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

Infectious viruses were recovered in Huh-7 cells from plasmids pBAC-MERS-⌬3, pBAC-MERS-⌬4ab, and pBAC-MERS-⌬5 with virus titers similar to that of the parental rMERS-CoV (around 10 6 PFU/ml). After one passage on fresh cell monolayers, the recombinant viruses were cloned by three plaque isolation steps and their genetic structure was confirmed by sequencing. All the deletion mutant viruses (rMERS-CoV-⌬3, rMERS-CoV-⌬4ab, and rMERS-CoV-⌬5) were identical to the parental virus (rMERS-CoV) in terms of CPE and plaque morphology (data not shown). The growth kinetics of these viruses were also similar, reaching maximum virus titers at 72 h postinfection (h.p.i.) (Fig. 3B ). In the case of rMERS-CoV-⌬4ab, the viral titer was around 10-fold lower than that obtained from the parental virus (Fig. 3B ). These data indicated that the proteins encoded by genes 3, 4a, 4b, and 5 were not essential for MERS-CoV replication in cell cultures.