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

Generation of a rMERS-CoV mutant lacking the structural E protein gene. Based on published data showing that the deletion of CoV E protein resulted in either replication-competent, propagation-defective viruses (24) or attenuated viruses (25, 26, 31) , a cDNA clone with the E gene deleted (pBAC-MERS-⌬E) was constructed from pBAC-MERS FL . The expression of the E gene was abrogated by the deletion of its TRS and coding sequence, with the exception of the 3= last 49 nucleotides, in order to preserve the expression of gene M (Fig. 4A ). To recover infectious virus, BHK cells were transfected with pBAC-MERS-⌬E or the full-length cDNA clone pBAC-MERS FL . Six h.p.t. the transfected cells were overlayed on Vero A66 cell monolayers, and at 72 h.p.t., the supernatants were harvested and serially passaged three times on fresh Huh-7 cells. Infectious rMERS-CoV was recovered with titers of around 10 6 PFU/ml, whereas visible plaques were not detected for rMERS-CoV-⌬E virus throughout these passages (data not shown). Since CPE was observed at passages 0 and 1, the cell supernatants from the different passages were titrated in Huh-7 cells by limiting dilution. In contrast to the wild-type virus, which was recovered with high titers (around 1 ϫ 10 6 50% tissue culture infection dose [TCID 50 ]/ml), the rMERS-CoV-⌬E was detected only at passage 0 with apparent low titers (around 2 ϫ 10 3 TCID 50 /ml) (Fig. 5A ). This apparent low titer was most probably due to the transfer of detached cells transfected with the pBAC-MERS-⌬E. These cells were taken with the supernatant used to infect the next cell monolayer and formed syncytia with the nontransfected cells, giving the impression of virus production. In fact, rMERS-CoV-⌬E virus was lost at subsequent passages in several independent experiments performed in two different cell lines, Vero A66 and Huh-7 cells (data not shown). The presence of viral proteins was analyzed by immunofluorescence microscopy in Huh-7 cells infected with either rMERS-CoV or rMERS-CoV-⌬E from passage 0. As expected, E protein was detected in cells infected with rMERS-CoV but not in those infected with rMERS-CoV-⌬E (Fig. 4B ). Viral N protein was detected in the cytoplasm of both rMERS-CoV-and rMERS-CoV-⌬E-infected cells (Fig. 4B) . Interestingly, whereas the N protein was detected all over the cell monolayer in rMERS-CoV-infected cells, it was only detected in small syncytia in cells infected with rMERS-CoV-⌬E. Altogether, these data suggested that E protein was required for efficient virus propagation.