title: 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.
authors.affiliation: Rega Institute for Medical Research, University of Leuven (KU Leuven), Leuven, Belgium. dirk.jochmans@rega.kuleuven.be
authors.forename: Dirk
authors.initials: D
authors.lastname: Jochmans
authors.forename: Pieter
authors.initials: P
authors.lastname: Leyssen
authors.forename: Johan
authors.initials: J
authors.lastname: Neyts
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D016923:Cell Death; D002470:Cell Survival; D002522:Chlorocebus aethiops; D058957:Coronavirus NL63, Human / Q000187:drug effects* / Q000502:physiology; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays; D054884:Host-Pathogen Interactions; D006801:Humans; D010455:Peptides / Q000737:chemistry; D059748:Proteolysis; D014709:Vero Cells
chemical_list: D000998:Antiviral Agents; D005456:Fluorescent Dyes; D010455:Peptides