At present the origins of major human pathogens associated with hepatic disease are poorly understood. The absence of such information pertaining to the evolutionary history of hepatitis B virus (HBV) and hepatitis C virus (HCV) and its genetically related viruses impacts upon the development of vaccines and effective eradication strategies. Studies are currently limited by the absence of historical samples from which to date the emergence of human infections and therefore the evolution of human hepatic viruses relies on epidemiological studies and genetic analysis of contemporary virus populations worldwide. Approximately one third of the world’s population is infected with HBV, and despite the availability of a vaccine, the virus is attributed with over 1 million deaths per year through liver disease. HBV variants infecting humans show genetic and antigenic heterogeneity and are currently classified into 8 genotypes A-H with nucleotide divergence of between 9-13%. In addition to these variants, recombination has been detected between genotypes A and D, and B and C, which can generate novel variants. The past 10 years has seen the detection of HBV in chimpanzees, gorillas and other non-human primates (NHPs) at frequencies comparable to those observed in regions of endemic human HBV infection. Despite the genetic divergence between human and NHP HBV variants the detection of recombination between human genotype C and chimpanzee and gibbon variants suggests that HBV can share hosts in nature. The evolutionary process that may have given rise to the distinct species-specific variants of NHP HBV within overlapping geographical regions has not been reconciled, with evidence supporting both allopatric speciation and co-speciation. HCV a member of the Flaviviridae family currently infects approximately 3% of the world’s population and is one of the major causes of chronic liver disease, hepatocellular carcinoma and liver cirrhosis. Human pegivirus (HPgV) a member of the Pegivirus genus of the Flaviviridae family infects approximately 5% of the world’s population, although it is of unknown disease association. Very recently, several studies of wild rodent and bat populations have revealed much greater viral diversity of members of both Hepacivirus and Pegivirus genera. Homologues of HCV have been detected in a range of species including domestic dogs (canine hepacivirus [CHV]) and horses (non-primate hepaciviruses [NPHV]). Similarly, several new pegiviruses have been described in horses (equine pegivirus, [EPgV] and Theiler’s Disease Associated Virus [TDAV]), several species of rodents (rodent pegivirus [RPgV]), and further species of bats (bat pegivirus, [BPgV]). Despite the differences in pathogenicity between HCV and HPgV infections, they share similar genomic organisation and are capable of establishing persistent infections in humans. Studies into bat, horse and rodent homologs of HCV and HPgV have yet to determine disease associations, transmission routes and seroprevalence. Studies presented within this thesis broaden our understanding of the clinical presentations and host range of NPHV and EPgV. Screening to determine the level of active and past infection to both viruses provides novel insight into infection frequencies, host range, disease progression and examines the correlation between infections and the presence or absence of hepatic disease. Research examining HBV variants circulating in NHPs in Cameroon provides novel evidence for the occurrence of recombination and cross species transmission between NHP variants of HBV and examines the role these findings play in expanding our understanding of the evolution of HBV.

Hepatitis C virus (HCV) has an internal ribosomal binding site (IRES) located near the 5ʹ end of its genome. The HCV IRES is capable of direct binding to the 40S small ribosomal unit and eukaryotic initiation factor eIF3, and can initiate translation after the assembly of the whole 80S ribosome. Various molecular types can act as IRES inhibitors. Small molecule compounds seem to be the most promising agent for use in the clinic. The main objective of the thesis was to develop a system for searching for small molecule compound inhibitors of HCV IRES in a library of chemical compounds. Several variants of vector carrying bicistronic cassettes were prepared. After validating their functionality by transient transfection of mammalian cell cultures, mammalian stable cell lines were established. These stable cell lines will allow for automatization of the search for small molecule compound inhibitors of HCV IRES. Our second objective was to study the variability of HCV IRES sequences in patient samples. The samples were analysed by temperature gradient gel electrophoresis (TGGE). Select specimen were sequenced, cloned into a vector with bicistronic cassette and analysed by flow cytometry. In this was we evaluated the effect of specific mutations in the HCV IRES sequence on the level of IRES dependent...

Dans le cadre de ce travail, nous avons synthétisé, isolé et caractérisé de nouvelles molécules amphiphiles dérivées d’acides nucléiques. Les modifications de ces structures ont été réalisées par voie chimique au moyen d’une réaction de chimie clic, la réaction de Huisgen. Les amphiphiles développés peuvent être classés dans deux catégories différentes : - Les amphiphiles de faible masse moléculaire qui dérivent de nucléosides et de glycosylnucléosides. Les propriétés d’auto organisation de ces composés ont été étudiées par différentes techniques, notamment de microscopie électronique et de diffraction des rayons X. La capacité de ces amphiphiles à former des gels a été évaluée dans différents solvants (eau et solvants organiques). Les propriétés de complexation des acides nucléiques de ces molécules ont également été mises en évidence. Les premiers résultats de transfection montrent que les glycosylnucléosides amphiphiles permettent l’internalisation des oligonucléotides à visée thérapeutique dans des cellules humaines de carcinome hépatocellulaire (Huh 7) en présence de sérum. - Les amphiphiles de masse moléculaire élevée qui dérivent d’oligonucléotides. La formation d’agrégats a été mise en évidence par différentes techniques telles que la microscopie électronique et la diffusion de la lumière. Les propriétés de reconnaissance associées à la séquence oligonucléotide ont été étudiées par des expériences de thermodénaturation. L’auto vectorisation de ces composés a pu être observée par microscopie d’épi-fluorescence et confocale. Cette auto vectorisation a également pu être quantifiée par cytométrie en flux sur une gamme variée de types cellulaires humains tels que les cellules épithéliales (Hela T4), les cellules gastriques (NCI) ou encore les cellules de carcinome hépatocellulaire (Huh-7). Ces travaux présentent également pour la première fois l’évaluation in cellulo d’oligonucléotides amphiphiles ciblant le virus de l’hépatite C. / *

Candida albicans belongs to serious human opportunistic pathogens, causing severe health complications to immunocompromised patients. To my best knowledge, it is the only organism that survives with unmethylated cap structures found on the 5'ends of mRNA molecules. Using functional assay, I demonstrated that orf19.7626 codes for C. albicans translation initiation factor 4E (Ca4E). We couldn't prove our hypothesis, that Ca4E could be responsible for the unmethylated cap recognition in our model organism S. cerevisiae. Candida sp. possesses also another rather unusual feature - ambiguous CUG codon. In most of the cases, CUG is decoded as a serine, but sometimes also as a leucine. This gives rise to a so called "statistical proteome". One CUG codon is also part of the mRNA coding for Ca4E protein, therefore two versions of Ca4E-Ca4ELeu and Ca4ESer -might occur in C. albicans simultaneously. Both of them are able to rescue deletion of S. cerevisiae eIF4E gene, but they confer temperature sensitivity to the heterologous host. This phenotype is more pronounced with the Ca4ELeu version. We observed milder temperature sensitive phenotype after co-expression of Ca4E together with C. albicans eIF4G (Ca4G). Conformational coupling between eIF4E and eIF4G leads to enhanced affinity of eIF4E to the cap...

A hepatite viral causada pelo vírus da hepatite C (HCV) é um problema à saúde mundial e afeta cerca de 170 milhões de pessoas. O caso crônico da doença resulta em cirrose hepática e a maioria dos pacientes tratados não desenvolve uma resposta imune satisfatória. A proteína não estrutural 3 (NS3) pode estimular uma resposta celular que auxilia a resposta nos infectados. Nosso trabalho desenvolveu duas pseudopartículas virais que carregam um material genético para a protease da NS3 do HCV. Um dos sistemas, a HCVpp é constituída por proteínas do vírus da leucemia murina e outras do HCV. E o outro sistema, a partícula viral é baseada no Semliki Forest Virus (SFV). As células HEK293T e BHK-21 foram transfectadas para a formação das pseudopartículas HCVpp-NS3p1a e SFV-NS3p1a, respectivamente. Essas partículas foram quantificadas pela presença do material genético da NS3 por qRT-PCR, atingindo uma produção aproximada de 4x105 partículas HCVpp-NS3p1a/mL e 2,5x107 partículas SFV-NS3p1a/mL. Essas partículas foram utilizadas para infecção de células HuH-7.0 e BHK-21. / Viral hepatitis caused by the hepatitis C virus (HCV) is a global health problem, affecting about 170 million people. The chronic case of the disease results in liver cirrhosis and most patients do not develop a satisfactory immune response. The nonstructural protein 3 (NS3) can stimulate a cellular response that helps answer the infected. Our work has developed two viral pseudoparticles who carry a genetic material for the HCV NS3 protease. One of the systems is constituted by the HCVpp proteins of murine leukemia virus and other HCV. The other system, the viral particle is based on the Semliki Forest Virus (SFV). The HEK293T and BHK-21 cells were transfected for forming the pseudoparticles HCVpp-NS3p1a and SFV-NS3p1a, respectively. These particles were quantified by the presence of genetic material of NS3 by qRT-PCR, reaching a production of about 4x105 HCVpp-NS3p1a particles/mL and 2,5 x107 SFV-NS3p1a particles /mL. These particles were used for infection of Huh-7.0 cells and BHK-21.

Sofosbuvir is a uridine based nucleotide inhibitor of the hepatitis C viral (HCV) polymerase that is the backbone of many treatment regimens. In combination with drugs targeting other viral enzymes (including the poorly potent guanosine analogue ribavirin or highly potent inhibitors of viral NS5A or protease) most patients clear virus and resistance to sofosbuvir is rare, allowing effective retreatment with sofosbuvir. Patients with Genotype 3 HCV respond less well than other genotypes and response is reduced in those previously exposed to interferon. Here we show that patientderived virus from patients with Genotype 3 HCV who relapse to sofosbuvir-based therapies have a reduced sensitivity to SOF in an in-vitro phenotyping assay. Analysis of viral sequencing data revealed two distinct polymorphisms (A150V and K206E) in the HCV polymerase that are associated with treatment failure and in-vitro; they reduce sofosbuvir sensitivity against genotype 3 hepatitis C virions. However both polymorphisms modify the cellular response to type I interferon and in cells lacking response to interferon the impact on sofosbuvir sensitivity is minimal. The A150V polymorphism reduces the response to interferon 70 fold whereas the K206E substitution has minimal effects on interferon in isolation but in combination with A150V reduces the response 100 fold. Preliminary data indicates that the A150V polymorphism interferes with the late response to type I interferons enabling the virus to overcome the induction of interferon-stimulated genes. These data indicate a complex interaction between direct acting antiviral drugs and the innate antiviral response.

Orientador: Paula Rahal / Banca: João Renato Rebello Pinho / Banca: Maurício Lacerda Nogueira / Resumo: O HCV é uma das maiores causas de doença do fígado, sendo estimado que mais de 2% da população mundial está infectada. Este vírus possui um genoma de RNA (+) fita simples, que devido à falta de atividade corretiva da polimerase viral apresenta variabilidade genética em vários níveis: genótipos, subtipos e quasispecies. O genótipo 1 é o mais prevalente no Brasil e no mundo, sendo preditivo de uma baixa resposta à terapia antiviral, que atualmente é baseada na administração de PEG-IFN e ribavirina. A variabilidade genética da região viral NS5A tem sido relacionada à sensibilidade ou resistência ao IFN. Este estudo teve como objetivo investigar se a possível relação entre a composição de quasispecies da NS5A e a resposta ao tratamento. Foram selecionados 12 pacientes, sendo 4 respondedores (R), 4 não respondedores (NR) e 4 respondedores ao final do tratamento (RFT). As amostras pré-tratamento destes pacientes foram amplificadas, clonadas e seqüenciadas, resultando em 165 seqüências da NS5A completa. Estas seqüências foram alinhadas, editadas e a construção da topologia da árvore filogenética foi realizada. A NS5A e suas regiões específicas CRS, PKR-binding, ISDR, NLS e V3 foram analisadas quanto às substituições e o grau de variabilidade genético. O grupo de pacientes RFT apresentou uma maior taxa de substituições sinônimas em relação aos demais grupos. Uma maior quantidade de mutações foi observada na região downstream à ISDR, principalmente na região V3. Nenhum sítio específico de mutação foi relacionado a um tipo particular de resposta, e não houve agrupamento filogenético das quasispecies de acordo com o tipo de resposta. Estes resultados sugerem que o número de mutações não é suficiente para predizer a sensibilidade ou resistência à terapia baseada em IFN, sendo necessário avaliar se estas mutações conservaram ou não as propriedades químicas dos aminoácidos. / Abstract: Hepatitis C virus (HCV) is major causes of liver desease and about 2% of world s population are infected. This virus is a single strain RNA genome of approximately 9.6 kb. Genetics variability of HCV exists at several different levels: genotypes, subtypes and quasispecies. The high mutation rates are related to the low fidelity of viral RNA polymerase. Genotype 1 HCV is the most prevalent in Brazil, as well as worldwide. Genotypes 1a and 1b are predictive of lower sustained virological response in peginterferon (PEG-IFN) plus ribavirin combination therapy. Genetic variability of viral NS5A has been related to IFN sensibility or resistance. To evaluate whether HCV NS5A quasispecies composition are related to responsiveness to combined PEG-IFN and ribavirin therapy, this study analyzed before treatment sample of 12 treated patients (4 sustained responders - SR, 4 non responders - NR and 4 end of treatment responder - ETR). Samples were amplified, cloned and sequenced, resulting in 165 sequences of complete NS5A. Sequences were aligned, edited and phylogenetical tree was constructed. Mutations and mean of genetic distance were analyzed to NS5A and specific regions CRS, PKR-binding, ISDR, NLS and V3. The number of synonymous substitutions per synonymous sites was higher in ETR patients than in other patient groups. Mutations were more common downstream ISDR, mainly concentrated in V3 domain. No single amino acid position or motif was associated with different responses to therapy in any NS5A regions analyzed and phylogenetic analysis did not show clustering of nucleotide sequences of viral isolates from SR, NR or ETR. These results suggest that number of mutations is not sufficient to predict sensibility or resistance to IFN based therapy. Other studies are necessary to evaluate whether chemical characteristics of amino acids were altered for the mutations. / Mestre

This thesis is concerned with the mathematical modelling for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) epidemics in Australia. There are two parts to this thesis. Part I is aimed at modelling the transmission of HIV and HCV via needle sharing among injecting drug users (IDUs). The dynamical model of an epidemic through needle sharing among IDUs is derived. This model reveals the correlation between needle sharing and the epidemic prevalence among IDUs. The simulations of HIV and HCV prevalence and incidence among IDUs in Australia are made with this model. The comparison of simulated results with literature estimates shows that the modelled results are consistent with the literature estimates. The effects of needle sharing and cleaning on HIV and HCV prevalence and incidence among IDUs in Australia are evaluated. Part II is devoted to modelling the spread of HIV in the general community in Australia. A mathematical model is formulated to assess the epidemiological consequences of injecting drug use and sexual transmission in Australia. The effects of highly active antiretroviral therapies (HAART) on the HIV epidemic are included. The modelled results are in broad agreement with the literature estimates and observed data. The long-term effects of HAART are also discussed.

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.

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.