(A) Alignment of top: dengue virus fusion loops; bottom: mosquito-borne flavivirus fusion loops, including yellow fever virus (YFV), Zika virus (ZIKV), West Nile virus (WNV), Kunjin virus (KUNV), Murray Valley encephalitis virus (MVEV), Japanese encephalitis virus (JEV), Usutu virus (USUV), and Saint Louis encephalitis virus (SLEV). Amino acids are colored by functional groups: negatively charged (red), positively charged (blue), nonpolar (yellow), polar (green), aromatic (pink), and sulfide (dark red). (B) Schematic of directed evolution procedure. Saturation mutagenesis libraries were used to produce viral libraries, which were passaged three times in either C6/36 or Vero 81 cells. At the end of the selection, viral genomes were isolated and mutations were identified by high-throughput sequencing. (C) Left: bubble plot of the sequences identified from either the unselected or selected (passage 3) C6/36 DENV libraries. Right: pie chart of the sequences from passage 3 C6/36 DENV libraries. (D) Structure of the DENV envelope with the fusion loop mutations highlighted in red. (E) Sequences of the fusion loop and furin cleavage site of DENV2, D2-FL, and D2-FLM.