科研之友
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摘要
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; 200 mu L cell culture; 2.10(4) cells/mL, IMDM 5% FBS medium). Cultures were subsequently incubated for 5 days at 35 degrees C after which 20 mu L of the peptide solution was added. Fluorescence was quantitated 2 hr after incubation at 37 degrees 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.
- 出版日期2012-8
- 单位河北医科大学; KU Leuven
