The confirmation of DENV infection.

L-02 cells were seeded into the wells containing coverslips in a 12-well plate and cultured for 24 hours. The cells in each well were infected with DENV1 or DENV2 stock at a mulplicity of infection (MOI) of 0.1, 0.5, and 1.0, respectively. At the time of 24, 48, and 72 hours after infection, the cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, then permeablized with 0.5% TritonX-100/PBS for 20 minutes and blocked with 10% FBS/PBS for 1 hour. The cells were then incubated for 2 hours with the primary rat monoclonal antibody specific to the nonstructural glycoprotein-1 (NS1) of DENV1 or DENV2 (Santa Cruz Biotechnology Inc., Dallas, TX) in 1:200 dilution. After washed with PBS for three times and 5 minutes for each with gentle shaking, the cells were incubated for 1 hour with a red fluorescence–labeled secondary goat anti-rat antibody (Santa) in 1:500 dilution. The cells were then washed with PBS as mentioned previously. The coverslips were taken out of the wells and rinsed with ddH₂O, air-dried, and mounted with the mounting oil containing 4′,6-diamidino-2-phenylindole to stain the nuclei. The virus particles stained with red fluorescence in the cells were visualized under a fluorescence microscope.