Figure 1.

Design and Qualification of ZIKV rvRNA

(A) Plasmid DNA encoding a Venezuelan equine encephalitis replicon derived from the vaccine strain TC-83 under the control of a T7 RNA polymerase promoter. Subgenomic, SG; gene of interest, GOI. (B) Design of replicating viral RNAs encoding pre-membrane (prM) and envelope (E) genes of ZIKV strain H/PF/2013 or secreted human embryonic alkaline phosphatase (SEAP). Supernatants of 293T cells transfected with ZIKV or SEAP rvRNA were analyzed for the presence of Zika virus-like particles following sedimentation through 30% sucrose by ultracentrifugation. (C) Western blot of pelleted samples under denaturing conditions using polyclonal antibodies against ZIKV demonstrated reactivity against proteins corresponding to prM (21 kDa), E (54 kDa), or unprocessed prM-E fusion (76 kDa). (D) Transmission electron microscopy of pelleted samples following negative staining.