2.4. Plaque Reduction Neutralization Test (PRNT)

A standard double-layer plaque assay (Shi et al., 2002) was performed to determine the PRNTs of each patient serum. We used ZIKV Puerto Rico strain PRVABC59 and DENV-2 New Guinea strain in the PRNT assay. Specifically, serial dilutions of serum samples (1/10 for the first dilution followed by serial 1/2 dilutions) were mixed with an equal amount of virus suspension containing 200 plaque-forming units (PFU) in 0.1 ml. After incubating the mixtures at 37 °C for 1 h, each virus-diluted serum sample (0.1 ml) was inoculated onto one well of a 6-well tissue culture plate containing a confluent monolayer of Vero cells. After incubating the plate at 37 °C for 1 h, an agar overlay was added to the infected cell monolayer, and the plate was further incubated at 37 °C. When virus plaques became visible, a second overlay containing neutral red was added, and plaques were counted. The antibody titer was determined as the serum dilution that inhibited 90% of the tested virus inoculum (PRNT₉₀).