Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate

Almazán, Fernando; DeDiego, Marta L.; Sola, Isabel; Zuñiga, Sonia; Nieto-Torres, Jose L.; Marquez-Jurado, Silvia; Andrés, German; Enjuanes, Luis

Indirect immunofluorescence assay. Vero A66 and Huh-7 cells were grown to 80% confluence on glass coverslips and infected with the recombinant MERS-CoVs. At 48 h.p.i., cells were fixed either with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min or with methanol at Ϫ20°C for 15 min. For N protein immunodetection, paraformaldehyde-fixed cells were permeabilized with 0.2% saponin in PBS containing 10% FBS for 20 min and incubated with MERS-CoV N protein pAb (dilution 1:200) in PBS containing 10% FBS at room temperature for 90 min. For E protein immunodetection, methanol-fixed cells were incubated with MERS-CoV E protein pAb (dilution 1:500) in PBS containing 10% FBS overnight at 4°C. Coverslips were washed 4 times with PBS and incubated at room temperature for 45 min with goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Invitrogen) diluted 1:500 in PBS containing 10% FBS. Nuclei were stained using DAPI (4=,6=-diamidino-2-phenylindole) (1:200, Sigma). To fully inactivate the samples' infectivity, methanol-fixed cells were treated with 4% paraformaldehyde in PBS as described above. Finally, coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and analyzed on a Leica SP5 confocal microscope. Images were acquired with the same instrument settings and analyzed with Leica software.