2.7. Plaque Reduction Neutralization Test

Plaque reduction neutralization test (PRNT) to measure ZIKV neutralizing antibody titers was performed. Vero cells were plated in 12 well plates 24 h before infection to have confluency of 90–100% at the time of infection. All serum samples were heat-inactivated at 56 °C for 30 min prior testing. The serum was diluted 1/100 followed by serial two-fold dilutions up to 1/12,800. Naïve mouse serum served as the negative control in these studies. An equal volume of virus suspension containing ~200 PFU was mixed with diluted serum samples and incubated at 37 °C for 1 h. After incubation, the mixture was used to infect the cells in the plates at 37 °C for 1 h with rocking every 10 min. Following infection, the inoculum was removed, and the cells were overlaid with medium containing 1% low gelling temperature (LGT) agarose in VGM as described for plaque assay. After incubation for 5 days at 37 °C, the plaques were counted manually. Antibody titers were determined as the reciprocal of the serum dilution that inhibited 50% of the virus infectivity (PRNT₅₀), and geometric mean titers (GMT) were calculated from the PRNT₅₀ values.