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

Recovery of recombinant viruses from the cDNA clones. To recover infectious virus, BHK cells were grown to 95% confluence in a 12.5-cm 2 flask and transfected with 6 g of the infectious cDNA clone using 18 g of Lipofectamine 2000 (Invitrogen) according to the manufacturer's specifications. At 6 h.p.t., cells were trypsinized, plated over a confluent monolayer of either Vero A66 or Huh-7 cells grown in a 12.5-cm 2 flask, and incubated at 37°C for 72 h. The cell supernatants were harvested and passaged once on fresh cells, and the recovered viruses were cloned by three rounds of plaque purification, following standard procedures.