Figure 6.

In vitro co-introduction of a coronavirus and a SAM vaccine encoding a coronavirus spike

(A) Schematic of experimental set-up (B) Vero cells were transfected with SAM vaccine encoding mCherry and infected with PEDV CV777 (0.1 TCID₅₀/cell, 24 h post transfection). (B) Fluorescence microscopy of co-transfected/infected Vero cell; cell nuclei stained with Hoechst, PEDV CV777 stained with mouse anti-PEDV spike, and SAM vaccine mCherry expression (recolored green). Merge showing co-expression of PEDV CV777 and SAM in syncytium (white line). (C) Vero cells were infected with PEDV CV777 or PEDV DR13 (0.1 TCID₅₀/cell) in the presence or absence of trypsin (n = 2). PEDV titers were determined based on serial dilutions of supernatants samples on Vero cells in the presence of trypsin (detection limit 3 log₁₀TCID₅₀/mL). D, Genome organization of PEDV CV777, three SAM vaccine constructs and a defective PEDV DR13 RNA construct. (E) Schematic of experimental set-up (F and G). The SAM vaccine or defective PEDV DR13 RNA (positive control) constructs were transfected into Vero cells, that were infected with PEDV CV777 (0.1 TCID₅₀/cell) 4 h prior or 24 h post transfection. After 72 h incubation in the presence of trypsin, supernatants were serial passaged a total of four times (every 3–4 days) on Vero cells in the presence or absence of trypsin, with CPE monitored by inverted light microscope. (F) Growth kinetics of PEDV CV777, with or without transfection with SAM vaccine constructs (n = 2, detection limit 3 log₁₀TCID₅₀/mL). (G) PEDV CV777 induced CPE in Vero cells with trypsin, but cannot replicate and induce CPE in Vero cells without trypsin. CPE in Vero cells without trypsin indicated the likely presence of a chimeric coronavirus. In the first experiment in 24-well plates, 1/2 wells showed CPE for the combination PEDV CV777 and the defective PEDV DR13 RNA (positive control). In the second experiment in 96-well plates, 4/96 wells showed CPE for PEDV CV777 and the defective PEDV DR13 RNA (positive control).