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

Construction of a full-length cDNA clone of MERS-CoV. Based on the data of the full-length sequence of the MERS-CoV-EMC12 strain (GenBank accession number JX869059) (15), a MERS-CoV infectious cDNA clone was assembled in BAC using a three-step strategy. In the first step, the restriction sites BamHI (genomic position 806), StuI (genomic positions 7,620 and 9,072), SwaI (genomic position 20,898), and PacI (genomic position 25,836), present in the viral genome, were selected (Fig. 1A) . Second, the intermediate plasmid pBAC-MERS-5=3= was constructed as the backbone for assembly of the full-length cDNA clone (Fig. 1B) . To generate this plasmid, two DNA fragments were generated by chemical synthesis (Bio Basic, Inc.). The first fragment contained the CMV promoter fused to the first 811 nucleotides of the viral genome flanked by SfoI and BamHI sites, and the other one contained a multicloning site with the restriction sites selected in the first step (BamHI, StuI, SwaI, and PacI) followed by the last 4,272 nucleotides of the viral genome joined to a 25-nt poly(A), HDV ribozyme, and BGH termination and polyadenylation sequences. The first DNA fragment was cloned into pBeloBAC11 ϪStuI (a pBeloBAC without the StuI restriction site) and digested with SfoI and BamHI to generate the plasmid pBAC-MERS-5=, and then the plasmid pBAC-MERS-5=3= was generated by cloning the second DNA fragment, digested with BamHI and SfiI, into pBAC-MERS-5= digested with the same restriction enzymes. Finally, the third step was the assembly of the full-length cDNA clone (pBAC-MERS FL ) by sequential cloning of four overlapping DNA fragments (MERS-1 to MERS-4) into the multicloning site of the intermediate plasmid pBAC-MERS-5=3= (Fig. 1C) . The overlapping DNA fragments flanked by the appropriate restriction sites were generated by chemical synthesis (Bio Basic, Inc.). In the case of fragment MERS-3, a silent mutation (T to C) was introduced at position 20,761 in order to eliminate the SwaI restriction site at position 20,760 and to use it as a genetic marker. The genetic integrity of the cloned DNAs was verified throughout the assembly process by extensive restriction analysis and sequencing.