A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE.
Identifieur interne : 001339 ( Main/Exploration ); précédent : 001338; suivant : 001340A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE.
Auteurs : Dirk Jochmans [Belgique] ; Pieter Leyssen ; Johan NeytsSource :
- Journal of virological methods [ 1879-0984 ] ; 2012.
Descripteurs français
- KwdFr :
- MESH :
- pharmacologie : Antiviraux.
- physiologie : Coronavirus humain NL63.
- Animaux, Cellules Vero, Colorants fluorescents, Coronavirus humain NL63, Humains, Interactions hôte-pathogène, Mort cellulaire, Peptides, Protéolyse, Survie cellulaire, Tests de criblage à haut débit.
English descriptors
- KwdEn :
- Animals, Antiviral Agents (pharmacology), Cell Death, Cell Survival, Chlorocebus aethiops, Coronavirus NL63, Human (drug effects), Coronavirus NL63, Human (physiology), Fluorescent Dyes (chemistry), High-Throughput Screening Assays, Host-Pathogen Interactions, Humans, Peptides (chemistry), Proteolysis, Vero Cells.
- MESH :
- chemical , chemistry : Fluorescent Dyes, Peptides.
- chemical , pharmacology : Antiviral Agents.
- drug effects : Coronavirus NL63, Human.
- physiology : Coronavirus NL63, Human.
- Animals, Cell Death, Cell Survival, Chlorocebus aethiops, High-Throughput Screening Assays, Host-Pathogen Interactions, Humans, Proteolysis, Vero Cells.
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.
DOI: 10.1016/j.jviromet.2012.04.011
PubMed: 22575574
Affiliations:
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Le document en format XML
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<term>Coronavirus humain NL63 (physiologie)</term>
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<term>Interactions hôte-pathogène</term>
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<term>Survie cellulaire</term>
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<front><div type="abstract" xml:lang="en">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.</div>
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