Dissertations / Theses on the topic 'Hepatitis G virus Victoria'

This thesis describes the genetic analysis of the heterogeneity of HGV/GBV-C and the characterization of the terminal regions of the viral genome. The sequence diversity across the HGV/GBV-C genome was significantly lower than that observed with HCV isolates. Comparative analysis of twenty-seven complete genome HGV/GBV-C sequences indicated the presence of four phylogenetic groups and this study demonstrated that these groupings could be reproduced by analysis of the 5'-untranslated region (5'-UTR) and of various sub-fragments. At the same time, the analysis of the 5'-UTR variability indicated the existence of group-specific polymorphisms, many of which are covariant and consistent with the proposed secondary structure of this region. An important difference between the polyproteins of HGV/GBV-C and HCV is the absence of a putative HGV/GBC-C core protein which is usually encoded at the 5'-end of the genome of flaviviruses. The buoyant density of HGV/GBV-C particles in human plasma was estimated to be between 1.07-1.12 g/ml, much lower than that of the other members of Flaviviridae family, except HCV. No HGV/GBV-C RNA was detected in fractions with densities higher than 1.17 g/ml which is expected for virus particles in immune complexes or in fractions with densities higher than 1.21 g/ml, the density range of HCV nucleocapsids. These biophysical properties correlate with the absence of a core-like protein in the genome of HGV/GBV-C isolates from different phylogenetic groups. The absence of the HGV/GBV-C nucleocapsid was also revealed by the sequence analysis data since no conserved open reading frame capable of encoding a core-like protein was identified. Generally, the untranslated regions at the 5' and 3' termini of a RNA virus genome contain regulatory elements important for viral RNA replication, transcription, translation and viral packaging. A comprehensive comparison and analysis of the primary sequence and secondary structure of the 3'-UTR of different HGV/GBV-C isolates allowed the construction of a common secondary structure model for this region and the identification of structural elements that may be involved in viral replication.

Die vorliegende Habilitationsschrift befasst sich schwerpunktmäßig vor allem mit der Klinik und Therapie der Hepatitis C. Evaluiert wurden: 1. verschiedene therapeutische Strategien, 2. die Ursachen der "Non-Response" auf eine anti-virale Therapie sowie 3. die klinische Relevanz der neu entdeckten Hepatitis-assoziierten Viren und 4. ihre Bedeutung bei Patienten mit akuter bzw. chronischer Lebererkrankung unklarer Ätiologie sowie bei Patienten vor und nach Lebertransplantation. Ad 1. Aus dem Vergleich verschiedener Therapie-Konzepte wie der Kurzzeit- Kombinationstherapie, Triple-Therapie, Hochdosis-Interferon?-Therapie und der Anwendung antiviraler Substanzen wie Ribavirin und Amantadin ergaben sich neue Erkenntnisse hinsichtlich relevanter prognostischer Parameter für die Therapieresponse. Ad 2. Analysiert wurden die möglichen molekularen Mechanismen der Therapieresponse bzw. Non-Response sowie der Stellenwert von Interaktionen bestimmter HCV-Proteine (NS5A, E2, sogenannte PKR-eIF2a Phosphorylisations-Homologie-Domäne [PePHD]) mit den Interferon? induzierten Effektorproteinen. Es konnte gezeigt werden, daß die Anzahl der Mutationen innerhalb des NS5A Proteins einen prognostischen Parameter darstellen hinsichtlich der Response auf eine Interferon?-Therapie. Dagegen spielen Mutationen innerhalb der PePHD-Region keine Rolle. Ad 3. Aus den Untersuchungen zur klinischen Relevanz der neu entdeckten Hepatitis-assoziierten Viren GB Virus-C/Hepatitis G Virus (GBV-C/HGV) und TT-Virus (TTV) ergaben sich keine Hinweise bzgl. eines Einflusses von GBV-C/HGV bzw. TTV-Infektionen auf den Verlauf der chronischen Hepatitis C. Die durchgeführten Verlaufsuntersuchungen bei koinfizierten Patienten sprechen dafür, daß es sich um Interferon-sensitive Viren handelt; jedenfalls beeinflussen sie nicht die IFN?-induzierte Response. Ad 4. Untersucht wurden ferner die Prävalenz, Transmission und Relevanz der GBV-C/HGV und TTV-Infektion im Hinblick auf ihre Hepatitis-induzierenden Eigenschaften. Die Ergebnisse belegen, dass beide Viren parenteral übertragen werden, und dass sie eine hohe Prävalenz bei Patienten mit parenteralen Risikofaktoren besitzen. Eine Hepatitis-induzierende Potenz dieser Viren konnten wir nicht beobachten; bei der Mehrzahl aller chronisch infizierter Personen ließen sich keine Zeichen einer chronischen Hepatitis finden.
The major goal of this thesis is the analysis of the clinical outcome of patients with Hepatitis C virus (HCV) infection and the response to therapy. Analysed were 1. different types of therapeutic strategies 2. causes responsible for ineffective antiviral therapy (non-response) 3. clinical relevance of the newly discovered hepatitis-associated viruses and 4. the role of these viruses in patients with acute or chronic hepatitis of unknown causes and in those receiving liver grafts. Ad 1. Compared were different therapeutic concepts such as short-term combination therapy, triple-therapy, high dose IFN?-therapy and the use of antiviral substances such as ribavirin and amantadine. It emerged that relevant prognostic parameters can be deduced with respect to the therapeutic response rate. Ad 2. Analysed were possible molecular mechanisms, which may interfere with response or non-response to antiviral therapy. In this respect, we focussed on the interaction of certain HCV-proteins as NS5A, E2, so-called PKR-eIF2a phosphorylisation-homology-domain (PePHD). with the interferon-?-induced effector proteins. There is evidence, that number of mutations within the NS5A proteins are of prognostic relevance with respect to the response to interferon?-therapy. In contrast, mutations within the PePHD-region do not play any role in this respect. Ad 3. We also studied the clinical relevance of the newly discovered viruses GBV-C/HGV and TTV, and found, that they have no impact concerning the course of chronic hepatitis C. These viruses are interferon-sensitive and do not influence the IFNa-response as it could be documented by following the course of co-infected patients. Ad 4. Our studies also focused on the prevalence, transmission and relevance of GBV-C/HGV and TTV infections with respect to their role as hepatitis-inducing agents. We can show that both virus types are parenterally transmitted. There is a high prevalence for both types in patients confronted with risk factors for parenteral factors. From analysis of many patients being chronically infected with these viruses it became quite clear that they lack any important potency to provoke chronic liver disease.

The hepatitis G virus and GBV-C are recently discovered variants of the same virus belonging to the family Flavivirus (HGV/GBV-C). Although initially thought to be a hepatitis virus, it has been shown to have no association with liver disease. No work has been performed on the prevalence or molecular characteristics of HGV/GBV-C in southern Africa. In addition, although it is clear that the liver is not the primary site of replication, there is no data on the sites of HGV/GBV-C replication in normal subjects. Thus, this study aimed to assess the prevalence of HGV/GBV-C carriage in the urban and rural adult Black communities of the Western and Eastern Cape Provinces of South Africa, and compare it to the prevalence of serological markers of the hepatitis viruses A-E. In addition, this study aimed to assess the molecular features of South African HGV/GBV-C isolates and demonstrate the organs where viral replication was present. The mean prevalences of antibodies to hepatitis A lgG, hepatitis B surface antigen and antibodies to hepatitis B surface antigen were 98%, 4.3% and 61.1 % respectively. The mean prevalence of antibodies to hepatitis C was 1.8%. No significant differences in prevalence were shown between the urban and rural regions for these viruses. The mean anti-hepatitis E prevalence varied from 5.8% to 19.1 % in the different regions. Those living in mud houses without access to chlorinated tap water had a significantly higher prevalence of antihepatitis E. No anti-hepatitis D positive samples were isolated. The overall prevalence of HGV/GBV-C was 26.9%, with rural communities having a significantly lower prevalence than urban communities. A significant relationship was observed between HGV/GBV-C infection with the use of illicit drugs, female gender, younger age and past blood transfusions. Phylogenetic analysis demonstrated a novel fourth South African HGV/GBV-C genotype, distinct from the previously described genotypes 1-3. In addition, certain isolates showed a major deletion in the highly conserved 5' non-coding region of HGV/GBV-C. Analysis of 23 tissue biopsies from infected cadavers suggested that the spleen and bone marrow were the primary sites of HGV/GBVC replication.

Un primer paso para conocer la entrada del virus de la hepatitis G en la célula es conocer dónde se encuentra el péptido de fusión. Por esta razón, éste fue el principal objetivo de la presente tesis. Dentro de la familia "Flaviviridae", los péptidos de fusión
descritos hasta el momento, se encuentran en la zona interna de la proteína estructural.
El virus de la hepatitis G se asemeja estructuralmente al virus de la hepatitis C, por ello,la búsqueda del péptido de fusión se centró en la proteína estructural E2 también presente en el virus de la hepatitis C. Las regiones escogidas dentro de la proteína estructural pertenecen a la región amino terminal, E2(7-26), y la zona interna (E2(279-298).
Estas secuencias fueron sintetizadas mediante metodología en fase sólida y se estudió la
interacción entre los péptidos y modelos de membrana de distinta complejidad (monocapas lipídicas, bicapas lipídicas). Las técnicas utilizadas para conocer la interacción entre ambos fueron las isotermas de Langmuir, la calorimetría diferencial de barrido, la espectroscopia de fluorescencia, la espectroscopia de UV y la microscopía.
Además se estudió la conformación adoptada por los péptidos por las técnicas de dicroísmo circular y espectroscopia de infrarrojos por transformada de Fourier. De todos los resultados obtenidos el péptido que interaccionó en mayor medida con los modelos de membrana, además de desestabilizar y producir fusión fue E2(279-298). Este péptido producía un cambio en su conformación al interaccionar con membranas fosfolipídicas(sobre todo en presencia de cargas negativas) hacia una estructura de tipo alfa-hélice. Este cambio hacia una estructura más ordenada podría proporcionar la conformación activa del péptido responsable de la desestabilización de las membranas.
The hepatitis G virus (GBV-C/HGV) is a enveloped RNA virus belonging to the "Flaviviridae" family. The natural history of the GBV-C/HGV infection is at present not fully understood and its potential to cause hepatitis in humans is questionable.
Elucidation of the mechanism of the fusion of enveloped viruses to target membranes has attracted considerable attention because of its relative simplicity and potential clinical importance. Apart from the functions of viral binding to target membranes and the activation of viral fusion proteins, usually only one viral protein is responsible for the membrane fusion step. However, the nature of the interaction of viral fusion proteins with membranes and the mechanism by which these proteins accelerate the formation of membrane fusion intermediates are poorly understood. In this sense, specialized
hydrophobic conserved domains ("fusion peptides") have been postulated to be absolutely required for the fusogenic activity.
The main objective of the present work was the knowlegment of the fusion peptide of the hepatitis G virus. For this purpose we have performed studies with different synthetic peptides belonging the envelope protein E2 of hepatitis G virus. The selected peptides were from the amino terminal part of the protein (E2(7-26)) and from the internal part (E2(279-298)). We have analysed lipid-peptide interactions depending on the degree of complexity of model membranes: monolayer studies (surface activity,
insertion of peptides into monolayers) and liposomes studies (differential scanning
calorimetry, fluorescence measurements). The peptides were compared for their ability
to interact and perturb membranes. In addition, they were also tested for their ability to
induce both leakage of vesicular contents and vesicle fusion as well as to lyse erythrocytes. Furthermore, we have studied the conformational behaviour of the peptides in water and in different membrane environments by Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD). The results obtained showed that the E2(279-298) sequence was able to interact, penetrate and permeabilize vesicles
bilayers in a higher extent than E2(7-26) sequence. Furthermore, the interaction with
membranes induced a change in the internal peptide to an alpha-helical conformation while
the amino terminal sequence did not. This indicate that this internal segment peptide
could be involved in the fusion of hepatitis G virus into cell membrane.

El contingut de la tesi radica fonamentalment en l’ús de models de membrana biològica per a l’estudi del comportament fisicoquímic de seqüències peptídiques derivades del GB-virus C (GBV-C). La concomitància d’aquest virus amb el virus causant del SIDA (HIV) ha demostrat disminuir notablement la virulència d’aquets darrer. És per això que és important conèixer el comportament de pèptids que són fragments de les proteïnes del GBV-C per a posteriori poder enfocar l’estudi de la inhibició de la capacitat infectiva del HIV per part del GBV-C. Es tracta d’un estudi bàsic però amb resultats que contribueixen al disseny de noves estructures peptídiques per a ser emprades com una nova estratègia per a enfocar el tractament de la SIDA. Finalment indicar que aquest tema s’engloba dins del camp de la nanotecnologia pel que fa a l’ús de liposomes en els distints estudis presentats. Els resultats obtinguts es presenten en les següents publicacions: 1. Alay, M.; Busquets, M.A., Haro, I.; Rojo, N.; Alsina, M.A.; Prat, J. Merocyanine 540 for spectroscopic analysis of the interaction of a peptide sequence of hepatitis G virus with liposomes. Luminescence, 17: 263-264 (2002). 2. Alay, M.; Prat, J.; Haro, I.; Rojo, N.; Alsina, M.A.; Busquets, M.A. Spectroscopic analysis of the interaction of a peptide sequence of Hepatitis G virus with bilayers. Talanta, 60: 269-277 (2003). 3. Alay, M.; Prat, J.; Alsina, M.A.; Busquets, M.A. Effect of merocyanine 540 on Langmuir-Blodgett films and liposomes of zwitterionic, anionic and cationic lipid composition. Journal de Physique IV. 113: 3 -6 (2004) 4. Alay, M.; Alsina, M.A.; Haro, I.; Prat, J.; Busquets, M.A. Analysis of the effect of a peptide sequence of the E2 protein (HGV/GBV-C) on the physicochemical properties of zwitterionic and negatively charged bilayers. Luminescence 20: 445-450 (2005) 5. Alay, M.; Alsina, M.A.; Haro, I.; Prat, J.; Busquets, M.A. Interaction of E2 (GBV-C/HGV)-derived peptides with liposomes: fluorescence anisotropy and fluorescence resonance energy transfer methods. Luminescence 21: 360 -361 (2006) 6. Alay, M.; Haro, I., Girona, V.; Prat, J.; Busquets, M.A. Interaction of two overlapped synthetic peptides from GB virus C with charged mono and bilayers. Colloids and Surfaces B-Biointerfaces105: 7 -13 (2013)

Background & Aims: Both Hepatitis C virus (HCV) and Hepatitis G virus (HGV) are single-stranded positive sense RNA viruses belonging to the Flaviviridae family. Epidemiological evidence has suggested an association between HCV infection and non Hodgkin lymphoma (NHL), but the association has mostly been seen in regions where the prevalence is high. Canadian studies have reported no significant association. The role of HGV infection in NHL has also been suggested, but there is little epidemiologic evidence. We investigated HCV, HGV and risk of NHL in a population-based case-control study in British Columbia, Canada. Methods: Cases were aged 20-79, diagnosed between March 2000 and February 2004, and residents in Greater Vancouver or Victoria. Cases with HIV or a prior transplant were excluded. Controls were chosen from the Provincial Health Insurance Client Registry and were age/sex/region frequency matched to cases. Results: Antibodies for HCV were measured in plasma of 795 cases and 697 control subjects. HCV seropositivity was 2.4% in cases and 0.7% in controls [odds ratio (OR)=3.4, (95%) confidence interval (CI)=1.3-9.1)]. The highest risks were associated with diffuse large B-cell lymphoma (OR=8.3, 95%CI=2.9-23.9), marginal zone lymphoma (OR=4.5, 95%CI=1.1-19.2) and small lymphocytic lymphoma/chronic lymphocytic leukemia (OR=6.9, 95%CI=1.3-36.8). HGV viremia was determined in plasma by the RT-PCR technique in 553 cases and 438 control subjects. The prevalence of HGV viremia was 4.5% in cases and 1.8% in controls (OR=3.2, 95%CI=1.4-7.3). The associations were strongest for cases with diffuse large B-cell lymphoma (OR=5.7, 95%CI=2.3-14.6), marginal zone lymphoma (OR=3.5, 95%CI=1.2-10.4) and other/unknown B-cell lymphoma (OR=4.9, 95%CI=1.3-17.6). Interpretation: Our results provide further evidence that exposure to HCV and HGV contribute to NHL risk. The associations were strongest for cases with diffuse large B-cell lymphoma and marginal zone lymphoma.
Medicine, Faculty of
Graduate

El virus de la hepatitis G (GBV-C/HGV) es un virus ARN perteneciente a la familia Flaviviridae. Su prevalencia, basada en la positividad del ARN, oscila entre el 1 y el 4% en la población general. Sin embargo este porcentaje aumenta hasta el 20-30% en los individuos expuestos a sangre o sus derivados, en individuos infectados por el virus de la hepatitis C o por el virus de la inmunodeficiencia humana (HIV), hecho que indica que se transmite principalmente por vía parenteral. Aunque no está claro su potencial patogénico, los estudios más recientes sugieren que la infección por este virus reduce la mortalidad en los pacientes infectados por el HIV, de ahí el interés en disponer de una herramienta que permita el diagnóstico de la infección por GBV-C/HGV de un modo rápido y sencillo.
Desde hace años, los péptidos sintéticos se vienen utilizando en sistemas de diagnóstico para muchas enfermedades, sin embargo, los péptidos lineales que mimetizan epítopos B son débilmente reconocidos por los anticuerpos, y por ello, existe la tendencia de utilizar combinaciones más complejas que permitan mejorar tanto la sensibilidad como la especificidad de los ensayos.

En esta tesis se han diseñado y sintetizado, utilizando la metodología de síntesis en fase sólida, construcciones peptídicas en las que se combinan regiones de proteínas de envoltura y no estructurales. Se han sintetizado tanto péptidos quiméricos, que contienen más de un epítopo (lineales y ramificados), como péptidos cíclicos en los que los epítopos sufren restricción de movilidad.
La capacidad antigénica de las construcciones sintéticas se ha evaluado utilizando principalmente la técnica del enzimoinmunoensayo (ELISA) aunque también se ha investigado la utilidad de la técnica de la resonancia del plasmón de superficie (SPR) para detectar la presencia de anticuerpos anti-GBV-C/HGV en muestras de suero de individuos pertenecientes tanto a los grupos de riesgo como en la población sana. Además, se ha realizado un estudio conformacional con la finalidad de establecer una correlación entre la estructura secundaria adoptada por los péptidos y su capacidad antigénica. Finalmente, se ha estudiado la capacidad inmunogénica de las construcciones peptídicas en animales de experimentación.

Los resultados obtenidos muestran, por un lado, que las construcciones en las que se combinan varios epítopos son las que presentan una mejor precisión diagnóstica, y por otro lado, que la introducción de restricción de movilidad permite incrementar la sensibilidad mostrada por la molécula precursora lineal.
"Design and synthesis of peptides for serodiagnose of the hepatitis G virus (GBV-C/HGV) infection".

The GB virus C, so called hepatitis G virus (GBV-C/HGV), is a single-strand RNA virus belonging to the Flaviviridae family. The prevalence rate of GBV-C/HGV in healthy blood donors is 1-4% in worldwide and about 20-35% in high risk populations, thus indicating that this virus is transmitted via the parenteral route. Although controversial data exit concerning the potential to cause hepatitis in humans recent studies suggest that coinfection with HIV is associated with prolonged survival. For this reason it would be interesting to find an easy tool to diagnose this apparently non-pathogenic virus.
In recent years, synthetic peptides that mimic specific epitopes of infectious agents have been used in diagnostic systems for various diseases. The main drawback of this approach is that peptides representing topographic B-cell epitopes are poorly recognised by antibodies. There is a tendency toward using chimeric to avoid those problems and to improve the sensitivity and specificity of the assays.
In this thesis, new putative epitopes located both in envelope and in nonstructural proteins of GBV-C/HGV were synthesized using solid-phase chemistry. The corresponding synthetic peptides, obtained in linear, multimeric and cyclic forms, were used as antigens in ELISA and in real-time bioespecific interaction measurements (SPR) to detect GBV-C/HGV-specific antibodies in different panels of human sera. Furthermore, CD and FT-IR have been used in conjunction to characterize the conformational changes therein with synthetic constructs that could explain their different antigenicity.
The results obtained showed, on one hand, that the combination of different antigens seems to be necessary to ensure good sensitivity and more specificity and, on the other hand, that cyclic compounds show higher ability to recognize anti-GBV-C/HGV antibodies than its parent peptide. Our results offer a new approach to develop new diagnostic peptide based biosensors for serodiagnosis of GBV-C/HGV infection.

Introdução: O vírus da hepatite G (GBV-C/HGV) é um flavivírus de genoma RNA de fita simples polaridade positiva, replicando em linfócitos, não sendo até hoje associado a qualquer patogenia humana. Estudos recentes demonstram que pessoas co-infectadas pelos vírus GBV-C/HGV e HIV têm menor progressão para AIDS e morte, embora alguns estudos tenham falhado em demonstrar tais efeitos. Objetivo: Determinar a soroprevalência e viremia (qualitativa) por GBV-C/HGV nas amostras de pacientes HIV+ e desenvolver uma metodologia de PCR em tempo real para fazer a determinação das cargas virais de GBV-C/HGV. Métodos: Avaliamos a presença de anticorpo e RNA do vírus GBV-C/HGV em 253 amostras de plasma de mulheres HIV positivas, coletadas entre 1997-99. Realizamos o ensaio imunoenzimático anti-E2 (EIA anti-HGenv kit, Roche(TM)), a reação em cadeia da polimerase (PCR), o NESTED PCR e, padronizamos um PCR em tempo real (RT-PCR), ensaio baseado no sistema Taqman, também aplicado nas mesmas amostras. A curva-padrão do ensaio foi feita com diluições seriadas de uma bolsa de plasma GBV-C/HGV+, e os resultados expressos em unidades aleatórias/mL. Resultados: Das 253 amostras testadas, 64 foram positivas para o anticorpo anti-E2 (25,3%), 36 RNA positivas no PCR convencional (14,2%) e, no NESTED PCR e PCR em tempo real 57 amostras foram concordantemente RNA positivas (22,5%), perfazendo um índice total de exposição de 48% (25.3 + 22.5). A carga viral teve uma média de 1.396 UA/mL (13.625 - 1.1UA/mL. As cargas virais encontradas não tiveram correlação direta com parâmetros laboratoriais da infecção pelo HIV como a carga viral e a contagem de células CD4. Conclusões: Foi obtida uma metodologia simples, rápida e de boa sensibilidade e especificidade, permitindo a quantificação do RNA do vírus GBV-C com reprodutibilidade. A metodologia permite análise simultânea de um grande número de amostras, sendo apropriada para estudos clínicos. A prevalência de exposição á este agente na população feminina HIV+ estudada é alta, provavelmente decorrente da via sexual comum de transmissão dos agentes.
Introduction: The Hepatitis G (GBV-C / HGV) agent is a flavivirus. Its genome is composed of single stranded RNA of positive polarity, replicating in lymphocytes. Up to now, it hasn\'t been implicated in any disease associated to human beings. Recent studies demonstrate that persons co-infected by the viruses GBV-C / HGV and HIV have a slower rate of progression to AIDS and death, although some studies have failed in demonstrating such effects. Objective: To determine the soroprevalence and viremia (qualitative) of GBV-C / HGV in samples from HIV-infected women. To develop a methodology of Real-Time PCR for GBV-C / HGV viral load determination. Methods: The presence of antibody and GBV-C/HGV RNA was evaluated in 253 plasma samples from HIV infected women, collected between 1997-99. An immunoenzymatic assay (EIA kit for anti-E2, Roche(TM)) was applied to obtain the seroprevalence while the polymerase chain reaction (PCR), nested PCR, and a standardized Taqman based real-time PCR (RT-PCR), were used in the assessment of viral RNA. For the viral load, a standard-curve was made with serial dilutions of a plasma bag containing GBV-C/ HGV RNA+, and results were expressed in random units/mL, in comparison to the reference matherial. Results: Of the 253 samples tested, 64 were positive for anti-E2 (25.3%), 36 RNA positive by PCR (14.2%), and 57 (22.5%) where both nested PCR and real-time PCR RNA positive, for a total exposure index of 48%. The mean viral load was of 1.396 RU/mL (13.625 - 1.1 RU/mL). GBV-C/HGV Viremia was not correlated to HIV laboratorial parameters, i.e. CD4 and HIV-RNA counts. Conclusions: A simple, fast and efficient system for the determination of GBV-C/HGV viral load was developed, presenting appropriate sensitivity and specificity, allowing reproducible quantification of GBV-C RNA in stored plasma samples. The methodology allows simultaneous analysis of a large number of samples, being appropriate for clinical studies. The prevalence of exposition to this agent in the studied female population is high, probably a consequence of the common sexual way of transmission of the agents.

A doença hepática crônica causada pelo vírus da hepatite C (HCV) tornou-se, nos últimos anos, uma das principais comorbidades dos pacientes portadores do vírus da imunodeficiência humana (HIV) nos países desenvolvidos. Os pacientes coinfectados com HIV/HCV apresentam uma progressão mais rápida para a cirrose e as suas complicações que os pacientes monoinfectados com HCV. Embora os mecanismos responsáveis por esta evolução não estejam totalmente esclarecidos, a expressão da molécula de HLA-G, um HLA de classe Ib não clássico, que tem propriedades bem reconhecidas na regulação negativa da resposta imune, pode estar relacionada à progressão da doença hepática. Os objetivos deste trabalho foram analisar o perfil de expressão de HLA-G em tecido hepático de pacientes coinfectados HIV/HCV e identificar possíveis variáveis do hospedeiro, do HCV e do HIV que possam estar relacionadas com a expressão de HLA-G na biópsia hepática. Para isso, 57 amostras de biópsia hepática de pacientes coinfectados com HIV/HCV, nas quais a imuno-histoquímica para HLA-G foi realizada, foram analisadas retrospectivamente quanto à expressão desta molécula no tecido hepático. Avaliaram-se também outras características histopatológicas da biópsia como grau de fibrose, atividade inflamatória, deposição de ferro e gordura. Determinou-se o polimorfismo de inserção ou deleção de 14 pares de bases da região 3` não traduzida do exon 8 do gene do HLA-G, que está relacionada com a produção de RNA-mensageiro, em 43 destes pacientes, além do polimorfismo de IL-28B, relacionado com a resposta ao tratamento do HCV, em 44 deles. Características bioquímicas e virológicas, tanto do HIV quanto do HCV também foram avaliadas. O genótipo 1 do HCV foi o mais prevalente (87,75%), especialmente o subgenótipo 1a (60%). A expressão do HLA-G foi observada em 38 (66,7%) amostras de fígado, e foi mais frequente em estágios moderados e severos de fibrose do que em estágios mais leves (94,1% x 55%, P < 0,01). Não houve relação entre a expressão do HLA-G e os outros parâmetros estudados. Embora a progressão para a cirrose no contexto da coinfecção por HIV/ HCV seja um processo complexo, modulado por muitos factores, a associação da intensidade de fibrose com a expressão do HLA-G pode indicar que a expressão desta proteína desempenha um importante papel nos mecanismos que contribuem para a progressão da doença, por meio da regulação negativa da resposta imune contra o HCV na coinfecção pelo HIV.
Chronic liver disease induced by hepatitis C virus (HCV) infection has recently become one of the most common comorbidities in patients who are infected with the human immunodeficiency virus (HIV) in developed countries. HIV/HCV coinfected patients show faster progression to cirrhosis and its complications than the HCV monoinfected patients. Even though the responsible mechanisms for this evolution have not been entirely clarified yet, the expression of the HLA-G molecule, a HLA from the non-classic Ib class, with well-known properties of negatively regulating the immune response, may be related to the liver disease progression. The aims of the present work were to analyze the HLA-G expression profile in the liver micro ambience of HIV/HCV coinfected patients and to identify possible host factors, HIV or HCV, that may be related to the HLA-G expression on the liver biopsy. For this purpose, 57 liver biopsies of HIV/HCV coinfect patients, in which immunohistochemistry for HLA-G had been performed, were retrospectively analyzed according the HLA-G expression on the hepatic tissue. Other histopathological features in the liver biopsies, such as fibrosis degree, inflammatory activity, iron deposition and fat were also evaluated. The polymorphism of insertion or deletion in 14-base pairs of the 3`non-translated region of exon 8 of the HLA-G gene, which is related to the production of HLA-G messenger RNA, was evaluated in 43 of the patients. Also, the polymorphism of IL-28B, related to the response to HCV treatment, was evaluated in 44 of them. Biochemical and virological features of HIV and HCV were also evaluated. The HCV genotype 1 was the most prevalent (87.75%), especially the subgenotype 1a (60%). The expression of HLA-G was observed in 38 (66.7%) samples of the liver biopsies, and it was most frequent in moderate and severe stages of fibrosis than in the mild stages (94.1% x 55%, P < 0.01). There was no established relationship between HLA-G and other parameters studied. Although the progression to cirrhosis in the context of HIV/HCV coinfection is a complex process modulated by many factors, the association of HLA-G expression with the intensity of the liver fibrosis may indicate the protein expression play an important role in the mechanisms that contribute to the progression of the disease, through the negative regulation of the immune response against HCV setting of a coinfection with HIV.

El GB virus C (GBV-C), también conocido como virus de la hepatitis G, es un virus humano no patogénico. Su infección es bastante frecuente, encontrándose incluso en personas sanas. Su prevalencia es mucho mayor en individuos considerados de riesgo, ya que comparte las vías de transmisión con otros virus, como por ejemplo con el virus de la hepatitis B, el virus de la hepatitis C o el virus de la inmunodeficiencia humana (VIH). Numerosos estudios asocian la coinfección del GBV-C y el VIH con una menor progresión de la enfermedad y con una mayor supervivencia de los pacientes una vez que el SIDA se ha desarrollado. Sin embargo, el mecanismo de acción aún no se ha determinado. De este modo, el estudio de la interacción entre ambos virus podría dar lugar a nuevos agentes terapéuticos para el tratamiento del SIDA. Por otro lado, en cuanto al diagnóstico, actualmente no existe ningún sistema comercial para detectar marcadores específicos de la infección causada por el GBV-C. De esta manera, en esta tesis se ha estudiado la capacidad de diversas moléculas peptídicas de regiones de la proteína de envoltura E2 y de la proteína no estructural NS4a del GBV-C para inhibir la entrada del VIH-1 a la célula, con el fin de conocer su potencial aplicación como agentes terapéuticos contra dicho virus. Asimismo, se ha estudiado la utilidad de dichas construcciones para desarrollar nuevos sistemas de diagnóstico de la infección causada por el GBV-C, empleando el ensayo inmunoenzimático de ELISA y la técnica de microarrays. En primer lugar, se ha realizado un estudio detallado de la proteína E2 del GVB-C mediante la preparación de microarrays peptídicos con 124 secuencias lineales de dicha proteína. Para ello, se han empleado sueros procedentes de pacientes infectados por el VIH-1, de los cuales se conocía la presencia o ausencia de anticuerpos anti-E2 del GBV-C, y sueros de personas voluntarias sanas. Este ensayo ha permitido identificar dominios potencialmente antigénicos de la proteína E2 del GBV-C. Teniendo en cuenta los resultados obtenidos y estudios previos realizados en el grupo de investigación, se ha llevado a cabo la síntesis de moléculas peptídicas derivadas del dominio N-terminal de la proteína E2 del GBV-C. Por un lado, se ha realizado la síntesis en fase sólida siguiendo una estrategia Fmoc/tBu, de péptidos lineales y de MAPs tetraméricos de tipo lineal homogéneo (con cuatro secuencias peptídicas iguales) y de tipo lineal heterogéneo (con secuencias peptídicas iguales dos a dos). También se han sintetizado péptidos cíclicos en solución mediante la reacción de transtioesterificación intramolecular conocida como Ligación Química Nativa. Por último, se ha llevado a cabo la formación de MAPs tetraméricos de tipo cíclico mediante ligación quimioselectiva en solución. Todas las moléculas sintetizadas se han caracterizado por HPLC, UPLC y espectrometría de masas (ESI y MALDI-TOF) y se han purificado por HPLC semipreparativo. Posteriormente, se han realizado ensayos de fusión celular con el fin de evaluar la actividad anti-VIH-1 de las moléculas peptídicas que derivan del dominio N-terminal de la proteína E2 del GBV-C. Las líneas celulares empleadas fueron la Hela-env y la TZM-bl. De este estudio pudo concluirse que las construcciones de tipo cíclico tienen mayor tendencia hacia una mayor inhibición de la entrada del VIH-1 en la célula. Se ha estudiado la capacidad para detectar anticuerpos anti-GBV-C de todas las secuencias y construcciones sintéticas mediante el ensayo inmunoenzimático de ELISA con sueros procedentes de pacientes con hepatitis C crónica, de pacientes sometidos a hemodiálisis, infectados con VIH-1 y sueros de donantes voluntarios sanos. Los resultados obtenidos demostraron la potencial utilidad diagnóstica de la región E2(7-26) del GBV-C. Por último, con un panel de sueros de pacientes infectados por el VIH-1, del que no se tenía información sobre la presencia o ausencia de anticuerpos anti-E2 del GBV-C, se ha evaluado el valor diagnóstico de los dominios peptídicos identificados mediante microarrays. La combinación de las secuencias peptídicas ensayadas ha permitido establecer una reactividad del 47% en cuanto a la detección de anticuerpos anti-E2 del GBV-C en personas infectadas por el VIH-1. Además, esta tecnología ha miniaturizado el ensayo inmunoenzimático de ELISA y ha demostrado la utilidad de los péptidos sintéticos como potenciales antígenos para el desarrollo de un sistema de diagnóstico de la infección del GBV-C.
GB virus C (GBV-C) (also formerly known as hepatitis G virus) is a non-pathogenic human virus. Its infection is more frequently in groups considered as high risk because it has similar routes of transmissions with other viruses such as hepatitis B virus, hepatitis C virus or human immunodeficiency virus (HIV). However, it is detected even in healthy people. There is a strong evidence for GBV-C association with ameliorated course of human immunodeficiency virus (HIV) disease, although its mechanism of action is yet to be determined. Thus, the study of the interaction between these viruses could give new therapeutic agents to HIV treatment. At present, there are no commercial systems to detect specific markers of GBV-C infection. In this thesis, the synthesis of branched peptide molecules was carried out by conjugating regions of the envelope protein E2 and the structural protein NS4 of the GBV-C virus. Afterwards, their antigenic capacity was evaluated. The ability of the synthesized molecules to inhibit cell fusion mediated by HIV-1 envelope protein was also studied in order to select potential inhibitors of virus entry into the cell. First of all, a detailed study of the E2 protein of GVB-C was carried out by preparing peptide microarrays with 124 linear sequences of this protein, using sera from patients infected with HIV-1 and healthy volunteers. Thus, potentially antigenic domains of the E2 protein of GBV-C were identified. After carrying out these assays and studying the accessibility profile of the potentially antigenic domain E2 (7-26), solid phase synthesis of linear, cyclic and branched peptides was carried out following an Fmoc/tBu strategy. Peptide constructions were characterized by HPLC, UPLC and mass spectrometry (ESI and MALDI-TOF) and purified by semipreparative HPLC. Subsequently, cell fusion assays were performed in order to evaluate the anti-HIV-1 activity of the peptide molecules derived from the N-terminal domain of the E2 protein of GBV-C. From this study, it could be concluded that the cyclic type constructions have a greater tendency towards a greater inhibition of HIV-1 entry in the cell. The ability to detect anti-GBV-C antibodies of all sequences and synthetic constructions were studied by the ELISA immunoenzymatic assay with sera from patients with chronic hepatitis C, patients undergoing hemodialysis, HIV-1 infected and sera from healthy volunteer donors. The results obtained demonstrated the potential diagnostic utility of the E2 region (7-26) of the GBV-C. Finally, the diagnostic value of the peptide domains identified by microarrays was evaluated with a panel of sera from patients infected with HIV-1. The combination of the peptide sequences studied allowed to establish a reactivity of 47% in the detection of anti-E2 antibodies of GBV-C in people infected with HIV-1. In addition, this technology has miniaturized the ELISA immunoenzymatic assay and it has demonstrated the usefulness of synthetic peptides as potential antigens for the development of a GBV-C infection diagnosis system.

碩士
國立臺灣大學
微生物學研究所
85
This study proceeded to the viral detection, epidemiological investigation, genome cloning and analysis of GB virus C/hepatitis G virus (GBV-C/ HGV). We detected serum viral RNA by reverse transcription-polymerase chain reaction (RT-PCR) and found higher GBV-C/HGV prevalences in drug abusers, hemophiliacs, patients with aplastic anemia, and HIV carriers. The result showed that the transmission of GBV-C/HGV is probably related to body fluid exchange. For the cloning of viral NS3 region, flanking primer method (Sorensen et al. , 1993) is useful for both 5'- and 3'-end extension except for Rapid Amplification of cDNA Ends (RACE) . We successfully obtained 582 base sequences from Taiwan strain NS3 region by these methods, and constructed an insert mutant for use in competitive PCR to quantitate GBV-C/HGV RNA. In the published reports, GBV-C/HGV genomes do not contain a full-length core gene. Since most virions are difficult to survive in absence ofnucleocapsid in the environment, the full-length core gene may exist but somehow wasn't cloned. We try to re-examine this hyposis but failed. Another interest is in some recipients who accepted GBV-C/HGV RNA positive blood didn't become infected. To investigate any viral difference, we compared the E1 and E2 region of viral RNA from donor sera and undertook the phylogenetic analysis. The results indicated that viral RNA from donor sera which cannot render recipients persistently infected did not belong to any specific genotypes or phylogenetic tree cluster. The possibilities of the other viral proteins or host neutralizing antibodies making such a difference were more likely. Another GB series virus- GB virus B (GBV-B)- causes hepatitis in tamarin and is more closely related to human hepatitis C virus (HCV) . We applied the sequences from GBV-B, HCV and Pestiviruses 5'-UTR consensus box to design two pairs of primers to do degenerated polymerase chain reaction for searching for the GBV-B related new human hepatitis virus. The strategy just began, still remained to be improved.

Recently a new Flavivirus, GB Virus C also referred to as Hepatitis G virus (GBV-C/HGV) was identified in humans with indeterminate hepatitis . Whilst in non-African countries this discovery led to an enormous enthusiasm to elucidate an association with liver disease, very little was known about the prevalence and pathogenicity of GBV-C/HGV infection in KwaZulu Natal, South Africa, where Hepatitis B Virus (HBV) infection is endemic and infection with the Human immunodeficiency virus (HIV) is a catastropic health problem. Sera from patients with liver disease (chronic liver disease [n = 98]; alcoholic liver disease [n = 50]); high risk groups (haemodialysis patients [n = 70]; HIV positive mothers and their babies [n = 75]) and control groups (alcoholics without liver disease [n = 35] and blood donors from the four racial groups [n = 232]) were screened for GBV-C/HGV RNA and Anti-E2 antibodies by reverse transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay (ELISA), respectively. Overall 43.9% (43/98) of patients with chronic liver disease; 60 % (30/50) of patients with alcoholic liver disease; 47.1% (33/70) of haemodialysis patients; 60% (21/35) of alcoholics without liver disease and 31.9% (74/232) of blood donors (Africans] 44/76; 5.9%); Asians (5/52; 9.6%); Whites (15/49; 30.6%) and "Coloureds" [mixed origin] (9/54; 16.6%)]) were exposed to GBV-C/HGV infection as determined by the detection of Anti-E2 &/or RNA in serum. There was a significant difference in the prevalence of GBV-C/HGV infection (RNA &/or anti E2) between African blood donors and the other racial groups (p < 0.001), between blood donors and haemodialysis patients (p = 0.02) and or patients with chronic liver disease (p =0.04). There was no significant difference in the prevalence of GBV-C/HGV between African blood donors (45/76, 59.2%) and alcoholics with and without liver disease (30/50, 60% and 21/35, 60%, respectively). Anti-E2 antibodies and GBV-C/HGV RNA were almost mutually exclusive. GBV-C/HGV infected dialysis patients tended to have had more transfusions (p = 0.03) and had a longer duration of dialysis than non infected patients, indicating that the majority of patients on maintenance haemodialysis acquire their GBV-C/HGV infection through the transfusions they receive. There was no evidence for in utero and/or intrapartum transmission of GBV-C/HGY. However, there is some mother-to-infant transmission of GBV-C/HGV, though it is very probable that in KZN GBV-C/HGV is transmitted by as yet undefined non-parenteral routes. Sequence and phylogenetic analysis of the 5' non-coding region (5' NCR) and E2 gene segments of the GBV-C/HGV genome identified an additional "genotype" (Group 5) of GBV-C/HGV that is distinct from all other known GBV-C/HGV sequences (Groups 1-4). Although there is a high prevalence of Group 5 GBV-C/HGV isolates in KZN, there was no significant difference in liver biochemistry between GBV-C/HGV infected and noninfected patients with liver disease or between blood donors in each of the four racial groups. There was no significant differences in CD4 (461.12 ± 163.28 vs 478.42 ± 181.22) and CD8 (680.83 ± 320.36 vs 862.52 ± 354.48) absolute cell counts between HIV positive patients co-infected with GBV-C/HGV and those not infected with GBV-C/HGV, respectively. However, significantly higher relative CD3 [80.0 ± 4.17% vs 70.99 ± 19.79%] (p = 0.015), gamma delta T cells (yLT) [3.22± 1.30% vs 2.15 ± 29.12%] (p = 0.052) and lower CD 30 [35.45 ± 17.86% vs 50.59 ± 9.20%] (p = 0.041) status were observed in GBV-C/HGV positive compared to GBV-C/HGV negative HIV infected patients, respectively. Although there is a high prevalence of novel Group isolates of GBV-C/HGV in KZN, the lack of elevated liver enzymes and clinical hepatitis in blood donors and haemodialysis patients suggests that GBV-C/HGV is not associated with liver disease. HBV and not GBV-C/HGV modifies the course of alcoholic liver disease. The relatively higher number of CD3 cells and increased yLT expression, together with a decrease in CD 30 cells tends to suggest an association with protection and or delayed progression of HIV disease in GBV-C/HGV infected patients. Whilst GBV-C/HGV is not associated with liver disease, it may be an important commensal in HIV infected patients.
Thesis (Ph.D.)-University of Natal, 2003.

Hepatitis B virus genotype G (HBV/G) is a unique genotype of HBV which contains a 36-nucleotide insertion in the Core gene as well as 2 mutations that lead to stop codons in the Pre-Core coding region. Chronic infection with HBV/G is not known to occur without a co-infecting HBV genotype, suggesting that it is defective on its own. This study aims to look at the replication capacity of HBV/G, HBV/A, and HBV/A/G recombinant strains circulating in Canada and to determine the relationship between co-infecting strains. Four full-length HBV genomes were isolated from 2 different patients and transiently transfected into the HepG2 human hepatoma cell line for phenotypic analysis of each strain. HBV/G, HBV/A and HBV/A/G recombinant strains were isolated from Patient 1, while a different HBV/A/G recombinant strain was isolated from Patient 2. HBV replication capacity was measured using a quantitative real time PCR assay. Markers of replication, such as secreted HBsAg and HBeAg, intracellular core particles and replicative DNA intermediates were measured by ELISA, Western blot and Southern blot, respectively. HBV/G demonstrated a higher replicative capability, relative to its co-infecting strains, while both HBV/A/G strains had levels of secreted HBV DNA greater than HBV/A alone, suggesting a modulating effect due to recombination. Replication marker levels revealed possible reasons for a co-infection requirement during HBV/G infection such as HBeAg for chronicity. These observations demonstrate the potential interactions of HBV/G with its co-infecting HBV genotype and provide the first reported phenotypic analysis of a HBV recombinant.

碩士
高雄醫學院
醫學研究所
87
HCV is the second cause of chronic hepatitis and hepatocellular carcinoma in Taiwan . To shed light on the status and clinical characteristics of the HGV infection in the HCV hyperendemic area, we studied the presence of HGV viremia and anti-E2 antibodies, the relation of HGV infection to the clinical characteristics, the factors influencing the seroconversion of anti-E2 antibodies and possible association of hepatitis B, C and G viruses among residents of Mashagou where is a highly endemic area for HCV located in the southwestern coastal area of Taiwan.as an HCV hyperendemic area model. Materials and Methods: Serum of two hundred residents of Mashagou were tested for alanine aminotransferase (ALT), HBsAg, second-generation HCV antibody (anti-HCV), HCV RNA and HGV RNA by nested RT-PCR using 5'untranslated region (5'UTR)-specific primers, and anti-E2 antibody by an enzyme linked immunosorbent assay. The prevalence of serum HGV RNA and anti-E2 antibodies were also investigated in 400 consecutive volunteer blood donors. Statistical analyses: Frequency was compared between groups using the chi-square test or Fisher's exact test, and group means were compared using the t test. Stepwise logistic regression method was used to analyze the study data. Results: The prevalence of HGV viremia, anti-E2 and HGV exposure among residents of Mashagou were significantly higher than those among volunteer blood donors (17.0% vs. 3.3%, 25.5% vs. 7.5% and 39.5% vs. 10.3%, respectively; all p<0.001). The prevalence of HGV exposure was significantly higher in individuals exposed to HCV than in those without HCV exposure (45.8% vs. 24.1%, respectively; p=0.005). None of sex, age, ALT levels, marker of chronic HBV infection and HCV genotype distribution was related to the exposure of HGV. The rate of anti-E2 seroconversion in Mashagou patients exposed to HGV (57.0%) was similar to that of blood donor (68.3%). Male had significant higher rate of anti-E2 seroconversion than female did (71.0% vs. 47.9%; p=0.04) in Mashagou rather than blood donors (male: 71.0%; female: 60.0%). None of age, ALT levels, markers of chronic HBV infection and HCV exposure was related to anti-E2 seroconversion. There was no difference of serum ALT levels between HGV viremic and non-viremic individuals in each group by the status of HBsAg and HCV RNA.The mean ALT level was significantly higher in those positive for HCV RNA with or without other viral markers (HBsAg and HGV RNA) than in those negative for HCV RNA. Based on multiple logistic regression analyses, significant factors associated ALT elevation was HCV RNA with odds ratio and 95% confidence interval of 6.96 and 2.60-18.7. Conclusions: In HCV hyperendemic area of Taiwan, HGV infection is closed associated with HCV infection. With minimal pathogenic-effect of HGV infection after exposure of HGV, HCV rather than HGV played the most important clinical hepatopathic role. HGV had lower persistent infection rate than HCV and there was no influence on the recovery from HGV infection by HCV infection. The reasons for the higher seroconversion rate in male than female in HCV endemic area can not be provided and need further evaluation.