Impact of mutations affecting 4′-fluorouridine susceptibility on fitness and treatment outcomes for Venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus (VEEV) is a prototypical encephalitic alphavirus. Members of the <i>Alphavirus</i> 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 for human use against any member of the <i>Alphavirus</i> genus. While a vaccine exists against chikungunya virus (CHIKV), a member of the arthritogenic alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic alphaviruses such as VEEV, Eastern equine encephalitis virus, and Western equine encephalitis virus. 4'-Fluorouridine (4'-FlU, EIDD-2749) was recently identified as a broad-spectrum antiviral against multiple RNA viruses, including alphaviruses. 4'-FlU can potently inhibit VEEV-TC83 replication, with submicromolar potency in cell culture. However, 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 reduce susceptibility to 4'-FlU, including P187A, Q191L, L289F, and T296I. We rebuilt each mutation in recombinant VEEV-TC83 and characterized the effects of these mutations on fitness and pathogenicity. In addition, we assessed the impact of mutations reducing sensitivity to 4'-FlU in a mouse model. Although mutations against 4'-FlU arise quickly <i>in vitro</i>, 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.IMPORTANCEVenezuelan equine encephalitis virus (VEEV) is a mosquito-spread virus that can cause encephalitis in people and animals. There are no FDA-approved countermeasures to treat VEEV infections in humans. 4'-Fluorouridine (4'-FlU) is currently being developed to treat multiple viral infections, including VEEV. A major problem with antivirals is the appearance of virus populations that are less susceptible to treatment. In this study, we treated infected mice with 4'-FlU and measured how well the compound inhibited virus replication and prevented severe disease. In addition, we identified mutations in VEEV's polymerase that confer reduced susceptibility to 4'-FlU. We then assessed if viruses encoding for these mutations were still pathogenic. Although VEEV can develop mild resistance to 4'-FlU <i>in vitro</i>, administration of 4'-FlU still reduced severe disease and prevented lethality in the animals infected with viruses that possess mutations that decrease susceptibility to 4'-FlU. These results suggest that 4'-FlU has strong potential as a future treatment for alphavirus infections.