The plaque assay is essential for virion quantitation but the classic protocol requires considerable efforts. A simplified dengue 96-well plaque assay with automated quantitation program is an alternative to access the level of infectious virus. Dengue plaque assay was simplified using LLC/MK2 cells and virus mixing simultaneously before semisolid addition. Results were obtained using a flatbed scanner and analysis by the self-written program optimized to manual reads. The newly developed microwell system was accurate to the standard assay because 19 independent titrations from all subtypes obtained from both systems differed less than a log10 p.f.u./ml with no significance (p>0.05) with good correlation (R2=0.9058). Coefficient of variations within and between assays, indicating assay reliability and repeatability, were 19.29%, and 12.50%, respectively. This method serves various experimental designs in drug discovery that requires viral titers assessment. Effective concentrations (EC90) results showed no significant difference between 24- and 96-well assays (p>0.05). Compound screening for potential antivirals and clinical isolate titrations were successfully arranged. The method contains distinguished features including protocol simplicity, less reagent consumption in microwell format, convenient and affordable data acquisition and analysis system.
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