{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,21]],"date-time":"2025-10-21T00:24:07Z","timestamp":1761006247236,"version":"build-2065373602"},"reference-count":30,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,10,18]],"date-time":"2025-10-18T00:00:00Z","timestamp":1760745600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NextGenerationEU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases","award":["PE00000007"],"award-info":[{"award-number":["PE00000007"]}]},{"name":"Horizon 2020 European Project","award":["N.101003551-EXSCALATE4CoV"],"award-info":[{"award-number":["N.101003551-EXSCALATE4CoV"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Testing medical countermeasures for SARS-CoV-2 transmission using vertebrates can be hindered by legislation regulating animal experimentation, high costs, and ethical concerns. To overcome these challenges, we propose a new Caenorhabditis elegans strain that constitutively expresses the human angiotensin-converting enzyme 2 receptor (ACE2). This resulted in significant impairment of reproduction and a defect in pharyngeal function compared to wild-type (WT) worms. SARS-CoV-2 infection was simulated by treating worms with the receptor-binding domain (RBD) of the spike protein, which caused dose-dependent and time-dependent pharyngeal impairment in ACE2 worms but not in WT worms. The toxicity of RBD was prevented by administering an anti-human ACE2 antibody, demonstrating that interactions with the ACE2 receptor are essential. The ACE2-expressing worm strain was further used for pharmacological research with Raloxifene. In vitro, 1\u20133 \u03bcM of Raloxifene reduced the entry of lentiviral particles carrying the Wuhan variant and B.1.1.7 UK and B.1.1.529 Omicron strains into HEK293-ACE2, in addition to particles expressing N501Y-mutated or P681H-mutated spike proteins. Raloxifene (0.1\u20131 \u03bcM) completely counteracted RBD toxicity in ACE2 worms, indicating that this strain offers a cost-effective in vivo screening platform for molecules with effects involving interactions with the ACE2 receptor.<\/jats:p>","DOI":"10.3390\/v17101387","type":"journal-article","created":{"date-parts":[[2025,10,20]],"date-time":"2025-10-20T08:18:54Z","timestamp":1760948334000},"page":"1387","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["SARS-CoV-2 Entry Can Be Mimicked in C. elegans Expressing Human ACE2: A New Tool for Pharmacological Studies"],"prefix":"10.3390","volume":"17","author":[{"given":"Margherita","family":"Romeo","sequence":"first","affiliation":[{"name":"Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6649-3846","authenticated-orcid":false,"given":"Sara","family":"Baroni","sequence":"additional","affiliation":[{"name":"Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6952-0069","authenticated-orcid":false,"given":"Maria Monica","family":"Barzago","sequence":"additional","affiliation":[{"name":"Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samuela","family":"Gambini","sequence":"additional","affiliation":[{"name":"Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7907-7139","authenticated-orcid":false,"given":"Ada","family":"De Luigi","sequence":"additional","affiliation":[{"name":"Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniela","family":"Iaconis","sequence":"additional","affiliation":[{"name":"EXSCALATE, Domp\u00e9 Farmaceutici SpA, Via Tommaso De Amicis, 95, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6830-2695","authenticated-orcid":false,"given":"Andrea Rosario","family":"Beccari","sequence":"additional","affiliation":[{"name":"EXSCALATE, Domp\u00e9 Farmaceutici SpA, Via Tommaso De Amicis, 95, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1769-3427","authenticated-orcid":false,"given":"Maddalena","family":"Fratelli","sequence":"additional","affiliation":[{"name":"Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2258-0531","authenticated-orcid":false,"given":"Luisa","family":"Diomede","sequence":"additional","affiliation":[{"name":"Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,18]]},"reference":[{"key":"ref_1","unstructured":"(2025, September 08). COVID-19 Deaths|WHO COVID-19 Dashboard. Available online: https:\/\/data.who.int\/dashboards\/covid19\/cases."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"323","DOI":"10.24272\/j.issn.2095-8137.2022.351","article-title":"Ferrets: A Powerful Model of SARS-CoV-2","volume":"44","author":"Zhao","year":"2023","journal-title":"Zool. Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e00509-17","DOI":"10.1128\/JVI.00509-17","article-title":"Caenorhabditis Elegans as an Emerging Model for Virus-Host Interactions","volume":"91","author":"Gammon","year":"2017","journal-title":"J. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"52340","DOI":"10.1074\/jbc.M308858200","article-title":"An Essential Role in Molting and Morphogenesis of Caenorhabditis Elegans for ACN-1, a Novel Member of the Angiotensin-Converting Enzyme Family That Lacks a Metallopeptidase Active Site","volume":"278","author":"Brooks","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Frand, A.R., Russel, S., and Ruvkun, G. (2005). Functional Genomic Analysis of C. Elegans Molting. PLoS Biol., 3.","DOI":"10.1371\/journal.pbio.0030312"},{"key":"ref_6","unstructured":"(2025, September 08). Available online: https:\/\/www.alliancegenome.org\/gene\/WB:WBGene00000039."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Sha, A., Liu, Y., and Zhao, X. (2023). SARS-CoV-2 and Gastrointestinal Diseases. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1177741"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1016\/j.stemcr.2024.03.008","article-title":"SARS-CoV-2 Tropism to Intestinal but Not Gastric Epithelial Cells Is Defined by Limited ACE2 Expression","volume":"19","author":"Fatykhova","year":"2024","journal-title":"Stem. Cell Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1007\/978-1-4419-8059-5_6","article-title":"Innate Immunity in C. Elegans","volume":"Volume 708","year":"2010","journal-title":"Invertebrate Immunity"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Natale, C., Barzago, M.M., and Diomede, L. (2020). Caenorhabditis Elegans Models to Investigate the Mechanisms Underlying Tau Toxicity in Tauopathies. Brain Sci., 10.","DOI":"10.3390\/brainsci10110838"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1007\/s12275-021-0617-7","article-title":"Raloxifene as a Treatment Option for Viral Infections","volume":"59","author":"Hong","year":"2021","journal-title":"J. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1038\/s41418-021-00844-6","article-title":"Repurposing the Estrogen Receptor Modulator Raloxifene to Treat SARS-CoV-2 Infection","volume":"29","author":"Allegretti","year":"2022","journal-title":"Cell Death Differ."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"101450","DOI":"10.1016\/j.eclinm.2022.101450","article-title":"A Phase 2 Randomized, Double-Blinded, Placebo-Controlled, Multicenter Trial Evaluating the Efficacy and Safety of Raloxifene for Patients with Mild to Moderate COVID-19","volume":"48","author":"Nicastri","year":"2022","journal-title":"eClinicalMedicine"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1038\/s41419-022-04961-z","article-title":"Characterization of Raloxifene as a Potential Pharmacological Agent against SARS-CoV-2 and Its Variants","volume":"13","author":"Iaconis","year":"2022","journal-title":"Cell Death Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1089\/ars.2016.6848","article-title":"Cardiac Light Chain Amyloidosis: The Role of Metal Ions in Oxidative Stress and Mitochondrial Damage","volume":"27","author":"Diomede","year":"2017","journal-title":"Antioxid. Redox Signal."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1007\/s11357-016-9936-8","article-title":"Age-Associated Vulval Integrity Is an Important Marker of Nematode Healthspan","volume":"38","author":"Leiser","year":"2016","journal-title":"Age"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Beeg, M., Baroni, S., Piotti, A., Porta, A., De Luigi, A., Cagnotto, A., Gobbi, M., Diomede, L., and Salmona, M. (2023). A Comprehensive Technology Platform for the Rapid Discovery of Peptide Inhibitors against SARS-CoV-2 Pseudovirus Infection. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241512146"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"56147","DOI":"10.18632\/oncotarget.11269","article-title":"OASIS 2: Online Application for Survival Analysis 2 with Features for the Analysis of Maximal Lifespan and Healthspan in Aging Research","volume":"7","author":"Han","year":"2016","journal-title":"Oncotarget"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1038\/s41423-020-0458-z","article-title":"Key Residues of the Receptor Binding Motif in the Spike Protein of SARS-CoV-2 That Interact with ACE2 and Neutralizing Antibodies","volume":"17","author":"Yi","year":"2020","journal-title":"Cell Mol. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1007\/978-1-4939-7471-9_4","article-title":"What Can We Learn About Human Disease from the Nematode C. Elegans?","volume":"Volume 1706","author":"DiStefano","year":"2018","journal-title":"Disease Gene Identification"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1261","DOI":"10.1002\/biot.201000183","article-title":"Modeling Human Diseases in Caenorhabditis elegans","volume":"5","author":"Markaki","year":"2010","journal-title":"Biotechnol. J."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Vallejo, J.A., Beceiro, A., Rumbo-Feal, S., Rodr\u00edguez-Palero, M.J., Russo, T.A., and Bou, G. (2015). Optimisation of the Caenorhabditis Elegans Model for Studying the Pathogenesis of Opportunistic Acinetobacter Baumannii. Int. J. Antimicrob. Agents, S0924857915002411.","DOI":"10.1016\/j.ijantimicag.2015.05.021"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1242\/dmm.006684","article-title":"Elegans: Model Host and Tool for Antimicrobial Drug Discovery","volume":"4","author":"Ewbank","year":"2011","journal-title":"Dis. Models Mech."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1080\/17460441.2017.1319358","article-title":"An Update on the Use of C. Elegans for Preclinical Drug Discovery: Screening and Identifying Anti-Infective Drugs","volume":"12","author":"Kim","year":"2017","journal-title":"Expert Opin. Drug Discov."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1728","DOI":"10.1128\/AAC.04198-14","article-title":"Insect-Derived Cecropins Display Activity against Acinetobacter Baumannii in a Whole-Animal High-Throughput Caenorhabditis Elegans Model","volume":"59","author":"Jayamani","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1016\/S0891-5849(03)00328-9","article-title":"An Automated High-Throughput Assay for Survival of the Nematode Caenorhabditis Elegans","volume":"35","author":"Gill","year":"2003","journal-title":"Free Radic. Biol. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e107821","DOI":"10.15252\/embj.2021107821","article-title":"TMPRSS2 Expression Dictates the Entry Route Used by SARS-CoV-2 to Infect Host Cells","volume":"40","author":"Koch","year":"2021","journal-title":"EMBO J."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"La\u017eeti\u0107, V., Blanchard, M.J., Bui, T., and Troemel, E.R. (2023). Multiple Pals Gene Modules Control a Balance between Immunity and Development in Caenorhabditis Elegans. PLoS Pathog., 19.","DOI":"10.1101\/2023.01.15.524171"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e54305","DOI":"10.15252\/embr.202154305","article-title":"ADAM10 and ADAM17 Promote SARS-CoV-2 Cell Entry and Spike Protein-mediated Lung Cell Fusion","volume":"23","author":"Jocher","year":"2022","journal-title":"EMBO Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"106505","DOI":"10.1016\/j.micpath.2023.106505","article-title":"Regulation of miR-61 and Col-19 via TGF-\u03b2 and Notch Signalling in Caenorhabditis Elegans against Klebsiella Aerogenes Infection","volume":"186","author":"Gowripriya","year":"2024","journal-title":"Microb. Pathog."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/10\/1387\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,20]],"date-time":"2025-10-20T08:29:39Z","timestamp":1760948979000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/10\/1387"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,18]]},"references-count":30,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["v17101387"],"URL":"https:\/\/doi.org\/10.3390\/v17101387","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,18]]}}}