{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T18:46:06Z","timestamp":1782413166498,"version":"3.54.5"},"reference-count":35,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,8,25]],"date-time":"2021-08-25T00:00:00Z","timestamp":1629849600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Fondation Dormeur, Vaduz","award":["ELISpot\/ImmunoSpot\u00ae S6 ULTIMATE UV Image Analyzer"],"award-info":[{"award-number":["ELISpot\/ImmunoSpot\u00ae S6 ULTIMATE UV Image Analyzer"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Currently, human infections with the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are accelerating the ongoing spread of the pandemic. Several innovative types of vaccines have already been developed, whereas effective options of antiviral treatments still await a scientific implementation. The development of novel anti-SARS-CoV-2 drug candidates demands skillful strategies and analysis systems. Promising results have been achieved with first generation direct-acting antivirals targeting the viral polymerase RdRp or the protease 3CLpro. Such recently approved or investigational drugs like remdesivir and GC376 represent a basis for further development and optimization. Here, we establish a multi-readout assay (MRA) system that enables the antiviral assessment and mechanistic characterization of novel test compounds, drug repurposing and combination treatments. Our SARS-CoV-2-specific MRA combines the quantitative measurement of several parameters of virus infection, such as the intracellular production of proteins and genomes, enzymatic activities and virion release, as well as the use of reporter systems. In this regard, the antiviral efficacy of remdesivir and GC376 has been investigated in human Caco-2 cells. The readouts included the use of spike- and double-strand RNA-specific monoclonal antibodies for in-cell fluorescence imaging, a newly generated recombinant SARS-CoV-2 reporter virus d6YFP, the novel 3CLpro-based FRET CFP::YFP and the previously reported FlipGFP reporter assays, as well as viral genome-specific RT-qPCR. The data produced by our MRA confirm the high antiviral potency of these two drugs in vitro. Combined, this MRA approach may be applied for broader analyses of SARS-CoV-2-specific antivirals, including compound screenings and the characterization of selected drug candidates.<\/jats:p>","DOI":"10.3390\/pathogens10091076","type":"journal-article","created":{"date-parts":[[2021,8,25]],"date-time":"2021-08-25T23:25:50Z","timestamp":1629933950000},"page":"1076","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs against SARS-CoV-2"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3011-8416","authenticated-orcid":false,"given":"Friedrich","family":"Hahn","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":"Sigrun","family":"H\u00e4ge","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":"Alexandra","family":"Herrmann","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":"Christina","family":"Wangen","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":"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":"Doris","family":"Jungnickl","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":"Julia","family":"Tillmanns","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\/0000-0003-4792-4991","authenticated-orcid":false,"given":"Kirsten","family":"Fraedrich","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\/0000-0001-7822-3835","authenticated-orcid":false,"given":"Klaus","family":"\u00dcberla","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":"Hella","family":"Kohlhof","sequence":"additional","affiliation":[{"name":"Immunic AG, 82166 Gr\u00e4felfing, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8873-3863","authenticated-orcid":false,"given":"Armin","family":"Ensser","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":[[2021,8,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1038\/s41579-020-00459-7","article-title":"Characteristics of SARS-CoV-2 and COVID-19","volume":"19","author":"Hu","year":"2021","journal-title":"Nat. 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