Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis

Abstract

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 polymoi]~h-ism (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.

Characterization of simple and complex hepatitis C virus quasispecies by heteroduplex gel shift analysis: correlation with nucleotide sequencing

“…Similarly, tracking HCV quasispecies during interferon therapy may improve our understanding of the problems of resistance and relapse during and after interferon therapy. In this respect, HCV quasispecies which are differentially sensitive to interferon therapy have been reported (Enomoto et al, 1994). It is also possible that GSA of one or several HCV regions may eventually provide prognostic information regarding response or resistance to various antiviral therapies.…”

“…In a similar fashion, GSA will allow faster evaluation of larger numbers of clones than nucleotide sequencing and should thus provide more accurate information about clonal frequency for future studies of the role of HCV quasispecies in pathogenesis. An alternative method for analysing HCV quasispecies variants is single-strand conformation polymorphism analysis (SSCP) as recently described by Enomoto et al (1994). This technique has the advantage of assessing overall genetic heterogeneity using direct PCR products.…”

“…Indeed, a recent report suggests that HCV quasispecies are differentially sensitive to interferon (Enomoto et al, 1994).…”

Mutations in nonstructural protein 5A gene and response to interferon in hepatitis C virus genotype 2 infection

“…36,57,58 Although HCV RNA was not detectable during treatment in most patients in this work, sequence determination of NS5A in the relapse phase is potentially important for more precise correlation between NS5A structure and interferon responses in a further analysis. However, it is not practical for the prediction of the interferon effect.…”

“…Extraction of RNA from serum with the modified acid guanidinium thiocyanate-phenol-chloroform (AGPC) method 35 using ISO-GEN (Wako, Osaka, Japan) and RT-PCR was performed as described previously 36 with a hot-start PCR technique using Ampliwax PCR Gem 50 (Takara, Kyoto, Japan). Briefly, RNA extracted from 15 µL of serum was reverse-transcribed to cDNA using random hexamers.…”