Against danoprevir, most genotypes check details developed substitutions at position 168, confirming the importance of this locus in the resistance mechanism against this class of macrocyclic inhibitors.26, 29 However, genotype 3a showed instead

substitutions at position 43, 77, and 80. Position 80 was described as a resistance locus against TMC435, another macrocyclic PI,33 findings that demonstrate that resistance towards macrocyclic inhibitors can not only be induced by a variety of viable changes but also that some confer resistance to all inhibitors of this class. Interestingly, substitutions at position 77 and 174 were both found in passaging with danoprevir and telaprevir, providing further evidence for the potential emergence of cross-resistance between structurally dissimilar PIs. This is further supported

see more by the occurrence of resistance mutations against danoprevir at position 36, 41, and 43 that have been previously identified in linear PIs such as boceprevir and telaprevir.34-36 Resistance loci 43, 41, and 138 observed against danoprevir,37 and at positions 36, 54, 155, 156 against telaprevir21 were reproduced in the in vitro assay, demonstrating its utility for exploring the range of reported resistance-associated mutations in each genotype as well detecting a number of further possible loci. To confirm that the observed substitutions confer increased resistance to PIs, they were assessed individually for PI susceptibility. Of the 29 tested, all conferred increased Carnitine palmitoyltransferase II resistance towards BILN 2061 or danoprevir. Generally, increases in resistance were higher with mutations in genotypes 1b, 4a, and 6a than genotypes 2a, 3a, or 5a, likely the result of the already intrinsically high resistance barrier of the latter genotypes.

Mutations generally conferred smaller increases in resistance against danoprevir than BILN 2061, an observation in agreement with the closer stereochemical fit of BILN 2061 to the NS3 protease compared with danoprevir.27 Our results are in agreement with previous studies showing the highest fold increase in resistance for D168A/V and A156V/T mutations.19, 22 Antiviral drug-resistant mutants vary in their replicative fitness relative to wildtype virus in the absence of drugs.19, 38, 39 Of major clinical concern are mutants that outgrow wildtype during treatment and still replicate to high levels and transmit further following the end of treatment. Assessment of the influence of a mutation on the viral replication kinetic is therefore necessary in the in vitro evaluation of PI resistance. In the current study we observed marked genotype-associated variability in the fitness cost of a range of different mutations. For example, the D168V mutation showed no effect on the replicative ability of the genotypes 1b and 4a recombinants (consistent with previous data obtained from genotype 1b22).