Quantification of West Nile Virus by Plaque-Forming Assay

The plaque-forming assay is a gold standard technique to determine the concentration of infectious viral particles. In this assay, lytic viruses infect and lyse the cells but are immobilized due to the presence of an agarose-containing overlay medium. The progeny viruses can only spread locally to and kill the adjacent cells and finally form a clear zone or plaque after staining the live cells. The number of plaques formed can be theoretically considered as the initial number of the infectious viral particles present in the sample and hence can be expressed as plaque-forming units (PFU) in a volume of the sample. Here, we provide a step-by-step method to carry out a plaque-forming assay to determine the titer of West Nile virus in a cell culture medium, which also can be adapted to other lytic viruses of eukaryotic cells.