**4.13 Plaque assay**

Plaque-based assays are the standard method used to determine virus concentration in terms of infectious dose. Viral plaque assays determine the number of plaqueforming units (pfu) in a virus sample, which is one measure of virus quantity. This assay is based on a microbiological method conducted in Petri dishes or multi-well plates like 6 well or 24 well etc. Specifically, a confluent monolayer of host cells is infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar or carboxymethyl cellulose, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when a virus infects a cell within the fixed cell monolayer [30]. Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. In research and development (R&D) based commercial and academic laboratories, the production of viral vaccines, recombinant proteins using viral vectors, viral antigens and clone screening, multiplicity of infection (MOI) optimization, and adaptation of methods to cell culture all require virus quantification. For quantification of virus incubated after infection at 37°C in a 5% CO2 incubator at 6, 12, 24, 36, 48, 60, and 72 h post-inoculation (hpi) based on the requirement to visualize plaques in wells [31, 32]. To quantify virus there are a lot of other methods used such as Focus forming assay (FFA), Endpoint dilution assay, Protein assays, Hemagglutination assay (HA), Bicinchoninic acid assay, Single radial immunodiffusion assay, Transmission electron microscopy (TEM).
