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.

Thesis (MPH)--University of Limpopo, 2010. / Introduction: Hepatitis B is a serious liver disease caused by the hepatitis B virus (HBV), with an estimated 360 million chronic infections worldwide, about a million of which die each year from chronic liver diseases. In South Africa (SA) over 50% of the population has been infected by HBV, and at least 3 million people are chronic HBV carriers. Chronic HBV carriers have the potential of transmitting HBV parenterally in the hospital setting, thus health care workers (HCWs) are at risk of contracting HBV, with the most likely exposure being via a needle stick injury (NSI). There is an effective vaccine against HBV which is recommended by the SA Department of Health, yet previous studies have shown that most HCWs are not vaccinated. Aim and objectives: The study aimed to investigate the knowledge, attitudes and practices regarding hepatitis B vaccination amongst HCWs in the Ekurhuleni Metro. Objectives were to determine: (1) the level of knowledge of HCWs about vaccination against HBV; (2) the attitudes of HCWs towards vaccination against HBV; (3) the practices of HCWs regarding HBV prevention and (4) the barriers to / predictors for effective HBV vaccination among HCWs at Ekurhuleni Metro Materials and Methods: This was a cross-sectional descriptive study which made use of a self-administered questionnaire that was sent to Ekurhuleni nurses and doctors who were working in 3 public hospitals, 7 district clinics, and 110 general practices. Results: Two hundred and fifteen questionnaires were distributed and 161 were returned giving an overall response rate of 74.9%. HCWs do not report their NSI; over a third [37.6% (41/81)] always reported the NSI; while 72% (116/161) of HCWs had been vaccinated, only 61.2% (71/116) of those vaccinated had received all 3 doses of the vaccine. For knowledge of HBV vaccination, 66.5% (107/161) scored poor; 31.7% (51/161) scored moderate; and 1.8% (3/161) scored high. For attitudes towards HBV vaccination, 0.6% (1/160) scored negative; 24.4% (39/160) scored neutral; and 74.5% (120/160) scored positive. A positive attitude score was a significant predictor for being vaccinated (OR=1.13, p=0.007) Conclusion: Guidelines should be put in place to increase vaccination uptake and reduce the risk of exposure to HBV infection by HCWs

microRNAs (miRNAs) are newly discovered small RNAs that repress gene expression and are evolving as clinical predictors for diagnosis and prognosis of human disease. Persistent hepatitis C virus (HCV) infection causes chronic inflammation and can lead to fibrosis, cirrhosis and liver cancer. HCV-associated liver injury is characterized by increased hepatocyte proliferation. Studies have demonstrated interactions between HCV and miRNAs. We investigated the role of miRNAs during HCV infection. We used global expression analysis to measure the expression levels of 380 miRNAs comparing HCV infected human livers with uninfected livers. We correlated the altered miRNA expression levels with clinical patient data, such as stage of fibrosis, liver transaminases, and viral load. We identified several miRNAs that correlated with these parameters, including miR-21. miR-21 correlated with stage of fibrosis, liver transaminases, and viral load. We used the mouse carbon tetrachloride model to induce fibrosis and identified correlations with miR-21 expression and stage of fibrosis. Furthermore, we identified, SMAD7, an inhibitor of fibrosis, as a novel target of miR-21 providing further evidence of miR-21's possible involvement in fibrosis development. In our patient samples, miR-21 expression also correlated with viral load. Establishing a further connection between miR-21 and HCV, we measured miR-21 expression levels in HCV replicons and infectious HCVcc tissue culture models. Altered expression of miR-21 varied depending on replicon genotype and tissue culture model preventing us from establishing a consistent relationship between miR-21 expression level and HCV infection. Interestingly, miR-21 inhibition in replicon cells resulted in decreased viral replication suggesting a possible miR-21 anti-viral effect. Hepatocyte replication is associated with chronic HCV-induced liver injury. To investigate whether miR-21 correlation with HCV and fibrosis was a consequence of hepatocyte proliferation caused by viral infection and fibrosis, we measured miR-21 expression during liver regeneration. miR-21 was up-regulated during the early proliferative phase of liver regeneration. We identified Pellino-1 (PELI1), an adapter molecule involved in Nuclear factor-ĸB (NF-κB) signaling, as a novel target of miR-21. During liver regeneration, PELI1 mRNA levels were reduced, consistent with miR-21 targeting. We also found miR-21 over-expression decreased the activity of a NF-κB reporter. These studies investigated the role of miR-21 during HCV viral infection, fibrosis, and liver regeneration. We found miR-21 correlates with fibrosis in both human patient samples infected with HCV and in a mouse carbon tetrachloride mouse model. miR-21 may be pro-fibrogenic by targeting SMAD7, an inhibitor of fibrosis. miR-21's correlation with HCV viral load may be an anti-viral response by the host, which was demonstrated by increased HCV replication upon inhibition of miR-21. Furthermore, miR-21 is associated with hepatocyte replication during liver regeneration and inhibits NF-κB signaling, possibly by targeting PELI1. These studies identified several new roles for miR-21 during HCV viral infection, fibrosis, and liver regeneration.

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.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma and liver cirrhosis worldwide. HBV vaccination can prevent new infections, but effective antiviral drugs are not available for a large number of HBV infected patients. To develop novel antiviral drugs, a better understanding of the regulation of HBV gene expression is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNAs from the nucleus of a cell to the site of incorporation into new HBV particles. The HBV post-transcriptional regulatory element (HBV PRE) is a cis acting RNA element found in all HBV transcripts. It has been reported to play an important role in the nuclear export of HBV mRNAs. Moreover, it has the ability to enhance expression of intronless as well as unspliced transcripts. Despite concerted investigations, the functional core element of HBV PRE remains unknown and the exact mechanism of how HBV PRE mediates nuclear export is unclear. This project first produced a complete HBV genome with comprehensive annotation of both coding regions and regulatory signals, which was then used for comparative genomic analysis. The functional elements of the HBV PRE were first subjected to analysis in silico. The HBV PRE is highly conserved among HBVs. Based on this sequence conservation and prediction of conserved RNA secondary structure, potentially functional HBV PRE elements including the previously reported elements (HBV SLα and HBV SLβ) were identified. Experimental deletion analysis of the HBV PRE sequence showed that the effect of each of these elements on the intronless reporter gene�s expression was similar to that of the entire full length HBV PRE. Thus, the results suggested that overall HBV PRE function was not due to additive effects from the individual elements. Surprisingly, a specific sub-section of HBV PRE decreased the level of reporter gene expression. This sub-section has not been identified previously, thus it is a novel HBV PRE inhibitory element. Further analyses using specific reporter assays revealed that the HBV PRE enhanced expression of an unspliced reporter gene whereas the RNA nuclear export elements of retroviruses, CTE (in MPMV) and RRE (in HIV-1) were not able to. Therefore, these results indicate that HBV PRE is involved in inhibition of splicing and it utilizes a different mechanism from CTE and RRE. Interestingly, HBV PRE was observed to be unable to enhance the expression of an intronless luciferase gene. Therefore, HBV PRE is not able to enhance cytoplasmic expression of all intronless transcripts. This project also addressed the idea that the RNA-binding protein, polypyrimidine tract binding protein (PTB) is a positive trans-acting factor for HBV PRE function. Transient expression of exogenous PTB in cultured cells showed no specific effect on constructs containing HBV PRE. Moreover, reduction of endogenous PTB by RNAi did not affect HBV PRE function. Therefore, the results presented in this project do not support the hypothesis that PTB plays a role in HBV PRE function. Given that HBV PRE is highly conserved and present in all HBV transcripts, it makes a good target site for novel molecular therapeutic treatments such as siRNA. To identify potential siRNA target sites within HBV PRE, an RNAi study using a plasmid expressing shRNA against HBV PRE was done. The results from the RNAi study revealed that the expression of a reporter gene could be significantly reduced by siRNA targeted to the HBV PRE. Overall, this project produced a highly annotated HBV genome that can be used as the reference sequence for comparative genomic analysis. Moreover, this work identified novel regulatory elements within HBV PRE that are likely to play an important role in HBV gene expression. Furthermore, the study also identified an excellent siRNA target site within HBV PRE that may inhibit HBV gene expression.

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?

This study tested the role of the calcium dependent cytosolic protein calpain in neutrophilic hepatitis. We hypothesized that inhibition of calpain would protect against LPS-induced neutrophilic liver damage. To test our hypothesis, a reliable LPS-mediated hepatitis model to investigate the mechanisms of hepatic neutrophil infiltration following LPS administration was developed by repeat intravenous injection of LPS at a dose of 10 mg/kg to rats. Blood was collected for hematologic and biochemical analysis and multiple organs including liver were collected for evaluation of histopathologic changes. Flow cytometry was employed to investigate L-selectin (CD 62L) and MAC-1 (CD11b/18) expression on neutrophils both in vivo and in vitro. Significant hematologic changes included neutrophilia, elevation in neutrophil to lymphocyte ratio with toxic changes and left shift. Biochemical changes were observed in several liver (AST, GGT) and kidney (BUN) parameters generally at the earliest time points. Histopathology revealed a time-dependent neutrophil and mononuclear infiltration around the periportal areas in the single dose study and mid-zonal inflammation with multifocal coagulative necrosis in the repeated dose study. CD 11b was up-regulated both in vitro and in vivo. After development of a suitable model, the first goal was to investigate the role of the intracellular enzyme calpain in the development of neutrophilic hepatitis and midzonal necrosis. A second goal was to compare the observed protective effects of calpain inhibition with a relatively selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine and an inhibitor of coagulation, heparin. When compared to rats administered LPS alone, administration of calpain 1 inhibitor prior to LPS significantly reduced hepatic iNOS expression, hepatic neutrophil infiltration and attenuated midzonal hepatic necrosis. Administration of heparin and aminoguanidine prior to LPS also decreased liver iNOS expression, hepatic neutrophil infiltration and liver pathology comparable to calpain inhibition. Blood neutrophil activation, as measured by the neutrophil adhesion molecule CD11b integrin, was upregulated in all the LPS treated groups regardless of inhibitor administration. We conclude that amelioration of liver pathology via calpain inhibition is likely dependent on the down-regulation of iNOS expression in the rat model of LPS mediated hepatitis.

Purpose: Egypt has the highest prevalence of chronic Hepatitis C virus (HCV) infections and also a high prevalence of female genital mutilation (FGM). The high prevalence chronic hepatitis C has been attributed to HCV transmission by contaminated injections for the control of schistosomiasis. HCV infection has not been well studied in the context of female genital mutilation (FGM). We sought to identify associations between FGM and HCV using the Egypt Demographic and Health Survey (EDHS), 2008. Methods: FGM was chosen as the main independent variable of interest. Other independent variables such as age, education, marital status, residence, beliefs associated with FGM, history of blood transfusion, surgery, sharing needles, and history of schistosomiasis were included in the analysis. Throughout the analysis, HCV infection was used as the main dependent variable. Results: Univariate analysis of FGM and HCV showed a statistically significant association with a Prevalence Odds Ratio of 4.82 (2.91 -7.96), after adjusting for age and schistosomiasis injection, the association between FGM and HCV remained statistically significant with an odds of 2.98 (1.76 – 5.05)Among the category for FGM performer and association with HCV infection, the OR was 4.28 (2.31 – 7.91) when the FGM was performed by a ghagaria, 3.68 (2.76 - 4.90) when the FGM was performed by daya, and 3.30 (1.81 -5.88) when the FGM was performed by a barber. Among other independent variables, a lack of education, rural residence, and having religious precepts for FGM had statistically increased odds of association with HCV infection. Conclusion: There is a statistically significant association between FGM and HCV infection. There are increased odds of HCV when the FGM is performed by providers other than doctors. Participants from a rural residence and who those who did not have any education were at increased odds of HCV. Subjects who believed in religious precepts for FGM and also who answered that FGM can continue had increased odds of association with HCV infections.

Hepatitis C is the most common blood-borne viral illness in the North America. Chronic hepatitis C infection may lead to cirrhosis of the liver, liver failure and liver cancer. In North America, injection drug use is the most important risk factor for infection and substance abusing populations are disproportionately affected by the disease. Antiviral therapy exists and approximately 50% of infected individuals can be cured. The aim of this thesis was to provide information to help clinicians and policy-makers minimize the impact of hepatitis C in substance abusers. The thesis is comprised of three studies. The first assessed the rate of progression to cirrhosis for those acquiring infection through injection drug use, using a meta-analysis of 44 studies from the published literature. We estimated that fibrosis progression occurs at a rate of 8.1 per 1000 person-years (95% Credible Region (CR), 3.9 to 14.7) corresponding to a 20-year cirrhosis prevalence of 14.8% (95% CR, 7.5 to 25.5). The second study measured the association between successful antiviral therapy and quality of life. We demonstrated that sustained responders to therapy had higher scores on the hepatitis-specific Medical Outcomes Survey Short-Form-36 (SF-36), Health Utilities Index Mark 2/3 (HUI2/3), and time-tradeoff (TTO) than treatment failures, an average of 3.7 years following antiviral therapy. The third study assessed rates of adherence to antiviral therapy and rates of sustained response in current or former iii substance abusers on methadone maintenance. We demonstrated that while use of illicit substances prior to therapy negatively affected adherence, rates of sustained response were comparable to non-substance abusing populations. Our work indicates the future burden of disease in current and former substance abusers, demonstrates that antiviral therapy can be successful in this population, and indicates that the benefits of successful therapy may extend beyond decreased disease burden to improved quality of life.