Assessing Genomic Mutations in SARS-CoV-2: Potential Resistance to Antiviral Drugs in Viral Populations from Untreated COVID-19 Patients

DOI record: { "DOI": "10.3390/microorganisms12010002", "ISSN": [ "2076-2607" ], "URL": "http://dx.doi.org/10.3390/microorganisms12010002", "abstract": "<jats:p>Naturally occurring SARS-CoV-2 variants mutated in genomic regions targeted by antiviral drugs have not been extensively studied. This study investigated the potential of the RNA-dependent RNA polymerase (RdRp) complex subunits and non-structural protein (Nsp)5 of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to accumulate natural mutations that could affect the efficacy of antiviral drugs. To this aim, SARS-CoV-2 genomic sequences isolated from 4155 drug-naive individuals from southern Italy were analyzed using the Illumina MiSeq platform. Sequencing of the 4155 samples showed the following viral variant distribution: 71.2% Delta, 22.2% Omicron, and 6.4% Alpha. In the Nsp12 sequences, we found 84 amino acid substitutions. The most common one was P323L, detected in 3777/4155 (91%) samples, with 2906/3777 (69.9%) also showing the G671S substitution in combination. Additionally, we identified 28, 14, and 24 different amino acid substitutions in the Nsp5, Nsp7, and Nsp8 genomic regions, respectively. Of note, the V186F and A191V substitutions, affecting residues adjacent to the active site of Nsp5 (the target of the antiviral drug Paxlovid), were found in 157/4155 (3.8%) and 3/4155 (0.07%) samples, respectively. In conclusion, the RdRp complex subunits and the Nsp5 genomic region exhibit susceptibility to accumulating natural mutations. This susceptibility poses a potential risk to the efficacy of antiviral drugs, as these mutations may compromise the drug ability to inhibit viral replication</jats:p>", "alternative-id": [ "microorganisms12010002" ], "author": [ { "affiliation": [ { "name": "Department of Clinical and Experimental Medicine, University Hospital of Messina, 98124 Messina, Italy" } ], "family": "Lombardo", "given": "Daniele", "sequence": "first" }, { "affiliation": [ { "name": "Department of Human Pathology, University Hospital of Messina, 98124 Messina, Italy" } ], "family": "Musolino", "given": "Cristina", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Clinical and Experimental Medicine, University Hospital of Messina, 98124 Messina, Italy" } ], "family": "Chines", "given": "Valeria", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Clinical and Experimental Medicine, University Hospital of Messina, 98124 Messina, Italy" } ], "family": "Caminiti", "given": "Giuseppe", "sequence": "additional" }, { "ORCID": "https://orcid.org/0009-0006-5434-6738", "affiliation": [ { "name": "Department of Clinical and Experimental Medicine, University Hospital of Messina, 98124 Messina, Italy" } ], "authenticated-orcid": false, "family": "Palermo", "given": "Claudia", "sequence": "additional" }, { "ORCID": "https://orcid.org/0000-0001-7721-6799", "affiliation": [ { "name": "Department of Clinical and Experimental Medicine, University Hospital of Messina, 98124 Messina, Italy" } ], "authenticated-orcid": false, "family": "Cacciola", "given": "Irene", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Clinical and Experimental Medicine, University Hospital of Messina, 98124 Messina, Italy" } ], "family": "Raffa", "given": "Giuseppina", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Clinical and Experimental Medicine, University Hospital of Messina, 98124 Messina, Italy" } ], "family": "Pollicino", "given": "Teresa", "sequence": "additional" } ], "container-title": "Microorganisms", "container-title-short": "Microorganisms", "content-domain": { "crossmark-restriction": false, "domain": [] }, "created": { "date-parts": [ [ 2023, 12, 19 ] ], "date-time": "2023-12-19T09:18:37Z", "timestamp": 1702977517000 }, "deposited": { "date-parts": [ [ 2023, 12, 19 ] ], "date-time": "2023-12-19T10:45:34Z", "timestamp": 1702982734000 }, "indexed": { "date-parts": [ [ 2025, 5, 9 ] ], "date-time": "2025-05-09T05:54:11Z", "timestamp": 1746770051090, "version": "3.37.3" }, "is-referenced-by-count": 4, "issue": "1", "issued": { "date-parts": [ [ 2023, 12, 19 ] ] }, "journal-issue": { "issue": "1", "published-online": { "date-parts": [ [ 2024, 1 ] ] } }, "language": "en", "license": [ { "URL": "https://creativecommons.org/licenses/by/4.0/", "content-version": "vor", "delay-in-days": 0, "start": { "date-parts": [ [ 2023, 12, 19 ] ], "date-time": "2023-12-19T00:00:00Z", "timestamp": 1702944000000 } } ], "link": [ { "URL": "https://www.mdpi.com/2076-2607/12/1/2/pdf", "content-type": "unspecified", "content-version": "vor", "intended-application": "similarity-checking" } ], "member": "1968", "original-title": [], "page": "2", "prefix": "10.3390", "published": { "date-parts": [ [ 2023, 12, 19 ] ] }, "published-online": { "date-parts": [ [ 2023, 12, 19 ] ] }, "publisher": "MDPI AG", "reference": [ { "DOI": "10.1038/s41580-021-00432-z", "article-title": "Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design", "author": "Malone", "doi-asserted-by": "crossref", "first-page": "21", "journal-title": "Nat. Rev. Mol. Cell Biol.", "key": "ref_1", "volume": "23", "year": "2022" }, { "DOI": "10.1038/s41467-021-26884-7", "article-title": "Organ-specific genome diversity of replication-competent SARS-CoV-2", "author": "Lambrechts", "doi-asserted-by": "crossref", "first-page": "6612", "journal-title": "Nat. Commun.", "key": "ref_2", "volume": "12", "year": "2021" }, { "DOI": "10.1371/journal.ppat.1009037", "doi-asserted-by": "crossref", "key": "ref_3", "unstructured": "Trypsteen, W., Van Cleemput, J., Snippenberg, W., and Gerlo SVandekerckhove, L. (2020). On the whereabouts of SARS-CoV-2 in the human body: A systematic review. PLoS Pathog., 16." }, { "DOI": "10.1146/annurev-biochem-052521-115653", "article-title": "The Life of SARS-CoV-2 Inside Cells: Replication-Transcription Complex Assembly and Function", "author": "Lou", "doi-asserted-by": "crossref", "first-page": "381", "journal-title": "Annu. Rev. Biochem.", "key": "ref_4", "volume": "91", "year": "2022" }, { "DOI": "10.1128/CMR.00109-21", "doi-asserted-by": "crossref", "key": "ref_5", "unstructured": "Tao, K., Tzou, P.L., Nouhin, J., Bonilla, H., Jagannathan, P., and Shafer, R.W. (2021). SARS-CoV-2 Antiviral Therapy. Clin. Microbiol. Rev., 34." }, { "DOI": "10.1038/s41594-021-00657-8", "article-title": "Molnupiravir: Coding for catastrophe", "author": "Malone", "doi-asserted-by": "crossref", "first-page": "706", "journal-title": "Nat. Struct. Mol. Biol.", "key": "ref_6", "volume": "28", "year": "2021" }, { "DOI": "10.1056/NEJMp2032369", "article-title": "FDA Approval of Remdesivir—A Step in the Right Direction", "author": "Rubin", "doi-asserted-by": "crossref", "first-page": "2598", "journal-title": "N. Engl. J. Med.", "key": "ref_7", "volume": "383", "year": "2020" }, { "DOI": "10.1136/bmj.m2610", "article-title": "COVID-19: Remdesivir is recommended for authorisation by European Medicines Agency", "author": "Wise", "doi-asserted-by": "crossref", "first-page": "m2610", "journal-title": "BMJ", "key": "ref_8", "volume": "369", "year": "2020" }, { "DOI": "10.1080/13543776.2021.1880568", "article-title": "SARS-CoV-2 RNA-dependent RNA polymerase as a therapeutic target for COVID-19", "author": "Vicenti", "doi-asserted-by": "crossref", "first-page": "325", "journal-title": "Expert Opin. Ther. Pat.", "key": "ref_9", "volume": "31", "year": "2021" }, { "DOI": "10.1038/s41586-020-2368-8", "article-title": "Structure of replicating SARS-CoV-2 polymerase", "author": "Hillen", "doi-asserted-by": "crossref", "first-page": "154", "journal-title": "Nature", "key": "ref_10", "volume": "584", "year": "2020" }, { "DOI": "10.1126/science.abb7498", "article-title": "Structure of the RNA-dependent RNA polymerase from COVID-19 virus", "author": "Gao", "doi-asserted-by": "crossref", "first-page": "779", "journal-title": "Science", "key": "ref_11", "volume": "368", "year": "2020" }, { "DOI": "10.1016/j.molcel.2021.01.035", "article-title": "Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication", "author": "Bravo", "doi-asserted-by": "crossref", "first-page": "1548", "journal-title": "Mol. Cell", "key": "ref_12", "volume": "81", "year": "2021" }, { "DOI": "10.1038/s41467-020-20542-0", "article-title": "Mechanism of SARS-CoV-2 polymerase stalling by remdesivir", "author": "Kokic", "doi-asserted-by": "crossref", "first-page": "279", "journal-title": "Nat. Commun.", "key": "ref_13", "volume": "12", "year": "2021" }, { "key": "ref_14", "unstructured": "Medicines and Healthcare Products Regulatory Agency (2021, November 04). Regulatory Approval of Lagevrio (Molnupiravir), Available online: https://www.gov.uk/government/publications/regulatory-approval-of-lagevrio-molnupiravir." }, { "key": "ref_15", "unstructured": "Food and Drug Administration (2021, December 23). Coronavirus (COVID-19) Update: FDA Authorizes Additional Oral Antiviral for Treatment of COVID-19 in Certain Adults, Available online: https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-oral-antiviral-treatment-covid-19-certain." }, { "DOI": "10.1038/s41594-021-00651-0", "article-title": "Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis", "author": "Kabinger", "doi-asserted-by": "crossref", "first-page": "740", "journal-title": "Nat. Struct. Mol. Biol.", "key": "ref_16", "volume": "28", "year": "2021" }, { "DOI": "10.1136/bmj.n2422", "article-title": "COVID-19: Molnupiravir reduces risk of hospital admission or death by 50% in patients at risk, MSD reports", "author": "Mahase", "doi-asserted-by": "crossref", "first-page": "n2422", "journal-title": "BMJ", "key": "ref_17", "volume": "375", "year": "2021" }, { "key": "ref_18", "unstructured": "European Medicines Agency (2023, January 21). Withdrawal of Marketing Authorizarion Application for LAGEVRIO, Molnupiravir, 200 mg Hard Capsules, EMEA/H/C/005789. Available online: https://www.ema.europa.eu/en/documents/withdrawal-letter/withdrawal-letter-lagevrio_en.pdf." }, { "key": "ref_19", "unstructured": "Food and Drug Administration (2021, December 22). Coronavirus (COVID-19) Update: FDA Authorizes First Oral Antiviral for Treatment of COVID-19, Available online: https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-first-oral-antiviral-treatment-covid-19." }, { "key": "ref_20", "unstructured": "European Medicines Agency (2022, January 27). COVID-19: EMA Recommends Conditional Marketing Authorisation for Paxlovid. Available online: https://www.ema.europa.eu/en/news/covid-19-ema-recommends-conditional-marketing-authorisation-paxlovid#:~:text=COVID%2D19%3A%20EMA%20recommends%20conditional%20marketing%20authorisation%20for%20Paxlovid,-Share&text=Update%3A%20Paxlovid%20is%20now%20authorised,Commission%20on%2028%20January%202022." }, { "DOI": "10.1038/s41467-020-16954-7", "article-title": "Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography", "author": "Kneller", "doi-asserted-by": "crossref", "first-page": "3202", "journal-title": "Nat. Commun.", "key": "ref_21", "volume": "11", "year": "2020" }, { "DOI": "10.1038/s41467-020-19662-4", "article-title": "Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site", "author": "Lee", "doi-asserted-by": "crossref", "first-page": "5877", "journal-title": "Nat. Commun.", "key": "ref_22", "volume": "11", "year": "2020" }, { "DOI": "10.1136/bmj.n2713", "article-title": "COVID-19: Pfizer’s paxlovid is 89% effective in patients at risk of serious illness, company reports", "author": "Mahase", "doi-asserted-by": "crossref", "first-page": "n2713", "journal-title": "BMJ", "key": "ref_23", "volume": "375", "year": "2021" }, { "DOI": "10.1038/s41586-020-2223-y", "article-title": "Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors", "author": "Jin", "doi-asserted-by": "crossref", "first-page": "289", "journal-title": "Nature", "key": "ref_24", "volume": "582", "year": "2020" }, { "key": "ref_25", "unstructured": "Vere Hodge, A., and Field, H.J. (2011). Genetics and Evolution of Infectious Disease, Elsevier." }, { "DOI": "10.2471/BLT.20.253591", "article-title": "Variant analysis of SARS-CoV-2 genomes", "author": "Koyama", "doi-asserted-by": "crossref", "first-page": "495", "journal-title": "Bull. World Health Organ.", "key": "ref_26", "volume": "98", "year": "2020" }, { "DOI": "10.1016/j.antiviral.2018.07.020", "article-title": "Comparison of antiviral resistance across acute and chronic viral infections", "author": "Mason", "doi-asserted-by": "crossref", "first-page": "103", "journal-title": "Antivir. Res.", "key": "ref_27", "volume": "158", "year": "2018" }, { "DOI": "10.3390/molecules27175413", "doi-asserted-by": "crossref", "key": "ref_28", "unstructured": "Patel, D., Ono, S.K., Bassit, L., Verma, K., Amblard, F., and Schinazi, R.F. (2022). Assessment of a Computational Approach to Predict Drug Resistance Mutations for HIV, HBV and SARS-CoV-2. Molecules, 27." }, { "DOI": "10.1186/s12967-020-02344-6", "article-title": "Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant", "author": "Pachetti", "doi-asserted-by": "crossref", "first-page": "179", "journal-title": "J. Transl. Med.", "key": "ref_29", "volume": "18", "year": "2020" }, { "DOI": "10.7717/peerj.9703", "article-title": "Mutation density changes in SARS-CoV-2 are related to the pandemic stage but to a lesser extent in the dominant strain with mutations in spike and RdRp", "author": "Eskier", "doi-asserted-by": "crossref", "first-page": "e9703", "journal-title": "PeerJ", "key": "ref_30", "volume": "8", "year": "2020" }, { "DOI": "10.1093/cid/ciaa1474", "article-title": "Emerging RNA-Dependent RNA Polymerase Mutation in a Remdesivir-Treated B-cell Immunodeficient Patient with Protracted Coronavirus Disease 2019", "author": "Martinot", "doi-asserted-by": "crossref", "first-page": "e1762", "journal-title": "Clin. Infect. Dis.", "key": "ref_31", "volume": "73", "year": "2021" }, { "DOI": "10.1371/journal.ppat.1009929", "doi-asserted-by": "crossref", "key": "ref_32", "unstructured": "Szemiel, A.M., Merits, A., Orton, R.J., MacLean, O.A., Pinto, R.M., Wickenhagen, A., Lieber, G., Turnbull, M.L., Wang, S., and Furnon, W. (2021). In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2. PLoS Pathog., 17." }, { "DOI": "10.1016/j.ebiom.2023.104559", "doi-asserted-by": "crossref", "key": "ref_33", "unstructured": "Ip, J.D., Chu, A.W.-H., Chan, W.-M., Leung, R.C.-Y., Abdullah, S.M.U., Sun, Y., and To, K.K.-W. (2023). Global prevalence of SARS-CoV-2 3CL protease mutations associated with nirmatrelvir or ensitrelvir resistance. EBioMedicine, 91." }, { "DOI": "10.1038/s41431-020-0596-x", "article-title": "Disruptive and avoidable: GDPR challenges to secondary research uses of data", "author": "Peloquin", "doi-asserted-by": "crossref", "first-page": "697", "journal-title": "Eur. J. Hum. Genet.", "key": "ref_34", "volume": "28", "year": "2020" }, { "key": "ref_35", "unstructured": "Italian Data Protection Authority (2016, December 15). Authorization n. 9/2016—General Authorization to Process Personal Data for Scientific Research Purposes. Available online: https://www.garanteprivacy.it/home/docweb/-/docweb-display/docweb/5805552." }, { "DOI": "10.1101/2021.01.16.425365", "doi-asserted-by": "crossref", "key": "ref_36", "unstructured": "De Sabato, L., Vaccari, G., Knijn, A., Ianiro, G., Di Bartolo, I., and Morabito, S. (2021). SARS-CoV-2 RECoVERY: A multi-platform open-source bioinformatic pipeline for the automatic construction and analysis of SARS-CoV-2 genomes from NGS sequencing data. bioRxiv." }, { "DOI": "10.1093/molbev/msy096", "article-title": "MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms", "author": "Kumar", "doi-asserted-by": "crossref", "first-page": "1547", "journal-title": "Mol. Biol. Evol.", "key": "ref_37", "volume": "35", "year": "2018" }, { "article-title": "Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees", "author": "Tamura", "first-page": "512", "journal-title": "Mol. Biol. Evol.", "key": "ref_38", "volume": "10", "year": "1993" }, { "DOI": "10.2307/2408678", "article-title": "Confidence limits on phylogenies: An approach using the bootstrap", "author": "Felsenstein", "doi-asserted-by": "crossref", "first-page": "783", "journal-title": "Evolution", "key": "ref_39", "volume": "39", "year": "1985" }, { "DOI": "10.1093/nar/gkab301", "article-title": "Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation", "author": "Letunic", "doi-asserted-by": "crossref", "first-page": "W293", "journal-title": "Nucleic Acids Res.", "key": "ref_40", "volume": "49", "year": "2021" }, { "DOI": "10.1002/cbic.202100346", "article-title": "Hidden in Plain Sight: Natural Products of Commensal Microbiota as an Environmental Selection Pressure for the Rise of New Variants of SARS-CoV-2", "author": "Dragelj", "doi-asserted-by": "crossref", "first-page": "2946", "journal-title": "Chembiochem", "key": "ref_41", "volume": "22", "year": "2021" }, { "DOI": "10.3389/fcimb.2022.736397", "doi-asserted-by": "crossref", "key": "ref_42", "unstructured": "De, R., and Dutta, S. (2022). Role of the Microbiome in the Pathogenesis of COVID-19. Front. Cell. Infect. Microbiol., 12." }, { "DOI": "10.1101/2023.11.16.567485", "doi-asserted-by": "crossref", "key": "ref_43", "unstructured": "Cao, B., Wang, X., Yin, W., Gao, Z., and Xia, B. (2023). Human microbiota is a reservoir of SARS-CoV-2 advantageous mutations. bioRxiv." }, { "DOI": "10.7717/peerj.9492", "article-title": "Identification of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2 and their implications on its protein structure", "author": "Chand", "doi-asserted-by": "crossref", "first-page": "e9492", "journal-title": "PeerJ", "key": "ref_44", "volume": "8", "year": "2020" }, { "DOI": "10.1002/jmv.26791", "article-title": "Mutations in SARS-CoV-2 Nsp7 and Nsp8 proteins and their predicted impact on replication/transcription complex structure", "author": "Reshamwala", "doi-asserted-by": "crossref", "first-page": "4616", "journal-title": "J. Med. Virol.", "key": "ref_45", "volume": "93", "year": "2021" }, { "DOI": "10.1038/s41598-021-91662-w", "article-title": "Concurrent mutations in RNA-dependent RNA polymerase and spike protein emerged as the epidemiologically most successful SARS-CoV-2 variant", "author": "Abdul", "doi-asserted-by": "crossref", "first-page": "13705", "journal-title": "Sci. Rep.", "key": "ref_46", "volume": "11", "year": "2021" }, { "DOI": "10.3390/biom11070919", "doi-asserted-by": "crossref", "key": "ref_47", "unstructured": "Mohammad, A., Al-Mulla, F., Wei, D.Q., Abubaker, J., and Remdesivir, M.D. (2021). Simulations Suggest a More Favourable Binding to SARS-CoV-2 RNA Dependent RNA Polymerase Mutant P323L Than Wild-Type. Biomolecules, 11." }, { "DOI": "10.1016/j.celrep.2023.113077", "article-title": "SARS-CoV-2 variants with NSP12 P323L/G671S mutations display enhanced virus replication in ferret upper airways and higher transmissibility", "author": "Kim", "doi-asserted-by": "crossref", "first-page": "113077", "journal-title": "Cell Rep.", "key": "ref_48", "volume": "42", "year": "2023" }, { "DOI": "10.1016/j.celrep.2020.107774", "article-title": "Structural and Biochemical Characterization of the Nsp12-Nsp7-Nsp8 Core Polymerase Complex from SARS-CoV-2", "author": "Peng", "doi-asserted-by": "crossref", "first-page": "107774", "journal-title": "Cell Rep.", "key": "ref_49", "volume": "31", "year": "2020" } ], "reference-count": 49, "references-count": 49, "relation": {}, "resource": { "primary": { "URL": "https://www.mdpi.com/2076-2607/12/1/2" } }, "score": 1, "short-title": [], "source": "Crossref", "subject": [], "subtitle": [], "title": "Assessing Genomic Mutations in SARS-CoV-2: Potential Resistance to Antiviral Drugs in Viral Populations from Untreated COVID-19 Patients", "type": "journal-article", "volume": "12" }