Mouse sera was heat-inactivated at 56 °C for 30 min. Sera then was diluted 2-fold in DMEM 2% FBS and incubated with 50 PFU of ZIKV strain PRVABC59 for 2 hours at 37 °C and 5% CO2. This was then added to a 24-well plate of confluent Vero cells. The plaque assay was continued as described above.
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