Non-isotopic detection of hepatitis C virus quasispecies by single strand conformation polymorphism

Abstract

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.

Significance of Pretreatment Analysis of Hepatitis C Virus Genotype 1b Hypervariable Region 1 Sequences To Predict Antiviral Outcome

“…Reverse transcription was carried out at 42°C for 30 min in the presence of 12.5 U of Moloney murine leukemia virus reverse transcriptase (Stratagene). For the nested PCR steps, an optimized genotype-independent set of primers specific for the amino-terminal region of the HCV E2 gene (HVR1) was used (17). The first round of PCR was carried out with the reverse transcription primer described above (20 pmol) as the outer antisense primer and with primer 5Ј-CGCATGGCGTGGGACATGATG-3Ј (20 pmol) as the outer sense primer.…”

Hepatitis C virus in the semen of men coinfected with HIV-1: prevalence and origin

“…RNA was extracted from 140 ml blood serum and from 500 ml seminal plasma using QIamp RNA Mini Kit (Qiagen) in the conditions described above. Nested PCR used the outer and inner HVR-1 primers, which generate a 307 basepair product (þ956 to þ1262) encompassing the C-terminal E1 transmembrane region and the Nterminal hypervariable E2 region [25].…”

Comparative sequence analysis of the core- and NS5-region of hepatitis C virus from tumor and adjacent non-tumor tissue

“…All patients were infected with subtype HCV-1b (classi®cation according to Simmonds et al [1993]) and tested negative for HBV-DNA by PCR in serum. Non-tumor and tumor tissues were resected surgically, frozen in liquid nitrogen, and stored at À 80 C. Before macroscopic separation of tumor and non-tumor tissue the PCR and DNA sequencing HCV-RNA was extracted from frozen tumor and corresponding non-tumor tissue by guanidinium thiocyanate-phenol-chloroform method [Chomczynski and Sacci, 1987] and was reverse transcribed into cDNA as described previously [Lee et al, 1997;Zeuzem et al, 1994] with the exception of using random hexamers instead of PCR-primer. Two-step PCR assays with nested primers corresponding to the N-terminal part of the core region and the NS5 region, respectively, were carried out as described elsewhere [Ru È ster et al, 1996].…”