Analysis of the post-immunization serum neutralizing activity against ZIKV. The sera neutralizing activity was measured using a plaque reduction neutralization test (PRNT) against the following Zika virus strains: H/PAN/2016/BEI-259634 and MR766.

Analysis of the serum neutralizing activity against ZIKV. The sera neutralizing activity was measured using a plaque reduction neutralization test (PRNT) against the following Zika virus strains: H/PAN/2016/BEI-259634 and MR766.

Plaque reduction neutralization test (PRNT).

To measure ZIKV neutralization by the obtained sera, PRNTs were performed. Briefly, 12-well cell culture plates were seeded with A549 cells to near confluence on the day of ZIKV infection. The sera were heat-inactivated at 56°C for 30 min. Sera were diluted 2-fold in fresh medium and mixed with equal volumes of ZIKV strain H/PAN/2016/BEI-259634 or MR766 containing approximately 50 PFU/well. The mixtures were incubated for 1 h at 37°C and used to infect cells. After 1 h, the inoculum was aspirated, and the cells were overlaid with 1% methylcellulose in MEM medium. The plates were incubated for 4 days at 37°C and stained with 0.5% crystal violet solution in 20% ethanol. Plaques were counted, and PRNT90 was estimated as the highest dilution that resulted in at least a 90% reduction in ZIKV plaques. The analysis was done twice. PRNT90 values estimated from each analysis are presented in the table.