Plaque Reduction Neutralization Assay

Heat-inactivated serum was diluted in DMEM. Diluted serum was incubated with 80 PFU of virus in 100 µl of DMEM for 1 hour at 37°C. The mixture was used to infect Vero cells in a 6-well plate. Cells were incubated at 37°C for 1 hour. Media was removed and overlaid with DMEM + 2% FBS + Penicillin/Streptomycin + 1% Low-melt agarose. The number of plaques was determined at 6–7 days post infection. The plaque reduction titer was determined based on the dilution of serum that reduced the number of plaques by one half of the input virus control.