Article
rMERS-CoVMockrMERS-CoV-GFP Mockb ca kb^0102030S
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F10MERS-CoVDNA fragmentsMERS-CoV-GFPDNA fragmentsMERS-CoV-EMCrMERS-CoV-GFPrMERS-CoV24 48 72 961234567log10(TCID50/ml)hours post-infection* ns ns ns
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ns ns ns *0Extended Data Fig. 1 | Generation of viral cDNA clones and recovery of
recombinant MERS-CoV and MERS-CoV-GFP. a, Schematic representation of
the genome organization of MERS-CoV (top) and MERS-CoV-GFP (bottom) with
8 and 10 viral subgenomic overlapping fragments used for TAR cloning,
respectively. b, Rescue of recombinant MERS-CoV and MERS-CoV-GFP. After
the delivery of viral RNAs into BHK-21 cells using electroporation, the cells were
co-cultured with Vero B4 cells, and supernatants containing recombinant
viruses that were produced were used to infect new Vero B4 cells. Infected cells
were visualized by bright-field microscopy for rMERS-CoV (top; 5 days after
infection), and by f luorescence microscopy for GFP expression of
rMERS-CoV-GFP (bottom, 3 days after infection). Mock, Vero B4 cells
inoculated with the supernatant of BHK-21 cells that were electroporated
without viral RNAs. Images are representative of two independent
experiments. Scale bars, 100 μm. c, Replication kinetics of MERS-CoV-EMC,
rMERS-CoV and rMERS-CoV-GFP. Vero B4 cells were infected (MOI = 0.01).
Cell-culture supernatants were collected at the indicated time points after
infection and titrated by TCID 50 assay. Data are the mean ± s.d. of three
independent biological replicates. Statistical significance was determined by
two-sided unpaired Student’s t-test without adjustments for multiple
comparisons. P values (from left to right): top, *P = 0.0332; ns, P = 0.3294; ns,
P = 0.2003; ns, P = 0.0966; middle, *P = 0.0457; ns, P = 0.1233; ns, P = 0.0838;
*P = 0.0199; bottom, ns, P = 0.3240; ns, P = 0.6641; ns, P = 0.1376; *P = 0.0427.
TCID50/ml, 50% tissue culture infectious dose per ml.