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

To further confirm the identity of the rMERS-CoV, the fulllength genome sequences of two independent clones rescued in Vero A66 and Huh-7 cells were analyzed. The recombinant viruses rescued in Huh-7 cells presented the same sequence as the cDNA clone, including the genetic marker at position 20,761. However, in the case of the viruses rescued in Vero A66 cells, several changes in the region of the accessory genes were detected in both clones. One of them presented a deletion of 179 nucleotides (from nucleotide 26,721 to 26,900) that disrupted gene 4b and eliminated the transcription-regulating sequence (TRS) and the first 20 amino acids of gene 5. The second clone presented a 1-base insertion at position 27,143 that changed the reading frame and promoted the expression of a truncated gene 5. Taking these data into consideration, two new virus clones rescued in Vero A66 cells were sequenced, and only one of them presented the wild-type sequence, suggesting that the MERS-CoV was more stable in Huh-7 cells. Therefore, Huh-7 cells were selected for further work.