 35 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines HCV-specific (Hepatis C virus-specific) protein and RNA expression in liver tissue from anti-HCV positive patients. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?