Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing
2016 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 126, 81-89 p.Article in journal (Refereed) PublishedText
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.
Hepatitis C virus, NS5A, Resistance, RAV, Deep sequencing, Pacific biosciences
IdentifiersURN: urn:nbn:se:uu:diva-279573DOI: 10.1016/j.antiviral.2015.12.005ISI: 000369680000010PubMedID: 26707078OAI: oai:DiVA.org:uu-279573DiVA: diva2:908399
FunderScience for Life Laboratory - a national resource center for high-throughput molecular bioscience