Plaque reduction neutralization assay

Serum samples and nasal swabs were further tested for neutralizing antibodies against MERS-CoV (Qatar15/2015 and EMC/2012 isolates) using a plaque reduction neutralization (PRNT) assay. PRNT assay was carried out using according to the previously published protocol [19] with some modification. Briefly, samples were first inactivated at 56°C for 30 min. Then, 50 μl of 2-fold serial dilutions of heat-inactivated serum were mixed 1:1 with virus (400 PFU) prior to over-layering onto Huh7 cells. After 8 h of infection, the cells were fixed and stained using mouse anti-MERS-CoV nucleocapsid protein (SinoBiological) and HRP-conjugated goat anti-mouse IgG1 (SouthernBiotech). The number of infected cells were detected using a precipitate-forming TMB substrate (True Blue, KPL) and counted using an ImmunoSpot® Image analyser (CTL Europe GmbH). The PRNT titre was calculated based on a 50% or greater reduction in infected cells counts.