For antiviral screening purposes, infection of cell cultures with the virus under study, should ideally result in the induction, within just a few days, of (nearly) complete CPE and allow the calculation of acceptable Z′ factors (>0.5). The human Corona virus (NL63) causes only limited CPE on different cell lines (Schildgen et al., J Virol Methods, 2006). Following infection of Vero118 cells, virus-induced CPE was too low to allow readout based on classical colorimetric methods (such as the MTS assay), even following prolonged incubation times (>7 days). In order to develop an antiviral screenings-assay against NL63, we explored whether a death-cell protease substrate could be used instead. The substrate used is a quenched peptide (bis-AAF-R110), that releases a fluorophore upon proteolytic-cleavage by proteases the latter released from death cells (Niles et al., Anal Biochem., 2007). After different rounds of optimization the following protocol was developed: Vero118 cells in 96-well plate format were infected with NL63 (MOI = 0.01, 200 μL cell culture, 2 × 104 cells/well, IMDM 5% FBS medium). Cultures were subsequently incubated for 5 days at 35 °C after which 20 μL of the peptide solution (16 μM final concentration) was added. Fluorescence was quantified 2 h after incubation at 37 °C. A roughly 3-fold increase in fluorescence intensity in the infected cultures was observed as compared to the uninfected cultures with a low well-to-well variability. Z′ factors calculated from different experiments were in the range of 0.6–0.8, indicating excellent assay quality. An anti-ACE-II polyclonal antiserum (that prevents coronavirus infection in cell cultures) was used as a positive control and allowed to validate the assay for antiviral screening purposes. In conclusion, in conditions where a viability staining is inadequate to quantify virus-induced CPE, a novel simple and convenient method that detects cell-death and that is suitable for high-throughput screening purposes can be employed.
Acknowledgements: Funding: This work was funded by EU FP7 project SILVER (260644).