Hepatitis C virus (HCV) infection is the most common blood-borne infection in the United States (U.S.). The largest increases of incidence for HCV infection are reported in the Appalachian region. This study aimed to 1) examine the prevalence and trends of HCV infection in the U.S. from 1999 to 2012; 2) investigate barriers to HCV infection treatment in Northeast Tennessee and the U.S.; and 3) study characteristics and risk factors for HIV-infection and HCV-infection in Northeast Tennessee. In the U.S., data were obtained from the National Health and Nutrition Examination Survey (NHANES) 1999-2012 to study the prevalence of HCV infection and barriers to treatment. In Northeast Tennessee, hepatitis C and HIV/AIDS data were obtained from National Electronic Disease Surveillance System (NEDSS) and enhanced HIV/AIDS Reporting System (eHARS). Descriptive statistics and multiple logistic regression models were used for analysis. Odds ratios (OR) and 95% confidence intervals (CI) were reported. There was an estimated 3.8 million people having HCV antibody in the U.S. in 2012. No significant change was found in the prevalence of HCV infection during 1999 – 2012. The leading barrier to the treatment was cost issues in the U.S. (50.0%) and Northeast Tennessee (25.0%), respectively. HCV patients without symptoms in Northeast Tennessee were more likely to be untreated (OR: 3.08, 95% CI: 1.10-8.60) and patients without health insurance in the U.S. were more likely to be untreated than their counterparts (OR: 3.38, 95% CI: 1.14-10.05). The incidence of acute hepatitis C peaked in 2012-2013 in Northeast Tennessee, while the incidence of HIV/AIDS increased by 100% from 2013 to 2015. More injection drug users (IDUs) and less men who have sex with men (MSM) were observed in patients with HCV infection than in those with HIV infection (IDUs: 50.63% vs.16.38% p

Hepatitis (HCV) is a communicable disease that impacts many Americans. The scholarly literature lacked the knowledge pertaining to the relationships between poverty and HCV diagnosis and prescription for HCV medication. The purpose of the study was to measure the magnitude and statistical significance of these relationships, as modeled by the health belief model and public health surveillance and action framework. Specifically, the study was designed to determine whether there is a statistically significant relationship between living below the poverty line and being diagnosed with HCV, as well as living being below the poverty line and being prescribed HCV medication. A total of 78 records of HCV-positive individuals from the National Health and Nutrition Examination Survey dataset were evaluated by applying the statistical procedure of odds ratio (OR) analysis. The results of the analysis revealed that (a) there was not a statistically significant relationship between being below the poverty line and being diagnosed with HCV, OR = 0.99 (SE = 0.38, z = -0.03, p = .974); and (b) there was not a statistically significant relationship between being below the poverty line and being prescribed HCV medications, OR = 0.32 (SE = 0.55, z = -0.66, p = .507). Numerous recommendations for improving measurements of the relationship between poverty and HCV are provided. This study may promote positive social change by indicating the importance of poverty as an agenda item for public health policy and practice.

Lorsque l'hépatite chronique C a occasionné une cirrhose et que, du fait de ses complications, le pronostic vital est en jeu au terme de quelques mois, la transplantation hépatique (TH) représente l'unique traitement efficace,. Malheureusement, la récidive quasi-systématique de la cirrhose C, après la transplantation hépatique, est la principale barrière à la survie du greffon. De nombreux facteurs ont été associés à la sévérité des récidives, et une implication des lymphocytes T régulateurs CD4+CD25+ (Treg) et de certains immunosuppresseurs a été suggérée. Par ailleurs, le patient transplanté peut également être confronté au problème du rejet aigu d’allogreffe, qui est partiellement contrôlé par les Treg et par une thérapie immunosuppressive rigoureuse. Paradoxalement, plusieurs études ont suggéré que certains immunosuppresseurs sont moins efficaces que d’autres dans la prophylaxie du rejet d’allogreffe et peuvent même être associés à des épisodes de rejet plus fréquents. Il existait donc un besoin urgent d’évaluer le rôle joué par les immunosuppresseurs sur les Treg dans la récidive de la fibrose C et dans le rejet du greffon. Dans un premier temps, nous avons confirmé l’implication des Treg dans la progression de la récidive de l’hépatite C. En effet, les marqueurs associés à cette population sont surexprimés dans le foie et dans le sérum de patients, 1 an et 5 ans après la TH, et ce proportionnellement à la sévérité de la récidive. Dans un deuxième temps, nous avons évalué l’effet d’immunosuppresseurs utilisés après la TH (cyclosporine A (CsA), tacrolimus, rapamycine et mycophénolate mofétif) sur l’activité des Treg. Nous avons ainsi montré que seule la CsA a une action inhibitrice sur l’activité des Treg, et ce, uniquement aux doses thérapeutiques de 20 et 40 ng/mL (doses administrées au long terme, 5 ans après la TH). Cette inhibition de l’activité des Treg par la CsA ne modifie pas leur phénotype (expression protéique ou génique), mais conduit à la sécrétion d’IL-2 et d’IFN-γ par les Treg, cytokines de la voie Th1. Le mécanisme immunosuppresseur de la CsA étant d’inhiber la transcription de l’IL-2, via la voie calcineurine/N-FAT, nous avons tenté d’identifier si elle agissait sur les Treg par cette voie ou par une voie indépendante de la calcineurine. Deux observations ont renforcé l’hypothèse d’un mode d’action calcineurine/N-FAT - indépendant : (i) le fait que le tacrolimus, qui a le même mécanisme immunosuppresseur que la CsA, n’inhibe pas l’activité des Treg et (ii) le fait que NIM811, un analogue de la CsA n’agissant pas sur la voie de la calcineurine, inhibe l’activité des Treg aux mêmes doses que la CsA. Cette hypothèse a par ailleurs été directement confirmée par l’absence de modification du profil de déphosphorylation du facteur de transcription N-FAT, en présence de CsA. Enfin, bien que les corticoïdes soient connus pour préserver l’activité des Treg et induisent leur prolifération in vitro, ils sont incapables de reverser l’effet inhibiteur de la CsA sur les Treg. Nos résultats suggèrent donc qu’une dose thérapeutique de CsA inhiberait l’activité des Treg CD4+CD25+. Les cellules T régulatrices jouent un rôle important dans la tolérance du greffon et dans la sévérité des récidives après la TH, leur inhibition par la CsA pourrait alors favoriser le rejet du greffon et diminuer la sévérité des récidives. Ces résultats sont importants dans la mesure où la transplantation hépatique est à l’heure actuelle la seule alternative de survie au stade du carcinome hépatocellulaire, et qu’il n’existe aucun traitement efficace contre le rejet du greffon ou la récidive de l’hépatite C. Le traitement immunosuppresseur idéal n’existe pas, cependant il ne devrait pas augmenter l’activité suppressive des Treg, au risque de favoriser la récidive de l’hépatite C, ni inhiber cette activité, au risque de favoriser le rejet du greffon. / Liver transplantation (LT) remains the only effective therapeutic approach for cirrhosis related HCC patients. Inevitable hepatitis C virus (HCV) recurrence after liver transplantation is a major barrier to the survival of a transplanted liver. It may be promoted by immunosuppression and the emergence of CD4+CD25+ regulatory T cells (Treg). Transplanted patients are also been confronted to allograft rejection, which is partially controlled by Treg cells and the administration of an immunosuppressive therapy. However, some immunosuppressive drugs have been associated with more frequent graft rejection. In this context, it was important to assess the effect of immunosuppressive drugs on regulatory T cells, both in HCV recurrence and graft rejection. We have first confirmed the implication of Treg cells in hepatitis C recurrence progression. Indeed, regulatory T cells markers are over-expressed, 1 and 5 years after LT, both in the liver and in periphery and proportionally of the recurrence severity. In a second time, we have analysed the effect of immunosuppressive drugs used after LT (cyclosporine A (CsA), tacrolimus, rapamycine and mycophenolate mofetil) on regulatory T cell activity. We have shown that only low concentrations of CsA (20 and 40 ng/mL) inhibit regulatory T cell activity (these doses are used 5 years after LT). It seems that CsA does not affect regulatory T cell phenotype (protein and gene expression) but lead to a secretion of Th1 cytokines in Treg cells : IL-2 and IFN-γ. As CsA, is known to inhibit IL-2 transcription through the calcineurin/N-FAT pathway, we have tried to identify if CsA inhibits Treg cells via this pathway or via a calcineurin -independent pathway. Two observations have confirmed the hypothesis of a calcineurin -independent pathway : (i) tacrolimus, which have the same immunosuppressive mechanism as CsA, could not inhibit Treg activity, and (ii) NIM811, a calcineurin - independent CsA analog, inhibits regulatory T cell activity at the same concentrations than CsA. Moreover, this hypothesis has been directly confirmed by the absence of of modification of the N-FAT dephosphorylation profile. Lastly, corticoids, known to preserve Treg activity, could induce Treg cell proliferation in vitro. However, they could not reverse the inhibitory effect of CsA on Treg cells. Our results suggest that a therapeutical dose of CsA could inhibit CD4+CD25+ regulatory T cell activity. Treg cells play an important role in graft tolerance and hepatitis C recurrence after LT, so their inhibition by CsA could favour graft rejection and decrease recurrence severity. These results are important, as liver transplantation iscurrently the only survival alternative for HCC related patients. The ideal immunosuppressive therapy does not exist, but it would not increase Treg activity, which may promote hepatitis C recurrence, neither abrogate this activity due to the risk of graft rejection.

L’infection par le virus de l’hépatite C (HCV) évolue dans la plupart des cas en hépatite chronique et peut conduire à une cirrhose ou un carcinome hépatocellulaire. Malgré les grandes avancées dans le traitement de l’hépatite C qui permettent d’inhiber ou même de bloquer l’évolution de cette infection vers la chronicité, l’absence de vaccin ainsi que sa répartition sur la surface du globe nous permet de classer cette pathologie en problème majeur de santé publique. La majorité des traitements actuels ciblent les protéines virales et leur fonction. Cependant un grand nombre de mécanismes du cycle viral de HCV reste à élucider.Comme pour la grande majorité des virus à ARN de polarité positive, la réplication de HCV a lieu dans des membranes cellulaires modifiées. Le remaniement de ces membranes est en lien étroit avec la voie de sécrétion précoce de la cellule. Il a été montré que GBF1, un facteur d’échange nucléotidique des protéines G de la famille Arf qui régulent la dynamique membranaire, est un facteur nécessaire à la réplication de HCV. L’inhibition de GBF1 par la bréfeldine A (BFA) inhibe la voie de sécrétion des protéines cellulaires néosynthétisées et inhibe aussi la réplication de HCV. Pour étudier le rôle de GBF1 pendant l’infection nous avons établi des lignées résistantes à la BFA. Deux de ces lignées étaient 100 fois plus résistantes que les lignées parentales à l’apoptose induite par la BFA, à l’inhibition de la sécrétion des protéines et à l’inhibition de l’infection par HCV. Ce phénotype était dû à une mutation ponctuelle dans le domaine catalytique sec7 de GBF1 de ces lignées. Un autre groupe de lignées était partiellement résistantes à l’inhibition de la sécrétion des protéines par la BFA tout en conservant un niveau d’infection proche de celui des lignées parentales dans les mêmes conditions. Ces résultats suggèrent que la fonction de GBF1 pendant l’infection HCV ne serait pas réduite à la régulation de la voie de sécrétion, évoquant ainsi un rôle additionnel de GBF1 nécessaire pour la réplication de HCV.Par ailleurs, nous avons pu montrer à l’aide des mutants de délétion de la protéine GBF1, que l’activité catalytique du domaine sec7 était nécessaire. Ceci suggère l’implication d’une protéine de la famille Arf dans l’activation de l’infection HCV via GBF1. L’implication de Arf dans l’infection HCV a été confirmée par la surexpression de dominants négatifs de la protéine Arf1 et par l’inhibition de l’activité de l’ArfGAP1 (régulateur des Arf) par l’inhibiteur spécifique QS11.Nous avons ensuite testé l’implication des différents Arf sensibles à l’inhibition par la BFA (Arf1, 3 ,4 et 5), dans l’infection HCV à l’aide de si-RNA. Il a été montré que ces protéines Arf possèdent des fonctions redondantes. Nos résultats confirment l’implication de Arf1 et indiquent que les 3 autres protéines sont aussi impliquées dans l’infection HCV. D’une manière intéressante, la déplétion combinée des Arf inhibe fortement l’infection HCV suggérant ainsi un rôle essentiel de certaines protéines Arf, probablement en activant des facteurs cellulaires nécessaires à l’étape de réplication. L’étude des facteurs cellulaires impliqués dans l’infection HCV permet de mieux comprendre l’étape de réplication et par conséquent le cycle viral de HCV. Par ailleurs, l’étude de ces facteurs pourrait permettre le développement éventuel de stratégies antivirales ciblant des facteurs de la cellule hépatique indépendamment du génotype viral, limitant ainsi le risque d’émergence de variants résistants au traitement. / The hepatitis C virus (HCV) infection progresses in most of the cases into a chronic hepatitis and can lead to cirrhosis or hepatocellular carcinoma. Despite the recent improvement of hepatitis C treatments, which inhibit or even block the progress of this infection into a chronic stage, a vaccine still not available and the worldwide distribution of the disease makes the hepatitis C a major public health problem. Most of the available treatments target viral proteins. However many mechanisms of the HCV life cycle remain unclear.As for many positive RNA viruses, HCV replication occurs in reorganized cellular membranes. These membrane rearrangements are closely linked to the early secretory pathway of the cell. It has been shown that GBF1, an exchange factor of small G proteins of the Arf family that regulates the membrane dynamics in the secretory pathway, is required for HCV replication. GBF1 inhibition by brefeldin A (BFA) inhibits the secretion of newly synthesized proteins and also inhibits HCV replication. To investigate the role of GBF1 in HCV infection, we isolated cell lines resistant to BFA. Two of these cell lines were 100 times more resistant than the parental cells to BFA-induced apoptosis, inhibition of proteins secretion and inhibition of HCV infection. This resistance was due to a point mutation in the catalytic sec7 domain of GBF1 of these cells. Another group of resistant cells was showing a partial resistance to the inhibition of proteins secretion while maintaining their sensitivity to the inhibition of HCV infection in the same conditions. These results suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication. Using GBF1 deletion mutants we showed that the catalytic activity of the sec7 domain of GBF1 is required for HCV infection. This suggests that the function of GBF1 during HCV replication is mediated by Arf activation. The involvement of Arf was confirmed with the overexpression of restricted mutants of Arf1 and by the inhibition of ArfGAP1, another regulator of Arf function. We then tested the possible involvement of different Arfs (Arf1, 3, 4 and 5) in HCV infection. It has been reported that Arfs have redundant functions. The results confirm the involvement of Arf1 and indicate that all the other BFA-sensitive Arfs (Arf3, Arf4 and Arf5) are also involved in HCV infection. The combined knockdown of Arfs strongly inhibited HCV replication, showing that the Arf proteins are working together in HCV replication probably by activating several host factors required for the virus life cycle.The study of cellular factors required for HCV infection is crucial to better understand the interaction of the virus with the host cell and thus the whole HCV life cycle. This could help to develop new therapies targeting the host cell, regardless of viral genotypes and reducing the risk of emergence of new resistant forms.

This thesis investigates the epidemiology of hepatitis B virus infection (HBV) and estimates HBV-related liver disease burden in Vietnam and Australia using a cross-sectional study design and mathematical modelling. A population-based seroprevalence survey was undertaken in rural Northern Vietnam. In a sample of 870 study participants, prevalence of anti-HBV core antibody (anti-HBc) and hepatitis B virus surface antigen (HBsAg) was 68.2% and 19.0%, respectively, and hepatitis B e antigen (HBeAg) was detected in 16.4% of the HBsAg-positive group. Factors associated with HBV infection (anti-HBc and/or HBsAg-positive) were age 60 years or older (adjusted odds ratio (AOR), 3.82; 95% CI, 1.35??10.80; P = 0.01), residence in Vu Thu district (AOR, 3.00; 95% CI, 2.16??4.17; P <0.001), hospital admission (AOR, 2.34; 95% CI, 1.33??4.13; P = 0.003) and history of acupuncture (AOR, 2.01; 95% CI, 1.29??3.13; P = 0.002). Household contact with a person with liver disease (AOR, 2.13; 95% CI, 1.29??3.52; P = 0.003), reuse of syringes (AOR, 1.81; 95% CI, 1.25??2.62; P = 0.002) and sharing of razors (AOR, 1.69; 95% CI, 1.03??2.79; P = 0.04) were independent predictors of HBsAg positivity. Alanine aminotransferase (ALT) level was elevated (>40 IU/L) in 43% of the HBsAg-positive group; the proportion of elevated ALT was higher in HBeAg-positive (65%) compared with HBeAg-negative (39%) (P = 0.02). Based on data from the seroprevalence study, other prevalence estimates and HBV natural history parameters, a mathematical model was used to estimate HBV-related liver disease burden in Vietnam. Estimated chronic HBV prevalence increased from 6.4 million cases in 1990 to around 8.4 million cases in 2005 and was projected to decrease to 8.0 million by 2025. Estimated HBV-related liver cirrhosis and hepatocellular carcinoma (HCC) incidence increased linearly from 21 900 and 9400 in 1990 to 58 650 and 25 000 in 2025. Estimated HBV-related mortality increased from 12 600 in 1990 to 40 000 in 2025. To estimate HBV-related HCC incidence among Australians born in the Asia-Pacific region (APR), a mathematical modelling was developed utilising HBV natural history parameters, HBV prevalence estimates in APR countries and immigration data. Chronic HBV cases among the APR-born population increased rapidly from the late 1970s, reaching a peak of 4182 in 1990. Chronic HBV prevalence increased to more than 53 000 in 2005. Estimates of HBV-related HCC increased linearly from one in 1960 to 140 in 2005, with a projected increase to 250 in 2025. Universal HBV vaccination programs in countries of origin had limited impact on projected HBV-related HCC to 2025. HBV-related HCC survival was analysed in a population-based linkage study in New South Wales (NSW), Australia. Between 1994 and 2002, 278 HCC cases notified to the NSW Cancer Registry were linked to chronic HBV infection notifications to the NSW Health Department. The majority of cases were male (83.5%) and overseas born (93.6%); Asian-born cases accounted for 72.1%. Median survival following HCC diagnosis was 15 months. HCC survival was poorer among older age groups (P <0.001), and among cases with regional spread (HR 3.23; 95% CI, 1.83??5.69; P <0.001) and distant metastases (HR 3.85; 95% CI, 2.44??6.08; P <0.001). Sex, region of birth, and study period (1994??1997 versus 1998??2002) were not associated with HCC survival. The results of these studies show that HBV infection remains a major public health challenge in highly endemic countries such as Vietnam. HBV-related liver disease burden in Vietnam was estimated to increase for at least two decades despite the introduction of a universal infant HBV-vaccination program. Similarly, HBV-related HCC among Australians born in the APR was estimated to continue to increase over the next two decades. Survival for HBV-related HCC even in settings such as Australia continues to be extremely poor. Strategies are required to expand HBV treatment to individuals with chronic HBV infection who are at greatest risk of progression to advanced liver disease.

This thesis examines the results of a hepatitis B vaccination questionnaire study that was completed by medical officers, nurses, carers of the developmentally disabled, and correctional officers in the Greater Western Sydney area of New South Wales, Australia. The main aim of the study was to contrast these four high risk occupational groups for their acceptance of hepatitis B vaccination, seroconversion status, and behavioural, attitudinal, motivational and institutional determinants of their vaccination status. The results of the thesis revealed that medical officers and nurses were the most likely to be tested and vaccinated for seroconversion, while DD carers and correctional officers had the largest number of Not Vaccinated respondents. The findings of the questionnaire are discussed in some detail. It is also suggested that groups differ in the degree of hepatitis risk anxiety they experience as well as the degree of control felt over their health status. A number of indications for personal, institutional and governmental interventions to increase vaccination levels are discussed. / Doctor of Philosophy (PhD)

Master of Dental Surgery / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

Bibliography: leaves 162-174.

This thesis explores the impact of hepatitis C on women’s childbearing decisions and experiences of motherhood. A partial grounded theory approach was used, in which 34 women living with hepatitis C participated in semi-structured interviews to determine the direct and indirect effects of hepatitis C on their own personal decisions regarding childbearing and to describe their lived experiences of motherhood. The qualitative interview data were analysed thematically, in which common themes were identified and explored.¶ Three key areas are explored: women’s social experience of hepatitis C; hepatitis C and childbearing decisions; and the meaning of motherhood for women with hepatitis C.¶ The interviews revealed that living with hepatitis C had direct effects on the childbearing decisions of women. The direct effects of the virus which impacted on women’s childbearing decisions included poor physical and emotional hepatitis C related health, the perceived risk of vertical transmission of hepatitis C, concerns their future hepatitis C related health might impact upon their mothering abilities, and childbearing can conflict with treatment for hepatitis C. However, of greater importance to these women, appeared to be the indirect effects of living with a virus which is so highly stigmatised within our society. In particular, hepatitis C is closely associated with injecting drug use, which means these women are often assumed to possess the stereotypical characteristics associated with injecting drug users. As a result, they experience widespread medical discrimination and social rejection. Hepatitis C also impacts indirectly upon a wide range of factors that most women in contemporary society take into consideration in their childbearing decisions, for example, available social support, financial security and age.¶ The experiences reported by these women are discussed in terms of their concordance or discord with prevailing theories of deviance, stigma and the social construction of motherhood. The interview data, considered in light of such theories reveal that possibly the greatest impact that hepatitis C can have upon women is to prevent them from achieving a legitimate adult female status through childbearing and becoming a ‘good mother’.¶ The implications of these findings are discussed in terms of public health and social policy.

Hepatitis C virus (HCV) replication is directed by NS5b, the viral RNA dependent RNA polymerase (RdRp). To date, our understanding of the HCV polymerase has come almost entirely from genotype 1. The aim of this study was to examine the influence of sequence variation in the polymerase region by characterising a polymerase derived from genotype 3a. The genotype 3a CB strain polymerase was cloned into the bacterial expression vector pTrcHis2C incorporating a hexahistidine tag to facilitate purification. An optimised process produced 2.5 mg of highly purified recombinant protein per litre of bacterial culture. The 3a preparation possessed an RdRp activity and could utilise both homopolymeric and heteropolymeric RNA templates. Optimal activity was seen at 30oC at pH 8 in reactions containing 160nM enzyme, 10??g/ml RNA template and 2.5mM MnCl2. Subsequently, three genotype 1b polymerases including the HCV-A, Con1 and JK1 strains were cloned for the comparison of activity under identical conditions. Steady state kinetic parameters for GMP incorporation revealed the 3a polymerase exhibited the highest activity, with an almost two fold higher catalytic efficiency (Kcat/Km) than HCVA-1b, primarily due to differences in Km for GTP (2.984??M vs 5.134??M). Furthermore, the 3a polymerase was 3.5 fold and 15 fold more active than JK1-1b and Con1-1b respectively. Improving our understanding of the influence of sequence difference on polymerase activity, particularly in the context of replication will be crucial to developing effective antiviral therapies.