Experimental evolution was performed using traditional serial passaging and DMS to identify molecular determinants of emergence. A: Traditional experimental evolution was performed by serially passaging MAYV at a MOI of 0.01 in BHK-21 cells (hamster) and two insect cell lines, Aag2 (Ae. aegypti) and U4.4 (Ae. albopictus). A total of ten passages were performed. B-D: Following one, five, and ten passages, the viral RNA was sequenced using Illumina NGS to identify potentially adaptive mutations. No high frequency variants were identified following passage one, and as such are not depicted here. E: The three MAYV DMS populations, along with WT MAYV, were used to perform three passages in Aag2 (F) and LLC-MK2 (G) cells. Following passage, the viral RNA was sequenced, and selection analyses were performed to identify enriched variants. The top three variants in each environment, based on selection strength, are presented for each graph. A and E were created with BioRender.com whose terms and conditions are at biorender.com/terms.