2.9. Plaque-Reduction Neutralization Test for Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

The serum samples from vaccinated mice were inactivated at 56 °C for 30 min. The samples were serially diluted from 1/40 to 1/640 with serum-free medium. The virus–serum mixture was prepared by mixing 125 PFU MERS-CoV with the diluted serum samples and incubated at 37 °C for 1 h. The virus–antibody mixture was inoculated into Vero cells. The plates were incubated for 1 h at 37 °C in 5% CO₂. After virus adsorption, agar overlay medium was added, and the plates were incubated at 37 °C in 5% CO₂ for four days. The cells were stained with 0.4% crystal violet solution (Sigma, St. Louis, MO, USA). Plaques were counted with the naked eye. The percentage neutralization represented the reduction value, which was calculated as 100× the number of plaques in the 100 PFU virus-infected well/ number of plaques in the virus–serum mixture-infected well.