The original aim of the project was to determine whether a non-poliovirus HEV could evolve to use the poliovirus receptor (PVR). A variety of methods were used to exploit aspects of the evolutionary capacity of viruses to achieve this goal. Although the aim was not attained, the investigation of recombinants between different HEVs yielded interesting results, which were pursued further. Two approaches were developed: in vitro generation of recombinant viruses and phenotypic analysis of such chimeras and the selection for recombinant viruses in vitro. In vitro generation of reciprocal recombinants between the structural and the non-structural coding region of coxsackievirus A21 (CVA21) and poliovirus type 3 (PV3) was initiated. Transfected and passaged chimeras did not produce infectious virions. Immunofluorescence analysis of VP1 protein expression suggested that the recombinants were not acytopathic. A series of assays were then carried out to investigate the nature of the defect. HeLa S10 translation/transcription reactions of the in vitro generated recombinants expressed the correct protein-processing pattern suggesting efficient processing occurred in vitro. Replication assays demonstrated that the chimeras were replication competent. Trans-encapsidation experiments were then carried out and preliminary results strongly suggested that the defect could lie at the packaging level. Selection of recombinants in vivo, without predetermining the crossover sites, was also conducted. Under the conditions used, recombinant between CVA21 and PV3 impaired genomes and echovirus 7 (EV7) and PV3 impaired genomes proved to be unsuccessful. Characterisation of the impaired parental genomes used for the experiment needs to be carried out. However, recent reports of recombinants between Sabin polioviruses and HEV-C confirm the possibility of such a recombination event occurring and emphasize concerns regarding the success of the polio eradication program.

Chronic hepatitis C virus (HCV) infection causes inflammation of the liver, which can lead to fibrosis and cirrhosis over time. Whether liver damage is a consequence of viral infection or is due to an immune mediated response is not clear. Steatosis is a histopathological feature often found in HCV infected patients. Steatosis is the accumulation of intracytoplasmic lipid droplets within hepatocytes. It has been linked to the progression of fibrosis (Adinolfi et al., 2001). Steatosis was found significantly more frequently in patients infected with HCV genotype 3 than those infected with genotype 1 (Mihm et al., 1997). Currently there is no cell-based method of investigating the life cycle of HCV genotype 3 and transgenic mice studies have been restricted to genotype 1 proteins. Three chimpanzees experimentally infected with HCV showed differential regulation of genes encoding enzymes concerned with lipid metabolism. Treatment of HCV genotype 1b replicon containing cells with cerulenin, which inhibits fatty acid synthase, reduced replication of HCV RNA in a dose dependent manner (Su et al., 2002). Polyunsaturated fatty acids (PUFAs) have recently been shown to inhibit replication of a genotype 1b sub-genomic replicon. PUFAs are essential and are known to down regulate lipogenic gene expression. However, the inhibitory effect of PUFAs on HCV RNA levels was thought to be independent of their inhibitory effect on fatty acid biosynthesis (Kapadia et al., 2005). To assess the effects of cerulenin and fatty acids on HCV genome replication we measured replication by northern blot analysis of total HCV RNA and using a replicon expressing luciferase. HCV protein production was measured by western blot using an antibody to the NS5A protein. To examine the effect on long chain fatty acid synthesis, we measured incorporation of 14C acetate into total cellular lipids. Toxicity was assayed using mitochondrial enzyme activity assays. Treating genotype 1b replicon cells with 30 μM cerulenin led to inhibition of fatty acid biosynthesis and a corresponding inhibition of HCV RNA replication. However, at this level of cerulenin, only 60 % of cells were viable. Inhibition of fatty acid biosynthesis was not observed at the lower non-toxic concentrations of 10 μM and 3 μM, although HCV replication was inhibited. These experiments were repeated using more frequent media changes and different suppliers of cerulenin. However, similar results were obtained. When a genotype 2a replicon expressing cell line (JFH1) was treated with cerulenin it was possible to inhibit both HCV RNA levels and fatty acid biosynthesis in a dose dependant manner. Furthermore cerulenin treatment of an alternative genotype 1b expressing cell line led to an inhibition of fatty acid synthesis in a dose dependent manner. We have studied the effects of the PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on JFH1 replicon (genotype 2) replication using both constitutive and transiently expressing systems. For a control, we used oleic acid, a monounsaturated fatty acid. DHA and EPA administered from 3 to 100 μM concentration showed a dose responsive reduction in replication. Fatty acid biosynthesis was also inhibited; however at the higher concentrations there were reductions in cell viability. Oleic acid did not effectively inhibit JFH1 replication even though, at higher concentrations, there was a small reduction in 14C acetate incorporation. Initial immunofluorescence data indicated that NS5A foci were not disrupted by treatment of cells with PUFAs and fluorescence recovery after photobleaching data indicated that PUFAs did not increase ER membrane fluidity. A genotype 3 genome was amplified and sequenced using reverse-transcription polymerase chain reaction (RT-PCR) from the serum of an HCV genotype 3ainfected patient. A majority sequence was assembled and amplification products were ligated into vectors, which were sequenced and mutated back to the majority sequence. The genotype 3 genome was modified by the exclusion of the structural genes and non-structural (NS) protein 2. A bicistronic replicon was created in which the HCV internal ribosome entry site (IRES) controlled expression of the selectable marker neomycin phosphotransferase and the encephalomyocarditis virus IRES controlled expression of the NS proteins. RNA replicons were transcribed and electroporated into HuH-7 cell lines. A transiently expressing replicon was made by replacing the neomycin gene with a firefly luciferase gene. Cells expressing neither the constitutively nor the transiently genotype 3 replicon sustained viral replication. In conclusion cerulenin inhibited HCV replication at levels, which did not inhibit fatty acid biosynthesis and were not toxic. There was toxicity at cerulenin concentrations, which inhibited fatty acid biosynthesis. Cerulenin inhibited replication but by a mechanism other than inhibition of fatty acid biosynthesis. Cells with different passage histories were shown to behave differently to each other in their response to drugs. The PUFAs, DHA and EPA exert an inhibitory effect on HCV replicon replication and fatty acid biosynthesis at non-toxic levels. Oleic acid did not inhibit HCV replication at equivalent concentrations. The mechanism behind PUFA inhibition of HCV RNA levels is still unknown. An attempt to create genotype 3 constitutively and transiently expressing replicon HuH-7 cell lines failed.

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.

Retroviruses must integrate their genome into the host DNA as a necessary step of their replication cycle. Normally, retroviruses integrate into somatic cells and are transmitted, from infected to uninfected hosts, as “exogenous” retroviruses. On rare occasions, they can infect germ line cells and become part of the host genome as “endogenous” retroviruses (ERVs), which are transmitted vertically to the offspring and inherited as Mendelian genes. During evolution, most ERVs have accumulated mutations that rendered them defective and unable to produce infectious viral particles. Some ERVs, however, have maintained intact open reading frames for some of their genes, and have been co-opted by the host as they fulfil important biological functions. Sheep betaretroviruses represent a unique model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) co-exists with the highly related endogenous JSRVs (enJSRVs). The sheep genome harbours at least twenty-seven enJSRV loci and, most likely, the process of endogenization is still occurring. During evolution, one of these enJSRV loci, enJS56A1, has acquired a defective and transdominant Gag polyprotein that blocks the late replication steps of related retroviruses, by a mechanism known as JSRV late restriction (JLR). Interestingly, enJSRV-26, a provirus that integrated in the sheep germ line less than two hundred years ago, possesses the unique ability to escape JLR. In this thesis, the molecular basis of JLR escape was investigated. The main determinant of JLR escape was identified in the signal peptide of enJSRV-26 envelope protein (SP26). A single amino acid substitution in SP26 was found to be responsible for altering its intracellular localization as well as its function as a post-transcriptional regulator of viral gene expression. Interestingly, interference assays demonstrated that enJSRV-26 relies on the presence of the functional signal peptide of enJS56A1 envelope protein (SP56) in order to escape JLR. In addition, the ratio between enJSRV-26 and enJS56A1 Gag polyproteins was found to be critical to elude JLR. Finally, sequence analyses revealed that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus, reinforcing the hypothesis that this locus has provided an evolutionary advantage to the host. This study unveils critical aspects of JLR that were previously unknown, and provides new insights on the molecular mechanisms governing the interplay between endogenous and exogenous sheep betaretroviruses.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Present estimates predict that between 120-130 million people worldwide are infected with HCV with the majority of all infections progressing to chronicity, ultimately leading to fibrosis, cirrhosis and hepatocellular carcinoma. The virus, which belongs to the family Flaviviridae, has a single-stranded RNA genome of positive polarity that codes for a unique polyprotein of approximately 3000 amino acids. The structural proteins E1 and E2 constitute the viral envelope glycoproteins. These glycoproteins have multiple functions in the viral life cycle such as promoting viral entry and fusion, assembly of infectious virions and aid in viral persistence through immune escape. Numerous cell culture-adaptive mutations have been reported within the HCV glycoproteins. The value of such mutations in understanding the virus interaction(s) with cellular receptors and neutralizing antibodies was first recognised from studies characterizing the E2 cell culture adaptive mutation G451R. This single mutation altered the affinity of HCV to the cell surface receptors CD81 and SRB1 as well as increasing its sensitivity to neutralizing antibodies targeting the viral glycoproteins. A striking observation from previously reported E2 cell culture adaptive mutations is their frequent occurrence within a highly conserved region of E2, spanning residues 412-423. Indeed, the long-term passaging of JFH1 infected cells here in this study also created an adapted virus with a substitution at residue 415. The aim of this study was to determine the phenotypic changes to viral entry caused by mutations in this region. To do this, four JFH1 viruses containing the mutations N415D, T416A, N417S and I422L were constructed and characterized. These mutant viruses were found to have very similar phenotypes to the G451R virus, suggesting all E2 adaptive mutations are selected to alter a specific function in viral entry. Residues 412-423 of HCV E2 also constitute the epitope of the in-house generated broadly neutralizing antibody AP33. ELISA binding and virus infection inhibition assays using AP33 with the E2 mutant viruses provided important information regarding the E2 contact residues of this antibody. In a separate study, intergenotypic chimeric JFH1 viruses were generated and characterised. Viable intra- and intergenotypic JFH1 chimeric viruses have previously been generated by different research groups by replacing the core to NS2 genes of JFH1 with those from different genotypes. Many of these chimeric viruses required numerous cell culture adaptive mutations to permit efficient infectious virus production. In the present study, 5 intergenotypic viruses were constructed by replacing the JFH1 envelope genes with those from other HCV genotypes. Despite these chimeric genomes replicating efficiently, none were capable of producing infectious virus. These viral genomes also failed to acquire infectivity during pro-longed cell passaging, suggesting that replacing the JFH1 envelope glycoproteins with those from other genotypes may confer total incompatibility for virus assembly. In addition to this work, the infectivity of a previously generated genotype 4a/JFH1 chimera was improved by repeatedly passaging the virus infected cells. The chimeric virus contained the core to NS2 genes of a genotype 4a strain in place of the those from the original JFH1 sequence. A total of six-adaptive mutations were identified throughout the adapted genome that enhanced infectivity by more than 100-fold. Achieving higher titers with this chimera permitted studies on its viral entry properties as well as its sensitivity to neutralizing antibodies. The ability of the adapted virus 4a/JFH1 virus to spread during multiple rounds of infection was greatly reduced compared to WT/JFH1 due to its inefficient cell-to-cell spread. The 4a/JFH1 virions were also highly sensitive to neutralizing antibodies targeting both linear and conformational E2 epitopes, suggesting that the glycoproteins are more exposed on the surface of this virus. In its totality, this study has provided key insights into the viral entry and antibodymediated neutralization properties of cell-culture adapted and intergenotypic chimeric forms of the JFH1 virus.

Haemorrhagic septicemia (HS), caused by the Gram-negative bacterium Pasteurella multocida serotype B:2, is an economically important disease responsible for morbidity and mortality of bovines, especially buffaloes, in countries of South or Southeast Asia and Africa. A feature of this disease is the rapid spread of infecting bacteria from the respiratory tract to the blood and lymph to cause a fatal septicemia. To pass into the blood stream, the bacteria must migrate through the epithelial layer into the pulmonary interstitium. Avian serogroup A strains of P. multocida have been reported to invade cultured mammalian cells, but the behaviour of other of serogroups has not been reported. The main object of the work was to confirm that HS strains of P. multocida B:2 have the capacity to invade and survive within cultured mammalian cells, such as J774.2 cells (mouse macrophage-like cell lines) and BL-3 cells (bovine lymphoma cell line). Invasion, defined as adhesion to, followed by uptake by, or entry into, J774.2 macrophage cells or BL-3 cells was determined by: (I) counting of viable intracellular bacteria after killing extracellular bacteria with polymyxin and gentamicin, (II) Transmission electronic microscopy. Comparison of the invasiveness of a B:2 HS strain and its aroA derivative JRMT12 with that of P. multocida A:3 and E. coli XL1-Blue, showed that both P. multocida B:2 strains invaded both types of mammalian cells more readily than P. multocida A:3 and that E. coli XL1-Blue was essentially non-invasive. Both strains of P. multocida B:2 could survive within J774.2 macrophage and BL-3 cells for at least 2 h. A longer-term survival experiment (up to 6 h incubation) indicated that the numbers of intracellular bacteria declined between 4 to 6 h post-infection. It was shown by TEM that a significant proportion of the P. multocida B:2 bacteria were found within vacuoles in the cytoplasm of the mammalian cells with some free in the cytoplasm. A much reduced invasion capacity of P. multocida A:3 and E. coli XL1-Blue was detected. Different effects on the appearance and viability of J774.2 and BL-3 cells were observed by the trypan blue method and TEM when exposed to the P. multocida B:2 strains. Evaluation of cytotoxicity of P. multocida B:2 stains with J774.2 cells by the MTT assay produced unsatisfactory results.

At the beginning of this study, knowledge of autotransporter proteins was in its infancy and the B. pertussis proteins pertactin, BrkA and tracheal colonisation factor (Tcf) had not been recognised as members of this family. However, the sequence similarity between these Bordetella proteins and a number of other proteins from different genera soon became apparent and the Bordetella proteins were also included in the autotransporter protein family. It was the similarity between pertactin and other B. pertussis proteins that, due to a mis-primed PCR amplification, led to the preliminary data for this project. A B. pertussis genomic library was screened, using this amplicon s a Southern blot probe, and a cosmid was identified and subcloned. Analysis of the sequence led to the identification of a larger open reading frame which had not been previously recognised. The cosmid did not contain the entire open reading frame and a further 200 bp was obtained using an adapter-linked single specific primer-PCR (SSP-PCR). An open reading frame was predicted and the amino acid similarity strongly suggests that this gene product represents another member of the autotransporter family. This protein was named Bap-5 as the fifth member of the Bordetella autotransporter protein family. Motif analysis revealed an RGD integrin-binding motif and an SGSG glycosaminoglycan-binding motif, which implicates this protein as a putative adhesin. Although not begun at the start of this study, the B. pertussis genome sequence was near completion during the final stages of this work and a sequence identical to bap-5 was located in the sequence data.

Despite the importance of host species choice of mosquito vectors to the epidemiology and control of malaria, our understanding of the ecological and evolutionary factors that drive the host species preference in these vectors is very limited. My PhD thesis aimed to experimentally investigate the potential ecological and evolutionary determinants of the host species choice of the African malaria vectors Anopheles arabiensis and An. gambiae s.s, which are amongst the most highly specialized and efficient malaria vectors in the world, and identify a control strategy that reduces their anthrophily. I used a unique semi-field system where these vectors were able to interact naturally with hosts of different species to establish whether their fitness depends on type of host species, they encounter and feed upon. My initial prediction was that highly host-specific feeding behaviour of these vectors is a product of natural selection whereby mosquito fitness is highest on their naturally preferred host types. This prediction was met in An. arabiensis, whose feeding success and lifetime egg production was predicted to be higher on their naturally preferred bovid hosts. However, I did not detect any association between the preference of An. gambiae s.s for humans and their lifetime reproductive success, although they obtain larger blood meals and survived longer on these naturally preferred human hosts. These findings suggest the role of host species on mosquito fitness varies between vector species. I then evaluated whether the host species-specific fitness of malaria vectors may be attributed to intrinsic defensive behaviours and haematological properties that make some host species being more beneficial than others. My initial prediction was that mosquito feeding success and fitness would be the highest in the absence of host defensive behaviours and, more specifically, that the least defensive host species would be the most highly preferred in nature. I have found that the feeding success (probability of obtaining a blood meal) of An. arabiensis is greater on host species with least effective defenses (e.g. bovids). However, this association was not apparent for anthrophilic An. gambiae s.s. Surprisingly, I found that the subsequent fitness (blood meal size and survival) of both vector species was generally greater on hosts who were free to exhibit defensive behaviours than those whose behaviours were restricted. These findings suggest that natural physical defensive behaviours made by hosts including humans may not impose strong fitness costs to malaria vectors. Therefore, I conclude that if natural host defensive behaviours shape the host species preference of malaria vectors they do so by influencing the probability of acquiring a blood meal but not the value of the blood meal if obtained. I also assessed whether the nutritive value of host blood, as determined by haematological properties of packed cell volume (PCV) and haemoglobin concentration (Hb), could explain variation in fitness of malaria vectors on different host species. I found that the PCV and Hb of host species that are commonly encountered by malaria vectors in their natural environments vary significantly. I further found that the variation in these haematological properties influence the feeding success (e.g. blood intake rate) of the anthrophilic An. gambiae s.s but not the An. arabiensis. Anopheles gambiae s.s obtain full blood meal faster on hosts with low and medium levels of PCV. Surprisingly, these haematological traits were predicted to have opposite effects on the survival of both vector species. The survival of An. gambiae s.s was positively correlated with host PCV, but negatively correlated with their Hb. In contrast, the survival of An. arabiensis was predicted to be positively correlated with host Hb, but negatively related with PCV. Overall, there was no clear evidence that haematological properties of the host species preferred by these mosquito vectors are optimal for their fitness. I then extended my investigations to a laboratory investigation to measure the impact of host species diversity on the fitness of An. gambiae s.s throughout their life. Under these conditions, I found that An. gambiae s.s had similar fitness after either feeding on a uniform (human-only) or mixed host species. These findings indicate that the blood composition of different species may be unlikely to reduce the fitness of An. gambiae s.s My PhD thesis also experimentally measured the impact of using simple intervention (e.g. an untreated bed net) on reducing the fitness of malaria vectors that acquire from human hosts. I found that the lifetime reproductive output of An. arabiensis on protected human was significantly lower than on bovid hosts. In contrast, the use of untreated nets by humans reduced survival of anthrophilic An. gambiae s.s, but the reduction was not predicted to be sufficient to significantly reduce the total lifetime reproductive output of these mosquitoes on human hosts than on animal alternatives. These findings suggest that the widespread use of simple untreated net may generate selection pressures for An. arabiensis to maintain their feeding on bovid hosts and to a lesser extent for An. gambiae s.s to reduce their anthrophily. The findings of my PhD research have implications for the epidemiology and control of malaria. I found that host species and their intrinsic properties may influence aspects of the feeding success, blood meal size and survival of malaria vectors which are the key determinants of malaria transmission intensity. I further demonstrate that selectively protecting humans with untreated nets may generate selection pressures for malaria vectors to reduce their anthrophily and consequently the transmission intensity of malaria. These findings suggest integrating existing interventions (e.g., use of untreated and insecticide treated bed nets) with environmental management that increases availability of an alternate host species (e.g. zooprophylaxis) may generate selection pressures for An. gambiae s.s to reduce their anthrophily, and An. arabiensis to maintain their feeding on alternative animal hosts (zoophily). Overall, I discuss the impacts of host species choice and intrinsic host factors on the fitness of African malaria vectors, the impacts of intervention on their fitness and their potential to select for a host shift, and the implications to epidemiology and control of malaria. I finally highlight gaps in the knowledge of the evolution of host species choice in malaria vectors where more research is required.

After the discovery of and development of diagnostic tests for hepatitis A and B it became apparent that a parenterally transmissible agent was responsible for cases of non-A non-B hepatitis. It was not until 1989, a further fifteen years later, that the agent responsible for most cases, the hepatitis C virus (HCV), was identified. This virus was initially thought to cause a mild self-limiting hepatitis. With the introduction of serological screening tests for HCV, it was soon apparent that it caused a chronic asymptomatic hepatitis which could be accompanied by significant fibrosis and sometimes cirrhosis and hepatocellular carcinoma. Epidemiological studies have since revealed that up to eighty percent of those infected develop chronic infection and that in the developed world hepatitis C virus infection is widespread. An estimated 0.5% of the UK population and 1.8% of the population in the USA are infected. It is now accepted that HCV can cause an asymptomatic indolent infection that can progress over decades with the development of cirrhosis. Hepatitis C infection is now established as the single most common condition referred to hepatologists and the leading indication for hepatic transplantation in Europe and the USA.Since the discovery of the hepatitis C virus much research has focussed on the epidemiology, natural history and treatment of the condition. The first chapter provides an overview of HCV and places in context the research contained in this thesis. In western countries the role of the healthcare setting in transmitting hepatitis C virus is poorly understood. We performed a large retrospective serological survey of hepatitis C virus infection in healthcare workers from theWest of Scotland. This revealed the overall prevalence of HCY infection in healthcare workers to be low regardless of involvement in exposure-prone procedures. This indicates that the risk of acquisition of hepatitis C virus infection by healthcare workers in an area with a large HCY infected intravenous drug using population is small and that the risk posed to patients by contact with the HCY infected healthcare workers is also low. Liver biopsy is the gold standard for assessing the extent of liver injury and determining prognosis in chronic hepatitis C. Non-invasive markers of liver injury have proved disappointing such that serial liver biopsies are required to monitor disease progression. In this thesis the hepatocellular enzyme a-glutathione stransferase is studied as a non-invasive marker of liver injury and as a means of assessing response to treatment with a - interferon. Disappointingly a-glutathione s-transferase performed poorly as a non-invasive marker of liver injury but showed some promise as a marker of response to interferon therapy. Three chapters of this thesis then focus on factors that may influence the natural history of chronic hepatitis C virus infection and in particular account for the variable rates of progression of liver fibrosis observed in chronic HCY. The role of iron and polymorphisms in the haemochromatosis gene (HFE) were studied. Carriage of HFE mutations was not related to the serum and liver markers of iron accumulation or the progression of liver fibrosis. Elevated liver iron concentrations were rarely observed, occurring in patients with more severe liver disease, and whether this was the cause or result of hepatocellular injury was unclear. Carriage of genetic polymorphisms in the renin-angiotensin system, which are associated with increased systemic renin-angiotensin system activity, were studied. This novel study was designed to explore whether these polymorphisms, known to influence the progression of renal and cardiac fibrosis in a number of cardiovascular diseases, influenced the progression of liver fibrosis in chronic HCY. In this study no association between these functional renin-angiotensin polymorphisms and the progression of liver fibrosis was observed. Hepatitis G virus infection was sought in a cohort of hepatitis C virus infected blood donors to investigate whether coinfection with this virus influenced the severity of hepatitis C virus related liver injury. Although hepatitis G virus co-infection was frequently observed it did not effect the severity of liver injury assessed biochemically and histologically. The factors that account for the variable rates of progression of chronic hepatitis C virus infection remain to be elucidated. Finally in this thesis the role of a - interferon therapy in the management of asymptomatic blood donors found to have hepatitis C virus infection at blood donation is studied. Most patients detected in this manner have only mild hepatitis with minimal fibrosis and little data exists as to whether they are appropriate candidates for treatment. In a randomised crossover study these patients were observed to tolerate a. - interferon therapy and have comparable response rates to other patient groups with chronic hepatitis C infection. These patients appear to be suitable candidates for treatment, although more data are required to establish what the prognosis is for these individuals if chronic HCV is left untreated.

No description available.