Etablierung einer PCR-unabhängigen Methode zum Nachweis des Hepatitis C-Virus und Studien zur Entwicklung eines Replikationssystems

Abstract

In this study a method for the specific detection of hepatitis C virus (HCV) negative and positive strand RNA by the hybrid detection assay (HDA) was developed. The principle of this method is a hybridization of the viral RNA with a specific single-strand DNA probe and the detection of the RNA:DNA-Hybrid using an anti-RNA:DNA hybrid antibody. The HDA was optimized for sensitivity and strand specificity with different amounts of synthetic RNA templates and reached a detection limit between 20 and 50 amol.Another intention of this work was the establishment of an efficient cell culture system for HCV. Various cell lines were infected with HCV and searched for the presence of HCV-RNA. Although negative strand RNA was detected by a Tth-based reverse-transcriptase polymerase chain reaction (RT-PCR) in U937- and FRhK4-cells, the development of an efficient in vitro system has been hampered.Nevertheless, this study provides evidence that the RNase L-Inhibitor (RLI) seems to support HCV-replication in vitro.