{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T01:18:26Z","timestamp":1785460706479,"version":"3.56.0"},"reference-count":34,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,27]],"date-time":"2025-12-27T00:00:00Z","timestamp":1766793600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Canadian Institutes of Health Research (CIHR)-funded Coronavirus Variants Rapid Response Network","award":["FRN-175622"],"award-info":[{"award-number":["FRN-175622"]}]},{"name":"CIHR Operating COVID-19 Rapid Research Funding Opportunity\u2014Therapeutics","award":["VR3-172626"],"award-info":[{"award-number":["VR3-172626"]}]},{"DOI":"10.13039\/501100000024","name":"CIHR","doi-asserted-by":"crossref","award":["PPE\u2013192112"],"award-info":[{"award-number":["PPE\u2013192112"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000024","name":"CIHR","doi-asserted-by":"crossref","award":["PPE-190337"],"award-info":[{"award-number":["PPE-190337"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000038","name":"NSERC","doi-asserted-by":"publisher","award":["RGPIN 2018-06335"],"award-info":[{"award-number":["RGPIN 2018-06335"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000024","name":"CIHR","doi-asserted-by":"crossref","award":["OV5-170349"],"award-info":[{"award-number":["OV5-170349"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000024","name":"CIHR","doi-asserted-by":"crossref","award":["VRI-173022"],"award-info":[{"award-number":["VRI-173022"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000024","name":"CIHR","doi-asserted-by":"crossref","award":["VS1-175531"],"award-info":[{"award-number":["VS1-175531"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000024","name":"CIHR","doi-asserted-by":"crossref","award":["PUU-177963"],"award-info":[{"award-number":["PUU-177963"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000024","name":"CIHR","doi-asserted-by":"crossref","award":["PTT-179810"],"award-info":[{"award-number":["PTT-179810"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000024","name":"CIHR","doi-asserted-by":"crossref","award":["MM1-181121"],"award-info":[{"award-number":["MM1-181121"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100021824","name":"Government of Saskatchewan","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100021824","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100000023","name":"Government of Canada","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100000023","id-type":"DOI","asserted-by":"crossref"}]},{"name":"College of Medicine (CoM) Devolved Scholarship"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The continuing spread of SARS-CoV-2 and the associated morbidity and mortality, especially in vulnerable populations, highlight the need for the development of antiviral therapeutics. Reverse genetics systems and reporter viruses are valuable for antiviral screening by simplifying methods to detect and quantify virus infections. This study aimed to generate wild-type and Nluc reporter full-length SARS-CoV-2 molecular clones and viruses as tools for high-throughput antiviral assays. The large SARS-CoV-2 genome (~30 kb) makes cDNA cloning and virus rescue technically challenging, so we opted to use cDNA chemical synthesis services to generate full-length wild-type and reporter Delta and Omicron clones. Clone-derived Delta and Omicron wild-type and reporter viruses were successfully rescued and showed replication kinetics comparable to patient-derived isolates. Nluc reporter viruses displayed stable luciferase expression that correlated with viral titres, supporting their reliability as replication substitutes. Antiviral assays measuring replication inhibition by Remdesivir, Molnupiravir, and Nirmatrelvir, based on Nluc expression, yielded IC50 values and selectivity indices consistent with published ranges. Finally, Delta Nluc viruses replicated in primary human bronchial epithelial cells, demonstrating the application of clone-derived viruses in physiologically relevant models. The SARS-CoV-2 cDNA clones and Nluc reporter viruses derived from DNA synthesis services provide a rapid, scalable reverse genetics platform for generating new viruses and developing assays to rapidly assess antiviral compounds against current and emerging SARS-CoV-2 variants or coronaviruses that may emerge in the future.<\/jats:p>","DOI":"10.3390\/v18010044","type":"journal-article","created":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T06:25:51Z","timestamp":1766989551000},"page":"44","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Developing Synthetic Full-Length SARS-CoV-2 cDNAs and Reporter Viruses for High-Throughput Antiviral Drug Screening"],"prefix":"10.3390","volume":"18","author":[{"given":"Megha","family":"Rohamare","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nidhi","family":"Kaushik","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"},{"name":"Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juveriya Qamar","family":"Khan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahrokh","family":"Balouchi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0454-9757","authenticated-orcid":false,"given":"Joaquin","family":"Lopez-Orozco","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert","family":"Kozak","sequence":"additional","affiliation":[{"name":"Biological Sciences, Sunnybrook Research Institute, Toronto, ON M4N 3M5, Canada"},{"name":"Shared Hospital Laboratory, Toronto, ON M1E 4B9, Canada"},{"name":"Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tom C.","family":"Hobman","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada"},{"name":"Department of Medical Microbiology & Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB T6G 2E1, Canada"},{"name":"Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8805-8068","authenticated-orcid":false,"given":"Darryl","family":"Falzarano","sequence":"additional","affiliation":[{"name":"Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada"},{"name":"Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6179-4516","authenticated-orcid":false,"given":"Anil","family":"Kumar","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5051-3088","authenticated-orcid":false,"given":"Joyce A.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Microbiology, and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"12910","DOI":"10.1074\/jbc.REV120.013930","article-title":"The molecular virology of coronaviruses","volume":"295","author":"Hartenian","year":"2020","journal-title":"J. Biol. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/S0140-6736(20)30183-5","article-title":"Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China","volume":"395","author":"Huang","year":"2020","journal-title":"Lancet"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1016\/S2213-2600(20)30076-X","article-title":"Pathological findings of COVID-19 associated with acute respiratory distress syndrome","volume":"8","author":"Xu","year":"2020","journal-title":"Lancet Respir. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e0422523","DOI":"10.1128\/spectrum.04225-23","article-title":"Tracking the prevalence and emergence of SARS-CoV-2 variants of concern using a regional genomic surveillance program","volume":"12","author":"Jung","year":"2024","journal-title":"Microbiol. Spectr."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1534\/genetics.119.302655","article-title":"Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans","volume":"213","author":"Sfarcic","year":"2019","journal-title":"Genetics"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1021\/acs.bioconjchem.6b00112","article-title":"NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence","volume":"27","author":"England","year":"2016","journal-title":"Bioconjugate Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3017","DOI":"10.1002\/jmv.27738","article-title":"Reverse genetics systems for SARS-CoV-2","volume":"94","author":"Wang","year":"2022","journal-title":"J. Med. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1761","DOI":"10.1038\/s41596-021-00491-8","article-title":"Engineering SARS-CoV-2 using a reverse genetic system","volume":"16","author":"Xie","year":"2021","journal-title":"Nat. Protoc."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1021\/acs.jafc.3c08327","article-title":"Rapid Construction of Recombinant PDCoV Expressing an Enhanced Green Fluorescent Protein for the Antiviral Screening Assay Based on Transformation-Associated Recombination Cloning in Yeast","volume":"72","author":"Zhang","year":"2024","journal-title":"J. Agric. Food Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1038\/s41586-020-2294-9","article-title":"Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform","volume":"582","author":"Labroussaa","year":"2020","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Khan, J.Q., Rohamare, M., Rajamanickam, K., Bhanumathy, K.K., Lew, J., Kumar, A., Falzarano, D., Vizeacoumar, F.J., and Wilson, J.A. (2023). Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology. Viruses, 15.","DOI":"10.1101\/2023.03.08.531833"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e0068721","DOI":"10.1128\/JVI.00687-21","article-title":"Construction of a noninfectious SARS-CoV-2 replicon for antiviral-drug testing and gene function studies","volume":"95","author":"Nguyen","year":"2021","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3859","DOI":"10.1128\/jb.172.7.3859-3867.1990","article-title":"Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers","volume":"172","author":"Durland","year":"1990","journal-title":"J. Bacteriol."},{"key":"ref_14","unstructured":"Bennett, J.E., Dolin, R., and Blaser, M.J. (2015). 148\u2014Hepatitis B Virus and Hepatitis Delta Virus. Mandell, Douglas, and Bennett\u2019s Principles and Practice of Infectious Diseases (Eighth Edition), W.B. Saunders."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"16273","DOI":"10.1038\/srep16273","article-title":"2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori","volume":"5","author":"Wang","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1007\/978-1-0716-3890-3_4","article-title":"Large-Scale Culture and Plasmid Preparation Procedure for Low-Yield Bacmids Containing Full-Length SARS-CoV-2 cDNAs","volume":"2813","author":"Rohamare","year":"2024","journal-title":"Methods Mol. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1016\/j.chom.2020.04.004","article-title":"An Infectious cDNA Clone of SARS-CoV-2","volume":"27","author":"Xie","year":"2020","journal-title":"Cell Host Microbe"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"422","DOI":"10.1016\/j.medj.2022.04.002","article-title":"Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, Beta, Delta, and Omicron variants","volume":"3","author":"Banerjee","year":"2022","journal-title":"Med"},{"key":"ref_19","unstructured":"Hierholzer, J., and Killington, R. (1996). Suspension assay method. Virology Methods Manual, Academic Press."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e13179","DOI":"10.1111\/irv.13179","article-title":"Use of whole genome sequencing to identify low-frequency mutations in SARS-CoV-2 patients treated with remdesivir","volume":"17","author":"Nirmalarajah","year":"2023","journal-title":"Influenza Other Respir Viruses"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Nasir, J.A., Kozak, R.A., Aftanas, P., Raphenya, A.R., Smith, K.M., Maguire, F., Maan, H., Alruwaili, M., Banerjee, A., and Mbareche, H. (2020). A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture. Viruses, 12.","DOI":"10.3390\/v12080895"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"768","DOI":"10.1093\/infdis\/jiab578","article-title":"Surface and Air Contamination With Severe Acute Respiratory Syndrome Coronavirus 2 From Hospitalized Coronavirus Disease 2019 Patients in Toronto, Canada, March\u2013May 2020","volume":"225","author":"Kotwa","year":"2022","journal-title":"J. Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1422","DOI":"10.1128\/JVI.76.3.1422-1434.2002","article-title":"Heterologous gene expression from transmissible gastroenteritis virus replicon particles","volume":"76","author":"Curtis","year":"2002","journal-title":"J. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1632","DOI":"10.1111\/febs.16662","article-title":"A guide to COVID-19 antiviral therapeutics: A summary and perspective of the antiviral weapons against SARS-CoV-2 infection","volume":"291","author":"Brady","year":"2024","journal-title":"FEBS J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"104786","DOI":"10.1016\/j.antiviral.2020.104786","article-title":"Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro","volume":"178","author":"Choy","year":"2020","journal-title":"Antivir. Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1038\/s41422-020-0282-0","article-title":"Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro","volume":"30","author":"Wang","year":"2020","journal-title":"Cell Res."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Tian, L., Pang, Z., Li, M., Lou, F., An, X., Zhu, S., Song, L., Tong, Y., Fan, H., and Fan, J. (2022). Molnupiravir and Its Antiviral Activity Against COVID-19. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.855496"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1038\/s41586-022-05514-2","article-title":"Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir","volume":"613","author":"Iketani","year":"2023","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"105970","DOI":"10.1016\/j.antiviral.2024.105970","article-title":"Nirmatrelvir and molnupiravir maintain potent in vitro and in vivo antiviral activity against circulating SARS-CoV-2 omicron subvariants","volume":"230","author":"Rosales","year":"2024","journal-title":"Antivir. Res."},{"key":"ref_30","first-page":"265","article-title":"Efecto citot\u00f3xico selectivo in vitro de muricin H (acetogenina de Annona muricata) en cultivos celulares de c\u00e1ncer de pulm\u00f3n","volume":"23","author":"Quispe","year":"2006","journal-title":"Rev. Peru. Med. Exp. Salud P\u00fablica"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"104974","DOI":"10.1016\/j.antiviral.2020.104974","article-title":"A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2","volume":"185","author":"Zhang","year":"2021","journal-title":"Antivir. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"001583","DOI":"10.1099\/jgv.0.001583","article-title":"SARS-CoV-2 replicon for high-throughput antiviral screening","volume":"102","author":"Zhang","year":"2021","journal-title":"J. Gen. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"105252","DOI":"10.1016\/j.antiviral.2022.105252","article-title":"Remdesivir, Molnupiravir and Nirmatrelvir remain active against SARS-CoV-2 Omicron and other variants of concern","volume":"198","author":"Vangeel","year":"2022","journal-title":"Antivir. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1038\/s41422-022-00618-w","article-title":"SARS-CoV-2 Omicron variant is highly sensitive to molnupiravir, nirmatrelvir, and the combination","volume":"32","author":"Li","year":"2022","journal-title":"Cell Res."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/1\/44\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,30]],"date-time":"2025-12-30T05:20:08Z","timestamp":1767072008000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/1\/44"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,27]]},"references-count":34,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["v18010044"],"URL":"https:\/\/doi.org\/10.3390\/v18010044","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,27]]}}}