Effects of 4-Fluorouridine Resistance on Fitness and Treatment Outcomes for Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a prototypical New World alphavirus. Members of the Alphavirus genus are found across the globe, transmitted by arthropod vectors, and cause significant disease burdens in humans and animals. There are currently no FDA-approved antivirals against any member of the Alphavirus genus. While a vaccine exists against Chikungunya virus, a member of the Old World alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic New World alphaviruses such as VEEV, Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). 4-Fluorouridine (4-FlU) was recently identified as a broad-spectrum antiviral against multiple RNA viruses, including alphaviruses. The emergence of antiviral resistance represents a hurdle for antiviral drug development and the implementation of effective treatment strategies. Here, we have identified novel mutations in the VEEV nsP4 RNA-dependent RNA polymerase that confer resistance to 4-FlU. We rebuilt each mutation in recombinant VEEV-TC83 and characterized the effects of resistance on fitness and pathogenicity. In addition, we assessed the impact of viral resistance on efficacy of 4-FlU in a mouse model. We show that although resistance mutations against 4-FlU arise quickly in vitro, treatment can still alleviate severe disease and lethal encephalitis. Together, these data highlight the promising therapeutic potential of 4-FlU for the treatment of alphavirus encephalitis.