Fig. 1.

Recombinant MERS-CoV design and replication kinetics. (A) Diagram of the MERS-CoV genome including the replicase locus (encoding 16 nsps), structural genes, and accessory genes is shown along with a diagram of each recombinant mutant constructed using the BAC reverse genetics system. (B) A549^DPP4 cells were infected at MOI = 5 with the indicated viruses, and protein lysates were harvested at 24 hpi. Expression of NS4a and NS4b was determined by SDS/PAGE and Western blot. (C) Vero cells were infected in triplicate at MOI = 1 with the indicated viruses. Supernatants were collected at the indicated hpi, and infectious virus was quantified by plaque assay. (D) A549^DPP4 cells were infected in triplicate with the indicated viruses at MOI = 1, and replication was quantified as in C. Data in C and D are from one representative of three independent experiments. (E) A549^DPP4 and (F) Calu-3 cells were infected in triplicate with the indicated viruses at MOI = 0.1, and replication was quantified as in C. All data in C–F are displayed as means ± SD. The dotted line in C–F indicates the limit of detection of the plaque assay. Statistical significance of each recombinant virus compared to WT MERS-CoV was calculated by repeated measures two-way ANOVA: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Data that were not statistically significant are labeled ns.