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-CoVs lacking accessory genes 3, 4a and 4b, and 5. The availability of the pBAC-MERS FL infectious clone opened the door to investigate the importance of accessory genes 3, 4a, 4b, and 5 for MERS-CoV replication. To this end, cDNA clones with genes 3 (pBAC-MERS-⌬3), 4a and 4b (pBAC-MERS-⌬4ab), or 5 (pBAC-MERS-⌬5) deleted were constructed from pBAC-MERS FL (Fig. 3A) . The expression of gene 3 was abrogated by deletion of its TRS and coding sequence, with the exception of the 3= last 41 nucleotides, containing the 4a TRS, in order to preserve the expression of gene 4a. In the case of genes 4a and 4b, the majority of both coding sequences was deleted, except for the last 81 nucleotides of gene 4b, which overlap the gene 5 TRS. Finally, the expression of gene 5 was avoided by deletion of its TRS and complete coding sequence.