Background: Hypervariable region 1 (HVR1) contained within envelope protein 2 (E2) gene is the most variable\npart of HCV genome and its translation product is a major target for the host immune response. Variability within\nHVR1 may facilitate evasion of the immune response and could affect treatment outcome. The aim of the study\nwas to analyze the impact of HVR1 heterogeneity employing sensitive ultra-deep sequencing, on the outcome of\nPEG-IFN-? (pegylated interferon ?) and ribavirin treatment.\nMethods: HVR1 sequences were amplified from pretreatment serum samples of 25 patients infected with genotype\n1b HCV (12 responders and 13 non-responders) and were subjected to pyrosequencing (GS Junior, 454/Roche).\nReads were corrected for sequencing error using ShoRAH software, while population reconstruction was done using\nthree different minimal variant frequency cut-offs of 1%, 2% and 5%. Statistical analysis was done using Mannââ?¬â??Whitney\nand Fisherââ?¬â?¢s exact tests.\nResults: Complexity, Shannon entropy, nucleotide diversity per site, genetic distance and the number of genetic\nsubstitutions were not significantly different between responders and non-responders, when analyzing viral\npopulations at any of the three frequencies (?1%, ?2% and ?5%). When clonal sample was used to determine\npyrosequencing error, 4% of reads were found to be incorrect and the most abundant variant was present at a\nfrequency of 1.48%. Use of ShoRAH reduced the sequencing error to 1%, with the most abundant erroneous\nvariant present at frequency of 0.5%.\nConclusions: While deep sequencing revealed complex genetic heterogeneity of HVR1 in chronic hepatitis C patients,\nthere was no correlation between treatment outcome and any of the analyzed quasispecies parameters.
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