Mouse Hepatitis virus (MHV) is a member of the coronavirus family in the order Nidovirales. The 32 kb genome contains cis-acting sequences necessary for replication of the viral genome. Those cis-acting sequences have been shown to bind host proteins, and binding of those proteins is necessary for virus replication. One of the cis-acting elements is the 3' terminal 42 nucleotide host protein binding element. Previous work has demonstrated that mitochondrial aconitase, mitochondrial heat shock protein 70, heat shock protein 60 and heat shock protein 40 bind to the 3' terminal 42 nucleotide host protein binding element. We demonstrated that RNA secondary structure of the 3' terminal 42 nucleotide host protein binding element is necessary for host protein binding in vitro. We also demonstrate that primary structure of the 3' terminal 42 nucleotide host protein binding element is necessary for viral replication by targeted recombination. DI replication assays infer that the 3' terminal 42 nucleotide host protein binding element plays a role in positive strand synthesis from the negative strand template. Current studies involve the infectious cDNA clone, which will provide definitive answers on the role of the 3' terminal 42 nucleotide host protein binding element in MHV replication.

Hepatitis C (HCV) is an infectious disease of the liver which affects more than 250,000 Canadians; the majority of those living with the disease have experience with injection drug use. Treatment for HCV involves a strict protocol, has only a 50% success rate and has harsh side effects. Interest in HCV treatment among people who use drugs is high, but actual uptake of treatment remains low. The objective of this research was to explore the barriers to accessing HCV treatment for individuals who were accessing methadone. A mixed methods approach was used; a cross sectional survey and an in-depth interview were administered to clients of a methadone maintenance program. The two sets of data identified three main barriers to HCV treatment; stigma, the toxicity of treatment, and day-to-day struggles. Future research should be conducted to further explore how stigma guides decisions around HCV treatment, particularly in a methadone treatment setting.

No description available.

Viral proteases are often considered to be attractive drug targets because of their crucial function in the viral replication machinery. In order to increase our knowledge of these important targets and to contribute to the discovery and development of new antiviral drugs, the proteases from hepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and their interactions with inhibitors and fragments have been characterized, using enzyme inhibition and SPR biosensor based interaction assay. The structure activity relationships and the resistance profiles of a series of HCV NS3 protease inhibitors based on either P2 proline or phenylglycine residues were analyzed using wild type genotype 1a and the major resistant variants A156T and D168V. The observed susceptibility to substitutions associated with these resistance variants was concluded to depend on the P2 and the P1 residue, and not only on the P2 residue as previously had been suggested. In order to be able to evaluate how the potency of inhibitors is affected by genetic variation, their effect was evaluated on wild type NS3 from genotype 1a, 1b and 3a as well as on the resistant variant R155K from genotype 1a. To enable a comparison of the inhibitory effect on the enzyme variants, the compounds were analyzed under conditions optimized for each variant. VX-950 was found to be the least susceptible compound to resistance and genetic variation. A more detailed analysis showed that the kinetic and mechanistic features of the inhibitors were significantly different for the different genotypes. The reversible non covalent macrocyclic inhibitor ITMN 191 was revealed to have favorable kinetics for all three genotypes. This is an advantage for the design of broad spectrum drugs. A fragment based procedure for identifying and validating novel scaffolds for inhibitors of HCMV protease was established. It identified fragments that may serve as starting points for the discovery of effective inhibitors against this challenging target.   The procedures developed for the evaluation and identification of novel HCV NS3 and HCMV protease inhibitors have contributed to a deeper understanding of protease-inhibitor interactions that is expected to have an impact on the design of novel antiviral drugs.

<p>Dengue viruses (DENV) are mosquito&#8208;borne flaviviruses that pose a continued and growing threat to global health. There are estimated to be 390 million DENV infections each year, and because there is no vaccine or approved therapeutic treatment, developing a small&#8208;molecule treatment is imperative. Possible small&#8208;molecule drug therapies for DENV could be immune system modulators, inhibitors of DENV&#8208;required host factor, or inhibitors of a viral gene product. In this study, we chose to take the latter approach and focused our drug discovery efforts on the most highly conserved flaviviral protein, non&#8208;structural protein 5 (NS5). NS5 contains two major domains, each with different enzymatic activities. The N&#8208;terminus has methyltransferase activity, and the C terminus, an RNA&#8208;dependent RNA polymerase (RdRp). The activities of both domains are purine&#8208;dependent, and therefore both domains contribute to the purine&#8208;binding properties of NS5. Inhibition of either of these domains in NS5 results in inadequate propagation of DENV, and the purine&#8208;binding domains present ideal drug targets for disrupting these activities. These factors make NS5 protein an ideal candidate target for our small&#8208;molecule library screen.</p><p>A high&#8208;throughput fluorescence&#8208;based screen was employed to identify anti&#8208;DENV compounds based on their ability to competitively bind NS5. The screen was performed by binding green fluorescent protein NS5 fusion protein (GFP&#8208;NS5) to immobilized ATP resin, and then performing parallel elutions using over 3,000 distinct compounds. One compound in particular, HS&#8208;205020, was able to competitively elute GFP&#8208;NS5 from the ATP resin and also exhibited antiviral activity in both the U937+DCSIGN human monocyte cell line and BHK&#8208;21 cells. Additionally, HS&#8208;205020 was able to inhibit DENV NS5 RNA polymerase activity in vitro. HS&#8208;205020 is chemically distinct from the majority of previously reported NS5 inhibitors, which are nucleoside analogs that can cause severe toxicity in animal studies. In contrast, over the concentration range that produced anti&#8208;DENV effects, HS&#8208;205020 showed comparable viabilities to ribavirin, an FDA approved hepatitis C virus (HCV) therapeutic. These findings support HS&#8208;205020 as a potential dengue antiviral candidate, and its chemical scaffold represents as an ideal starting compound for future structure&#8208;activity relationship studies.</p> / Dissertation

This thesis explores the argument that inherent in medical treatment interventions for chronic hepatitis C, there are certain implicit ontological assumptions about the relationship between the body, disease, and society. Focusing primarily on biomedical practices, it is argued that these assumptions might have a profound effect on the world-views of patients undergoing them. This in turn, might have far-reaching sociological implications. Using a methodology specifically developed for the purpose of explicating the ontological assumptions inherent in medical treatment, the visual and narrative texts produced by thirteen hepatitis C positive participants are examined. A deconstructive analytical approach is then applied to these texts as they relate to the treatment interventions pursued by participants. An exploration of the way participants engage with, negotiate, and/or resist the discourses and assumptions inherent in biomedicine, traditional Chinese medicine, and to some extent naturopathy, is conducted. Two broad ways in which the participants visualise the relationship between disease and their bodies, relating to treatment undertaken, are identified. The possible social implications of these are then suggested. The first, and predominant view, is aligned with biomedicine. The relationship between disease and the body is antagonistic in this view. It is suggested that this way of seeing might naturalise xenophobic attitudes and perpetuate social conflict. The marginal view is related to non-biomedical treatments for hepatitis C. The relationship in this case is the result of a negotiated accommodation with the disease. It is suggested that such a view might allow for non-resistant social tolerance of that which is perceived of as new and different. This qualitative study contributes to the body of knowledge in the field of the sociology of health and illness in two ways: Firstly, it proposes a methodology that may be taken up or adapted for future sociological research, and secondly, it suggests something of the social and political nature of treatment decisions made by people living with chronic hepatitis C.

Hepatitis B virus (HBV) causes an estimated 600,000 deaths annually, largely due to hepatocellular carcinoma (HCC). HBx, a promiscuous transactivator, is a viral oncoprotein, but its exact functions are poorly understood. Many studies have suggested that NF-κB signaling mediates HBx functions, but the underlying molecular mechanisms remain yet to be elucidated. Here, we provide evidence that HBx-mediated NF-κB activation depends on the physical interaction between HBx and a transcription factor, p65. In the cytoplasm, HBx-p65 interaction may promote IκBα phosphorylation and subsequent p65 nuclear localization. A cytokine assay using qPCR and RT-PCR indicates that HBx is associated with a unique profile of cytokine mRNA expression. As shown by chromatin immunoprecipitation (ChIP), HBx in the nuclues can be recruited to the gene promoter by p65. These findings support the importance of HBx-p65 interaction and suggest that it is potentially a promising target of novel therapeutics for HBV-associated liver diseases, including HCC.

Background: Chronic HBV within the Canadian Arctic is considered endemic (>2% prevalence). To control endemic rates in Nunavut, a vaccination program was initiated approximately 20 years ago, targeted at newborns and grade school students, as an interim catch-up program, such that all individuals born after 1980 are potentially vaccinated. This study investigates the efficacy of these programs and is the first seroepidemiological survey to determine HBV prevalence in Nunavut in the post-vaccination era. Methods: Anonymized serum specimens scheduled for destruction following routine medical testing were collected from individuals granting consent. Specimens were tested for antibodies to HBV (anti-HBs, anti-HBc) and hepatitis C virus. Anti-HBc positive samples were further tested for surface antigen (HBsAg) positivity, and HBV DNA was extracted from HBsAg positive samples in order to perform molecular characterization. Results: 4802 specimens were collected according to the age distribution of Nunavut, with vaccine age cohort specimens comprising just over half of all collected specimens. Overall anti-HCV+ was 0.55%, with all positivity observed among those aged 24 to 69 years old. Total anti-HBc+ prevalence was 9.40%; however, a 10-fold decrease in the rate of HBV exposure was noted among those born after 1980 compared to those born before (1.89% vs 20.1%, p<0.001). HBsAg positivity was primarily documented in individuals born before 1980 (2.55%), although cases still occurred among the vaccine age cohort (0.21%). HBV subgenotype B5 (HBV/B5), known to be unique among Inuit and Alaska Native people, was the most prevalent genotype observed (82%). Vaccine-based antibody as the sole serological marker was evident in the vaccine age cohort, although the rate of decay with increasing age was much greater than anticipated. Conclusion: Nearly two decades after the advent of HBV vaccination in Nunavut, HBV prevalence has decreased to 1.17%, indicating a non-endemic or low risk prevalence. However, the persistence of infection and a lower than expected prevalence of vaccine-based immunity in the vaccine age cohort will require further investigation to understand the causes and consequences.

The separation, identification, and characterisation of the proteins present in a tissue or biological sample is called ‘proteomics’. This technique can be used for example to identify biomarkers and investigate signalling pathways. Increasingly, proteomics is being applied to the analysis of virus related samples; here two such examples are described. Presently there is no reliable non-invasive way of assessing liver fibrosis. Here a novel 2D-PAGE based proteomics study was used to identify potential fibrosis biomarkers. Serum from patients with varying degrees of hepatic scarring induced by infection with the hepatitis C virus (HCV) was analysed. Several proteins associated with liver scarring and/or viral infection were identified. The most prominent changes were observed when comparing serum samples from cirrhotic patients with healthy controls: Expression of inter-α-trypsin inhibitor heavy chain H4 fragments, α1 antichymotrypsin, apolipoprotein L1 (Apo L1), prealbumin and albumin was decreased in cirrhotic serum, whereas CD5 antigen like protein (CD5L) and β2 glycoprotein I (β2GPI) increased. In general, α2 macroglobulin (a2M) and immunoglobulin components increased with hepatic fibrosis whereas haptoglobin and complement components (C3, C4 and factor H-related protein 1) decreased. Novel proteins associated with HCV-induced fibrosis include the inter-alpha-trypsin inhibitor heavy chain H4 fragments, complement factor H-related protein 1, CD5L, Apo L1, β2GPI and the increase in thiolester cleaved products of a2M. The relationship between these changes is discussed. One of the accessory genes of the HIV viral genome encodes for the Nef protein. Nef is present in lipid rafts and increases viral replication within infected host cells by binding to a guanine nucleotide exchange factor, Vav. This leads to activation of a GTPase, Cdc42, however, the signalling pathway is poorly understood. 2D-PAGE based proteomics was used to identify differentially expressed raft-associated proteins by comparing T cells in the presence and absence of Nef. A ubiquitin conjugating enzyme UbcH7, which acts in conjugation with c-Cbl, was absent from the rafts of Nef-transfected cells. Vav ubiquitination was also absent from these rafts. In collaboration with Dr. Alison Simmons and Prof. Andrew McMichael the absence of UbcH7 in rafts was found to be caused by β-Pix forming a ternary complex with c-Cbl and activated Cdc42. Vav ubiquitination was restored and viral replication was diminished when β-Pix was knocked down providing a new candidate target for inhibiting HIV replication. This thesis demonstrates the use of proteomics in providing novel information for virus related samples. This influential technology benefits in both biomarker discovery to aid clinicians with early diagnosis of diseased individuals and in the elucidation of novel signalling pathways in infected cells to provide new candidate targets.

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.