Quasispecies analysis in hepatitis C virus infection by fluorescent single strand conformation polymorphism.
Hepatitis C virus (HCV) results frequently in chronic hepatitis and its sequelae liver cirrhosis and hepatocellular carcinoma. Interferon-alpha is at present the most effective treatment, resulting in a sustained response in about 20-25% of patients. HCV genotype, titer and quasispecies determine the success of treatment. In this study, fluorescent single strand conformation polymorphism (f-SSCP) was evaluated for the analysis of HCV quasispecies. Two sera from a chronically HCV-infected patient, obtained 6 years apart, were examined. The hypervariable region I (HVRI) of the HCV genome was amplified by reverse transcription and PCR. The PCR products were cloned and sequenced or fluorescein-labeled and subjected to f-SSCP. Both methods demonstrated a single HCV species in the early serum and multiple quasispecies in the late serum. Single clones of the heterogeneous virus population were used to optimize conditions for f-SSCP. The most important factors were the gel temperature and virus titer. At the optimal running temperature one base exchange in 218 bases was detectable. Repeat extractions and amplifications gave identical results. Dilution of the serum containing multiple 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.
Peters
T
T
University Hospital, Department of Medicine II, Freiburg, Germany.
Schlayer
H J
HJ
Hiller
B
B
Rösler
B
B
Blum
H
H
Rasenack
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012367:RNA, Viral / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014759:Viral Envelope Proteins / Q000235:genetics
D004279:DNA, Viral; C087035:E1 protein, Hepatitis C virus; D012367:RNA, Viral; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus