OBJECTIVES: Direct-acting antiviral agents (DAAs) against hepatitis C virus 
(HCV) have recently been developed and are ultimately hoped to replace 
interferon-based therapy. However, DAA monotherapy results in rapid emergence of 
resistant strains and DAAs must be used in combinations that present a high 
genetic barrier to resistance, although viral kinetics of multidrug-resistant 
strains remain poorly characterized. The aim of this study is to track the 
emergence and fitness of resistance using combinations of telaprevir and NS5A or 
NS5B inhibitors with genotype 1b clones.
METHODS: HCV-infected chimeric mice were treated with DAAs, and resistance was 
monitored using direct and ultra-deep sequencing.
RESULTS: Combination therapy with telaprevir and BMS-788329 (NS5A inhibitor) 
reduced serum HCV RNA to undetectable levels. The presence of an NS3-V36A 
telaprevir resistance mutation resulted in poor response to telaprevir 
monotherapy but showed significant HCV reduction when telaprevir was combined 
with BMS-788329. However, a BMS-788329-resistant strain emerged at low 
frequency. Infection with a BMS-788329-resistant NS5A-L31V mutation rapidly 
resulted in gain of an additional NS5A-Y93A mutation that conferred telaprevir 
resistance during combination therapy. Infection with dual NS5AL31V/NS5AY93H 
mutations resulted in poor response to combination therapy and development of 
telaprevir resistance. Although HCV RNA became undetectable soon after the 
beginning of combination therapy with BMS-788329 and BMS-821095 (NS5B 
inhibitor), rebound with emergence of resistance against all three drugs 
occurred. Triple resistance also occurred following infection with the 
NS3V36A/NS5AL31V/NS5AY93H triple mutation.
CONCLUSIONS: Resistant strains easily develop from cloned virus strains. 
Sequential use of DAAs should be avoided to prevent emergence of 
multidrug-resistant strains.