A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE.
Identifieur interne : 000D16 ( PubMed/Corpus ); précédent : 000D15; suivant : 000D17A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE.
Auteurs : Dirk Jochmans ; Pieter Leyssen ; Johan NeytsSource :
- Journal of virological methods [ 1879-0984 ] ; 2012.
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
Links to Exploration step
pubmed:22575574Le document en format XML
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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></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">22575574</PMID><DateCompleted><Year>2012</Year><Month>10</Month><Day>01</Day></DateCompleted><DateRevised><Year>2019</Year><Month>12</Month><Day>10</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1879-0984</ISSN><JournalIssue CitedMedium="Internet"><Volume>183</Volume><Issue>2</Issue><PubDate><Year>2012</Year><Month>Aug</Month></PubDate></JournalIssue><Title>Journal of virological methods</Title><ISOAbbreviation>J. Virol. Methods</ISOAbbreviation></Journal><ArticleTitle>A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE.</ArticleTitle><Pagination><MedlinePgn>176-9</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.jviromet.2012.04.011</ELocationID><Abstract><AbstractText>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.</AbstractText><CopyrightInformation>Copyright © 2012 Elsevier B.V. All rights reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Jochmans</LastName><ForeName>Dirk</ForeName><Initials>D</Initials><AffiliationInfo><Affiliation>Rega Institute for Medical Research, University of Leuven (KU Leuven), Leuven, Belgium. dirk.jochmans@rega.kuleuven.be</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Leyssen</LastName><ForeName>Pieter</ForeName><Initials>P</Initials></Author><Author ValidYN="Y"><LastName>Neyts</LastName><ForeName>Johan</ForeName><Initials>J</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2012</Year><Month>04</Month><Day>28</Day></ArticleDate></Article><MedlineJournalInfo><Country>Netherlands</Country><MedlineTA>J Virol Methods</MedlineTA><NlmUniqueID>8005839</NlmUniqueID><ISSNLinking>0166-0934</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000998">Antiviral Agents</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005456">Fluorescent Dyes</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D010455">Peptides</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D000998" MajorTopicYN="N">Antiviral Agents</DescriptorName><QualifierName UI="Q000494" MajorTopicYN="Y">pharmacology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D016923" MajorTopicYN="N">Cell Death</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002470" MajorTopicYN="N">Cell Survival</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002522" MajorTopicYN="N">Chlorocebus aethiops</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D058957" MajorTopicYN="N">Coronavirus NL63, Human</DescriptorName><QualifierName UI="Q000187" MajorTopicYN="Y">drug effects</QualifierName><QualifierName UI="Q000502" MajorTopicYN="N">physiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005456" MajorTopicYN="N">Fluorescent Dyes</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D057166" MajorTopicYN="Y">High-Throughput Screening Assays</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D054884" MajorTopicYN="N">Host-Pathogen Interactions</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010455" MajorTopicYN="N">Peptides</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D059748" MajorTopicYN="N">Proteolysis</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D014709" MajorTopicYN="N">Vero Cells</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2011</Year><Month>11</Month><Day>17</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2012</Year><Month>04</Month><Day>06</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2012</Year><Month>04</Month><Day>23</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2012</Year><Month>5</Month><Day>12</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2012</Year><Month>5</Month><Day>12</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2012</Year><Month>10</Month><Day>2</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">22575574</ArticleId><ArticleId IdType="pii">S0166-0934(12)00138-3</ArticleId><ArticleId IdType="doi">10.1016/j.jviromet.2012.04.011</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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