The study protocol was approved by the Human Ethics Review Committee of the institution. Blood samples were frozen at −80°C within 4 hours of collection and were not thawed until used for testing. Anti-HCV, HCV RNA, HCV genotype, and aa substitutions of the HCV-1b core region were assayed using stored frozen sera. HCV genotype was determined by PCR using a mixed

primer set derived from nucleotide sequences of the NS5 region.24 HCV RNA quantitative click here analysis was measured by branched DNA assay v. 2.0 (Chiron), AMPLICOR GT HCV Monitor v. 2.0 using the 10-fold dilution method (Roche Molecular Systems, Pleasanton, CA), or COBAS TaqMan HCV test (Roche Diagnostics, Tokyo, Japan). High viral load of viremia levels was defined as branched DNA

assay ≥1.0 Meq/mL, AMPLICOR GT HCV Monitor ≥100 × 103 IU/mL, or COBAS TaqMan HCV test ≥5.0 log IU/mL. Low viral load was defined as branched DNA assay <1.0 Meq/mL, AMPLICOR GT HCV Monitor <100 × 103 IU/mL, or COBAS TaqMan HCV test <5.0 log IU/mL. In the present study, aa substitutions of the core region of HCV-1b were analyzed by direct sequencing. HCV RNA was extracted from serum samples Opaganib manufacturer and reverse transcribed with random primer and MMLV reverse transcriptase (Takara Syuzo, Tokyo, Japan). Nucleic acids of the core region were amplified by nested PCR using the following primers. The first-round PCR was performed with CE1 (sense, 5′-GTC TGC GGA ACC GGT GAG TA-3′, nucleotides: 134-153) and CE2 (antisense, 5′-GAC GTG GCG TCG TAT TGT CG-3′, nucleotides: 1096-1115) primers, and the second-round PCR with CC9 (sense, 5′-ACT GCT AGC CGA GTA GTG TT-3′, nucleotides: 234-253) and CE6 (antisense, 5′-GGA GCA GTC GTT CGT GAC AT-3′, nucleotides: 934-953) primers. All samples were initially denatured BCKDHB at 95°C for 2 minutes. The 35 cycles of amplification were set as follows: denaturation for 30 seconds at 95°C, annealing of primers for 30 seconds at 55°C, and extension for 1 minute at 72°C with an additional 7 minutes for extension. Then 1 μL of

the first PCR product was transferred to the second PCR reaction. Other conditions for the second PCR were the same as the first PCR, except that the second PCR primers were used instead of the first PCR primers. The amplified PCR products were purified by the QIA quick PCR purification kit (Qiagen, Tokyo, Japan) after agarose gel electrophoresis and then used for direct sequencing. Dideoxynucleotide termination sequencing was performed with the Big Dye Deoxy Terminator Cycle Sequencing kit (Perkin-Elmer, Tokyo, Japan). With the use of HCV-J (accession no. D90208) as a reference,25 the dominant sequence of 1-191 aa in the core protein of HCV-1b was determined by direct sequencing and then compared with the consensus sequence constructed on 50 clinical samples to detect substitutions at aa 70 of arginine (Arg70) or glutamine/histidine (Gln70/His70) and aa 91 of leucine (Leu91) or methionine (Met91).