MERS-CoV Pseudovirus Neutralization Assay

This was done as previously described with some modifications.^{38,39 (link)} Briefly, 293T cells were co-transfected with a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and a plasmid encoding MERS-CoV (EMC-2012 strain) S protein using the calcium phosphate method. Cells were changed into fresh DMEM 8 h later, and pseudovirus-containing supernatants were harvested 72 h post-transfection for single-cycle infection of Huh-7 cells. The pseudovirus was incubated with serially diluted mouse sera at 37°C for 1 h before adding to the cells preplated in 96-well culture plates. Twenty-four hours later, cells were refed with fresh medium, which was followed by lysing cells 72 h later using cell lysis buffer (Promega, Madison, WI) and transferring the lysates into 96-well luminometer plates. Luciferase substrate (Promega) was added to the plates, and relative luciferase activity was determined in an Infinite 200 PRO Luminator (Tecan). MERS pseudovirus neutralization was calculated and expressed as 50% neutralizing antibody titer, NT₅₀.^{40 (link)}