MERS-CoV Pseudovirus Neutralization Assay

MERS-CoV neutralization assay was performed as previously described [43 (link),44 (link)]. Briefly, 293T cells in 10 cm² dishes were transiently co-transfected with a pcDNA3.1-MERS-CoV-S plasmid and a PNL4-3.luc.RE plasmid encoding an Env-defective luciferase-expressing HIV-1genome. After 48 h post-transfection, the produced pseudovirus was harvested from the supernatant, and filtered through 0.45 μm sterilized membrane. The MERS-CoV pseudovirus was incubated with four inhibitors at 37 °C for 30 min, and then pseudovirus and inhibitors were added to DPP4-expressing Huh-7 cells (10⁴/well) preplated in 96 well tissue culture plates for 6 h. After 12 h, fresh medium was added to the plates and incubated for another 48 h. Cells were lysed with lysis reagent (Promega, Madison, WI, USA) and lysates were transferred into 96-well Costar flat-bottom luminometer plates (Corning, Corning, NY, USA). Luciferase substrate was added and the readings were recorded with an Ultra 384 Microplate Reader (Tecan, Männedorf, Switzerland).