A Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene.
Identifieur interne : 000071 ( Hal/Checkpoint ); précédent : 000070; suivant : 000072A Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene.
Auteurs : Liang Shen [République populaire de Chine] ; Yang Yang [République populaire de Chine] ; Fei Ye [République populaire de Chine] ; Gaoshan Liu [République populaire de Chine] ; Marc Desforges [Canada] ; Pierre J. Talbot [Canada] ; Wenjie Tan [République populaire de Chine]Source :
- Antimicrobial Agents and Chemotherapy [ 0066-4804 ] ; 2016-07-01.
Abstract
Human coronaviruses (HCoVs) cause 15-30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43), which express the Renilla luciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. The rOC43-ns2DelRluc was comparable with its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replication in vitro, with an IC50 of 0.33 μM. However, ribavirin showed inhibition on HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50 of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded RNA-activated protein kinase (PKR) and DEAD-box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high throughput antiviral screening and quantitative analysis of viral replication.
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DOI: 10.1128/aac.00814-16
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However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43), which express the Renilla luciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. The rOC43-ns2DelRluc was comparable with its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replication in vitro, with an IC50 of 0.33 μM. However, ribavirin showed inhibition on HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50 of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded RNA-activated protein kinase (PKR) and DEAD-box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high throughput antiviral screening and quantitative analysis of viral replication.</p> </div></front></TEI><hal api="V3"> <titleStmt> <title xml:lang="en">A Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene.</title> <author role="aut"> <persName> <forename type="first">Liang</forename> <surname>Shen</surname> </persName> <idno type="halauthorid">1191657</idno> <affiliation ref="#struct-463335"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Yang</forename> <surname>Yang</surname> </persName> <idno type="halauthorid">98598</idno> <affiliation ref="#struct-463335"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Fei</forename> <surname>Ye</surname> </persName> <idno type="halauthorid">1370022</idno> <affiliation ref="#struct-463335"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Gaoshan</forename> <surname>Liu</surname> </persName> <idno type="halauthorid">1370023</idno> <affiliation ref="#struct-463335"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Marc</forename> <surname>Desforges</surname> </persName> <idno type="halauthorid">752119</idno> <affiliation ref="#struct-54664"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Pierre J</forename> <surname>Talbot</surname> </persName> <idno type="halauthorid">752125</idno> <affiliation ref="#struct-54664"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Wenjie</forename> <surname>Tan</surname> </persName> <idno type="halauthorid">1370024</idno> <affiliation ref="#struct-463335"></affiliation> </author> <editor role="depositor"> <persName> <forename>Michel</forename> <surname>Courcelles</surname> </persName> <email type="md5">235ee71abfb5f3fb19e1b204192e01ee</email> <email type="domain">iaf.inrs.ca</email> </editor> <funder>This work, including the efforts of Wenjie Tan, was funded by Megaproject for Infectious Disease Research of China (2014ZX10004001 and 2013ZX10004601) and National Key Plan for Scientific Research and Development of China (2016YFD0500301)</funder> </titleStmt> <editionStmt> <edition n="v1" type="current"> <date type="whenSubmitted">2016-08-03 22:35:46</date> <date type="whenModified">2018-10-08 17:44:05</date> <date type="whenReleased">2016-08-25 11:17:13</date> <date type="whenProduced">2016-07-01</date> <ref type="externalLink" target="https://aac.asm.org/content/aac/60/9/5492.full.pdf"></ref> </edition> <respStmt> <resp>contributor</resp> <name key="157387"> <persName> <forename>Michel</forename> <surname>Courcelles</surname> </persName> <email type="md5">235ee71abfb5f3fb19e1b204192e01ee</email> <email type="domain">iaf.inrs.ca</email> </name> </respStmt> </editionStmt> <publicationStmt> <distributor>CCSD</distributor> <idno type="halId">pasteur-01351547</idno> <idno type="halUri">https://hal-riip.archives-ouvertes.fr/pasteur-01351547</idno> <idno type="halBibtex">shen:pasteur-01351547</idno> <idno type="halRefHtml">Antimicrobial Agents and Chemotherapy, American Society for Microbiology, 2016, 60 (9), pp.5492-5503. ⟨10.1128/aac.00814-16 ⟩</idno> <idno type="halRef">Antimicrobial Agents and Chemotherapy, American Society for Microbiology, 2016, 60 (9), pp.5492-5503. ⟨10.1128/aac.00814-16 ⟩</idno> </publicationStmt> <seriesStmt> <idno type="stamp" n="RIIP">Institut Pasteur RIIP (Réseau International)</idno> <idno type="stamp" n="INRS-IAF" corresp="RIIP">Institut Armand Frappier</idno> </seriesStmt> <notesStmt> <note type="audience" n="2">International</note> <note type="popular" n="0">No</note> <note type="peer" n="1">Yes</note> </notesStmt> <sourceDesc> <biblStruct> <analytic> <title xml:lang="en">A Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene.</title> <author role="aut"> <persName> <forename type="first">Liang</forename> <surname>Shen</surname> </persName> <idno type="halauthorid">1191657</idno> <affiliation ref="#struct-463335"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Yang</forename> <surname>Yang</surname> </persName> <idno type="halauthorid">98598</idno> <affiliation ref="#struct-463335"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Fei</forename> <surname>Ye</surname> </persName> <idno type="halauthorid">1370022</idno> <affiliation ref="#struct-463335"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Gaoshan</forename> <surname>Liu</surname> </persName> <idno type="halauthorid">1370023</idno> <affiliation ref="#struct-463335"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Marc</forename> <surname>Desforges</surname> </persName> <idno type="halauthorid">752119</idno> <affiliation ref="#struct-54664"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Pierre J</forename> <surname>Talbot</surname> </persName> <idno type="halauthorid">752125</idno> <affiliation ref="#struct-54664"></affiliation> </author> <author role="aut"> <persName> <forename type="first">Wenjie</forename> <surname>Tan</surname> </persName> <idno type="halauthorid">1370024</idno> <affiliation ref="#struct-463335"></affiliation> </author> </analytic> <monogr> <idno type="halJournalId" status="VALID">10566</idno> <idno type="issn">0066-4804</idno> <idno type="eissn">1098-6596</idno> <title level="j">Antimicrobial Agents and Chemotherapy</title> <imprint> <publisher>American Society for Microbiology</publisher> <biblScope unit="volume">60</biblScope> <biblScope unit="issue">9</biblScope> <biblScope unit="pp">5492-5503</biblScope> <date type="datePub">2016-07-01</date> <date type="dateEpub">2016-07-05</date> </imprint> </monogr> <idno type="doi">10.1128/aac.00814-16 </idno> <idno type="pubmed">27381385</idno> </biblStruct> </sourceDesc> <profileDesc> <langUsage> <language ident="en">English</language> </langUsage> <textClass> <classCode scheme="halDomain" n="sdv">Life Sciences [q-bio]</classCode> <classCode scheme="halDomain" n="sdv.bbm">Life Sciences [q-bio]/Biochemistry, Molecular Biology</classCode> <classCode scheme="halDomain" n="sdv.gen">Life Sciences [q-bio]/Genetics</classCode> <classCode scheme="halTypology" n="ART">Journal articles</classCode> </textClass> <abstract xml:lang="en"> <p>Human coronaviruses (HCoVs) cause 15-30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43), which express the Renilla luciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. The rOC43-ns2DelRluc was comparable with its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replication in vitro, with an IC50 of 0.33 μM. However, ribavirin showed inhibition on HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC50 of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded RNA-activated protein kinase (PKR) and DEAD-box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high throughput antiviral screening and quantitative analysis of viral replication.</p> </abstract> </profileDesc> </hal></record>Pour manipuler ce document sous Unix (Dilib)
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