Plaque reduction neutralization tests

ZIKV plaque reduction neutralization tests (PRNTs) were conducted according to previously published protocols^18,19. Briefly, ZIKV MEX-I-44 isolated in Tapachula, Mexico in 2016 was obtained from The University of Texas Medical Branch, Galveston, TX and cultured to passage 8 in Vero cells. Serum specimens were incubated for one hour at serial dilutions of 1:10, 1:20…1:320 with a previously frozen virus stock of known plaque forming unit (PFU). Samples were then inoculated in duplicate onto a mono-layer of Vero cells grown on 6-well plates and allowed to incubate for an additional hour. Infectious material was then removed and replaced with a 1:1 mixture of Vero media and Avicel before being incubated for 4 days. To read plaques, the Avicel layer was fixed with 10% neutral buffered formalin. Finally, the formalin-Avicel layer was removed and the monolayer was stained with crystal violet, washed with tap water and allowed to dry before plaques were counted manually.

Percent reduction in observed plaques and a PRNT90 cutoff were used for interpretation. A PRNT90 titer is the dilution of a sample at which a 90% reduction in possible plaques is observed. The maximum number of potential plaques was obtained for each run using a corresponding back-titration and a linear model was fit to the observed number of plaques for each dilution. A PRNT90 titer was derived for each sample using the linear model and the equation for a straight line in the statistical program R²⁰. For samples that were positive but above the resolution of the PRNT assay the value of the greatest number of possible plaques for that run, as determined by the back titration, was assigned for each dilution for use with the linear model.