Nucleic acid-based detection and characterization of hepatitis C virus

Sammanfattning: Nucleic acid-based methods were developed to detect and characterize the HCV genome in immunocompetent and immunodeficient patents of whom some were IFN-a treated. HCV RNA was found in most sera (95%), livers (97%) and PBMC (100%) of patients with confirmed chronic hepatitis, but also in sera (21%) of patients with indeterminate confirmatory antibody test, especially those with c22 antigen reactivity. Minus-stranded HCV RNA was found in almost all livers, but also in PBMC (53%) and sera (35%). The quantification assay, based on competitive PCR, was found to be sensitive and suitable to follow the viral load during IFN-c therapy. The pretreatment HCV RNA level was lower in patients with sustained response than in those with no or non-sustained response. HCV was misclassified as genotype Ib in mixed infections by a widely used type-specific PCR due to mispriming of one primer. Concordant results were found between phylogenetic analysis of short core and NS5 sequences. Using a direct sequencing assay, the mutation rate of the hyper variable region I (HVRI) was as high as O.1-0.2 substitutions/genome site/year in immunocompetent patients. During 15 months IFN-o~ therapy, the heterogeneity and mutation rate of HVRI decreased significantly. The heterogeneity and the mutation rate of patients with a gamma-globulinemia or AIDS was substantially lower or absent. As determined by both direct sequencing and single strand poly-morphism analysis, the heterogeneity was decreased in liver transplantat patients with advanced immunosuppression during the early post-LTx period and increased when the dosages of the immuno-suppressive drugs were decreased. By our developed nucleic acid-based assays, important information was obtained. Thus, HCV replicates not only in the liver, but probably also in PBMC. The pretreatment HCR RNA level seems to be predictive for the response to IFN-c-therapy. Phylogenetic analysis of short core and NS5 sequences is suitable for HCV genotyping. Both selection of HCV strains and general suppression of the viral replication may occur during IFN-c~therapy. An intact immune response is crucial for the development of a high heterogeneity in HVR 1, although also other factors may contribute in shaping the viral population.