Enteroviruses 71 (EV71) is a single-stranded, neurotrophic RNA virus responsible for the numerous outbreaks of hand, foot, and mouth disease (HFMD) in the Asia-Pacific regions. HFMD primarily affects children to cause range of infection, from mild symptoms to acute flaccid paralysis, and hemorrhage. Despite increased incidence of EV71 epidemics globally and research against EV71 becoming prioritized, no antiviral agent against EV71 has yet been licensed and approved worldwide. In this chapter, detailed EV71 antiviral screening techniques are described, including plaque assay which determines viral titers through the use of a semisolid overlay, carboxymethyl cellulose to allow even viral spread and infection across the host cellular monolayers as well as a crystal violet, a distinct counterstain to visualize circular regions of infectious zones-plaques. qRT-PCR is used to quantify the viral genomic RNA in the infected samples and MTS cell viability assay to quantify the cell viability after infection or toxicity of the compound on the cells. Furthermore, various antiviral inhibition assays including prophylactic, post infection, and virucidal assays are demonstrated for estimation of the antiviral activity of potential antiviral drugs against EV71. These methods can be effectively utilized in virology laboratories for effective high-throughput screening of antiviral molecules against EV71 that can assist in the future development of antiviral drugs.
Keywords: Antiviral assays; Cytopathic effect; Enterovirus 71; High-throughput assay; MTS assay; Plaque assay; RNA copy number; RT-PCR; Virus quantification.