Current methods for studying RNA binding proteins (RBPs) combine the use of ultraviolet (UV) crosslinking and immunoprecipitation (CLIP) to analyze RNA-protein interactions. An underexplored alternative approach is using site-specific incorporation of photoactivatable non-canonical amino acids (ncAAs) to enhance the crosslinking efficiency of many CLIP protocols. This thesis describes the incorporation of the photo-crosslinking unnatural amino acid p-azido-L-phenylalanine (AzF) into the Hepatitis C Virus (HCV) non-structural protein 3 helicase (NS3h) for photo-crosslinking and in vitro analysis of the potential binding sites found within the HCV RNA genome. From the five potential sites identified from the NS3h crystal structure for AzF incorporation, two sites, E503AzF and Q580AzF, allowed for nucleic acid photo-crosslinking with fluorescently labelled DNA substrates. We further tested if these mutations adversely affected NS3h and binding activity through a molecular beacon helicase assay and fluorescence polarization methods. We found that E503AzF unexpectedly had a faster unwinding rate than wild type (WT) NS3h and managed to have a similar binding affinity to the tested DNA substrate. Finally, we found that there was a 5-fold increase in the photo-crosslinking efficiency of nucleic acids for E503AzF NS3h mutant compared to our WT NS3h at 254 nm UV light. We are currently working on methods for our CLIP-based protocol to ensure quality RNA footprint generation and purification from photo-crosslinked NS3h. Other work contained in this thesis consists of using Prevotella sp. P5-125 Cas13b (PspCas13b), a clustered regularly interspaced short palindromic repeats (CRISPR) RNA-targeting system, which has been previously shown to knockdown viral RNA and mRNA through designable guide CRISPR RNA (crRNA). Here we incorporated the photo-crosslinking ncAA AzF into PspCas13b to irreversibly bind the crRNA in an attempt to enhance knockdown efficiency and longevity of viral and mRNA targets. We were able to design a crRNA that produced significant knockdown targeting the luciferase mRNA of a luciferase rennilla reporter system. When targeting an HCV subgenomic replicon luciferase reporter system, knockdown was not observed. Additionally, the WT PspCas13b had photo-crosslinking to the bound crRNA and requires further optimization for future use.

Hepatitis C virus (HCV) infection is common among injection drug users (IDUs). There is accumulating evidence that circulating microRNAs (miRNAs) are related to HCV infection and disease progression. The present study was undertaken to determine the in vivo impact of heroin use on HCV infection and HCV-related circulating miRNA expression. Using the blood specimens from four groups of study subjects (HCV-infected individuals, heroin users with/without HCV infection, and healthy volunteers), we found that HCV- infected heroin users had significantly higher viral load than HCV-infected non-heroin users (p=0.0004). Measurement of HCV-related circulating miRNAs in plasma showed that miRs-122, 141, 29a, 29b, and 29c were significantly increased in the heroin users with HCV infection, whereas miR-351, an HCV inhibitory miRNA, was significantly decreased in heroin users as compared to control subjects. Further investigation identified a negative correlation between the plasma levels of miR-29 family members and severity of HCV infection based on aspartate aminotransferase to platelet ratio index (APRI). Heroin use and/or HCV infection also dysregulated a panel of plasma miRNAs. Taken together, these data for the first time revealed in vivo evidence that heroin use and/or HCV infection alter circulating miRNAs, which provides a novel mechanism for the impaired innate anti-HCV immunity among IDUs. Recent studies revealed that extracellular miRNAs were able to incorporate into cell-derived exosomes as a method of cell-to-cell interaction. Exosomes are a class of cell-released small vesicles that mediate intercellular communication by delivering functional factors to recipient cells. During HCV infection, the interaction between liver resident macrophages and hepatocytes is important for host defense and viral elimination, triggered by innate immune activation, especially Toll like receptors (TLR). In our study, we explored the role of macrophage-derived exosomes in the transmission of innate immune responses against HCV infection in hepatocytes, and the involvement of exosomal miRNAs in transferring the anti-HCV activities. We reported that upon TLR3 activation, macrophages shed exosomes that were able to attenuate HCV-JFH1 infection in Huh7 cells. We further demonstrated that exosomes from poly I:C treated macrophages were internalized by Huh7 cells, which induced the intercellular anti-HCV responses (type I interferon, interferon stimulated genes, etc.) and thus drastically inhibited HCV infection in Huh7 cells. Moreover, using an in vitro macrophage and Huh7 cell co-culture model, we also found exosomes mediated HCV suppression in Huh7 cells after TLR3 activation. The presence of exosome inhibitor in co-culture compromised the anti-HCV activity by TLR3-activated macrophages. Interestingly, the miRNA-29 family, which was reported to suppress HCV infection, was significantly increased in the macrophage exosomes after TLR3 activation. The inhibition of miRNA-29 partially compromised the anti-HCV activity of TLR3-activated macrophages, indicating the potential involvement of exosomal miRNAs in the transmission of anti-HCV activity from macrophages to Huh7 cells through exosomes. In conclusion, this study proposed an antiviral mechanism of TLR3 activation that involves the intercellular communication between immune cells and hepatic parenchymal cells via exosomes, and exosomal miRNAs. This discovery sheds light on exploiting the therapeutic potential of new drugs against HCV infection. / Pathology

This thesis aimed to describe the epidemiology of HCV in South Australian prisons - prevalence, transmission and risk factors. This thesis also aimed to determine the impact of incarceration on reported risk behaviours. A related objective was to evaluate the epidemiological effectiveness of the ELISA - 3 HCV antibody test using PCR as the gold standard. Finally, this thesis aimed to explore the potential for minimising HCV risk in the South Australian prison population. Methods: Two case note audits were conducted at each of eight publicly operated SA prisons ( in summer and winter ) to identify any documented HCV - antibody test results. Prisoners recruited at entry to prison were offered tests for HCV - antibody and completed a pre - entry risk factor survey. Participants completed additional risk factor surveys and ( if HCV - negative at last test ) underwent further antibody tests at three - monthly intervals for up to 15 months. A sample of participants also provided blood specimens for HCV - RNA testing. Limited stakeholder consultations with prison officers and nurses were also conducted. Quantitative data were analysed using univariate and multivariate techniques. Results: 1347 case notes were audited in summer, and 1347 in winter and an overall HCV prevalence of 42 % was estimated. In both univariate and multivariate analyses, HCV prevalence was significantly higher in female prisoners ( 65 % ), those aged above 28 years ( 48 % ), and in Indigenous prisoners originating from metropolitan areas ( 56 % ). Indigenous prisoners originating from remote areas had significantly lower HCV prevalence ( 20 % ). 666 prisoners were recruited at entry, and 42 % were estimated to be HCV - antibody positive. Three seroconversions were noted in 151 initially HCV - seronegative negative individuals followed up for a median time of 121 days - a rate 4.6 per 100 person years - but community exposure could not be ruled out. Overall agreement between HCV - antibody and HCV - RNA assays was 86 % ( 100% in the HCV negative samples ) - kappa = 0.71. Injecting history was highly prevalent in prison entrants ( 70 % ) and both community and prison injecting ( but not tattooing ) were independent predictors of entry HCV status. Prison history was also independently associated with entry HCV status. Injecting in prison during the study was infrequently reported, but significantly more likely in those testing HCV - antibody positive at prison entry ( risk ratio = 2.48, P = 0.046 ). Stakeholders were most supportive of strategies to increase education and to minimise risks associated with hair clippers, but did not support most other suggested preventive strategies. Other issues related to communicable diseases and infection control were explored in the stakeholder interviews. Conclusions: HCV prevalence in South Australian prisoners is extremely high and may have contributed to a ' ceiling effect ' , minimising the seroconversion rate observed in this population. Injecting is relatively infrequently reported in prison, but more likely in those already infected with HCV. Thus, contaminated injecting equipment represents a significant threat to other prisoners and prison staff. Strategies aimed at reducing HCV risk in prisons, which address the concerns of those expected to implement them, are proposed in this thesis. / Thesis (Ph.D.)--School of Population Health and Clinical Practice, 2006.

This thesis aimed to describe the epidemiology of HCV in South Australian prisons - prevalence, transmission and risk factors. This thesis also aimed to determine the impact of incarceration on reported risk behaviours. A related objective was to evaluate the epidemiological effectiveness of the ELISA - 3 HCV antibody test using PCR as the gold standard. Finally, this thesis aimed to explore the potential for minimising HCV risk in the South Australian prison population. Methods: Two case note audits were conducted at each of eight publicly operated SA prisons ( in summer and winter ) to identify any documented HCV - antibody test results. Prisoners recruited at entry to prison were offered tests for HCV - antibody and completed a pre - entry risk factor survey. Participants completed additional risk factor surveys and ( if HCV - negative at last test ) underwent further antibody tests at three - monthly intervals for up to 15 months. A sample of participants also provided blood specimens for HCV - RNA testing. Limited stakeholder consultations with prison officers and nurses were also conducted. Quantitative data were analysed using univariate and multivariate techniques. Results: 1347 case notes were audited in summer, and 1347 in winter and an overall HCV prevalence of 42 % was estimated. In both univariate and multivariate analyses, HCV prevalence was significantly higher in female prisoners ( 65 % ), those aged above 28 years ( 48 % ), and in Indigenous prisoners originating from metropolitan areas ( 56 % ). Indigenous prisoners originating from remote areas had significantly lower HCV prevalence ( 20 % ). 666 prisoners were recruited at entry, and 42 % were estimated to be HCV - antibody positive. Three seroconversions were noted in 151 initially HCV - seronegative negative individuals followed up for a median time of 121 days - a rate 4.6 per 100 person years - but community exposure could not be ruled out. Overall agreement between HCV - antibody and HCV - RNA assays was 86 % ( 100% in the HCV negative samples ) - kappa = 0.71. Injecting history was highly prevalent in prison entrants ( 70 % ) and both community and prison injecting ( but not tattooing ) were independent predictors of entry HCV status. Prison history was also independently associated with entry HCV status. Injecting in prison during the study was infrequently reported, but significantly more likely in those testing HCV - antibody positive at prison entry ( risk ratio = 2.48, P = 0.046 ). Stakeholders were most supportive of strategies to increase education and to minimise risks associated with hair clippers, but did not support most other suggested preventive strategies. Other issues related to communicable diseases and infection control were explored in the stakeholder interviews. Conclusions: HCV prevalence in South Australian prisoners is extremely high and may have contributed to a ' ceiling effect ' , minimising the seroconversion rate observed in this population. Injecting is relatively infrequently reported in prison, but more likely in those already infected with HCV. Thus, contaminated injecting equipment represents a significant threat to other prisoners and prison staff. Strategies aimed at reducing HCV risk in prisons, which address the concerns of those expected to implement them, are proposed in this thesis. / Thesis (Ph.D.)--School of Population Health and Clinical Practice, 2006.

This thesis aimed to describe the epidemiology of HCV in South Australian prisons - prevalence, transmission and risk factors. This thesis also aimed to determine the impact of incarceration on reported risk behaviours. A related objective was to evaluate the epidemiological effectiveness of the ELISA - 3 HCV antibody test using PCR as the gold standard. Finally, this thesis aimed to explore the potential for minimising HCV risk in the South Australian prison population. Methods: Two case note audits were conducted at each of eight publicly operated SA prisons ( in summer and winter ) to identify any documented HCV - antibody test results. Prisoners recruited at entry to prison were offered tests for HCV - antibody and completed a pre - entry risk factor survey. Participants completed additional risk factor surveys and ( if HCV - negative at last test ) underwent further antibody tests at three - monthly intervals for up to 15 months. A sample of participants also provided blood specimens for HCV - RNA testing. Limited stakeholder consultations with prison officers and nurses were also conducted. Quantitative data were analysed using univariate and multivariate techniques. Results: 1347 case notes were audited in summer, and 1347 in winter and an overall HCV prevalence of 42 % was estimated. In both univariate and multivariate analyses, HCV prevalence was significantly higher in female prisoners ( 65 % ), those aged above 28 years ( 48 % ), and in Indigenous prisoners originating from metropolitan areas ( 56 % ). Indigenous prisoners originating from remote areas had significantly lower HCV prevalence ( 20 % ). 666 prisoners were recruited at entry, and 42 % were estimated to be HCV - antibody positive. Three seroconversions were noted in 151 initially HCV - seronegative negative individuals followed up for a median time of 121 days - a rate 4.6 per 100 person years - but community exposure could not be ruled out. Overall agreement between HCV - antibody and HCV - RNA assays was 86 % ( 100% in the HCV negative samples ) - kappa = 0.71. Injecting history was highly prevalent in prison entrants ( 70 % ) and both community and prison injecting ( but not tattooing ) were independent predictors of entry HCV status. Prison history was also independently associated with entry HCV status. Injecting in prison during the study was infrequently reported, but significantly more likely in those testing HCV - antibody positive at prison entry ( risk ratio = 2.48, P = 0.046 ). Stakeholders were most supportive of strategies to increase education and to minimise risks associated with hair clippers, but did not support most other suggested preventive strategies. Other issues related to communicable diseases and infection control were explored in the stakeholder interviews. Conclusions: HCV prevalence in South Australian prisoners is extremely high and may have contributed to a ' ceiling effect ' , minimising the seroconversion rate observed in this population. Injecting is relatively infrequently reported in prison, but more likely in those already infected with HCV. Thus, contaminated injecting equipment represents a significant threat to other prisoners and prison staff. Strategies aimed at reducing HCV risk in prisons, which address the concerns of those expected to implement them, are proposed in this thesis. / Thesis (Ph.D.)--School of Population Health and Clinical Practice, 2006.

Host-pathogen interactions in Hepatitis C Virus infection: Deciphering the role of host proteins and microRNAs Hepatitis C virus (HCV) is a positive sense single stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV genome consists of a single open reading frame flanked by highly structured 5‟ and 3‟ untranslated regions (UTRs) at both ends. Unlike cellular mRNAs, HCV RNA translation is independent of the cap structure and is mediated by an internal ribosomal entry site (IRES) present in the 5‟UTR. HCV replication begins with the synthesis of a complementary negative-strand RNA using the positive strand RNA genome as a template catalyzed by the NS5B RNA dependent RNA polymerase (RdRp). The de novo priming of HCV RNA synthesis by NS5B occurs at the very end of the 3‟UTR. The 3‟UTR is organized into highly structured regions namely the variable region, poly U/UC region and the 3‟X region. These regions contain cis-acting elements that determine the efficiency of viral replication. In addition, the interaction of trans-acting factors with the 3‟ UTR is also important for regulation of HCV replication. HCV 3‟UTR interacts with several cellular proteins such as the human La protein, polypyrimdine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) and Human antigen R (HuR). However, the molecular basis of regulation of viral replication by these proteins is not well understood. Many proteins that are hijacked by HCV as well as other cytoplasmic RNA viruses, such as La, PCBP2, HuR and PTB are RNA binding proteins (RBPs). They are involved in post transcriptional regulation of cellular gene expression. Thus the subversion of these proteins by the virus can affect their normal physiological functions. In addition to proteins, recent reports also describe the involvement of non-coding RNAs including microRNAs (miRNA) and long non coding RNAs (lncRNA) in HCV infection. miRNAs can either directly bind to the HCV genome and regulate its life cycle or indirectly modulate the expression of host proteins required by the virus. miRNAs that are differentially regulated in virus infected tissues or body fluids of infected patients can also serve as biomarkers for diagnosis of various stages of the disease. Hence, it was planned to study the role of host proteins and miRNAs in the HCV life cycle and pathogenesis to have novel insights into the biology of HCV infection. Riboproteomic studies have identified several host proteins that directly interact with the 5‟ and/or 3‟UTRs of the HCV RNA. One of the RNA binding proteins that predominantly interact with the 3‟UTR of HCV RNA was found to be HuR. In the present study, we have extensively characterized the interaction between HuR and HCV 3‟UTR and studied its functional implications in HCV life cycle along with other host factors. Characterizing the HCV 3’UTR–HuR interaction and its role in HCV replication HuR is a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress. Whole genome siRNA knockdown and other studies have suggested that HuR is essential for HCV replication. However, the molecular mechanism of its involvement in this process was not clear. We observed that siRNA mediated knockdown of HuR reduces the HCV RNA and protein levels. Immunofluorescence studies indicated that HuR relocalizes from the nucleus to the cytoplasm in HCV infected cells. Through confocal microscopy and GST pulldown assays, we have demonstrated that HuR co localizes with the viral polymerase, NS5B and directly interacts with the NS5B protein. Membrane flotation assays showed that HuR is present in the detergent resistant membrane fractions which are the active sites of HCV replication. In addition to the interaction of HuR with the viral protein NS5B, we also characterized its interaction with the viral RNA. Direct UV cross linking assays and UV cross linking immunoprecipitation assays were performed to demonstrate the interaction of HuR with the HCV 3‟UTR. The RRM3, hinge region and RRM1 of HuR were found to be important for binding. Further, we observed that HuR competes with PTB for binding to the 3‟UTR when cytoplasmic S10 extracts or recombinant proteins were used in UV cross linking assays. In contrast, the addition of HuR facilitated the binding of La protein to the HCV 3‟UTR in the above assays. Competition UV cross linking assays indicated that both HuR and PTB bind to the poly U/UC region of the 3‟UTR while La binds to the variable region. HuR and La showed higher affinities for binding to the 3‟UTR as compared to PTB in filter binding assays. Since HuR and PTB interact with the same region on the 3‟UTR and HuR showed ~4 fold higher affinity for binding, it could displace PTB from the 3‟UTR. Next, we investigated the roles of HuR, PTB and La in HCV translation and replication in cell culture using three different assay systems, HCV sub genomic replicon, HCV bicistronic SGR-JFH1/Luc replicon as well as the infectious HCV full length RNA (JFH1). Results clearly indicated that HuR and La are positive modulators of HCV replication. Interestingly, PTB facilitated HCV IRES mediated translation but appeared to have a negative effect on HCV replication. The positive effectors, HuR and La showed significant co localization with one another in the cytoplasm in immunofluorescence studies. GST pulldown and coimmunoprecipitation experiments indicated protein-protein interactions between HuR and La but not between HuR and PTB. Through quantitative IP-RT assays, we demonstrated that the overexpression of HuR in HCV RNA transfected cells increases the association of La with the HCV RNA while HuR knockdown reduces the association of La with the HCV RNA. Previous studies in our laboratory have shown that La helps in HCV genome circularization. The addition of HuR significantly increased La mediated interactions between the 5‟UTR and the 3‟UTR of HCV RNA as monitored by 5‟-3‟ co precipitation assays, suggesting a possible mechanism by which cooperative binding of HuR and La could positively regulate HCV replication. Taken together, our results suggest a possible interplay between HuR, PTB and La in the regulation of HCV replication. Studying the role of HuR- associated cellular RNAs in HCV infection HuR belongs to the category of mRNA turnover and translation regulatory proteins (TTR-RBPs), which are capable of triggering rapid and robust changes in cellular gene expression. HuR plays a role in several post transcriptional events such as mRNA splicing, export, stability and translation. In the present study, we have investigated the possible consequences of relocalization of HuR on cellular processes in the context of HCV infection. We observed that 72h post transfection of infectious HCV-JFH1 RNA, there is an increase in the mRNA levels of some of the validated targets of HuR including the vascular endothelial growth factor A (VEGFA), dual specificity phosphatise 1 (MKP1) and metastasis - associated lung adenocarcinoma transcript (MALAT1). IP-RT assays demonstrated that the association of HuR with VEGFA and MKP1 was higher in HCV-JFH1 RNA transfected cells as compared to the mock transfected cells indicating that increase in HuR association could probably help in stabilization of these mRNAs. Interestingly, we observed that the association of HuR with the lncRNA MALAT1 decreases in the presence of HCV RNA, while its RNA levels increased. Earlier it has been reported that MALAT1 interacts with HuR and was predicted to interact with La. We confirmed the interaction of both HuR and La proteins with MALAT1 RNA in vitro and in the cell culture system. Results from our time course experiments suggest that relocalization of HuR and La upon HCV infection might decrease their association with the nuclear retained MALAT1 RNA leading to significant reduction in MALAT1 RNA levels at the initial time points. However at later time points, MALAT1 was found to be unregulated through activation of the Wnt/beta-catenin pathway as demonstrated using a chemical inhibitor against β-catenin. Since MALAT1 is a known regulator of epithelial mesenchymal transition (EMT) and metastasis, we further studied the physiological consequence of the observed increase in MALAT1 levels upon HCV infection. Cell migration and cell invasion studies suggested that the knockdown of MALAT1 led to the inhibition of HCV- triggered wound healing and matrigel invasion and also rescued the down regulation of E-Cadherin protein levels, an EMT marker. Our study highlights the importance of the lncRNA, MALAT1 in HCV infection and suggests its possible involvement in HCV induced HCC. Investigating the role of miRNAs in HCV pathogenesis and replication miRNAs can also regulate HCV infection and pathogenesis in multiple ways. It is known that under disease conditions, there is aberrant expression of intracellular as well as circulating miRNAs. We have investigated the expression profile of 940 human miRNAs in HCV infected patient serum samples to identify the differentially regulated miRNAs. miR-320c, miR-483-5p and the previously reported miR-125b were found to be upregulated in the serum of cirrhotic and non-cirrhotic HCV infected patient serum samples. All three miRNAs were also unregulated in the cell culture supernatant of HCV infected cells as well as within the HCV infected cells. miR-483-5p was specifically enriched in the exosomes isolated from patient serum samples. Knockdown of miR-320c and miR-483-5p did not have significant effect on HCV replication while knockdown of miR-125b affected HCV replication through regulation of one of its target genes, HuR. We observed that with time, miR-125b levels in HCV-JFH1 RNA transfected cells increase while the HuR protein levels decrease. Using luciferase reporter constructs, we demonstrated that the decrease in HuR protein levels is indeed mediated by miR-125b. Mutations in the target site of miR-125b in the HuR 3‟UTR prevented the down regulation of luciferase activity. Next we tested the effect of silencing miR-125b on HCV replication. Knockdown of miR-125b prevented the reduction in HuR protein levels but with no significant effect on HCV replication. It appeared that the HuR protein already present in the cytoplasm could be sufficient to support HCV replication. Hence similar experiments were carried out in cells depleted of HuR using either siRNA against HuR or a chemical inhibitor of nucleocytoplasmic transport of HuR, Leptomycin B. We observed that when the intracellular levels of HuR are reduced using either of the two approaches, there is a decrease in HCV replication. This is in accordance with the results obtained in the first part of the thesis. However when miR-125b was silenced in HuR depleted cells, we noticed an upregulation in the HuR protein levels by western blot analysis and a consequent increase in HCV RNA levels as quantified by qRT-PCR. From our findings, we can conclude that miR-125b mediated regulation of HuR plays an important role in HCV replication. We hypothesize that this could be a cellular response to HCV infection to which the virus responds by inducing protein relocalization. Altogether, these studies outline the importance of host factors including cellular proteins and non-coding RNAs in the regulation of HCV life cycle and pathogenesis. Results reveal the mechanistic insights into how HCV infection triggers host defense pathways, which are evaded by the virus by counter strategies.

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.

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.

Hepatitis C Virus (HCV) infects 170 million people worldwide. The current treatment regimen, which is combination therapy with pegylated interferon (PegIFN) and Ribavirin (Rib), cures only 50% of the patients infected with the most prevalent HCV genotype. Therefore, there is a pressing need to understand the molecular mechanism of interferon resistance and to develop a prognostic tool to predict who will respond to treatment before initiation of therapy. It has been firmly established that the virus-host interaction plays an important role in determining treatment outcomes. My thesis investigated the host factors that are involved in interferon resistance with an aim to provide insights into the molecular mechanism of IFN resistance. cDNA microarray analysis identified 18 differentially expressed hepatic genes from pretreatment liver tissues of responders (Rs) and non-responders (NRs). Based on the differential expression levels of these 18 genes, a prognostic tool was developed to predict who will respond to therapy, with a positive predicting value (PPV) of 96%. Most of these 18 genes are interferon stimulated genes (ISGs) and they are more highly expressed in NR livers, indicating that preactivation of interferon signaling in the pre-treatment liver tissues contributes to NR. 3 out of the 18 genes are involved in an ubiquitin-like ISG15/USP18 signaling pathway that plays an important role in interferon response. Over-expression of USP18 and ISG15 in the pretreatment liver tissues of NR promotes HCV production and blunts interferon anti-HCV activity. There exists a distinct cell-type specific ISG activation in the pretreatment liver tissues of Rs and NRs. Up-regulation of the two ISGs that I tested (ISG15 and MxA) was found mainly in hepatocytes in NRs while ISG activation was preferentially observed in macrophages in Rs. Taking all these data together, pre-activation of interferon signaling and cell-type specific gene activation in the pretreatment liver tissues of patients infected with HCV are associated with treatment non-response. HCV exploits the host interferon system to favour its persistence by enhanced replication /secretion stimulated by a few ISGs (ISG15, USP18) in response to IFN. The developed prognostic tool can be used to stratify patients for treatment and the novel insights of the molecular mechanism of IFN resistance in HCV patients offer potential drug targets for future development.