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

Rescue of infectious rMERS-CoV from the cDNA clone in Vero A66 and Huh-7 cells. Infectious viruses were recovered from the full-length cDNA clone, using susceptible Vero A66 and Huh-7 cells, with titers of around 10 6 PFU/ml at 72 h posttransfection (h.p.t.). The recovered viruses were cloned by three rounds of plaque purification, and their phenotypic and genotypic properties were determined. Viruses rescued from both cell lines (rMERS-CoV) induced a clear cytopathic effect (CPE), characterized by the induction of cell fusion, which was more apparent in Huh-7 cells (Fig. 2 ). In addition, the replication of the rMERS-CoV was confirmed by indirect immunofluorescence microscopy using a nucleocapsid (N) protein-specific antibody, showing a cytoplasmic staining pattern in both cell lines (Fig. 2) .