HCV PI-naive patients referred to our hospital between 2010 and 2011 were included in the study. Patients were stratified according to HCV genotype and a comparable number of patients infected with HCV genotypes 1a, 1b, 2, 3 and 4 were sequentially enrolled in the study. Most (75%) were treated with pegylated Interferon-α and Ribavirin, while none had ever been treated with a PI for hepatitis C. For NS3 sequencing, surplus serum samples were prospectively collected from each patient. HCV genotypes were defined using the Versant HCV Genotype 2.0 Assay LiPA (Siemens Healthcare Diagnostic Inc., Tarrytown, NY USA). The NS3 region was sequenced to further subtype HCV strains and identify genotypes 1a/1b. Data were analyzed with the Blast program (http://blast.ncbi.nlm.nih.gov). The study was approved by the Ethics Committee of the Fondazione IRCCS Policlinico San Matteo (protocol no. 20080009620). Informed consent was obtained from all subjects prior to enrollment.
Viral RNA was extracted from serum samples using the automatic Easy Mag extractor (Biomerieux, Lyon, France), and full-length HCV NS3/4A sequences were amplified using a nested RT-PCR. In detail, the primers used respectively in PCR and nested PCR, spanning NS3/4A aa from 1 to 181, were as follows: 1a-Forward outer 5′-GACATCATCAACGGCTTGCCCG-3′ and 1a-Reverse outer 5′-GAGTACGTGATGGGGCTGCCAG-3′, 1a-Forward inner 5′-GGAATGGTCTCCAAGGGGTGGA-3′ and 1a-Reverse inner 5′-CATGGGCCTTGGACATGTAAGC-3′ for genotype 1a; 1b-Forward outer 5′-CGAGACCTTGCGGTGGCAGT-3′ and 1b-Reverse outer 5′-CAGCCGTYTCCGCTTGGTCC-3′, 1b-Forward inner 5′-CATCACCTGGGGGGCAGACACC-3′ and 1b-Reverse inner 5′-GTCAGTTGAGTGGCACTCATCAC-3′ for genotype 1b; 2abc-Forward outer 5′-GGCACHTAYATCTATGACCA-3′ and 2-Reverse outer 5′-CAGYCCRATGGAGARGAARGTCA-3′, 2-Forward inner 5′-GTYCTRATGTTGGGRTTGATBCC-3′ and 2-Reverse inner 5′-TASGCCCCAAAMCCMAGSGTGG-3′ for genotype 2; 3-Forward outer 5′-GTCTCTGCRCGATTAGGCCGTGA-3′ and 3-Reverse outer 5′-CAGTTTRGCACCAGTTGTAACG-3′, 3-Forward inner 5′-GTTGGGACCTGCTGATGACTA-3′ and 3-Reverse inner 5′-CCCAGTGCGGATGTTGGGGT-3′ for genotype 3; finally, 4-Forward outer 5′-GGGYAATGARATMYTGCTCGG-3′ and 4-Reverse outer 5′-GCCAGGAACTTMCCRTABGT-3′, 4-Forward inner 5′-GGAGRCTBCTYGCBCCCAT-3′ and 4-Reverse inner 5′-GAGTAYGTGATYGGCGC-3′ for genotype 4. The PCR products in the first round were obtained by using the following conditions: 15’ at 45°C for the reverse transcription followed by 10’ at 94°C, and then 50 cycles at 94°C for 1’, 55°C for 1’ and 72°C for 70”, with an extension at 72°C for 10’. Three microliters from the first PCR reaction were used in the nested PCR with the following conditions: denaturation step at 94°C for 10’ and then 30 cycles at 94°C for 1’, 52°C for 1’ and 72°C for 70”, with an extension at 72°C for 10’.
Direct sequencing of PCR products was performed using an automatic sequencer (ABI PRISM 3100 genetic analyzer DNA Sequencer, Applied Biosystems, Foster City, CA, USA) and the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA).
Only variants present in more than 5% of the patient virus populations for each HCV genotype group were considered in the genotypic resistance analysis .
Nucleotide sequences were assembled using the Sequencer 4.6 (Gene Codes Corp., Ann Arbor, MI) software program. To obtain a detailed subtyping of HCV strains, nucleotide sequences were aligned with confirmed references of different subtypes using the ClustalW method which is embedded in the Mega 5 package .
The phylogeny of the sequences was constructed using the Neighbour Joining method. The nucleotide substitution model was selected according to Akaike Information Criterion scores. A Neighbour Joining tree was constructed with MEGA 5 software  setting the Tamura 3-parameter as an evolutionary model with an heterogeneous rate among sites using gamma distribution for the relative rate. Branch support was assessed by bootstrap analysis with 1000 replicates. Bootstrap values of 70% were used as the cut off point for cluster analysis. The sequences reported in this study have been submitted to the GenBank database under accession numbers J × 170910 to J × 171065.
The GenBank accession numbers for reference sequences used to determine the HCV genotypes were as follows: M62321 (subtype 1a), NC004102 (subtype 1a; H77-US1977), D90208 (subtype 1b), D14853 (subtype 1c), D00944 (subtype 2a), D10988 (subtype 2b), D50409 (subtype 2c), AB031663 (subtype 2 k), D17763 (subtype 3a), D49374 (subtype 3b), D63821 (subtype 3 k), Y11604 (subtype 4a), GU085486 (subtype 4a), FJ025855 (subtype 4b), FJ025854 (subtype 4b), FJ462436 (subtype 4c) FJ462437 (subtype 4d), EU392172 (subtype 4d), EU392170 (subtype 4f), FJ462432 (subtype 4 g), FJ462438 (subtype 4 k), EU392171 (subtype 4 k) EU392173 (subtype 4 k), FJ839870 (subtype 4 l), FJ462433 (subtype 4 m), FJ462441 (subtype 4n), FJ462440 (subtype 4o), FJ462431 (subtype 4p), FJ462434 (subtype 4q) FJ462439 (subtype 4r) FJ839869 (subtype 4 t), Y13184 (subtype 5a), Y12083 (subtype 6a), D84262 (subtype 6b), D84263 (subtype 6d), D63822 (subtype 6 g), D84265 (subtype 6 h), D84264 (subtype 6 k).