In vitro neutralization assay (i) For the plaque reduction neutralization test (PRNT), eight 3fold serial dilutions of mAbs (from 200 mg/ml to 0.1 mg/ml) were mixed with an equal volume of 200 pfu of DENV-2 (16681) and incubated at 4uC for 1 h. The final concentration of mAbs at the PRNT ranged from 100 to 0.05 mg/ml. Antibody-virus mixtures (100 ml) were added to BHK-21 cells at 80%-90% confluency in 12-well plates. After absorption of virus for 2 h, BHK-21 cells were washed and 2 ml of 1% (w/v) carboxyl methyl cellulose (Sigma-Aldrich) in MEM plus 2% (v/v) FBS was layered onto the infected cells. After incubation at 37uC for 5 to 7 days, the viral plaque that had formed on the cell monolayer was fixed by 1 ml 3.7% formaldehyde (Sigma-Aldrich) at RT for 1 h. The cells were then stained with 1% crystal violet. Percentage of plaque reduction was calculated as: %Inhibition = 1002[(plaque number incubated with mAb/plaque number without mAb)6100]. (ii) For flow cytometry, serial dilutions of DB32-6 mAb were incubated with DENV-2 (16681, NGC, PL046 and Malaysia 07587) at MOI of 0.5 at 4uC 1 h before adding BHK-21 cells. After 2 h absorption, the monolayers were washed and incubated with MEM (Gibco) plus 2% (v/v) FBS at 37uC for 2 days. The cells infected with DENV-2 were washed and fixed with 3.7% formaldehyde at 4uC for 10 min. They were then permeabilized in PBS supplemented with 1% FBS, 0.1% saponin (Sigma) at 4uC for 10 min. For staining, cells were incubated with 4G2 at a concentration of 1 mg/ml at 4uC for 30 min. After two washes, R-Phycoerythrin (PE)conjugated AffiniPure F(ab9) 2 fragment goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratorie) diluted 1:250 was then added at 4uC for 30 min followed by two washes and analyzed by flow cytometry. % Infection = (the intensity of cells incubated with mAb/without mAb)6100.