Non-isotopic detection of hepatitis C virus quasispecies by single strand conformation polymorphism.
In patients infected with the hepatitis C virus (HCV), a heterogeneous population of viruses, so-called quasispecies exists in vivo. The hypervariable regions (HVR) within the second envelope gene (HCV-E2) show particularly highly intratypic variability and are considered to be the target of neutralizing antibodies. The aims of the study were to optimize a genotype-independent primer set for amplification of HVR-1 and to establish a sensitive SSCP analysis for rapid and non-isotopic detection of predominant serum HCV quasispecies. Using the optimized SSCP technique, changes of quasispecies composition were investigated in five chronically infected patients with HCV before and during interferon-alpha treatment. HCV genotyping was performed by sequence and phylogenetic analysis. In addition, serial viremia and serum alanine aminotransferase (ALT) levels were determined. The SSCP analysis was performed at two time points before and during interferon-alpha therapy, respectively. Four patients showed an alteration of the SSCP pattern during the first three months of interferon-alpha therapy, whereas in one patient the SSCP pattern changed before therapy and remained stable during treatment with interferon-alpha. The present approach for non-isotopic analysis of single strand conformation polymorphism provides a direct, rapid, and sensitive technique for detection of the heterogeneous population of HCV quasispecies of different genotypes. Using this test procedure, investigations of large cohorts of patients with chronic hepatitis C can be undertaken.
Lee
J H
JH
Medizinische Klinik II, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt a.M., Germany.
Stripf
T
T
Roth
W K
WK
Zeuzem
S
S
D000410:Alanine Transaminase / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral; D016174:Hepacivirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000821:virology*; D006801:Humans; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D018807:Polymorphism, Single-Stranded Conformational; D012689:Sequence Homology, Nucleic Acid; D014759:Viral Envelope Proteins / Q000235:genetics
D017931:DNA Primers; D004279:DNA, Viral; C087035:E1 protein, Hepatitis C virus; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D000410:Alanine Transaminase