Abstract
Determination of hepatitis C virus (HCV) genotypes plays an important role in the direct-acting agent era. Discrepancies between HCV genotyping and serotyping assays are occasionally observed. Eighteen samples with discrepant results between genotyping and serotyping methods were analyzed. HCV serotyping and genotyping were based on the HCV nonstructural 4 (NS4) region and 5′-untranslated region (5′-UTR), respectively. HCV core and NS4 regions were chosen to be sequenced and were compared with the genotyping and serotyping results. Deep sequencing was also performed for the corresponding HCV NS4 regions. Seventeen out of 18 discrepant samples could be sequenced by the Sanger method. Both HCV core and NS4 sequences were concordant with that of genotyping in the 5′-UTR in all 17 samples. In cloning analysis of the HCV NS4 region, there were several amino acid variations, but each sequence was much closer to the peptide with the same genotype. Deep sequencing revealed that minor clones with different subgenotypes existed in two of the 17 samples. Genotyping by genome amplification showed high consistency, while several false reactions were detected by serotyping. The deep sequencing method also provides accurate genotyping results and may be useful for analyzing discrepant cases. HCV genotyping should be correctly determined before antiviral treatment.
Highlights
Hepatitis C virus (HCV) is a positive-stranded RNA virus ~9.6 kb in length, and it belongs to the genus Hepacivirus within the Flaviviridae family [1]
In 17 of 18 samples, both results of cloned sequencing in the core and nonstructural 4 (NS4) regions were consistent with the HCV genotyping by 5 -untranslated region (5 -UTR), but not with those of HCV serotyping
Results of HCV Genotyping by Deep Sequencing in NS4 Regions Genotyping results investigated by deep sequencing of HCV NS4 regions using the MiSeq
Summary
Hepatitis C virus (HCV) is a positive-stranded RNA virus ~9.6 kb in length, and it belongs to the genus Hepacivirus within the Flaviviridae family [1]. The HCV genome encodes at least three structural proteins: core and two envelope glycoproteins (E1 and E2); and seven nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). At least seven HCV genotypes and 67 subtypes have been characterized. HCV isolates differ by >15% among different genotypes or subtypes [3]. HCV genotypes and subtypes are distributed differently among different areas of the world. Several methods have been established to determine HCV genotypes, such as sequence analysis [6], restriction fragment length polymorphism [7], hybridization of PCR products with specific probes [8] and next-generation sequencing [5]
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