A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals.

The spread of highly pathogenic avian influenza across geographical and species barriers underscores the increasing need for novel antivirals to compliment vaccination and existing antiviral therapies. Identification of new antiviral lead compounds depends on robust primary assays for high-throughput screening (HTS) of large compound libraries. We have developed a cell-based screen for potential influenza antivirals that measures the cytopathic effect (CPE) induced by influenza virus (A/Udorn/72, H3N2) infection in Madin Darby canine kidney (MDCK) cells using the luminescent-based CellTiter Glo system. This 72 h assay is validated for HTS in 384-well plates and performs more consistently and reliably than methods using neutral red, with Z values>0.8, signal-to-background>30 and signal-to-noise>10. In a blinded pilot screen (n=10,781) at 10 microM concentration, four compounds (with previously demonstrated efficacy against influenza) inhibited viral-induced CPE by >50%, with EC50/CC50 values comparable to those determined by other cell-based assays, thereby validating this assay accuracy and ability to simultaneously evaluate compound cellular availability and/or toxicity. This assay is translatable for screening against other influenza strains, such as avian flu, and may facilitate identification of antivirals for other viruses that induce CPE, such as West Nile or Dengue.