Replication capacity of minority variants in viral populations can affect the assessment of resistance in HCV chimeric replicon phenotyping assays.
OBJECTIVE
Drug-resistant minority viral variants can pre-exist in the viral quasispecies of chronically infected hepatitis C virus (HCV) patients and can emerge gradually upon drug treatment. When heterogeneous clinical samples are tested for drug susceptibility in a chimeric replicon-based phenotyping assay, biphasic dose-response curves may be observed. The effect of drug-resistant minority viral variants on the biphasic phenotype of mixtures was assessed in detail.


METHODS
Susceptibility of mutant/wild-type mixtures containing minorities of NS3 mutants with different replication capacities and susceptibilities to protease inhibitors were tested in a transient replicon assay. The contribution of both variants in the mixture to the overall replication level was described with an E(max) model.


RESULTS
The 90% and 99% effective concentrations (EC(90) and EC(99), respectively) provide a more accurate measure of the susceptibility of the population than the determination of EC(50) values. Reduced susceptibility at the EC(50) level correlated with the replication capacity of the NS3 mutant in the mixture. Using replication-enhanced mutant/wild-type mixtures demonstrated that the relative difference between the replication capacity of the variants present in the mixture results in biphasic dose-response curves. Modelling revealed that in mixtures containing wild-type and resistant variants with low replication capacity, the contributions of the wild-type variants are higher than expected from the replication level of the replicons transfected alone.


CONCLUSIONS
Differences in the replication capacity of variants present in HCV replicon-based phenotype assays can lead to biphasic dose-response curves. Using EC(90) or EC(99) values increases the sensitivity of the assay to minor variants.
Janssen Infectious Diseases BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium. tverbinn@its.jnj.com
Thierry
T
Verbinnen
Tom
T
Jacobs
Leen
L
Vijgen
Hugo
H
Ceulemans
Johan
J
Neyts
Gregory
G
Fanning
Oliver
O
Lenz
D000998:Antiviral Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D024882:Drug Resistance, Viral; D016174:Hepacivirus / Q000187:drug effects* / Q000302:isolation & purification / Q000502:physiology*; D019698:Hepatitis C, Chronic / Q000821:virology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D050505:Mutant Proteins / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D050505:Mutant Proteins; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases