A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE

Abstract

For antiviral screenings 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 (HCoV-NL63) causes only limited CPE on different cell lines (Schildgen et al., 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). To develop an antiviral screenings-assay against HCoV-NL63, we explored whether a dead-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 dead cells. Following different rounds of optimization a screening protocol was developed: Vero118 cells in 96-well plate format were infected with HCoV-NL63 (MOI=0.01; 200μL cell culture; 2.10(4)cells/mL, IMDM 5% FBS medium). Cultures were subsequently incubated for 5 days at 35°C after which 20μL of the peptide solution was added. Fluorescence was quantitated 2 hr 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-ACE2 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 quantitate 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.