Project Summary Live-attenuated vaccines are the gold standard for preventing viral illness but they can revert to virulence, sometimes causing severe or fatal disease. The mosquito-borne alphaviruses chikungunya (CHIKV) and Venezuelan equine encephalitis (VEEV) are RNA viruses that produce a debilitating arthritic syndrome or encephalitis, respectively. Both have repeatedly emerged to produce millions of human cases worldwide and CHIKV has expanded into the Americas since 2013, indicating increasing need for vaccines. Although live-attenuated candidate human vaccines have been developed for both viruses, no licensed vaccines currently exist, in part due to adverse events in clinical trials caused by revertant mutations. There is therefore a need to increase the genetic stability of live-attenuated virus vaccine candidates to improve safety. The goal of this project is to develop safe and effective CHIKV and VEEV vaccines to prevent millions of human infections globally each year. Virus variants that mutate less frequently would accrue fewer mutations that confer virulence, and may therefore serve as safer live-attenuated vaccine candidates. This project will use high fidelity variants we already identified and characterized inserted into candidate CHIKV and VEEV vaccines to understand: 1) stability, infectivity, and potential for reversion; 2) whether incorporation of high fidelity mutations improves vaccine safety while maintaining or increasing immunogenicity in established mouse models; and 3) mechanism(s) of attenuation. This approach represents the first use of fidelity modulation to increase safety of live alphavirus vaccine candidates. If successful, this strategy will potentially lead to a broader application of fidelity variants in improving vaccine safety that can be used for other live-attenuated alphavirus vaccine platforms and possibly other RNA viruses.