At least one sfRNA is necessary for efficient ZIKV infection and transmission in A. aegypti mosquitoes. (A) Schematic overview of experimental design. (B) Female A. aegypti mosquitoes were fed with an infectious blood meal containing 1 × 106 FFU/ml of ZIKVAr WT or mutant viruses producing different qualities of sfRNAs. Mutant ZIKVAr ΔSLI, ΔSLII, or ΔSLI-II was used as indicated. Engorged females were incubated for 14 days at 28°C, and infection and transmission rates were determined by viral genome RT-qPCR of whole body or saliva. Results from a representative experiment are shown. Statistics were performed by Fisher’s exact test on cumulative data analyzing each condition in pairs. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C) ZIKV mutants produce different kinds of sfRNAs in infected A. aegypti mosquitoes. Northern blots of RNA samples prepared from individual infected mosquitoes are shown.