The WT and the mutant infectious clones were linearized by Sac II digestion and used as templates for in vitro transcription [70] . RNAs were electroporated into BHK cells and cells were cultured overnight at 37uC followed by 6 d at 28uC in MEM containing 2% FBS and 10 mM HEPES, pH 8.0. Progeny virus in the medium was quantitated by infectious center assay on indicator BHK cells, using mouse polyclonal anti-DENV2 antibody. To detect primary infection, aliquots of the electroporated cells were plated on coverslips, cultured 3 d at 37uC, and processed for immunofluorescence microscopy as above.