The role of mixed infections with different hepatitis C virus (HCV) genotypes in viral persistence, treatment effects, and tissue tropism is unclear. Next-generation sequencing (NGS), which is suitable for analysis of large, genetically diverse populations offers unparalleled advantages for the study of mixed infections. The aim of the study was to determine, using two different deep sequencing strategies (pyrosequencing - 454 Life Sciences/Roche and reversible terminator sequencing-by-synthesis by Illumina), the origin of a novel HCV genotype transiently detectable during antiviral therapy (pre-existing minor population vs. de novo superinfection). Secondly, we compared 5' untranslated region (5'-UTR) variants obtained by the two NGS approaches. 5' UTR amplification products from 9 samples collected from genotype 1b infected patient before, during, and after treatment (4 serum and 5 peripheral blood mononuclear cell - PBMC - samples) were subjected to the next-generation sequencing. The sequencing revealed the presence of two (454/Roche) and one (Illumina) genotype 4 variants in PBMC at Week 16. None of these variants were present either in the preceding or following samples as revealed by both platforms. 454/Roche sequencing detected 24 different 5'-UTR variants: 8 were present in serum and PBMC, 4 only in serum and 12 only in PBMC. Illumina sequencing detected 11 different 5'-UTR variants: 5 in serum and PBMC, 4 only in serum and 2 only in PBMC. Six variants were identical for both sequencing platforms. The difference in variants number was primarily due to variability in two 5'-UTR homopolymeric regions. In conclusion, longitudinal analysis of HCV variants, employing two independent deep sequencing methods, suggests that the transient presence of a different genotype strain in PBMC was a result of superinfection and not a selection of pre-existing minor variant.
Keywords: 5’ untranslated region; Hepatitis C virus; Mixed infection; Next-generation sequencing; Peripheral blood mononuclear cells.