At a state university in the Pacific Northwest, the authors surveyed 477 matriculating college students about their immunization status, knowledge, self-perceived risk, and health risk behaviors for hepatitis A and B vaccinations. Review of immunization records revealed that 10% of these students were completely immunized for hepatitis A and 58.8% were completely immunized for hepatitis B. Although their knowledge about hepatitis A and B was very good, there were no significant differences in immunization status or health risk behaviors based on their knowledge or their self-perceived risk of hepatitis A and B. About 66% of students perceived they had no risk or low risk for hepatitis A and B and only 5% felt they had a high risk. In spite of the low self-perceived risk, health risk behaviors for hepatitis A and B were common in this population of college students. In the past twelve months, 56.2% of these students reported being sexually active (vaginal, oral, anal sex), including 20.5% who had two or more sexual partners. Students with multiple sexual partners reported having unprotected sex at a high frequency: 61.3% vaginal, 81.1% oral-genital, and 13.2% anal. The non-sexual health risk behaviors were also common in this population: 53.2% had body piercing/tattoos, and 26.5% traveled internationally to regions with intermediate to high rates of hepatitis A (Africa, SE Asia, Central/South America). These students who traveled internationally were more likely to be immunized for hepatitis A, although 70% remained unimmunized. In addition, students with multiple sexual partners or with a body piercing/tattoo were more likely to be immunized for hepatitis B. However, about one-third of these students with multiple sexual partners or body piercing/tattoo have not completed the hepatitis B series. The theory of planned behavior was applied to determine factors that are associated with college students' decision to be vaccinated for hepatitis A and B. For hepatitis A and B vaccinations, important attitude constructs identified were 'vaccine effectiveness, vaccine adverse effects, anticipation anxiety, inconvenience, and vaccine expense.' The subjective norm constructs with the greatest influence for college students were: 'parents, family doctor, and student health services staff'. The significant perceived behavioral constructs were 'parents reminder, student health services (SHS) reminder, and SHS hepatitis vaccine clinic.' In conclusion, there is still a sizable portion of unvaccinated college students who possess health risk behaviors for hepatitis A and B, such as multiple sexual partners, unprotected sexual behaviors, body piercing/tattoos, and international travel. With a greater understanding of college students' attitudes, subjective norms, and perceived behavioral control college health services could develop more effective strategies to educate unvaccinated college students about these risks and motivate them to be immunized. / Graduation date: 2003

Hepatitis C, a life threatening disease, caused by the hepatitis C virus (HCV) currently affects over 170-200 million people worldwide (~3% of global human population), more than five times the percentage of total HIV infections. HCV infection has been shown to be a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation in the U.S. HCV has escaped every therapeutic target to date by means of its error-prone RNA polymerase, which allows it to mutate prolifically. The current standard anti-HCV therapy, which is pegylated interferon a combined with ribavirin, is difficult to tolerate, and more than 50% of HCV patients are refractory to it. No protective vaccine or therapeutic antibody is available, making the need for the development of an efficacious immunoprophylactic and therapeutic agent imperative. HCV is an enveloped virus with a positive sense RNA genome of ~9.6 kilobases (kb), which carries a large open reading frame (ORF), flanked by 5'- and 3'- untranslated regions (UTRs). Interestingly, within the highly mutational HCV RNA, there are a limited number of 100% conserved and functionally vital motifs, located in the 5' UTR, coding region and in the 3' UTR. Within the HCV genome, these motifs have been proposed to be involved in multiple exclusive interactions with each other and furthermore, these interactions have been demonstrated to be essential for HCV replication and/or translation of the viral proteins. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD; / Dissertation;

Cellular immune responses to Hepatitis C (HCV) epitopes are crucial for successful host response to HCV infection. We investigated a platform to assess specific and global immune responses in HCV infection. We identified 57 HCV peptides from literature (24 of CD4+, 33 of CD8+ specificity) and tested them in two peptide pools to assess specific response in non-transplanted and post-liver transplant (LT) patients. Robust interferon-gamma (IFN) response to CD4+ peptide and mitogen stimulation was seen in sustained virological clearance. IFN response to the CD4+ peptide pool could differentiate between SVR and NR with 82% accuracy. In patients with recurrent HCV post-LT, HCV-specific responses were attenuated, but global immune responses were preserved. Significantly lower specific (CD4+) and global immune responses (mitogen response) were observed in patients with advanced allograft disease (fibrosis score>2). Quantiferon-HCV may identify patients likely to respond to anti-HCV treatment, as well as post-LT patients with aggressive HCV recurrence. / Experimental Surgery

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

Context & Rationale: Individuals with similar CD4 cell counts and RNA levels can vary considerably with regards to clinical progression. This variation is likely the result of a complex interplay between viral, host and environmental factors. This study aimed to characterize and identify predictors associated with disease progression to AIDS or death in Saskatoon, Saskatchewan. Methods: This is a retrospective cohort study of 343 seroprevalent HIV positive patients diagnosed from Jan 2005 to Dec 2010. Of these, 73 had an estimated seroconversion date. Data was extracted from medical charts at two clinics specialized in HIV/AIDS care. Disease progression was measured as time from HIV diagnosis (or seroconversion) to immunological AIDS and death. The Cox hazard model was used. Results: The 3-year and 5-year immunological AIDS free probability was 53% and 33%, respectively. The 3-year and 5-year survival probability was 89% and 77%, respectively. Among the seroconversion cohort, the 3-year immunological AIDS free probability was 76%. Due to multicollinearity, separate models were built for IDU, hepatitis C and ethnicity. A history of IDU (HR, 3.0; 95%CI, 1.2-7.1), hepatitis C coinfection (HR, 2.9; 95%CI, 1.2-6.9), baseline CD4 counts (HR, 0.95; 95%CI, 0.92-0.98, per ever 10 unit increase), ever on ART, and year of diagnosis were significant predictors of progression to immunological AIDS among the seroprevalent cohort. Age at diagnosis, sex and ethnicity were not. For survival, only treatment use was a significant predictor (HR, 0.34; 95%CI, 0.1-0.8). Hepatitis C coinfection was marginally significant (p=0.067), while a history of IDU, ethnicity, gender, age at diagnosis, and year of diagnosis were not. Among the seroconversion cohort, no predictors of progression to immunological AIDS were identified. Ethnicity, hepatitis C coinfection and history of IDU could not be assessed. Conclusion: Our study found that IDU, HCV coinfections, baseline CD4 counts, and ART use were significant predictors of disease progression. This highlights the need for increased testing and early detection and for targeted interventions for these particularly vulnerable populations to slow disease progression.

Context & Rationale: Individuals with similar CD4 cell counts and RNA levels can vary considerably with regards to clinical progression. This variation is likely the result of a complex interplay between viral, host and environmental factors. This study aimed to characterize and identify predictors associated with disease progression to AIDS or death in Saskatoon, Saskatchewan. Methods: This is a retrospective cohort study of 343 seroprevalent HIV positive patients diagnosed from Jan 2005 to Dec 2010. Of these, 73 had an estimated seroconversion date. Data was extracted from medical charts at two clinics specialized in HIV/AIDS care. Disease progression was measured as time from HIV diagnosis (or seroconversion) to immunological AIDS and death. The Cox hazard model was used. Results: The 3-year and 5-year immunological AIDS free probability was 53% and 33%, respectively. The 3-year and 5-year survival probability was 89% and 77%, respectively. Among the seroconversion cohort, the 3-year immunological AIDS free probability was 76%. Due to multicollinearity, separate models were built for IDU, hepatitis C and ethnicity. A history of IDU (HR, 3.0; 95%CI, 1.2-7.1), hepatitis C coinfection (HR, 2.9; 95%CI, 1.2-6.9), baseline CD4 counts (HR, 0.95; 95%CI, 0.92-0.98, per ever 10 unit increase), ever on ART, and year of diagnosis were significant predictors of progression to immunological AIDS among the seroprevalent cohort. Age at diagnosis, sex and ethnicity were not. For survival, only treatment use was a significant predictor (HR, 0.34; 95%CI, 0.1-0.8). Hepatitis C coinfection was marginally significant (p=0.067), while a history of IDU, ethnicity, gender, age at diagnosis, and year of diagnosis were not. Among the seroconversion cohort, no predictors of progression to immunological AIDS were identified. Ethnicity, hepatitis C coinfection and history of IDU could not be assessed. Conclusion: Our study found that IDU, HCV coinfections, baseline CD4 counts, and ART use were significant predictors of disease progression. This highlights the need for increased testing and early detection and for targeted interventions for these particularly vulnerable populations to slow disease progression.

Viral hepatitis is a disease caused by a virus, or a virus-like particle and is extremely infectious. Infection can occur when the virus is present in minute quanities. Outbreaks of this disease have been associated with water supplies contaminated with raw sewage. Detection of viral hepatitis in water samples, at present, consists of testing the serum of persons who have contracted the disease after consuming water suspected of containing hepatitis virus. Since this method of determination is time consuming and subjective, an alternative method of detecting the presence of hepatitis virus in water was explored in this research.Radioim munoassay (RIA), used for the detection of Australia antigen (HAA) in the serum of humans, utilizes a tagging system in which Australia antigen antibody is bound with iodine-125. This technique, a combination of chemistry and immunology, can detect less than one- billionth of a gram of HAA in serum and requires approximately four hours to complete the testing. RIA detection of HAA is presently limited to detection of HAA in serum samples.This research used RIA as a detection system to test water samples collected from local water sources. Control serum samples collected from a local hospital were tested for HAA. Fifteen water samples and two serum samples tested were positive upon initial testing for Australia antigen. HAA was also detected in water samples in control situations in which sera with a high titer to HAA were diluted in water and then tested using RIA.Ball State UniversityMuncie, IN 47306

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.