QUASI-SPECIES ANALYSIS IN HEPATITIS-C VIRUS-INFECTION BY FLUORESCENT SINGLE-STRAND CONFORMATION POLYMORPHISM

Hepatitis C virus (HCV) results frequently in chronic hepatitis and it s sequelae liver cirrhosis and hepatocellular carcinoma. Interferon-al pha is at present the most effective treatment, resulting in a sustain ed response in about 20-25% of patients. HCV genotype, titer and quasi species determine the success of treatment. In this study, fluorescent single strand conformation polymorphism (f-SSCP) was evaluated for th e analysis of HCV quasispecies. Two sera from a chronically HCV-infect ed patient, obtained 6 years apart, were examined. The hypervariable r egion I (HVRI) of the HCV genome was amplified by reverse transcriptio n and PCR. The PCR products were cloned and sequenced or fluorescein-l abeled and subjected to f-SSCP. Both methods demonstrated a single HCV species in the early serum and multiple quasispecies in the late seru m. Single clones of the heterogenous virus population were used to opt imize conditions for f-SSCP. The most important Factors were the gel t emperature and virus titer. At the optimal running temperature one bas e exchange in 218 bases was detectable. Repeat extractions and amplifi cations gave identical results. Dilution of the serum containing multi ple quasispecies resulted in a 'loss' of species. Provided the running temperature is optimal and virus titer is sufficient, f-SSCP is shown to be Fast and reliable for HCV quasispecies analysis. Copyright (C) 1997 Elsevier Science B.V.