Nucleic acid-based detection and characterization of hepatitis C virus

Abstract: GENERAL Nucleic acid-based methods weredeveloped to detect and characterizethe HCV genome in immunocompe-tent and immunodeficient pahents ofwhom some were IFN-a treated.HCV RNA was found in most sera(95%), livers (97%) and PBMC(100%) of patients with conflrmedchronic hepatitis, but also in sera(21%) of patients with indeterminateconfirmatory antibody test, especial-ly those with c22 antigen reactivity.Minus-stranded HCV RNA wasfound in almost all livers, but alsoin PBMC (53%) and sera (35%).The quantification assay, based oncompetitive PCR, was found to besensilive and suitable to follow theviral load during IFN-c therapy.The pretreatment HCV RNA levelwas lower in patients with sustainedresponse than in those with no ornon-sustained response.HCV was misclassified as genotypeIb in mixed infections by a widelyused type-specific PCR due tomi.spriming of one primer.Concordant results were foundbetween phylogenetic analysis ofshort core and NS5 sequences.Using a direct sequencing assay, themutation rate of the hypervariableregion I (HVRI) was as high asO.1-0.2 substitutions/genome site/year in immumocompetent patients.During 15 months IFN-o~ therapy,the heterogeneity and mutation rateof HVRI decreased significantly.The heterogeneity and the mutationrate of patients with agamma-globulinemia or AIDS wassubstantially lower or absent. Asdetermined by both directsequencing and single strand poly-morphism analysis, the hetero-geneity was decreased in livertransplantat patients with advancedimmunosuppression during theearly post-LTx period and increasedwhen the dosages of the immuno-suppressive drugs were decreased.By our developed nucleic acid-based assays, important informationwas obtained. Thus, HCV replicatesnot only in the liver, but probablyalso in PBMC. The pretreatmentHCR RNA level seems to bepredictive for the response to IFN-oltherapy. Phylogenetic analysis ofshort core and NS5 sequences issuitable for HCV genotyping. Bothselection of HCV strains andgeneral suppression of the viralreplication may occur during IFN-c~therapy. An intact immune responseis crucial for the development of ahigh heterogeneity in HVR 1,although also other factors maycontribute in shaping the viralpopulation.Key words: HCV, PCR, genotype, HVRI, heterogeneity, IFN-a, immunodeficiency