Impact of Mutations Affecting 4'-Fluorouridine Susceptibility on Fitness and Treatment Outcomes for Venezuelan Equine Encephalitis Virus

Abstract

Venezuelan equine encephalitis virus (VEEV) is a prototypical encephalitic 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 (CHIKV), a member of the arthitogenic alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic alphaviruses such as VEEV, Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). 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 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. ImportanceVEEV is one of several mosquito-spread viruses that can cause serious brain infections in people and animals. Unlike CHIKV, against which an approved vaccine exists, there are no countermeasures to prevent or treat VEEV infections or block its close relatives, EEEV and WEEV. 4-FlU inhibits many different RNA viruses, including VEEV, and is currently being developed to treat multiple viral infections. A major problem with small-molecule antivirals is the appearance of virus populations that are less susceptible to treatment. In this study, we identified mutations in VEEVs RNA-dependent RNA polymerase (nsP4) that confer reduced susceptibility to 4-FlU. We then engineered these mutations into full-length infectious clones and assessed if viruses encoding for these mutations were still pathogenic. In addition, we treated infected mice with 4-FlU and measured how well the compound inhibited virus replication and prevented severe disease, even when mice were infected with viruses harboring mutations that reduced susceptibility to 4-FlU. Although VEEV can develop moderate resistance to 4-FlU in vitro, 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 like VEEV encephalitis.