Important factors in reliable determination of hepatitis C virus genotype by use of the 5' untranslated region.

Accurate genotyping of hepatitis C virus (HCV) is important for determining the optimal regimen, dose, and duration of antiviral therapy for chronic HCV infection, as well as for estimating the response rate. The 5' untranslated region (UTR) of HCV RNA is used in commercial genotyping, but the probes and the lengths of the amplicons are proprietary and vary among the assays. In this study, factors involved in the reliable determination of HCV genotypes utilizing the 5' UTR were evaluated. Serum samples from four subjects with chronic HCV infection and disparate results on commercial genotyping and four controls were analyzed. HCV RNA was extracted from serum samples, and the 5' UTR and NS5B region were sequenced. Ten clones from each region were compared to prototype sequences and analyzed for genotype assignment using five programs. The results were compared to those from commercial assays. 5' UTR sequences were sequentially shortened from either the 5' end, the 3' end, or both ends, with genotyping of the resultant fragments. Sequences were obtained for the 5' UTR in all eight subjects and for the NS5B region in five subjects. The genotype assignments were identical between the two regions in the five subjects with complete sequencing. Genotyping by sequencing gave different results than those from the commercial assays in the four experimental samples but agreed in the four controls. Shortening of the sequences affected the results, and the results for sequences of <200 bases were inaccurate. Neither the Hamming distance nor the quasispecies affected the results. Sequencing of the HCV 5' UTR provided reliable genotyping results and resolved discrepancies identified in commercial assays, but genotyping by sequencing was highly dependent upon sequence length.