An antiviral drug screening system for enterovirus 71 based on an improved plaque assay: A potential high-throughput method

J Med Virol. 2019 Aug;91(8):1440-1447. doi: 10.1002/jmv.25463. Epub 2019 Apr 1.


Plaque assay plays an irreplaceable role in a variety of virological studies, including determining titers of viruses. Our previous study showed that a simple and highly repeatable plaque assay could be used for enterovirus 71 (EV-A71). Now, we show that using a subclone of a clinical EV-A71 isolate and a rhabdomyosarcoma cell line (RD), a plaque assay based on an EV-A71/RD model could exhibit the most rapid formation of plaques (<2 days), with much higher repeatability and consistency. Inspired by a plaque inhibitory test for testing ribavirin and interferon, as well as a plaque reduction neutralization test, this modified method has been used to establish a convenient system by using 96-well plates for screening anti-EV-A71 drugs from a 130-compound library containing multiple types of inhibitors. Nine candidate effective compounds for EV-A71 have been screened out, and among them, nobiletin (flavonoid) was found to be a novel effective compound at the concentration of 10 μM. Our findings imply that this improved method based on an EV-A71/RD model proved to be a potential high-throughput method in screening novel antiviral drugs for EV-A71. Undoubtedly, this method can also be applied to other viruses that can produce an obvious cytopathic effect.

Keywords: antiviral drug screening; enterovirus 71; plaque assay; plaque inhibitory test.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiviral Agents / pharmacology*
  • Cell Line
  • Drug Evaluation, Preclinical / methods*
  • Enterovirus A, Human / drug effects*
  • Flavones / pharmacology
  • High-Throughput Screening Assays / methods*
  • Humans
  • Reproducibility of Results
  • Viral Plaque Assay / methods*


  • Antiviral Agents
  • Flavones
  • nobiletin