DESCRIPTION (provided by applicant): Chikungunya (CHIKV) is a reemerging mosquito-borne virus that originates in an enzootic African cycle but periodically initiates an urban cycle involving human amplification hosts and the urban vector mosquitoes, Aedes aegypti and recently, A. albopictus. Since 2004, Spread via infected travelers has introduced CHIKV into Asia, Europe, Oceania and the Americas with millions of cases of severe, debilitating, often chronic arthralgia. A hallmark of recently emerged strains in the Indian Ocean Lineage (IOL) has been the evolution of a series of A. albopictus-adaptive substitutions in the envelope glycoproteins. These mutations increase transmission efficiency by this species in the Indian Ocean basin and Asia. A pattern of IOL substitutions focused near the fusion loop of the E1 protein (residue 226) and in the acid sensitive region of E2 suggests an effect on CHIKV entry into mosquito cells by altering the pH threshold for conformational changes in the viral spikes. This pattern allowed us to predict additional adaptive E2 substitutions, never detected in nature, and to show that a combination of these substitutions will soon result in even more efficient transmission by A. albopictus. However, the dependence of these "second-step" E2 mutations on E1-226, and their phenotypes in the genetic background of the Asian lineage recently introduced into the Americas, remain unknown. Furthermore, the history of vector-specific adaptation of the Asian Lineage has never been explored. Therefore, the vector-adaptive trajectory of the Asian CHIKV lineage now in the Americas and its effect on spread and endemicity remains unpredictable despite major public health implications. Critical gaps in understanding include: 1) Has/will the Asian lineage (further) adapt for A. aegypti transmission in the Americas, and will such adaptation affect transmission by A. albopictus?; 2) Can A. albopictus-adaptive E2 substitutions increase Asian strain infectivity for A. albopictus or A. aegypti? and; 3) Can we predict additional vector-adaptive CHIKV evolution as spread continues in the Americas? We address these critical questions using 4 specific aims: 1. Characterize the vector-adaptive evolutionary history of the Asian CHIKV lineage since 1958, and use the findings to predict the future evolutionary trajectory of this strain in the Americas. . Follow the evolution of both Asian and IOL CHIKV strains in Asia and the Americas to identify additional vector-adaptive evolution. 3. Determine the effects of "second-step" vector-adaptive E2 substitutions on infectivity for A. albopictus or A. aegypti in the Asian backbone. 4. Use a prospective approach with deep sequencing of experimentally manipulated CHIKV strains from the Americas to identify potential adaptive mutations that could affect vector usage in the Western Hemisphere. The results will not only improve prediction of imminent and future CHIKV epidemics, but also will inform surv...