Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis.
Hepatitis C virus (HCV) populations in vivo consist of heterogeneous mixtures of genetically different but closely related variants defined as a 'quasispecies'. The longitudinal fluctuation of HCV quasispecies populations in chronic hepatitis C has not been elucidated. Serial plasma samples were obtained from four patients with chronic hepatitis C (two patients without any treatment and two patients treated with interferon), and cDNA fragments containing the 5'-terminal region of the E2 gene of HCV were amplified from plasma RNA using PCR. Since conventional cloning of PCR products detects only a small part of the entire population, PCR products of each sample were separated by electrophoresis using single-strand conformation polymorphism (SSCP) analysis, which can distinguish DNA fragments of the same size as different electrophoretic bands depending on their sequence-specific conformation. Separated DNA fragments were recovered from SSCP bands in gels and their nucleotide sequences determined. SSCP electrophoresis separated PCR products into bands with different mobility. Sequence analysis of these bands confirmed that HCV populations in each patient are composed of quasispecies with different E2-hypervariable regions (HVR), which are known to contain antibody epitopes. Different patterns of variation in the HVR of quasispecies were observed in individual patients with different clinical features over time during chronic infection. Following interferon treatment, some quasispecies disappeared during the treatment and reappeared after the end of the treatment, whereas other quasispecies in the same patient remained during the treatment suggesting that the sensitivity to interferon is different among quasispecies.
Enomoto
N
N
Second Department of Internal Medicine, Faculty of Medicine, Tokyo Medical and Dental University, Japan.
Kurosaki
M
M
Tanaka
Y
Y
Marumo
F
F
Sato
C
C
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics; D006526:Hepatitis C / Q000382:microbiology*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012367:RNA, Viral; D011994:Recombinant Proteins