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

The BAC clone carrying the MERS-CoV infectious cDNA was generated in several steps (Fig. 1) . After selection of appropriate restriction sites in the viral genome (Fig. 1A) , the intermediate plasmid pBAC-MERS-5=3= (Fig. 1B) was generated as the backbone to assemble the full-length cDNA clone. This plasmid contained the first 811 nucleotides of the viral genome fused to the CMV promoter, a multicloning site containing the restriction sites selected in the first step (BamHI, StuI, SwaI, and PacI), and the last 4,272 nucleotides of the genome, followed by a 25nucleotide (nt) poly(A) stretch, the hepatitis delta virus (HDV) ribozyme, and the bovine growth hormone (BGH) termination and polyadenylation sequences. Finally, the full-length MERS-CoV infectious cDNA clone (pBAC-MERS FL ) was assembled by sequential cloning of four chemically synthesized overlapping DNA fragments (MERS-1 to MERS-4) into the plasmid pBAC-MERS-5=3= (Fig. 1C) . The full-length clone sequence was identical to that reported for the MERS-CoV-EMC12 strain (15) , with the exception of a silent point mutation (T to C) introduced in the cDNA at position 20,761 (Fig. 1C ). This mutation, which eliminates an additional SwaI restriction site at position 20,760, was introduced to facilitate the cloning process and was used as a genetic marker to identify the virus recovered from the cDNA clone.