The codon usage of DV2, DV3 and DV4 prME genes differed substantially from that of mammalian cells and, therefore, was optimized as described for DV1 by replacing more than 70% of the codons ( Figure S1 ). RSPs of the four serotypes were first produced in 293T cells by transient expression of prME genes. Particles were purified and detected by Western blotting using either the anti-E 4E11 monoclonal antibody or a mixture of sera from four patients who were infected by all dengue serotype ( Figure 8A-B) . With the anti-E 4E11 antibody, monomeric and dimeric forms of the DV1 E protein could be readily detected whereas weaker signals were observed for the other three serotypes, because of limited affinity of the antibody ( Figure 8A ) [32] . E proteins of all DVs were detected using the mixture of sera ( Figure 8B ) with a signal of similar intensity, which suggested that RSPs of four serotypes could be generated to comparable levels. Monomeric E proteins of four serotypes displayed slightly different electrophoresis mobility, which could be due to a differential level of N-glycosylation. E glycoproteins of DV1 and DV3 have two N-glycosylation sites at Asn 67 and Asn 153, whereas those of DV2 and DV4 have only one at Asn 67 [33] . Dimeric E protein was observed in DV1, DV3 and DV4 but not in DV2. Besides E, the prM protein was also detected in RSPs produced by transient transfected 293T cells. Recently, we have established HeLa-prME and 293T-prME cell lines of DV1, DV2 and DV3 using the same procedure described for DV1 HeLa-prME. We have compared the maturation of RSPs produced by both cell types using SDS-PAGE and silver staining of the gel ( Figure 8C ). Supernatants from parental HeLa and 293T cells were used as controls in the experiment. We found that, whereas only a small fraction of prM was cleaved in the RSPs produced by 293T-prME cell lines, cleavage of prM was much more effective in RSPs from the HeLa-prME cell lines. This result indicates that efficacy of maturation is cell type dependent. RSPs of the four serotypes were further analyzed by sucrose gradient. As for DV1, RSPs of DV2, DV3 and DV4 were concentrated in fractions containing 20% to 30% sucrose ( Figure 8D) . Altogether, our results demonstrated that the strategy of codon optimization was successful in leading to the production of RSPs of all serotypes with comparable efficacy and similar sedimentation properties.