5' NC region of the HCV genome complexity detected by SSCP and RFLP analysis

Comparison between different HCV isolates from different host pointed to the region-depended variability of HCV. There are several advantages in using the 5' NC region of the HCV genome for genotyping and reflecting quasispecies nature of the HCV. The aim of the presented study was to establish the degree of complexity of 5' NC region HCV genome and grouping of hepatitis C isolates by restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) by the use of reverse - transcription - polymerase chain reaction products (RT-PCR). Representatives were checked by line probe assay. Isolated RNA preparations from whole blood or serum samples obtained from 55 patients with chronic HCV infection were used for cDNA synthesis. The nested polymerase chain reaction, RFLP experiments, SSCP analysis, the line probe assay and the phylogenetic analysis have shown that the applied methods RFLP and SSCP applied simultaneously to the analysis of the 5' NC virus region provided good evidence, that the region is highly complex. This phenomenon was examined by INNO-LIPA HCV II tests, used normally for genotyping, and it was shown, that the applied RFLP and SSCP methods alone are sufficient to describe the heterogeneity of this region.

Keywords: hepatitis C virus (HCV), Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Single Strand Conformation Polymorphism (SSCP), Restriction Fragment Length Polymorphism (RFLP), genetic heterogeneit