Deng Xue-Long. / "January 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 196-230). / 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. / Abstracts in English and Chinese.

Hepatitis C virus is a pathogen causing severe hepatic diseases. Though HCV genotype 6a circulated prevalently in Hong Kong, its sequence information is greatly in deficient. The molecular characteristics and epidemiology of HCV 6a were thus extensively investigated in this thesis. The distribution of HCV genotypes in Hong Kong was analyzed from 1055 samples by the Ohno's method. HCV 6a accounted for 23.6% HCV infections in the general population and 58.5% in the injecting drug-users. It is prevalent in Hong Kong, associated with younger age, injecting drug-user status and the male gender. Fourteen independent HCV 6a isolates were sequenced for their full-genomes. They share a sequence homology of 94.5% between each other. HCV 6a had undergone high frequency of recombination. Four (28.5%) of 14 isolates were found having recombination with other strains in different genomic regions. Evolutionary pressure on HCV 6a genomes was analyzed by evaluating dS/dN ratio. NS4A, NS4B and NS3 showed higher dS/dN ratios (16.94-29.30) indicating a purification effect, whilst NS2, E2 and p7 showed lower dS/dN ratios (2.35-7.33) indicating a positive selection effect. This pattern of evolutionary pressure distribution alongside genomic regions was similar to the observations in HCV 1b. However HCV 6a eISDR experienced less extent of positive selection than HCV 1b eISDR (dS/dN = 12.82 vs 4.96) did. Evolutionary history of HCV 6a was inferred by Bayesian coalescent analysis. Twenty-six heterochronic, 513-bp HCV 6a partial-NS5A sequences and 63 HCV 1b sequences were analyzed. The time of exponential growth of HCV 6a in Hong Kong was postulated as during 1986 to 1994, overlaps with the time 1987 to 1997 when the second Vietnamese Boat People influx event occurred. Rooted phylogenetic analysis showed that Vietnamese HCV 6a strains were ancestors of the Hong Kong strains. Hence, a hypothesis was raised that HCV 6a outbreak in Hong Kong may be related to the Vietnamese Boat People influx event. The sequence variations within the eISDR of HCV 6a and HCV 1b were explored for correlation with the outcome of IFN-alpha/ribavirin combination treatment. Twenty-five HCV 6a patients and 37 HCV 1b patients were recruited. Three amino acid variations I2160V, V2256I, and I2292V were significantly correlated with the treatment outcome (P < 0.05) for HCV 6a. Three variations R2260H, V2268I and S2278T (P = 0.023-0.076) were weakly correlated with the outcome for HCV 1b. None of correlated variations located within the previously defined ISDR. These pieces of information can be helpful for predicting the outcome before the commencement of treatment. / Zhou Xiaoming. / "May 2006." / Adviser: Paul K. S. Chan. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0207. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 178-186). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Made available in DSpace on 2015-04-22T22:14:03Z (GMT). No. of bitstreams: 1 Myuki Alfaia Esashika Crispim.pdf: 2246402 bytes, checksum: d9773c605258310c1244020f1e5cb467 (MD5) Previous issue date: 2007-09-20 / Hepatitis B and D are endemic in the Western Brazilian Amazon region , but few studies have been conducted to investigate the genetic variability of both viruses. The hepatitis B virus (HBV) has a high genetic variability, with eight different genotypes defined (A-H). In present classification hepatitis D virus (HDV) is also supposed to present eight different genotypes (I-VIII). The aim of this study was to describe the genotypes of the virus B and D of the Western Brazilian Amazon region. We selected 190 samples of chronic carriers with HBV and 50 of them presented double infection with HDV. The serum samples of HBV were submitted to the genotyping through Polymerase Chain Reaction (PCR), with type-specific primers . In the reactive samples for HDV RNA by RT-PCR was used the genotyping by Restriction Fragment Lenght Polymorphism (RFLP). The genotype A of HBV was detected as the most frequent, in 91 participants (56,5%), following by genotype F, in 41 (25,5%), and genotype D, in 29 (18,0%). In the HDV genotyping, we found only the genotype III. This study showed that the genotypes A, D and F of VHB and the genotype III of HDV represented the predominant genotypes in the Western Brazilian Amazon. / As hepatites B e D são endêmicas na Amazônia Ocidental Brasileira, mas poucos estudos têm buscado investigar a variabilidade genética de ambos os vírus. O vírus da Hepatite B (VHB) tem uma alta variabilidade genética, sendo definidos oito genótipos distintos (A-H). O vírus da hepatite D (VHD), na atual classificação, também é sugerido apresentar oito genótipos (I-VIII). O presente estudo teve o objetivo de descrever os genótipos do vírus B e D na Amazônia Ocidental Brasileira. Selecionamos 190 amostras de portadores crônicos do vírus da hepatite B, sendo que, destas, 50 apresentavam infecção dupla com o VHD. As amostras de soro do VHB foram submetidas a genotipagem por meio da Reação em Cadeia da Polimerase (PCR), com iniciadores tipo específicos. Em amostras reativas para o VHD RNA por RT-PCR foi realizada a genotipagem por Análise do Polimorfismo do Tamanho de Fragmentos de Restrição (RFLP). Foi detectado o genótipo A como o mais freqüente em 91 participantes (56,5%), seguido pelo F em 41(25,5%), e o D em 29 (18,0%). Na genotipagem para o VHD encontramos somente o genótipo III. Este estudo mostrou que os genótipos A, D e F do VHB e o genótipo III do VHD, representam os genótipos predominantes na Amazônia Ocidental Brasileira.

This thesis examines cancer and mortality rates among people diagnosed with hepatitis B (HBV) and C (HCV) infection in New South Wales (NSW) from 1990 through 2002, by linking hepatitis notifications with the NSW Central Cancer Registry (CCR) and National Death Index. Of the 39101 HBV, 75834 HCV and 2604 HBV/HCV co-infection notifications included 1052, 1761 and 85 were linked to cancer notifications and 1233, 4008 and 186 were linked to death notifications respectively. Of 2072 hepatocellular carcinoma (HCC) notifications to the CCR 323, 267 and 85 were linked to HBV, HCV and HBV/HCV co-infection notifications. Incidence of HCC was 6.5, 4.0 and 5.9 per 1000 person years for HBV, HCV and HBV/HCV co-infected groups. Risk of HCC in those diagnosed with hepatitis was 20 to 30 times greater than the standard population. There was a marginally statistically significant increased risk of immunoproliferative malignancies associated with HCV infection (SIR=5.6 95% CI 1.8 ???17.5). Risk of death for those with hepatitis was significantly greater, 1.5 to 5 fold, than the general population with the greatest risk among those with HBV/HCV co-infection. The primary cause of HBV deaths was liver related, particularly HCC, whereas in the HCV groups drug related deaths were most frequent. Among people with HCV, risk of dying from drug related causes was significantly greater than from liver related causes (p=0.012), with the greatest increased risk in females age 15- 24 years (SMR 56.9, 95%CI 39.2???79.9). Median age at diagnosis of HCC varied markedly by country of birth and hepatitis group: HBV 66, 63 and 57years ; HCV 51, 68 and 71 years; unlinked 69, 70 and 64 years for Australian, European, and Asian-born groups, respectively (P<0.0001 for all groups). While the risk of cancer, particularly HCC, is elevated among people with HBV and HCV infection, the absolute risk remains low. Young people with HCV face a higher mortality risk from continued drug use than from liver damage related to their HCV infection. The influence of IDU in the epidemiology of HCC in New South Wales was possibly reflected in the varying distributions of age and country of birth.

This thesis examines cancer and mortality rates among people diagnosed with hepatitis B (HBV) and C (HCV) infection in New South Wales (NSW) from 1990 through 2002, by linking hepatitis notifications with the NSW Central Cancer Registry (CCR) and National Death Index. Of the 39101 HBV, 75834 HCV and 2604 HBV/HCV co-infection notifications included 1052, 1761 and 85 were linked to cancer notifications and 1233, 4008 and 186 were linked to death notifications respectively. Of 2072 hepatocellular carcinoma (HCC) notifications to the CCR 323, 267 and 85 were linked to HBV, HCV and HBV/HCV co-infection notifications. Incidence of HCC was 6.5, 4.0 and 5.9 per 1000 person years for HBV, HCV and HBV/HCV co-infected groups. Risk of HCC in those diagnosed with hepatitis was 20 to 30 times greater than the standard population. There was a marginally statistically significant increased risk of immunoproliferative malignancies associated with HCV infection (SIR=5.6 95% CI 1.8 ???17.5). Risk of death for those with hepatitis was significantly greater, 1.5 to 5 fold, than the general population with the greatest risk among those with HBV/HCV co-infection. The primary cause of HBV deaths was liver related, particularly HCC, whereas in the HCV groups drug related deaths were most frequent. Among people with HCV, risk of dying from drug related causes was significantly greater than from liver related causes (p=0.012), with the greatest increased risk in females age 15- 24 years (SMR 56.9, 95%CI 39.2???79.9). Median age at diagnosis of HCC varied markedly by country of birth and hepatitis group: HBV 66, 63 and 57years ; HCV 51, 68 and 71 years; unlinked 69, 70 and 64 years for Australian, European, and Asian-born groups, respectively (P<0.0001 for all groups). While the risk of cancer, particularly HCC, is elevated among people with HBV and HCV infection, the absolute risk remains low. Young people with HCV face a higher mortality risk from continued drug use than from liver damage related to their HCV infection. The influence of IDU in the epidemiology of HCC in New South Wales was possibly reflected in the varying distributions of age and country of birth.

La surinfection par HDV/ HBV est la forme la plus grave d'hépatite virale chronique et affecte entre 15-20 millions de patients au niveau mondial. HDV n'est pas susceptible aux traitements anti-HBV et le taux de réponse à l'IFNα est <25%. Malgré une progression plus rapide de la maladie hépatique, la majorité des patients présente une suppression de la réplication du HBV. Les détails des interactions entre HDV, HBV et le système immunitaire inné des cellules infectées restent inconnus. Les objectifs de ces travaux de thèse ont été: i) l'étude de l'infection par HDV et son interaction avec la réponse innée cellulaire; ii) l'identification de nouvelles stratégies thérapeutiques anti-HDV; iii) l'exploration de l'interaction entre HDV et HBV. L'approche expérimentale a été basée sur l'infection de cellules dHepaRG, capables d´entretenir des cycles réplicatifs complets de HBV et HDV et ayant une réponse immunitaire innée physiologique. Nous avons observé que: i) l'infection par HDV est associée à un réplication forte dans un nombre limité de cellules, et à une induction de l'expression des ISGs; ii) le traitement des cellules infectées par HDV avec de l'IFNα ne conduit pas à une induction accrue des ISGs et a une faible activité antivirale. Quelques agonistes de PRR, notamment activant la voie NF-kB, induisent une forte diminution de la réplication de HDV; iii) malgré le faible nombre de cellules infectées, HDV et ses protéines induisent une diminution de la réplication de HBV. Ces travaux ouvrent des perspectives importantes concernant la caractérisation de la pathogénèse de l'hépatite delta et l'identification de nouvelles stratégies thérapeutiques immuno modulatrices / HDV/HBV superinfection is the most aggressive form of chronic viral hepatitis and is estimated to affect 15-20 million patients worldwide. HDV is not susceptible to available direct anti-HBV drugs and sustained response to IFNα therapy occurs in less than 1/4 of patients. Despite the faster progression of liver disease, most HDV/ HBV infected patients present a suppression of HBV replication. The details of the interactions between HDV, HBV and the host cell innate immune response remain largely unexplored and research efforts have been limited by the lack of infection models. The aims of this thesis work were: i) to study HDV infection and the interplay with the host innate immune response; ii) to identify novel therapeutic strategies for the inhibition of HDV; iii) to further explore HDV/ HBV interference. The experimental strategy was based on infection of dHepaRG cells, which are known to be permissive to both HBV and HDV full replicative cycles and to present physiological innate immune responses. We observed that: i) HDV infection is associated with a strong, yet transient replication, a potent induction of the expression of ISGs; ii) IFN-α treatment of HDVinfected cells does not induce a further increase of ISG expression and has a modest antiviral activity. Conversely, some PRR agonists, in particular those inducing the NFkB pathway, induce a strong decline in HDV replication; iii) despite the low number of coinfected cells, HDV as well as its encoded proteins exert a repressive effect on HBV replication. Our work opens an array of perspectives on the pathogenesis of hepatitis delta and the identification of novel immune modulatory therapeutic strategies

Introduction Hepatitis E is a worldwide distributed disease, which is caused by the hepatitis E virus (HEV). In addition to humans, domestic pigs, wild boars, rabbits and dromedaries can be subclinically infected as reservoir animals with the zoonotic HEV genotypes 3, 4 and 7. In addition, HEV and HEV-like viruses have been described sporadically in other mammals, as well as in birds and fish, although their distinct role as reservoirs or carriers of the virus is still unclear. Aims The aim of the study was therefore to analyse in more detail the importance of different mammalian species, which do not belong to the known HEV reservoirs, for the epidemiology of HEV infections, thus enabling a better assessment of the risk of virus transmission by these animal species. Material and Methods Fourteen non-human primate species and 66 other mammal species, as well as Norway rats (Rattus norvegicus) and feeder rats (Rattus norvegicus forma domestica) from German zoos were selected for the investigations. In total 259 individual non-human primate sera and 244 individual mammalian sera of clinically healthy zoo animals were analysed for the presence of HEV-specific antibodies (ab) using a species-independent double-antigen sandwich ELISA. The non-human primate sera were additionally examined using a commercial human ELISA. Real-time reverse-transcription (RT)-PCR, nested broad-spectrum RT-PCR and a rat HEV-specific RT-PCR were used to detect the HEV genome in sera of mammals and rat liver samples. A commercial and an in-house method were used for the DNA sequencing. Results HEV-specific ab were detected in 3.9% (10/259) of the non-human primate sera (4 species) and 11.5% (28/244) of the mammalian sera (16 species). The highest detection rates were recorded with 33.3% (9/27) in porcines and with 27.0% (10/37) in carnivores. HEV-RNA was detected in a clinically healthy female Syrian brown bear (Ursus arctos syriacus) and in 8 of the investigated Norway rats. Sequence analysis identified the virus as rat HEV; the viruses from the bear and the free-ranging rats from the same zoo showed a high nucleotide sequence identity (94.6%–97.8%). Because of the small number of samples due to the small populations within the individual zoos, further statistical evaluations were not carried out. Conclusions The results show that non-human primates in zoos may be infected with HEV or HEV-like viruses; however, the low ab detection rates together with the negative genome detection argue against a high risk of virus transmission to humans. The study in other zoo-housed mammalian species was able to significantly increase the number of animal species with indications of HEV infections. In most animal species, only rare evidence and low detection rates were available, which can best be explained by “spillover-infections”. In addition to the expected high detection rate in porcine species, the high percentage of HEV antibody-positive carnivores is remarkable. Their role as possible HEV reservoir animals should therefore be clarified in further investigations. The detection of rat HEV in the serum of the bear and its high nucleotide sequence identity with the HEVs of the pest rodents provides first evidence of transmission of this virus species between rodents and carnivores.:List of content List of figures List of tables List of abbreviations 1 General introduction 1.1 Discovery of HEV 1.2 Taxonomy 1.3 Morphology 1.4 Genomic organisation 1.5 Viral replication 1.6 Hepatitis E in humans 1.7 Tools for HEV diagnosis 1.8 Therapy 1.9 Animal infections with HEV and HEV-like viruses 1.10 Experimental infections of animals 1.11 Geographical distribution 1.12 Transmission pathways 1.13 Epidemiology 1.14 Prevention 2 Aims of the study 3 Publications 3.1 Publication I 3.1.1 Summary of Publication I 3.1.2 Key messages of Publication I 3.1.3 Own contribution to Publication I 3.2 Publication II 3.2.1 Summary of Publication II 3.2.2 Key messages of Publication II 3.2.3 Own contribution to Publication II 3.3 Publication III 3.3.1 Summary of Publication III 3.3.2 Key messages of Publication III 3.3.3 Own contribution to Publication III 4 General discussion 4.1 HEV infections in various animal species 4.2 Prevalence of natural HEV infections in non-human primates 4.3 Prevalence of natural HEV infections in other zoo-housed mammals 4.4 Transmission pathways of HEV in a zoo-setting 4.5 Risk of virus transmission from zoo animals to humans 5 Conclusion and perspectives 6 Summary 7 Zusammenfassung 8 References List of animals investigated in the study List of publications Acknowledgements / Einleitung Hepatitis E ist eine durch das Hepatitis E-Virus (HEV) verursachte, weltweit verbreitete Erkrankung. Neben dem Menschen können Hausschwein, Wildschwein, Kaninchen und Dromedar als Reservoirtiere subklinisch mit den zoonotischen HEV-Genotypen 3, 4 und 7 infiziert werden. Darüber hinaus wurden HEV und HEV-ähnliche Viren vereinzelt bei weiteren Säugetieren, sowie Vögeln und Fischen beschrieben, wobei deren genaue Rolle als Reservoir oder Überträger des Virus bislang unklar ist. Ziele Ziel der Arbeit war es deshalb, die Bedeutung verschiedener Säugetierarten, die nicht zu den bekannten HEV-Reservoiren gehören, für die Epidemiologie der HEV-Infektionen besser zu erfassen und dadurch das Risiko einer Virusübertragung durch diese Tierarten besser abzuschätzen. Material und Methoden Vierzehn Affenarten und 66 weitere Säugetierarten, sowie Wanderratten (Rattus norvegicus) und Futterratten (Rattus norvegicus forma domestica) aus deutschen Zoos wurden für die Untersuchungen ausgewählt. Insgesamt wurden 259 individuelle Affenseren und 244 individuelle Säugerseren klinisch gesunder Zootiere mittels eines Spezies-unabhängigen Doppel-Antigen-Sandwich-ELISAs auf das Vorhandensein von HEV-spezifischen Antikörpern (AK) untersucht. Die Affenseren wurden zusätzlich mittels eines kommerziellen humanen ELISAs untersucht. Real-time reverse-transcription (RT)-PCR, nested broad-spectrum RT-PCR sowie eine Ratten-HEV-spezifische RT-PCR wurden für den HEV-Genomnachweis in Seren der Säuger und in Ratten-Lebern verwendet. Für die DNA-Sequenzierungen wurden eine kommerzielle und eine In-house-Methode verwendet. Ergebnisse In 3,9% (10/259) der Affenseren (4 Arten) und 11,5% (28/244) der Säugerseren (16 Arten) wurden HEV-spezifische AK nachgewiesen. Die höchsten Nachweisraten wurden mit 33,3% (9/27) in Schweineartigen und 27,0% (10/37) in Fleischfressern ermittelt. HEV-RNA wurde in einer klinisch gesunden Syrischen Braunbärin (Ursus arctos syriacus), sowie in 8 der untersuchten Wanderratten nachgewiesen. Die Sequenzanalyse identifizierte das Virus als Ratten-HEV; die Viren aus der Bärin und aus den wildlebenden Ratten desselben Zoos zeigten eine hohe Nukleotidsequenz-Identität (94,6%–97,8%). Weitergehende statistische Auswertungen wurden wegen der geringen Probenzahlen aufgrund der kleinen Populationen innerhalb der einzelnen Zoos nicht durchgeführt. Schlussfolgerungen Die Ergebnisse zeigen, dass Affen in Zoos mit HEV oder HEV-ähnlichen Viren infiziert sein können, jedoch sprechen die geringen AK-Nachweisraten zusammen mit den negativen Genomnachweisen gegen ein hohes Übertragungsrisiko auf den Menschen. Die Studie an anderen Säugetierarten in Zoos konnte die Zahl der Tierarten mit Hinweisen auf HEV-Infektionen deutlich erhöhen. Bei den meisten Tierarten lagen nur seltene Nachweise und niedrige Detektionsraten vor, die am besten durch „Spillover-Infektionen“ erklärt werden können. Neben der erwarteten hohen Nachweisrate bei Schweineartigen ist der hohe Prozentsatz an HEV AK-positiven Fleischfressern bemerkenswert, weshalb deren Rolle als mögliche HEV-Reservoirtiere in weiteren Untersuchungen geklärt werden sollte. Der Ratten-HEV-Nachweis im Serum der Bärin, sowie dessen hohe Nukleotidsequenz-Identität zu den HEVs der Schadnager geben erstmals Hinweise auf eine Übertragung dieser Virusart zwischen Nagern und Fleischfressern.:List of content List of figures List of tables List of abbreviations 1 General introduction 1.1 Discovery of HEV 1.2 Taxonomy 1.3 Morphology 1.4 Genomic organisation 1.5 Viral replication 1.6 Hepatitis E in humans 1.7 Tools for HEV diagnosis 1.8 Therapy 1.9 Animal infections with HEV and HEV-like viruses 1.10 Experimental infections of animals 1.11 Geographical distribution 1.12 Transmission pathways 1.13 Epidemiology 1.14 Prevention 2 Aims of the study 3 Publications 3.1 Publication I 3.1.1 Summary of Publication I 3.1.2 Key messages of Publication I 3.1.3 Own contribution to Publication I 3.2 Publication II 3.2.1 Summary of Publication II 3.2.2 Key messages of Publication II 3.2.3 Own contribution to Publication II 3.3 Publication III 3.3.1 Summary of Publication III 3.3.2 Key messages of Publication III 3.3.3 Own contribution to Publication III 4 General discussion 4.1 HEV infections in various animal species 4.2 Prevalence of natural HEV infections in non-human primates 4.3 Prevalence of natural HEV infections in other zoo-housed mammals 4.4 Transmission pathways of HEV in a zoo-setting 4.5 Risk of virus transmission from zoo animals to humans 5 Conclusion and perspectives 6 Summary 7 Zusammenfassung 8 References List of animals investigated in the study List of publications Acknowledgements

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include a diverse group of pathogens that cause a wide array of diseases that can range from sub-clinical to lethal. These viruses infect humans and mammals as well as a variety of other hosts. For their successful replication, viruses interact closely with host cells from the binding to the receptor to the exit as complete viral progenies. During the events, viruses are dependent on host factors for receptor bindings, genome synthesis, and trafficking of viral genome and proteins. Thus there have been major efforts on the studies of understanding the virus-host interactions in the field of virology. In my PhD program, I have studied the host factors that regulate the replication of viruses using porcine reproductive and respiratory syndrome virus (PRRSV) and hepatitis C virus (HCV). I found that modulation of either the viral receptor or cellular signaling pathways had pronounced effects in the replication of PRRSV or HCV respectively. Using PRRSV, I found that the modulation of the level of the putative receptor CD163 on cells with cytokines significantly influence virus replication, suggesting the importance of cytokine presence in environments to determine the replication and pathogenicity of PRRSV via receptor expression in vivo. With HCV, I found that the enhancement of the virus replication occurs through the activation of the epidermal growth factor receptor/extracellular signal-regulated kinase pathway by bile acids which are abundant in the liver where the virus targets in vivo. Furthermore, I found that the bile acid-mediated signaling pathway significantly inhibited the antiviral activities against HCV. These results indicate the importance of environmental factors such as bile acids and signaling pathways in the replication and pathogenicity of HCV in vivo.

The work described in this thesis investigates the lipid metabolism of human hepatocytes in the context of Hepatitis C Virus (HCV) infection. This includes lipoprotein signalling and cholesterol metabolism targeted analysis of gene expression as well as the influence of polyunsaturated ER targeting liposomes (PERLs) on infection. These analyses indicate that HCV suppresses the expression of key regulators throughout the cholesterol biosynthesis pathway. This effect was quantified and the influence of liposome treatment evaluated. The latter resulted in the formulation of the hypothesis that PERL treatment interfers with virus-induced abberations of the cholesterol biosynthesis pathway and normalises the expression of four genes directly involved in cholesterol regulation. In addition, the lipidome of isolated lipid droplet was analysed by mass spectrometry. These data, combined with microscopy data suggest that PERLs interfere with S-palmitoylation of the HCV core protein resulting in dissociation of core from lipid droplets. This is likely to interrupt the viral assembly process, leading to inhibition of the production of infectious viral particles. Further described here are two different yet unsuccessful approaches to fluorescently label HCV RNA for live cell microscopy studies, namely an MS2 coat protein mediated approach, and Alexa®UTP labelling.

Thesis (PhD (Med))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background and Objective: The most common cause of severe proteinuria/nephrotic syndrome (NS) in children worldwide is minimal change disease (MCD). This is also the pattern observed in white and Indian children in South Africa (SA). By contrast, black and mixed race/coloured children of Southern Africa in the 1960s to 1990s were shown to have a different pattern of NS. One of the main differences was the frequency of hepatitis B virus (HBV) associated glomerulonephritis, usually membranous glomerulonephritis (MGN). The objective of this project was a clinicopathological study of this subgroup of nephrotic children to document the disease further and in particular to seek correlations between pathological and clinical features including prognosis. A central focus was to document the detailed ultrastructural examination of the renal biopsies of these children and to correlate the spectrum of pathological features with demographic, clinical, laboratory and prognostic features. The hypothesis was that the clinicopathological features of HBV MGN in children differed substantially from idiopathic MGN in general (children and adults) and also from HBV MGN in adults and that HBV MGN in children should be viewed as a distinct disease. Patients and methods: The childhood (12 years and younger) patient cohort was 309 children with severe proteinuria/nephrotic syndrome who presented at Tygerberg Hospital (TBH) over a 21 year period from 1974-1995, including 67 children from Namibia. The study group was 71 children with HBV MGN who were followed up to 2005. The comparative adult group was 45 adults with MGN of whom 12 had HBV MGN and 33 idiopathic MGN. (A comparison could not be made with idiopathic MGN in childhood as this centre only had 2 such patients during the study period.) Demographic, clinical, laboratory and renal pathology data were collected, compared and correlated. Results: HBV associated MGN was the most frequent cause of NS in the Namibian subgroup, 25/67 (37%) and the third most frequent, 71/309 (23%) in the childhood cohort as a whole. The MGN group was 86% (71/83) of the total HBV childhood nephrotic cohort, by far the dominant subgroup. The average age of the 71 children with HBV MGN was 6.0 years (range 2-12 years) at presentation and boys comprised 80% of the group. Hepatitis B envelope antigen (HBeAg) was identified in the serum of 87% of children tested. Laboratory features different from idiopathic MGN included more prominent haematuria, mildly raised serum transaminases and more frequently lowered serum C3 and C4 levels. Light microscopic examination of renal biopsies showed mesangial proliferation in all patients but with minimal glomerular sclerosis and interstitial disease. On ultrastructural examination mesangial and subendothelial deposits were common and prominent as was mesangial interposition. The MGN of HBV in children therefore frequently showed mesangiocapillary glomerulonephritis (MCGN) features in addition to the subepithelial deposits of MGN. The subgroup of 23 whose renal biopsies displayed severe mesangial interposition in addition to the subepithelial deposits of MGN were termed the mixed HBV MGN-mesangiocapillary GN group. Virus like bodies and tubuloreticular inclusion bodies were both found in more than 80% of biopsies of childhood HBV MGN. HBeAg was identified in the subepithelial deposits in the glomeruli. This was the first time this feature was demonstrated in Africa. The 46 South African children with HBV MGN showed a cumulative remission rate of 25% at 2 years and 52% at 4 years. Seven of the children (10%) of the total cohort developed chronic renal failure (CRF). Age of 6 years and above at presentation and severe mesangial deposits on biopsy correlated with fewer remissions and poorer outcome. In 3 patients the interval between the diagnosis of HBV MGN and the onset of CRF was more than 19 years with the longest being 23 years. The 358 cases of childhood HBV MGN from Southern Africa constitute 37% of the reported childhood patients. Comparative data A comparison was made between the 71 children with HBV MGN, 12 adults with HBV MGN and 33 adults with idiopathic MGN. The main differences were that both HBV MGN groups included only coloured and black patients and were more predominantly male while the idiopathic MGN group included all races. In the HBV patients, haematuria was more frequent and severe, liver enzymes were frequently raised and C3 more frequently reduced than in the idiopathic cohort. Both groups of adult MGN patients had normal C4 levels while the childhood HBV MGN group had reduced C4 levels. The immune complex pattern in both of the HBV MGN adult and childhood groups on biopsy was similar with more mesangial and subendothelial deposits as well as mesangial interposition than the idiopathic group. Despite this similarity between the two HBV groups, both adult groups showed more glomerular sclerosis and interstitial disease than the childhood group. The clinical outcome of the children’s cohort was better than the other 2 groups with remission (52%) more frequent at 4 years (p< 0.01) and better renal and patient survival. Including the 83 cases from this series, at least 1243 renal biopsy proven cases of HBV MGN have been reported in the English literature; children (80%) and adults (20%). The male gender predominance in both age groups for HBV MGN is similar (children 79%; adults 84%) and significantly greater than for idiopathic MGN. Conclusions: The findings confirm that HBV MGN in children is a distinct form of GN which broadens the classical morphologic description of MGN by often including a number of mesangiocapillary GN features. The subgroup of renal biopsies with the most severe mesangiocapillary GN features was classified as the mixed HBV MGNmesangiocapillary GN group. The MGN spectrum as a whole comprised 86% of the HBV positive childhood group. HBV MGN was the most frequent association with NS/severe proteinuria in the Namibian subgroup (37%) and the third largest group (19%) in the SA children. It showed a relatively high spontaneous remission rate but at least 10% of the children developed renal failure. Age of 6 years and above at presentation and severe mesangial deposits on biopsy correlated with fewer remissions and poorer outcome. Extended follow up (more than 15 years) was required to demonstrate renal failure in some patients in the poor outcome group. Urbanisation, associated with lower HBV carrier rates, and HBV vaccination (initiated routinely in 1995 in SA), have already lead to a sharply decreasing incidence of this disease in SA. HBV MGN has been a valuable and possibly unique model of human GN and MGN in particular in that the HBeAg has been identified in both the serum and glomeruli enabling confirmation of the aetiological role of HBeAg. / AFRIKAANSE OPSOMMING: Agtergrond en Doelwit: Die algemeenste oorsaak van erge proteïenurie/nefrotiese sindroom (NS) in kinders wêreldwyd is minimale veranderingsiekte. Hierdie patroon kom ook voor in blanke- en Indiër kinders in Suid-Afrika. In teenstelling hiermee is aangetoon dat swart en kleurling/gemengde ras kinders in Suider Afrika tussen die jare 1960s tot 1990s ’n ander patroon van nefrotiese sindroom gehad het. Een van die hoof verskille was die algemene voorkoms van hepatitis B virus (HBV) geassosieerde glomerulonefritis, gewoonlik membraneuse glomerulonefritis (MGN). Die doelwit van hierdie projek was ’n klinies-patologiese studie van hierdie subgroep van nefrotiese kinders ten einde die siekte verder te beskryf en veral om korrelasies te tref tussen patologiese en kliniese kenmerke insluitende prognose. Die gedetaileerde ultrastrukturele ondersoek van die kinders se nierbiopsies en die korrelasie van die spektrum patologiese kenmerke met demografiese, kliniese, laboratorium en prognostiese kenmerke was ‘n sentrale fokusarea. Die hipotese was dat die klinies-patologiese kenmerke van HBV MGN in kinders wesenlik van idiopatiese MGN in die algemeen verskil (in kinders en volwassenes) en ook van HBV MGN in volwassenes, en dat die beeld in kinders as ’n afsonderlike siekte beskou behoort te word. Pasiënte en metodes: Die kinder kohort (12 jaar en jonger) was 309 kinders met erge proteïenurie/nefrotiese sindroom wie in Tygerberg Hospitaal (TBH) behandel was oor ‘n 21 jarige periode vanaf 1974 tot 1995, insluitende 67 kinders van Namibië. Die studiegroep was 71 kinders met HBV MGN wie waar moontlik tot 2005 opgevolg was. Die vergelykende volwasse groep was 45 volwassenes met MGN van wie 12 HBV MGN gehad het en 33 idiopatiese MGN. (’n Vergelyking met idiopatiese MGN in kinders kon nie gedoen word nie omdat hierdie sentrum net twee sulke pasiënte tydens die studietyd behandel het.) Demografiese, kliniese, laboratorium en nierpatologie inligting is versamel, vergelyk en gekorreleer. Resultate: HBV geassosieerde MGN was die algemeenste oorsaak van NS in die Namibiese subgroep, 25/67 (37%) en die derde mees algemeen, 71/309 (23%) in die kinder kohort as geheel. Die MGN groep was 86% (71/83) van die totale HBV kinder nefrotiese kohort en verreweg die oorheersende subgroep. Die gemiddelde ouderdom van die 71 kinders met HBV MGN by presentering was 6.0 jaar (reikwydte 2-12 jaar) en seuns het 80% van die groep behels. Hepatitis B omhullingsantigeen (envelope antigen- HBeAg) is aangetoon in die serum van 87% van die kinders wie daarvoor getoets is. Laboratoriumkenmerke wat van idiopatiese MGN verskil het, het ingesluit meer prominente hematurie, gering verhoogde serum transaminases en meer dikwels verlaagde serum C3 en C4 vlakke. Ligmikroskopiese ondersoek van die nierbiopsies het mesangiale proliferasie in elke pasiënt getoon, maar met minimale glomerulêre sklerose en interstisiële siekte. Met ultrastrukturele ondersoek was mesangiale en subendoteliële neerslae asook mesangiale interposisie algemeen. Die MGN van HBV in kinders het dus dikwels kenmerke van mesangiokapillêre glomerulonefritis getoon bo en behalwe die subepiteliële neerslae van MGN. Die ondergroep van 23 van wie die nierbiopsies erge mesangiale interposisie aangetoon het asook die subepiteliale neerslae van MGN is die gemengde HBV MGN-mesangiokapillêre GN groep genoem. Virustipe liggaampies en tubuloretikulêre insluitingsliggaampies is in meer as 80% van die biopsies bevestig. HBeAg was in die subepiteliële neerslae identifiseer. Dit was die eerste keer dat hierdie kenmerk in Afrika identifiseer is. Die 46 Suid-Afrikaanse kinders het ’n kumulatiewe remissie koers van 25% teen 2 jaar en van 52% teen 4 jaar getoon. Sewe van die kinders (10%) van die hele kohort het kroniese nierversaking (KNV) ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale neerslae in ‘n biopsie het met minder remissies en ’n swakker uitkoms gekorreleer. Drie pasiënte het meer as 19 jaar na aanvanklike voordoening ooglopende KNV ontwikkel, waarvan 23 jaar die langste interval was. Die 358 gevalle van kinderjare HBV MGN van Suidelike-Afrika maak 37% uit van die gerapporteerde kinder pasiënte. Vergelykende data ’n Vergelyking is getref tussen die 71 kinders met HBV MGN, 12 volwassenes met HBV MGN en 33 volwassenes met idiopatiese MGN. Die hoof verskille was dat beide HBV groepe net kleurling en swart pasiënte ingesluit het en meer oorwegend manlik was, terwyl die idiopatiese groep alle rasse ingesluit het. In die HBV pasiënte was hematurie meer algemeen en erg, lewer ensieme meer dikwels verhoog en C3 meer dikwels verlaag as in die idiopatiese kohort. Beide groepe van volwasse MGN pasiënte het normale C4 vlakke getoon terwyl die kindergroep met HBV MGN verlaagde C4 vlakke bewys het. Die immuunkompleks patroon in biopsies van die HBV MGN volwasse en kindergroepe was soortgelyk met meer mesangiale en subendoteliële neerslae asook meer mesangiale interposisie as in die idiopatiese groep. Ten spyte van hierdie ooreenkoms tussen die twee HBV groepe, het die twee volwasse groepe meer glomerulêre sklerose en interstisiële siekte as die kindergroep vertoon. Die kliniese uitkoms van die kinderkohort was beter as die ander twee groepe met remissie (52%) wat meer algemeen was teen 4 jaar (p< 0.01) en met beter nier- en pasïent oorlewing. Ingeslote die 83 gevalle van hierdie reeks, is ten minste 1243 nierbiopsie bewysde gevalle van HBV MGN in kinders (80%) en volwassenes (20%) in die Engelse literatuur gerapporteer. Die manlike oorheersing in beide ouderdomsgroepe van HBV MGN is soortgelyk (kinders 79%; volwassenes 84%) en betekenisvol meer as vir idiopatiese MGN. Gevolgtrekkings: Die bevindinge bevestig dat HBV MGN in kinders ’n afsonderlike vorm van GN is wat die klassieke beskrywing van MGN verbreed deur die algemene insluiting van ’n aantal mesangiokapillêre GN kenmerke. Die ondergroep van nier biopsies met erge mesangiokapillêre GN kenmerke is as die gemengde HBV MGNmesangiokapillêre GN groep geklassifiseer. Die MGN spektrum in geheel het 86% van die HBV positiewe kindergroep behels. HBV MGN was die mees algemene assosiasie met NS/erge proteïenurie in die Namibiese subgroep (37%) en die derde grootse groep (19%) onder die SA kinders. Die siekte het ’n relatiewe hoë spontane remissiekoers getoon, maar ten minste 10% van die kinders het nierversaking ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale neerslae in ‘n nierbiopsie het met minder remissies en ’n slegter uitkoms gekorreleer. Uitgebreide opvolg (meer as 15 jaar) was nodig om nierversaking in sommige van die swak uitkomsgroep aan te toon. Verstedeliking is geassosieerd met laer HBV draersyfers en hierdie faktor saam met algemene HBV inenting in die kinderjare (wat in 1995 in SA begin was), het ’n skerp daling in die voorkoms van hierdie siekte in SA teweeg gebring. HBV MGN is ’n waardevolle en moontlik unieke model van menslike GN en MGN, veral omdat die HBeAg in beide die serum en glomeruli identifiseer kon word om die etiologiese rol van HBeAg te bevestig.