Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFNα/RBV therapy.

M Holysz,P Migdalski,W H Trzeciak,K Bialas,D Kmieciak

doi:10.1007/s13353-014-0256-3

M Holysz, P Migdalski + Show 3 more

Open Access

https://doi.org/10.1007/s13353-014-0256-3

Copy DOI

Abstract

A modified method which can be used for the rapid screening of mutations in the protein kinase R-binding domain (PKR-BD) region and the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is described. This method is based on a high-resolution melting (HRM) technique used for genotyping single nucleotide polymorphisms and allows the detection of single nucleotide substitutions in the DNA sequence by measuring its Tm. The modified method, in addition to precisely measuring the Tm, allows the recording of the melting curve of the investigated cDNA fragment, which can provide provisional information about the number of different quasi-species present in the sample. The HRM analysis of the amplified cDNAs encoding the PKR-BD and HVR1 allowed the detection of partial replacement of HCV-1b by HCV-1a subspecies in one of our patients, as well as evaluation of the effectiveness of pegylated interferon α/ribavirin (PEG-IFNα/RBV) therapy. The HRM technique has never been used for the rapid screening of sequence variations in these regions and may be used for a similar purpose in any viral genome.

Highlights

Routine therapy of hepatitis C virus (HCV), based on pegylated interferon α combined with ribavirin (PEG-IFNα/ RBV), enables the achievement of a sustained virologic response (SVR) in only about 50 % of patients suitable for this type of treatment (Fried et al 2002)
high-resolution melting (HRM) analysis was applied to screen for alterations in the nucleotide sequences of cDNAs encoding the protein kinase R-binding domain (PKR-BD) region and the hypervariable region 1 (HVR1) in our patient
These two regions were chosen because the PKR-BD regions contains an interferon sensitivity-determining region (ISDR) and mutations in this region might play a crucial role in the resistance to PEGIFNα/RBV therapy, whereas the HVR1 is highly variable and mutations in this region reflect the ineffectiveness of therapy (Kmieciak et al 2006)

Summary

Introduction

Routine therapy of hepatitis C virus (HCV), based on pegylated interferon α combined with ribavirin (PEG-IFNα/ RBV), enables the achievement of a sustained virologic response (SVR) in only about 50 % of patients suitable for this type of treatment (Fried et al 2002). The serine protease inhibitors telaprevir (TVR) or boceprevir (BEC) are currently added to standard PEG-IFNα/RBV treatment, resulting in the improvement of SVR to over 70 % (Kozielewicz et al 2012). The amplified cDNA is cloned, sequenced, and converted into an amino acid (aa.) sequence of viral protein. This method is laborious, time-consuming, and expensive, and there is a need for a method which would allow one to distinguish between the subspecies of the virus rapidly and at a low cost

Methods

Results

Discussion

Conclusion