To generate DV RSPs, we first transfected HeLa cells with native DV1 prME gene derived from the DV1 FGA/NA d1d [24] strain, which contained the signal sequence of vesicular stomatitis virus G (VSV-G) envelope glycoprotein inserted in frame upstream of the prME cDNA. 48 hours post-transfection, expression of dengue E protein was monitored by flow cytometry on permeabilized transfected HeLa cells with the 4E11 monoclo-nal antibody against dengue E protein. E protein was detected in transfected cells at low expression level ( Figure 1A) . Analysis of the prME gene sequence revealed that 15% of nucleotide triplets were rarely used in mammalian cells so that its codon usage was not adapted for efficient expression in this cell system. Thus we designed and synthesized an optimized DV1 prME (prMEopt) gene in which the rarely used codons were replaced with those preferred by mammalian cells, without changing the amino acid sequence ( Figure S1 ). More than 70% codons were modified for prME optimization. In addition, we also removed the negative cisacting sites (such as splice sites, poly(A) signals, etc) which might have negatively influenced expression. The optimized gene resulted in high and stable expression levels in transfected mammalian cells ( Figure 1B ). Both the mean fluorescence intensity (MFI) and percentage of positive cells increased by almost 3 fold. E protein was hardly detected without cell permeabilization, indicating that its localization was intracellular and was not expressed at cell surface (data not shown).