Development of infectious Zika virus (ZIKV) with discreet xrRNA1 structural mutation. (A) The 3′ UTR of ZIKV encodes two host exoribonuclease resistant RNA structures (xrRNAs). (B) The tertiary structure of ZIKV xrRNA1 has been previously described, revealing that a single cytosine at position 10,415 (green) of the crystalized RNA fragment is necessary for stabilizing the phosphate backbone kink (blue) which protects the viral 3′ UTR (red) from exoribonuclease degradation. We produced a ZIKV mutant (called X1) in which the cytosine at position 10,415 of xrRNA1 has been replaced with a guanine (C), weakening the tertiary structure of the RNA. A wild type (WT) ZIKV clone with no mutations (A) was also produced alongside the X1 mutant as a positive control. The viral genomes of either X1 or the WT clone were transfected into Vero cells. At 12 days post transfection, the amounts of both viral genome (D) and infectious virus (E) rescued from the X1 transfection were comparable to those rescued from cells transfected with WT ZIKV RNA (n = 6).