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HCMV infections, especially those in high-risk patients, still raise many unanswered questions, so current investigations are focused on viral pathogenesis, vaccine development, and options for antiviral drug targeting. To this end, the use of suitable viral strains as well as recombinant reporter constructs in cultured cells and model systems has specific significance. We previously reported on the application of various herpesviruses that express green, red, or related fluorescent proteins, especially in the fields of virus\u2013host interaction and antiviral research. Here, we characterized a recombinant version of the clinically relevant and cell type-adaptable HCMV strain TB40, which expresses firefly luciferase as a quantitative reporter of viral replication (TB40-FLuc). The data provide evidence for five main conclusions. First, HCMV TB40-FLuc is employable in multiple settings in primary human cells. Second, viral reporter signals are easily quantifiable, even at early time points within viral replication. Third, the FLuc reporter reflects the kinetics of viral intracellular replication, cascade-like viral IE-E-L protein production, and progeny release. Fourth, as relates to specific applications of the TB40-FLuc system, we demonstrated the reliability of quantitative antiviral compound determination in multi-well formats and its independence from fluorescence-based measurements in the case of autofluorescent inhibitors. Finally, we illustrated increased reporter sensitivity in comparison to other recombinant HCMVs. In essence, recombinant HCMV TB40-FLuc combines several molecular properties that are considered beneficial in studies on viral host tropism, replication efficiency, and antiviral drug assessment.<\/jats:p>","DOI":"10.3390\/pathogens13080645","type":"journal-article","created":{"date-parts":[[2024,7,31]],"date-time":"2024-07-31T10:34:43Z","timestamp":1722422083000},"page":"645","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Establishment of a Luciferase-Based Reporter System to Study Aspects of Human Cytomegalovirus Infection, Replication Characteristics, and Antiviral Drug Efficacy"],"prefix":"10.3390","volume":"13","author":[{"given":"Julia","family":"Tillmanns","sequence":"first","affiliation":[{"name":"Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-N\u00fcrnberg (FAU), 91054 Erlangen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jintawee","family":"Kicuntod","sequence":"additional","affiliation":[{"name":"Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-N\u00fcrnberg (FAU), 91054 Erlangen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonia","family":"Ehring","sequence":"additional","affiliation":[{"name":"Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-N\u00fcrnberg (FAU), 91054 Erlangen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Endrit","family":"Elbasani","sequence":"additional","affiliation":[{"name":"Institute of Virology, Hannover Medical School (MHH), 30625 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eva Maria","family":"Borst","sequence":"additional","affiliation":[{"name":"Institute of Virology, Hannover Medical School (MHH), 30625 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Debora","family":"Obergf\u00e4ll","sequence":"additional","affiliation":[{"name":"Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-N\u00fcrnberg (FAU), 91054 Erlangen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Regina","family":"M\u00fcller","sequence":"additional","affiliation":[{"name":"Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-N\u00fcrnberg (FAU), 91054 Erlangen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3011-8416","authenticated-orcid":false,"given":"Friedrich","family":"Hahn","sequence":"additional","affiliation":[{"name":"Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-N\u00fcrnberg (FAU), 91054 Erlangen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manfred","family":"Marschall","sequence":"additional","affiliation":[{"name":"Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-N\u00fcrnberg (FAU), 91054 Erlangen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,7,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1093\/infdis\/144.1.47","article-title":"Mechanisms of immunosuppression in cytomegalovirus mononucleosis. 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