A comprehensive method for quantifying human cytomegalovirus plaque assays

Human cytomegalovirus (hCMV) is an important human pathogen accounting for significant morbidity in immunocompromised individuals from neonates to cancer patients. To effectively produce progeny and establish a new infection, hCMV produces dozens of proteins and manipulates the expression of thousands of host genes. When studying the effect of these viral or host genes on the production of new infective progeny, the plaque assay is the gold standard method employed. This assay is carried out by incubating serial dilutions of the supernatant to be investigated on a monolayer of permissive cells. The experimental setup includes covering the monolayer with an overlay, thereby allowing the virus to spread only through cell-to-cell contact. Subsequently, the areas of cell death, or plaques, are quantified by fixing and staining with crystal violet. Here we take advantage of the fluorescent nature of crystal violet and image hCMV plaques in a variety of modalities. Following the acquisition of these images, we developed a broadly applicable pipeline to use open access software ImageJ to quantify the number and size of the hCMV plaques. The use of this method can provide a standardized way to objectively count and quantify the infectious progeny produced, allowing researchers to better understand hCMV.