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Antimicrob Agents Chemother. 2016 Aug 22;60(9):5492-503. doi: 10.1128/AAC.00814-16. Print 2016 Sep.

Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene.

Author information

1
Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
2
Laboratory of Neuroimmunovirology, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
3
Laboratory of Neuroimmunovirology, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada pierre.talbot@iaf.inrs.ca tanwj28@163.com.
4
Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China pierre.talbot@iaf.inrs.ca tanwj28@163.com.

Abstract

Human coronaviruses (HCoVs) cause 15 to 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. rOC43-ns2DelRluc was comparable to 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 a 50% inhibitory concentration (IC50) of 0.33 μM. However, ribavirin showed inhibition of 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.

PMID:
27381385
PMCID:
PMC4997878
[Available on 2017-03-01]
DOI:
10.1128/AAC.00814-16
[Indexed for MEDLINE]
Free PMC Article

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