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.

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.

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.