Method for determining drug resistance mutations in any of the non-structural protein regions NS3 to NS5B of hepatitis C virus (HCV) for genotypes 1 to 6

Method for determining drug resistance mutations in any of the non-structural protein regions NS3 to NS5B of Hepatitis C Virus (HCV) for genotypes 1 to 6, more in particular for subtype specific genotypes 1a, 1b, 2a, 2b, 3a, 4a and 4d, present in a sample comprising:

• a) obtaining said sample from a patient,
• b) extracting viral genetic material from said sample,
• c) amplification of the NS5B region of HCV to generate a DNA amplicon of 388 base pairs by using primers having the sequences selected from the group consisting of SEQ ID NO's 1-5,
• d) sequencing of the amplicon to obtain a sequence of 329 base pairs by using the sequences selected from the group consisting of SEQ ID NO's 3-5,
• e) performing phylogenetic tree analysis using the 329 base pair sequence information of NS5B to obtain HCV-subtype information in said patient sample,
• f 1) using subtype-specific primers having the sequences selected from either the group consisting of SEQ ID NO's 6-9, 42-45, 104-107, 120-123, 145-148 or 180-183 for the generation of a DNA amplicon comprising the non-structural protein NS3 (N-terminal 181 amino acids),
• g 1) sequencing the NS3 amplicon to obtain a sequence of 543 base pairs by using the sequences selected from the group consisting of SEQ ID NO's 8 and 9;
• 43 and 45-46;
• 104 and 106;
• 120 and 122;
• 146 and 148 or 180 and 182
• h) aligning the sequence obtained in step (g 1), (g 2), (g 3) or (g 4) with a reference or wild-type HCV sequence,
• i) determining drug resistance mutation(s) in the viral genetic material present in patient sample,
• j) generating a NS3 amplicon starting from the DNA amplicon comprising the NS3 (N-terminal 181 amino acids) as obtained in step (f 1) using primers having the sequence of SEQ ID NO 11 and 12,
• k) inserting, by InFusion™ cloning or in vitro recombination, said amplicon obtained in step (i) into a NS3 deleted replication incompetent marker containing shuttle vector having the sequence of SEQ ID NO 10 to obtain a NS3 replication competent recombinant HCV replicon,
• l) generating RNA, by in vitro transcription, from said HCV replicon obtained in step (k)
• m) transfecting said RNA into suitable cells,
• n) determining, based on the expression of the marker gene, the EC50 value and/or fold change as a measure for the presence of drug resistance mutations in a sample.