Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-␣), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV. IMPORTANCEViral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV, we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness. Selection of viral mutants resistant to antiviral agents is a major problem for the successful treatment of viral diseases. In the case of RNA viruses, high mutation rates during genome replication provide viral populations with an ample reservoir of phenotypic variants, including mutants that can escape selective constraints. Resistance to a single drug that targets a viral protein develops at a rate that depends on the genetic barrier (number and types of mutations needed to acquire resistance) and the phenotypic barrier (fitness cost) imposed by the resistance mutations (1-16). When drug resistance mutations do not entail a significant fitness cost-either because the mutations per se do not critically affect viral functions or because compensatory mutations are acquired-they may reach detectable levels despite no prior exposure of the viral population to the drug (1, 16-27).Control of hepatitis C virus (HCV) infections is hampered by the complexity of HCV quasispecies replicating in the liver (16,28,29). Directly acting antiviral agents (DAAs)-some currently in use and others under development-offer great promise for control of HCV either as a substitute for or complement of the standard-of-care (SOC) therapy based on treatment using a combination of pegylated alpha interferon (IFN-␣) and ribavirin (30)(31)(32)(33)(34)(35)(36). Combinations that include the polymerase inhibitor sofosbuvir have produced sustained viral responses that in some cases have been higher than 90% in clinical trials (37-40), but the possible impact of resistance mutations is not known; sofosbuvir resistance substitution S282T in NS5B is present in the ...

Mutant spectrum dynamics (changes in the related mutants that compose viral populations) has a decisive impact on virus behavior. The several platforms of next generation sequencing (NGS) to study viral quasispecies offer a magnifying glass to study viral quasispecies complexity. Several parameters are available to quantify the complexity of mutant spectra, but they have limitations. Here we critically evaluate the information provided by several population diversity indices, and we propose the introduction of some new ones used in ecology. In particular we make a distinction between incidence, abundance and function measures of viral quasispecies composition. We suggest a multidimensional approach (complementary information contributed by adequately chosen indices), propose some guidelines, and illustrate the use of indices with a simple example. We apply the indices to three clinical samples of hepatitis C virus that display different population heterogeneity. Areas of virus biology in which population complexity plays a role are discussed.