Focus reduction neutralization test (FRNT) was carried out in Vero cells seeded at a density of 25,000 cells in 96-well plate 24 hours before infection as described earlier (71, 72). Serum samples from immunized mice were heat activated at 56°C for 30 min and were twofold serially diluted (from 1:12.5 to 1:3200) in serum-free DMEM. After decomplementation and dilution of serum, 50 μl of each dilution was thoroughly mixed with 50–plaque-forming unit DENV (DENV-1, DENV-2, DENV-3, and DENV-4) diluted in DMEM and incubated at 37°C for 2 hours. Next, Vero cells were washed with serum-free DMEM and infected in duplicate with 45 μl of the neutralization mixture followed by another incubation for the next 2 hours. After incubation, the viral inoculum was removed and overlaid with 200 μl of 1.5% carboxymethylcellulose (Sigma-Aldrich) in serum-free DMEM and incubated at 37°C for 4 to 5 days, depending on the serotype. After incubation, the overlay medium was carefully removed followed by three washes with 1X PBS. The cells were further fixed, permeabilized with 0.2% Triton X-100, and stained with anti-dengue monoclonal antibody (GeneTex) for 1 hour at 37°C. After washing with 1X PBS, the stained cells were incubated with HRP-linked anti-mouse secondary antibody for 1 hour at 37°C. The cells were lastly washed with 1X PBS and developed using DAB (3,3′-diaminobenzidine) substrate. Viral foci were counted manually, and the percentage of foci reduction against control serum was calculated. Neutralizing antibody titer (FRNT50) values were determined as the reciprocal of the highest dilution that resulted in 50% decrease in focus-forming units and analyzed using GraphPad Prism software.

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