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Open Access Research article

Deep sequencing of hepatitis C virus hypervariable region 1 reveals no correlation between genetic heterogeneity and antiviral treatment outcome

Kamila Caraballo Cortés1*, Osvaldo Zagordi2, Karol Perlejewski1, Tomasz Laskus1, Krzysztof Maroszek1, Iwona Bukowska-Ośko1, Agnieszka Pawełczyk1, Rafał Płoski3, Hanna Berak4, Andrzej Horban45 and Marek Radkowski1

Author Affiliations

1 Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, 3c Pawińskiego Street, 02-106 Warsaw, Poland

2 Institute of Medical Virology, University of Zurich, Winterthurerstrasse, 190 8057 Zurich, Switzerland

3 Department of Medical Genetics, Medical University of Warsaw, 3c Pawińskiego Street, 02-106 Warsaw, Poland

4 Hospital for Infectious Diseases, 37 Wolska Street, 01-201 Warsaw, Poland

5 Clinics of Infectious Diseases, Medical University of Warsaw, 37 Wolska Street, 01-201 Warsaw, Poland

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BMC Infectious Diseases 2014, 14:389  doi:10.1186/1471-2334-14-389

Published: 13 July 2014

Abstract

Background

Hypervariable region 1 (HVR1) contained within envelope protein 2 (E2) gene is the most variable part of HCV genome and its translation product is a major target for the host immune response. Variability within HVR1 may facilitate evasion of the immune response and could affect treatment outcome. The aim of the study was to analyze the impact of HVR1 heterogeneity employing sensitive ultra-deep sequencing, on the outcome of PEG-IFN-α (pegylated interferon α) and ribavirin treatment.

Methods

HVR1 sequences were amplified from pretreatment serum samples of 25 patients infected with genotype 1b HCV (12 responders and 13 non-responders) and were subjected to pyrosequencing (GS Junior, 454/Roche). Reads were corrected for sequencing error using ShoRAH software, while population reconstruction was done using three different minimal variant frequency cut-offs of 1%, 2% and 5%. Statistical analysis was done using Mann–Whitney and Fisher’s exact tests.

Results

Complexity, Shannon entropy, nucleotide diversity per site, genetic distance and the number of genetic substitutions were not significantly different between responders and non-responders, when analyzing viral populations at any of the three frequencies (≥1%, ≥2% and ≥5%). When clonal sample was used to determine pyrosequencing error, 4% of reads were found to be incorrect and the most abundant variant was present at a frequency of 1.48%. Use of ShoRAH reduced the sequencing error to 1%, with the most abundant erroneous variant present at frequency of 0.5%.

Conclusions

While deep sequencing revealed complex genetic heterogeneity of HVR1 in chronic hepatitis C patients, there was no correlation between treatment outcome and any of the analyzed quasispecies parameters.

Keywords:
Hypervariable region 1; Ultra-deep sequencing; Treatment; Genetic heterogeneity; Hepatitis C virus; Quasispecies; Pyrosequencing