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Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology. Univ Tsukuba, PhD Program Human Biol, Sch Integrat & Global Majors, Tsukuba, Ibaraki 3058577, Japan..
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
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2016 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 126, 81-89 p.Article in journal (Refereed) Published
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Abstract [en]

Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naive-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naive patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual.

Place, publisher, year, edition, pages
2016. Vol. 126, 81-89 p.
Keyword [en]
Hepatitis C virus, NS5A, Resistance, RAV, Deep sequencing, Pacific biosciences
National Category
Infectious Medicine
Identifiers
URN: urn:nbn:se:uu:diva-279573DOI: 10.1016/j.antiviral.2015.12.005ISI: 000369680000010PubMedID: 26707078OAI: oai:DiVA.org:uu-279573DiVA: diva2:908399
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2016-03-02 Created: 2016-03-02 Last updated: 2017-11-30Bibliographically approved

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Bergqvist, AndersAmeur, AdamLennerstrand, Johan

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