title: High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.
abstract: Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection.
authors.affiliation: Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden Department of Clinical Microbiology, Infectious Diseases, Umeå University, Umeå, Sweden.
authors.forename: Md Koushikul
authors.initials: MK
authors.lastname: Islam
authors.affiliation: Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Baudin
authors.affiliation: Department of Chemistry, Umeå University, Umeå, Sweden Laboratories for Chemical Biology Umeå, Chemical Biology Consortium Sweden, Solna, Sweden.
authors.forename: Jonas
authors.initials: J
authors.lastname: Eriksson
authors.affiliation: Department of Chemistry, Umeå University, Umeå, Sweden Laboratories for Chemical Biology Umeå, Chemical Biology Consortium Sweden, Solna, Sweden.
authors.forename: Christopher
authors.initials: C
authors.lastname: Öberg
authors.affiliation: Innate Immunity Laboratory, Max Planck Institute of Biochemistry, Martinsried, Munich, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Habjan
authors.affiliation: Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig University Gießen, Gießen, Germany.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Weber
authors.affiliation: Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Överby
authors.affiliation: Department of Clinical Microbiology, Infectious Diseases, Umeå University, Umeå, Sweden.
authors.forename: Clas
authors.initials: C
authors.lastname: Ahlm
authors.affiliation: Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden magnus.evander@umu.se.
authors.forename: Magnus
authors.initials: M
authors.lastname: Evander
mesh_terms: D000072283:A549 Cells; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001549:Benzamides / Q000737:chemistry / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D006835:Hydrazones / Q000737:chemistry / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016865:Reassortant Viruses; D012296:Rift Valley fever virus / Q000187:drug effects* / Q000254:growth & development; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014779:Virus Replication / Q000187:drug effects; D000097573:Red Fluorescent Protein
chemical_list: D000998:Antiviral Agents; D001549:Benzamides; D006835:Hydrazones; D008164:Luminescent Proteins; D054852:Small Molecule Libraries; D000097573:Red Fluorescent Protein
keywords: Rift Valley fever; antiviral; cell-based assay; high-throughput screening; recombinant virus