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Studies on the hepatitis C virus (HCV) life cycle have been aided by the development of in vitro systems that permit replication of the viral RNA genome and virus particle production. However, the exact functions of the viral proteins, particularly those engaged in RNA synthesis, are poorly understood. It is thought that NS4B, one of the replicase components, induces the formation of replication complexes (RCs) derived from host cell membranes. These RCs appear as punctate foci at the endoplasmic reticulum (ER) membrane and incorporate the viral and cellular proteins necessary for HCV RNA synthesis. To gain insight into the nature of RCs, green fluorescent protein (GFP) was inserted into the coding region of NS5A, one of the HCV-encoded replicase components. The impact of the GFP insertion was examined in the context of a subgenomic replicon (SGR) based on JFH1, a genotype 2a HCV strain that exhibits efficient RNA replication in cell culture. The resulting construct was capable of robust replication and allowed characterisation of NS5A in live cells that synthesised viral RNA. NS5A displayed a diffuse, ER-like distribution and was also observed in foci. These foci are presumed to represent RCs and NS5A was relatively immobile at these sites. This result was confirmed using SGRs harbouring a photoactivatable derivative of GFP (PAGFP). Utilising plasmid-encoded HCV polyproteins, it was apparent that the targeting of NS5A to these structures was dependent on NS4B. Removal of the NS4B coding region resulted in a diffuse, ER-like distribution of NS5A, with little evidence of the protein within RCs. NS5A was mobile under these conditions, suggesting that the dynamics of NS5A are linked to focus formation by NS4B. To further investigate these findings, a panel of 15 alanine substitutions was constructed in the C-terminal region of NS4B. Transient replication assays revealed that five mutants were incapable of replication, two displayed an attenuated phenotype, and eight exhibited replication levels comparable to the wild-type (wt) genome. Of the five non-replicating mutants, two were defective in their ability to produce foci, while one failed to generate any foci. Thus, the C-terminus of NS4B is important for RC formation. Loss of NS4B foci correlated with decreased NS5A located in these structures. Furthermore, NS5A hyperphosphorylation was reduced for mutants compromised in foci production. This suggests that the membranous changes induced by NS4B provide a favourable environment for post-translational modifications of NS5A. Interestingly, the remaining two non-replicating mutants displayed no impairment in foci production and the characteristics of NS5A were also unaltered. Therefore, in addition to producing the cellular environment for HCV genome synthesis, NS4B is likely to play a more direct role in RNA replication. HCV RCs are believed to be relatively enclosed structures that permit limited exchange of materials with the cytoplasm. In support of this hypothesis, previous reports have shown that NS5A is the only replicase component capable of restoring replication to defective genomes when supplied in trans. In those studies, SGRs harbouring replication-lethal NS4B mutations could not be rescued by trans-complementation. Utilising the five novel non-replicating genomes described above, the potential to trans-complement NS4B in transient replication assays was re-examined. Wt protein produced from a functional HCV replicon could trans-complement defective NS4B expressed from two of the five mutants. Moreover, active replication could be reconstituted from two defective viral RNAs harbouring mutations within NS4B and NS5A. These findings have important implications for our understanding of RC formation. Genome-length JFH1 RNA produces infectious virus particles in Huh-7 cells. Using this system, it has become increasingly apparent that some HCV-encoded replication components are also involved in virus assembly and release. To determine whether NS4B had any influence on these latter stages of the virus life cycle, the NS4B mutations that did not block RNA replication were introduced into the full-length JFH1 genome. While the majority of mutants had no effect on virus production, one mutant consistently enhanced infectious virus titres by up to five-fold compared to wt JFH1. Interestingly, introduction of the same mutation into a chimeric J6-JFH1 genome resulted in repressed virion production. Together, these results suggest that NS4B contributes to virus assembly and release in a genotype-specific manner. In an attempt to identify novel cellular proteins involved in HCV genome replication, a siRNA library targeting 299 nucleotide-binding proteins was screened. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tri-cistronic SGRs. While the U2OS cell line supported HCV RNA replication less efficiently compared to Huh-7 cells, this cell type was efficiently transfected with siRNA. Consequently, increased gene-silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells may be a suitable alternative to Huh-7 cells for HCV-related siRNA studies. For the library screen, all siRNAs were tested in both cell lines, and cell viability measurements allowed specific effects on viral RNA synthesis to be characterised. The screen identified several cellular proteins that enhanced and suppressed HCV RNA replication. This study provides an important framework for more detailed analyses of these proteins in the future.

The epidemiology and natural history of Hepatitis C has been studied in a large geographically determined population (Trent HCV study). It has previously been suggested that patients with Hepatitis C and a persistently normal Alanine aminotransferase (PNALT) represent a group of patients with mild disease and at low risk of disease progression. Patients with PNALT were, therefore, compared to those with an elevated ALT. The majority of patients initially fulfilling the definition of a PNALT had an abnormal ALT within 3 years of follow-up. They also demonstrated similar rates of fibrosis progression as a sub-group of HCV infected patients with an elevated ALT who were re-biopsied prior to any institution of therapy. They, therefore, warrant the same consideration with regard to treatment. The morbidity and mortality associated with Hepatitis C with severe fibrosis was assessed in a group of patients with a liver biopsy demonstrating Ishak fibrosis stage  4. A worse prognosis than previously reported was observed for this patient population. Once decompensation develops, HCV infection is associated with a high mortality rate. Indicators of poor synthetic liver function and hypergammaglobulinaemia were important prognostic factors for mortality, while combination antiviral therapy was associated with improved survival. The majority of HCV infected patients (75%) diagnosed with hepatocellular carcinoma (HCC) were known to have cirrhosis at least 6 months prior to diagnosis of HCC and were, therefore, amenable to surveillance. There was a variable application of surveillance, however, and no significant improvement in survival was demonstrated. Age, duration of infection and immunoglobulin G levels were associated with an increased risk of HCC in cirrhotic patients in the univariate analysis. Achieving an SVR was associated with a reduced risk. No variable in cirrhotic patients was shown to be independently associated with HCC in the multivariate analysis. A comparison of disease progression and treatment outcome in White and Asian (Indian subcontinent) patients was made. Asian patients generally presented at an older age and with more severe disease on biopsy. The patient’s ethnic group was not associated with the likelihood of either an SVR or completion of therapy. Instead cirrhosis and a raised GGT were associated with a failure to achieve SVR in the multivariate analysis. The platelet count is a surrogate marker for the severity of liver fibrosis and correlates with the Ishak fibrosis stage. An analysis of factors associated with an SVR was performed. In the multivariate model, age at start of treatment was the only independent predictor of SVR in Genotype 1, while estimated duration of infection and Ishak stage were predictors in genotype 2/3 patients. The platelet count was not an independent predictor of SVR or completion of therapy.

Hepatitis C Virus (HCV) exists as a population of sequence variants that evolves during infection adapting to host pressures. The main targets for the immune response are the envelope glycoproteins E1 and E2, which also mediate viral cell entry. The first hypervariable region (HVR1) of E2, previously implicated in the outcome of acute infection, has been a focus of many studies. However more broadly neutralising antibodies tend to target epitopes outside this region, yet evolution of full length E1E2 heterodimer is poorly understood. The HCV transmission and window period as well as seroconversion are the evolutionary events shaping primary infection hence influencing outcome of acute infection. However, due to the asymptomatic character of the early phases of HCV infection, evolutionary data describing this interval is still lacking depth. Defining the genetic and phenotypic characteristics of HCV population of sequence variants that establish infection in a new host would aid vaccine and new therapy design. This study aimed to identify patterns of HCV envelope glycoprotein evolution upon transmission and during early phases of disease. We studied this in three settings: experimental transmission of immunocompromised mice by known inoculum; occurrence of horizontal transmission in a haemodialysis unit between hypothesised source and index case individuals; and unrelated cases of acutely infected HCV patients. The single genome amplification (SGA) approach was utilised, which allowed us to accurately assign linkage between substitutions and determine the frequency distribution of E1E2 variants in analysed viral populations. Data from the first experimental setting indicates that a selective sweep occurs upon HCV transmission, with selective amplification of envelope sequence variants that possess fitness advantage at entry level. Molecular determinants associated with this enhanced infectivity have also been identified. In further part of the project we confirmed a horizontal infection in haemodialysis unit with use of phylogenetic methods and suggested revision of current safety guidelines. Analysis of sequences from the last setting showed that indeed HVR1 might not be a good enough indicator of evolutionary events in the acute phase, as linked substitutions occur also outside this region. Seroconversion is associated with increasing population diversity indicating role of antibodies in driving HCV evolution, which is host specific.

Background: Hepatitis C virus infection affects more than 170 million people worldwide and is frequently associated with chronic liver disease and hepatocellular carcinoma. No protective vaccine is yet available and the current standard of care, consisting of pegylated interferon alpha and ribavirin, has limited efficacy. Ribavirin is a key component of any effective anti-HCV regimen. However, accumulation of ribavirin in the red cell compartment not only reduces drug efficacy as a result of diversion to extra-hepatic sites but also produces haemolytic anaemia which can lead to dose reduction or discontinuation of treatment. Lipid or polymer based nanoparticles can be used to deliver therapeutic agents, such as drugs or small interfering RNAs (siRNAs) directly to their site of action. We therefore elected to develop new antiviral strategies based on the targeted delivery of ribavirin to hepatocytes, coupled with the identification of new therapeutic targets. In order to inform the rational use of direct intracellular delivery of ribavirin, we enquired whether variation in expression of the ribavirin transporter may determine drug uptake and permit the identification of individuals who would benefit from these alternative approaches to treatment. Aims: The aims of this study were to: • identify host proteins involved in virus replication • demonstrate reduction of viral replication by modulation of host gene expression • develop and test a nanoparticle based system for the delivery of therapeutic molecules, including siRNAs either alone or in combination with ribavirin. • assess the relationship between ribavirin uptake by primary human hepatocytes and expression of ribavirin receptors Methods: A subgenomic HCV replicon system was established to study the virus-host relationship and identify host proteins supporting viral replication by using stealth siRNA. Viral RNAs were in vitro transcribed and transfected into Huh7 cells and expression assessed using engineered GFP as a reporter gene. siRNAs were co-transfected with viral RNAs using a nucleofector. Modulation of host gene expression was measured by both quantitative RT-PCR and protein blotting. Liposomal nanoparticles containing ApoB-100 duplexes were supplied by Lipoxen. Primary human hepatocytes were isolated by a modified two step collagenase perfusion method and cultured on collagen coated plates. HPLC and real time PCR conditions were used to measure and correlate drug uptake and receptor expression respectively. Equilibrative nucleoside transporter (ENT1) gene was analysed by direct sequencing. Results: A JFH1 (HCV genotype 2a) virus based subgenomic replicon system was successfully established. Using this model system, host proteins VAP-A and STAT3 were shown to positively regulate virus replication while ACTN1 had no effect. Liposomes failed to deliver either siRNA targeted at apoB-100 or ribavirin and this was found to be due to structural instability of the delivery vehicle. In contrast, fluorescently labelled liposomes were stable and could be taken up by human hepatocyte cell lines under optimised conditions. A protocol capable of efficient isolation and culture of hepatocytes from human donor was validated. Data from primary human hepatocytes show that ENT1 expression was highly variable in different sets of primary livers and correlated strongly with ribavirin uptake. Strikingly, Huh7 cells did not take up ribavirin despite expressing wild type ENT1. It was also found that interferon alpha does not modulate ENT1 expression and therefore ribavirin uptake, suggesting it to be a highly unlikely mode of synergism between the two drugs. Conclusion: Modulation of host proteins VAP-A and STAT3 inhibited viral replication, confirming that host genes can be used as a potential target to inhibit viral replication. Liposomes used in this study were, however, found to be ineffective vehicles for the delivery of ribavirin or siRNA, as the majority of drug leaked before cellular uptake. Polymer based nanoparticles are currently being assessed for antiviral drug delivery. Variation in ENT1 expression may account for differences in response rate in patients receiving anti-HCV therapy. Results in the Huh 7 cell line suggest that, while ENT1 is necessary, other factors are also required to mediate ribavirin uptake.

Copies of author's previously published articles contained in back cover pocket. / Bibliography: leaves 129-152. / xiv, 152, [60] leaves, [28] leaves of plates : ill. (some col.) (some folded) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines hepadravirus and HDV replication and gene expression with particular emphasis on the block(s) preventing HBV infection invitro, the extent of the helper function provided by HDV by HBV and the mechanism of HDV RNA replication. / Thesis (Ph.D.)--University of Adelaide, Depts. of Microbiology and Immunology, 1992

Complex patterns of HCV infection are increasingly reported, particularly in highly exposed individuals, with multiple and variable subtype profiles seen in many chronic patients. This study aims to address some of the questions arising from this increasingly diverse and dynamic picture, both within hosts and at a population level. In Chapter 2 I find evidence for a highly dynamic infection profile in acute HCV, both in terms of viral load and the dominant subtype. I extrapolate these observations from individual patients to formulate a model of HCV transmission across a high-risk population in order to predict the impact of current and anticipated interventions in Chapters 3 and 4. I show that antiviral therapy and a putative vaccination can still have a significant impact on HCV prevalence at the population level, even when the latter offers only partial protection and in the epidemiological background of ongoing exposure. Thus, in an epidemic with more than one circulating strain it will be crucial for any individual or combination of interventions to target all variants present. In Chapter 5 I demonstrate that early viral load kinetics of patients initiating treatment are indicative of treatment outcome. Strain differences are also evident in the virologic response to treatment with hard-to-treat genotype 1 exhibiting a slower rate of viral load decline than genotypes 2 and 3.

Hepatitis C virus (HCV) is a major global pathogen estimated to infect over 170 million people worldwide. A recent study has shown that vaccination with adenoviral vectors, based on rare human and simian serotypes encoding the non-structural (NS) proteins of HCV, induces highly potent, multi-specific and durable T cell responses in healthy human volunteers. In this thesis I assess the safety and immunogenicity of these vaccines (ChAd3–NSmut and Ad6-NSmut), for the first time in HCV infected patients. This work also explores whether vaccine-induced T cell responses target in vivo circulating HCV antigens and common naturally occurring epitope variants. Patients with treatment naive chronic genotype 1 HCV infection were vaccinated (i.m.) with ChAd3-NSmut and Ad6-NSmut in a heterologous prime boost schedule, either with or without current IFN and ribavirin (IFN/RBV). Epitope-specific T cell responses were defined by fine mapping using HCV peptides. Circulating viral genomic sequence was determined in vaccinated patients at baseline and at any point of viral relapse. Cross-reactivity of vaccine-induced T cell responses was determined in T cell assays, using peptides corresponding to both circulating host virus and common population HCV epitope variants. An in vitro dendritic cell /T cell priming model was used to identify possible candidates for a cross-reactive vaccine immunogen at the most immunodominant epitope, NS3₁₄₀₆. 33 patients were vaccinated. Vaccination was well tolerated. At the highest vaccine dose (2.5 x 10¹⁰vp) vaccine-induced T cell responses were detectable in 11/20 patients receiving concurrent IFN/RBV and 2/4 patients receiving vaccination alone. In total 14 antigenic targets were identified, 2 of which have not previously been described. However, T cell responses were of lower magnitude and more narrowly focused than those observed in healthy volunteers vaccinated with the same regimen. Analysis of viral sequence showed that in many cases vaccine-induced T cells did not target the circulating virus. At the most immunodominant epitope (NS3₁₄₀₆), T cells induced by vaccination failed to target common circulating genotype 1 HCV variants. An in vitro model suggested that in order to target all genotype 1 sequences at this epitope, it would be necessary to insert both a genotype 1a and 1b version of this epitope into a vaccine immunogen. Vaccination with adenoviral vectors induces T cell responses in patients with chronic HCV infection, however immune responses are attenuated compared with healthy volunteers. Ultimately a successful therapeutic or prophylactic vaccine strategy will rely on inducing responses that target conserved or cross-reactive epitopes.