Differentiation of hepatitis C virus subtypes 1a and 1b based on analysis of NS5B and 5′UTR region sequences using realtime PCR

Relevance. The hepatitis C virus has high genetic variability and is represented by a number of genotypes and subtypes that differ in their geographical distribution. Timely and highly accurate detection of the hepatitis C virus (HCV) followed by genotyping is a prerequisite for starting anti-HCV therapy. The molecular biological method—polymerase chain reaction (PCR)—is currently recognized as the “gold standard” for HCV genotyping. Aim. To study the nucleotide sequences of the NS5B and 5'UTR regions of HCV for the differentiation of subtypes 1a and 1b of genotype 1 (GT), followed by testing of the developed test system based on real-time PCR (RT-PCR). Materials and methods. The study examined 270 blood plasma samples infected with the hepatitis C virus. PCR results were confirmed by comparing the group of samples studied with data obtained using commercially available genotyping PCR kits, which allow differentiation between subtypes 1a and 1b. The specificity of the PCR system was evaluated on 150 hepatitis C virus samples, 100 of which belong to GT 3, and the remaining 50 to GT 2. Results and discussion. Of the 120 blood plasma samples tested, a large percentage of samples were identified as subtype 1b (95 %) and 1a (5 %), which corresponds to the data on the determination of subtypes 1a and 1b using the genotyping kits used in this study. Sequencing of the NS5B and 5'UTR regions also revealed that three samples contained the recombinant form HCV 2k/1b, one of which had previously been identified as GT 2. A specificity test showed that the PCR system under development using the NS5B and 5'UTR regions did not cross-react with 100 GT 3 samples and 49 GT 2 samples. Conclusion. The data obtained confirm the possibility of performing analysis using NS5B/5'UTR regions and the effectiveness of the method for determining subtypes 1a and 1b of hepatitis C virus genotype 1 in clinical practice. In addition, the use of the proposed PCR system made it possible to identify the recombinant variant HCV 2k/1b.

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