FIG 6.
Replication kinetics of MERS-CoV on BHK cells expressing DPP4 of livestock species. Camel (green circles), cow (green squares), goat (green triangles), or sheep (green inverted triangles) DPP4 and rhesus macaque (red squares), ferret (blue squares), or mouse (blue triangles) DPP4 were expressed on BHK cells. As a control, human (red) or hamster (blue) DPP4 was expressed on BHK cells. Twenty-four hours posttransfection, cells were inoculated with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 hpi, and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations. (B) BHK cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 h posttransfection using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. (C) Expression of DPP4 mRNA was measured via qRT-PCR. Relative fold increase was calculated by the comparative CT method (35), where DPP4 expression is normalized to HPRT.