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

Baby hamster kidney cells (BHK-21) were obtained from American Type Culture Collection (ATCC CCL-10). Human liverderived Huh-7 cells were kindly provided by R. Bartenschlager (University of Heidelberg, Germany). African green monkey kidney-derived Vero A66 cells were kindly provided by A. Carvajal (University of Leon, Spain). In all cases, cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 25 mM HEPES, 1% nonessential amino acids (Sigma), and 10% fetal bovine serum (FBS) (BioWhittaker). Virus titrations were performed on Vero A66 or Huh-7 cells following standard procedures and using closed flasks or plates sealed in plastic bags. For plaque assays, infected cells were overlaid with DMEM containing 0.6% low-melting agarose and 2% FBS, and at 72 h.p.i., cells were fixed with 10% formaldehyde and stained with crystal violet. For 50% tissue culture infectious dose (TCID 50 ) assays, CPE was recorded at 72 h.p.i. All work with infectious virus was performed in biosafety level 3 facilities by personnel wearing positive-pressure air-purifying respirators (highefficiency particulate Air-Mate).