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Evaluation of antiviral resistant hepatitis B virus subpopulations in patients with chronic hepatitis B by using terminal restriction fragment length polymorphism

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Abstract

Antiviral therapies with nucleotide analogues (NA) is crucial in the treatment of chronic hepatitis B as it substantially protects patients from the complications of the disease . However in most of the available NA therapies, resistance emerges in the patients’ HBV populations. Therefore, detection of antiviral resistance as early as possible by means of genotypically monitoring the patients’ HBV pool during NA therapy is critical to manage treatment regime. In this research study we have investigated the sensitivity and specificity of the terminal restriction fragment length polymorphism (T-RFLP) method in detecting HBV subpopulations carrying antiviral resistance mutations. For this aim, differentiation of mutant strains from wild type strains was demonstrated by PCR-RFLP method. With using recombinant plasmids containing mutant and wild type HBV genomes, we constructed artificial HBV genome populations in order to determine the sensitivity of PCR-T-RFLP method in detecting antiviral resistant minor HBV populations. Finally by comparing with the DNA sequencing method, we demonstrated the specificity of T-RFLP method in genotyping HBV populations. As a result we showed that T-RFLP is able to detect HBV subpopulations representing as low as 1 % of the whole viral population. Additionally T-RFLP showed 100 % concordance with the DNA sequencing method in genotyping HBV populations. As a conclusion, considering the other genotyping methods used in evaluating HBV populations, T-RFLP showed high sensitivity and specificity profiles in detecting antiviral resistant HBV subpopulations. Therefore T-RFLP method can be easily employed in genotypic evaluation of patients’ HBV populations during the course of antiviral treatment.

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Acknowledgments

I am grateful to The Scientist Supporting Unit (BİDEB) of The Scientific and Technological Research Council of Turkey (TUBİTAK) for being supported by their National Graduate Studies Scholarship Program (code: 2210). I am grateful to Dr. A. Mithat Bozdayi for his valuable mentorship for my graduate research studies at Ankara University Institute of Hepatology. I am thankful to Dr. Ersin Karatayli who is a specialist at Ankara University Institute of Hepatology for his help regarding the construction of plasmid vectors containing hepatitis B virus full genomes.

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Authors and Affiliations

  1. Department of Biology, Faculty of Science, Ankara University, Tandogan, 06100, Ankara, Turkey

    Ergin Şahin

  2. Institute of Hepatology, Ankara University, Ankara, Turkey

    Ergin Şahin

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  1. Ergin Şahin
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Correspondence to Ergin Şahin.

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Suppl. Fig. 1

PCR-T-RFLP assay with the artificially constructed HBV genome populations that contain mutant(N236T) HBV subpopulations at various ratios. Three electropherograms represent the T-RFLP assay results of artificially constructed HBV genome populations of which mutant (N236T) to wild type ratios were adjusted to 5%, 1%, and 0.66% from top to the bottom respectively. Peaks (blue color) that identify the mutant (N236T) HBV subpopulations representing the 5% and 1% of the total HBV population were indicated with black arrows. The peaks that represent wild type(236N) HBV populations are indicated with red arrows (JPEG 1630 kb)

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Şahin, E. Evaluation of antiviral resistant hepatitis B virus subpopulations in patients with chronic hepatitis B by using terminal restriction fragment length polymorphism. VirusDis. 26, 267–275 (2015). https://doi.org/10.1007/s13337-015-0282-8

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