title: Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene.
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.
authors.affiliation: Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Shen
authors.affiliation: Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ye
authors.affiliation: Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China.
authors.forename: Gaoshan
authors.initials: G
authors.lastname: Liu
authors.affiliation: Laboratory of Neuroimmunovirology, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
authors.forename: Marc
authors.initials: M
authors.lastname: Desforges
authors.affiliation: Laboratory of Neuroimmunovirology, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada pierre.talbot@iaf.inrs.ca tanwj28@163.com.
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Talbot
authors.affiliation: 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.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Tan
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D018352:Coronavirus Infections / Q000188:drug therapy*; D028962:Coronavirus OC43, Human / Q000187:drug effects* / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D012141:Respiratory Tract Infections / Q000188:drug therapy / Q000821:virology; D012254:Ribavirin / Q000494:pharmacology; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D012254:Ribavirin; D002738:Chloroquine; D008156:Luciferases