Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

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published_or_final_version / Public Health / Master / Master of Public Health

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M.Sc.(Med.), Faculty of Health Sciences, University of the Witwatersrand, 2011 / Hepatitis 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.

Members 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.

Hepatitis 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.

by Chan Lik-yuen, Henry. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 192-217). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Worldwide there are 350 million people with chronic hepatitis B infection and globally it causes up to half of the liver cancer deaths and one third of deaths from cirrhosis. Only a fraction of sufferers will develop these complications. Various studies have implicated socio-demographic, biochemical and viral factors in disease progression but research has been limited to local populations in endemic countries. Our aim was to study the prevalence and factors associated with advanced disease of hepatitis B infection in immigrants living in East London. I completed a retrospective analysis of notes and electronic health records of 1209 immigrant patients attending hospitals in East London, 217 of whom were from Bangladesh and Pakistan. Screening of volunteers attending local mosques using oral mucosal transudate swabs and national statistics data allowed us to calculate prevalence rates in these populations. Those 13 patients from Bangladesh and Pakistan admitted over 30 months with decompensated disease were men aged > 40. Age, sex, ALT, smoking, alcohol and diabetes were significant predictors for cirrhosis and decompensated disease but not viral markers. Similar analyses were performed for other ethnicities with similar outcomes. The scale of under diagnosis of hepatitis B for all ethnicities was estimated and the reasons explored. This work has scrutinised the epidemiology of chronic hepatitis B in East London and the difficulties encountered exploring it. We provide differing results to published studies and suggestions for how this domain can be examined further.