Further functional investigation revealed that knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells, concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium Conversely, overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta 1 (IFN-beta1). In this connection, IFN-beta1-mediated 2', 5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover, GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p= 0.019) as compared with their counterpart pre-treatment liver biopsies. / Hepatitis B virus (HBV) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although considerable progress has been made over the past decade, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. / In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via IFN-beta1-OAS-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection. / In this study, we applied a two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach to globally analyze the host early response to HBV by using an inducible HBV-producing cell line HepAD38. Twenty-three proteins were identified as differentially expressed, with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, as well as in HBV-infected human liver biopsies by immunohistochemistry. / Ma, Yan. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-129). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lau, Chi Chiu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 133-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Zhu, Ranxu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The use of mathematical models in studying disease epidemics can be diverse, from the focused study of the role of a single determinant of the epidemic, or to the overall estimation of morbidity and mortality. In using simple deterministic models, a balance is struck between biological and social complexities, and the high data input demands of mathematical models. This thesis aims to apply the use of deterministic mathematical models to the studies of HIV and hepatitis C epidemiology in the Asia-Pacific region. In Australia, about 85% of reported HIV cases are among homosexual men. Casual homosexual partnerships made up 40% of incident HIV cases in 1995 increasing to 65% in 2004. In the state of New South Wales, it was estimated that over 7,500 people were living with HIV/AIDS in 2005, increasing to over 10,000 by 2016 with existing levels of intervention. Intervention measures were estimated to have prevented some 44,500 cases, the majority being among injecting drug users through the Needle and Syringe Programmes. Models for the HIV epidemics in developing countries were also developed incorporating multiple routes of HIV transmission. For Papua New Guinea, it was estimated 64,000 people were living with HIV/AIDS in 2005, rising to over 500,000 by 2025 with current levels of intervention. High levels of interventions, in particular increased condom use, will be required to achieve a stabilisation or reduction in HIV prevalence. In East Timor, the HIV epidemic is still in the early stages with 138 people estimated to be living with HIV/AIDS, rising to 5,000 by 2025 with minimal intervention. For HCV, models of the epidemic in Australia showed HCV incidence peaking in 1999, followed by a decline reaching 9,700 incident cases in 2005. Of 197,000 estimated chronic HCV cases in 2005, 58% had stage F 0/1 liver disease, 15% F 2/3 liver disease, and 2% HCV-related cirrhosis. Models estimated 210 and 105 people developed HCV-related liver failure and hepatocellular carcinoma, respectively. Comparisons of modelled HCV long-term sequelae projections with linkage data showed relatively good agreement, despite discrepancies in liver-related deaths. To decrease the number of chronic HCV, at least a tripling of treatment coverage would be required. These models provide estimates of the current levels of epidemics as well as projections of future scenarios under different intervention strategies, which have an important role in the planning of strategies, as well as assessment of previous epidemic conditions.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 268-300.

published_or_final_version / Medicine / Master / Doctor of Medicine

The use of mathematical models in studying disease epidemics can be diverse, from the focused study of the role of a single determinant of the epidemic, or to the overall estimation of morbidity and mortality. In using simple deterministic models, a balance is struck between biological and social complexities, and the high data input demands of mathematical models. This thesis aims to apply the use of deterministic mathematical models to the studies of HIV and hepatitis C epidemiology in the Asia-Pacific region. In Australia, about 85% of reported HIV cases are among homosexual men. Casual homosexual partnerships made up 40% of incident HIV cases in 1995 increasing to 65% in 2004. In the state of New South Wales, it was estimated that over 7,500 people were living with HIV/AIDS in 2005, increasing to over 10,000 by 2016 with existing levels of intervention. Intervention measures were estimated to have prevented some 44,500 cases, the majority being among injecting drug users through the Needle and Syringe Programmes. Models for the HIV epidemics in developing countries were also developed incorporating multiple routes of HIV transmission. For Papua New Guinea, it was estimated 64,000 people were living with HIV/AIDS in 2005, rising to over 500,000 by 2025 with current levels of intervention. High levels of interventions, in particular increased condom use, will be required to achieve a stabilisation or reduction in HIV prevalence. In East Timor, the HIV epidemic is still in the early stages with 138 people estimated to be living with HIV/AIDS, rising to 5,000 by 2025 with minimal intervention. For HCV, models of the epidemic in Australia showed HCV incidence peaking in 1999, followed by a decline reaching 9,700 incident cases in 2005. Of 197,000 estimated chronic HCV cases in 2005, 58% had stage F 0/1 liver disease, 15% F 2/3 liver disease, and 2% HCV-related cirrhosis. Models estimated 210 and 105 people developed HCV-related liver failure and hepatocellular carcinoma, respectively. Comparisons of modelled HCV long-term sequelae projections with linkage data showed relatively good agreement, despite discrepancies in liver-related deaths. To decrease the number of chronic HCV, at least a tripling of treatment coverage would be required. These models provide estimates of the current levels of epidemics as well as projections of future scenarios under different intervention strategies, which have an important role in the planning of strategies, as well as assessment of previous epidemic conditions.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Hepatitis C virus (HCV) represents a global health threat. HCV is a blood-borne positive-strand RNA virus belonging to the Flaviviridae family that infects ~160 million people worldwide. About 70% of infected individuals fail to clear the virus and subsequently develop chronic hepatitis, frequently leading to liver cirrhosis and in some cases hepatocellular carcinoma. Therapeutic options for HCV infection are still limited and a protective vaccine is not yet available. Currently available therapies include administration of pegylated alpha interferon in combination with ribavirin. The recently approved protease inhibitors Boceprevir and Telaprevir are also included in the treatment regimen. However, limitations to the treatment with direct-acting antivirals (DAAs) are associated with severe side effects and low sustained virological response (SVR) rates that vary depending on the virus and host genotype. The replication step of the viral life cycle is mostly targeted by majority of DAAs. Recent findings have suggested that a combination of entry inhibitors together with DAAs exhibit a synergistic effect in the treatment of HCV. Therefore, identification of efficient HCV entry inhibitors is of high priority In vitro studies have shown that HCV attachment and subsequent entry into the host cells is mediated by E1 and E2 viral envelope proteins. HCV entry requires interaction with a number of receptors which include CD81, scavenger receptor B1 (SR-B1) and the tight junction proteins, claudin 1 (CLDN1) and occludin (OCLN). Since the E2 glycoprotein is reported to interact directly with cellular receptors, it is an attractive target for neutralisation. The present study focuses on the establishment and characterisation of entry inhibitors as antivirals for HCV. The thesis is presented in three chapters: Chapter 1- ‘Introduction’, provides a brief overview on HCV genotypes, genome organisation, life cycle including details on the entry process and therapies used for the treatment of HCV. Chapter 2 describes the generation of monoclonal antibodies (mAbs) against HCV envelope proteins as potent anti-viral agents for the prevention of HCV infection. Data on the identification and characterization of the neutralizing epitopes of HCV envelope proteins have been presented. Chapter 3 includes isolation of entry inhibitors of HCV from natural sources and identification and characterization of the active components exhibiting antiviral property. A number of studies have reported the role of neutralizing antibodies in the course of HCV infection and emerging data suggest protective effect of antibodies against HCV infection. Most of the ongoing studies are based on HCV genotype 1a which is prevalent globally. However in India, the prevalent genotype is 3a. Therefore, we established a panel of mAbs against HCV-LPs comprising of core-E1-E2 derived from genotype 3a as described in chapter 2. HCV-LP based system has been used in this study since it mimics the biophysical conformation, morphology and antigenic properties of the native virion and represents a model system for studies on viral binding and entry. MAbs were characterised and analyzed for their ability to prevent viral binding and entry into host cells. Three mAbs namely E3D8, H6D3 and A10F2 were identified to recognize the E2 viral glycoprotein which significantly inhibited HCV-LP binding to Huh7 cells in vitro. The neutralizing epitopes corresponding to the mAbs were identified using overlapping truncated fragments and synthetic peptides of the E2 protein. Our experiments suggest that the epitopes recognised by the inhibitory mAbs are unique and different from those reported till now. The synergistic effect of a combination of mAbs on virus neutralization has shown promising results for treatment of viral infections. Since in the present study the epitopes recognised by the mAbs are non-overlapping, we went ahead to determine whether a combination of these mAbs would enhance the ability to block HCV-LP binding. Indeed, flow cytometry and fluorescence microscopy studies revealed that a combination of the antibodies efficiently blocked the binding of HCV-LP to human hepatoma cells. More importantly and of relevance is the observation that the mAbs in combination inhibited viral infection (JFH1 strain) and replication in permissive human hepatocytes as determined by real time RT-PCR. Phytochemicals present in plants have been considered as conducive for prevention of several viral infections and are found to be promising antiviral agents. Natural products which are biologically active disclose drug-like properties since they are small molecules and can be easily metabolised and absorbed by the body. In our study as described in chapter 3, we evaluated extracts from Indian medicinal plants and fruits which are known to have hepato-protective effect, for natural potent attachment and entry inhibitors for HCV. Flow cytometric analysis suggested that the root extract of the herb Boerhavia diffusa and fruit extract of Prunus domestica exhibited high antiviral activity by inhibiting the binding of Hepatitis C virus like particles (HCV-LPs) to the human hepatoma cells. We went on to isolate, identify and confirm the active principles to be Boeravinone H, a dehydrorotenoid, (from Boerhavia diffusa) and Rutin, a flavonoid, (from Prunus domestica) by LC-ESI-MS, NMR, UV and IR spectral analysis. Our study revealed that the compounds block the attachment as well as entry step probably by targeting the viral particle. We also assessed the efficiency of these small molecules (Boeravinone H and Rutin) to inhibit HCV negative strand synthesis post entry by real time RT-PCR. Results suggest significant inhibition of viral entry and infection in the HCV cell culture (ex vivo). To our knowledge it is the first report on Boeravinone H and Rutin as entry inhibitor for HCV. In conclusion, our findings support the potential of employing a cocktail of neutralizing mAbs and antiviral agents from natural source in the management of HCV infection.

Ho Kar Fai, William. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 121-129). / Abstracts in English and Chinese. / Abstract --- p.I / Acknowledgement --- p.V / Table of Contents --- p.VI / Abbreviations --- p.VIII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The recent situation of hepatitis B infection and HBV-induced HCC in Hong Kong / Chapter 1.2 --- Natural history of HBV infection in human / Chapter 1.3 --- The genomic organization of HBV / Chapter 1.4 --- Potential oncogenic mechanism of HBV-induced hepatocarcinogenesis / Chapter 1.5 --- Aim of the present study / Chapter Chapter 2 --- Materials and methods --- p.16 / Chapter 2.1 --- Transformation in E.coli for subtracted normal-counterpart library / Chapter 2.2 --- PCR amplification of subtracted clones / Chapter 2.3 --- Sequencing of subtracted clones with dye-terminator cycle sequencing technology / Chapter 2.4 --- Sequence analysis and database construction / Chapter 2.5 --- Molecular cloning and characterization of novel gene / Chapter 2.6 --- In silico structural and functional analysis of Z313 / Chapter 2.7 --- Cloning and sequencing analysis of zinc finger protein 313 (Z313) / Chapter 2.7.1 --- PCR amplification of target gene -Z313 / Chapter 2.7.2 --- Mini-preparation of plasmid DNA / Chapter 2.7.3 --- Cycle sequencing of cloned cDNA -Z313 with dye-primer technology / Chapter 2.8 --- Multiple Tissue Northern (MTN) blot hybridisation / Chapter 2.9 --- RT-PCR analysis of Z313 / Chapter 2.10 --- Subcellular localization study of Z313 by Green Fluorescent Protein (GFP) / Chapter 2.10.1 --- Directional cloning of Z313 into pEGFP-Cl / Chapter 2.10.2 --- Mini-preparation of plasmid DNA / Chapter 2.10.3 --- Transient transfection of plasmids in different cell lines / Chapter 2.10.4 --- Microscope observation of GFP transfected cells / Chapter Chapter 3 --- Results --- p.49 / Chapter 3.1 --- PCR selection of subtracted clones for sequencing analysis / Chapter 3.2 --- Partial sequencing of selected subtracted clones / Chapter 3.3 --- DNA homology searching using program - BLASTN / Chapter 3.4 --- Catalogue of the 467 ESTs from the subtracted normal-counterpart library / Chapter 3.5 --- Classification and frequency of the subtracted normal-counterpart cDNA clones / Chapter 3.6 --- Identification of putative differentially expressed genes in HCC surrounding normal liver / Chapter 3.7 --- Categorization of ESTs exclusively appeared in the subtracted normal- counterpart library / Chapter 3.8 --- In silico structural and functional analysis of zinc finger protein313 (Z313) / Chapter 3.9 --- Molecular cloning of zinc finger protein 313 (Z313) / Chapter 3.10 --- Northern analysis of zinc finger protein 313 (Z313) / Chapter 3.11 --- RT-PCR analysis of zinc finger protein 313 (Z313) / Chapter 3.12 --- Subcellular localization study of zinc finger protein 313 (Z313) / Chapter Chapter 4 --- Discussion --- p.104 / Chapter 4.1 --- EST analysis on the subtracted normal-counterpart cDNA clones / Chapter 4.1.1 --- Characterization of ESTs generated from the subtracted normal-counterpart library / Chapter 4.1.2 --- Putative differentially expressed genes in HCC surrounding normal liver related to hepatocellular carcinoma / Chapter 4.2 --- Molecular cloning and characterization of zinc finger protein313 (Z313) / Chapter 4.3 --- Future aspects / References --- p.121