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Cell. Infect. Microbiol."],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3389\/fcimb.2026.1818664","type":"journal-article","created":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T05:41:30Z","timestamp":1777009290000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["A comprehensive method for quantifying human cytomegalovirus plaque assays"],"prefix":"10.3389","volume":"16","author":[{"given":"Laurel E.","family":"Kelnhofer-Millevolte","sequence":"first","affiliation":[{"name":"Molecular and Cellular Biology, Graduate Program, University of Washington and Fred Hutchinson Cancer Center","place":["Seattle, WA, United States"]},{"name":"Medical Scientist Training Program, University of Washington","place":["Seattle, WA, United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel H.","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Pharmacology at the University of Miami Miller School of Medicine","place":["Miami, FL, United States"]},{"name":"Cancer Epigenetics Group at the Sylvester Comprehensive Cancer Center at the University of","place":["Miami, FL, United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lea S.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Pharmacology at the University of Miami Miller School of Medicine","place":["Miami, FL, United States"]},{"name":"Cancer Epigenetics Group at the Sylvester Comprehensive Cancer Center at the University of","place":["Miami, FL, United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daphne C.","family":"Avgousti","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Pharmacology at the University of Miami Miller School of Medicine","place":["Miami, FL, United States"]},{"name":"Cancer Epigenetics Group at the Sylvester Comprehensive Cancer Center at the University of","place":["Miami, FL, United States"]},{"name":"Human Biology Division, Fred Hutchinson Cancer Center","place":["Seattle, WA, United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2026,4,24]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1128\/msphere.00590-18","article-title":"Genotypic and phenotypic diversity of herpes simplex virus 2 within the infected neonatal population","volume":"4","author":"Akhtar","year":"2019","journal-title":"mSphere"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.02267-21","article-title":"Human cytomegalovirus UL138 protein inhibits the STING pathway and reduces interferon beta mRNA accumulation during lytic and latent infections","volume":"12","author":"Albright","year":"2021","journal-title":"mBio"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002878","article-title":"The human cytomegalovirus assembly compartment: A masterpiece of viral manipulation of cellular processes that facilitates assembly and egress","volume":"8","author":"Alwine","year":"2012","journal-title":"PloS Pathog."},{"key":"B4","doi-asserted-by":"publisher","first-page":"515","DOI":"10.6061\/clinics\/2015(07)09","article-title":"Cytomegalovirus infection in transplant recipients","volume":"70","author":"Azevedo","year":"2015","journal-title":"Clinics"},{"key":"B5","doi-asserted-by":"publisher","first-page":"533","DOI":"10.1128\/cmr.11.3.533","article-title":"Quantitation of cytomegalovirus: Methodologic aspects and clinical applications","volume":"11","author":"Boeckh","year":"1998","journal-title":"Clin. 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