Molnupiravir increases SARS‐CoV‐2 genome diversity and complexity: A case‐control cohort study
Cesare Ernesto Maria Gruber, Fabio Giovanni Tucci, Emanuela Giombini, Valentina Mazzotta, Pietro Giorgio Spezia, Martina Rueca, Ilaria Mastrorosa, Lavinia Fabeni, Giulia Berno, Ornella Butera, Silvia Rosati, Eliana Specchiarello, Fabrizio Carletti, Daniele Focosi, Emanuele Nicastri, Enrico Girardi, Andrea Antinori, Fabrizio Maggi
Journal of Medical Virology, doi:10.1002/jmv.29642
Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed
effective transmission of newly drug-induced possibly emerging variants. In conclusions, our data confirm the suspect that molnupiravir-generated variants can be generated also in immunocompetent outpatients 33 and highlight the importance of genomic surveillance in the characterization of new, potentially transmissible drug-derived variants.
AUTHOR CONTRIBUTIONS Cesare Ernesto Maria Gruber, Emanuela Giombini, Martina Rueca, and Daniele Focosi conceived the study and developed the study protocol. Enrico Girardi and Fabrizio Maggi provided oversight and supervision. Martina Rueca, Lavinia Fabeni, Giulia Berno, Ornella Butera, Eliana Specchiarello, and Fabrizio Carletti did the molecular assays and sequencing. Valentina Mazzotta, Ilaria Mastrorosa, Silvia Rosati, Emanuele Nicastri, and Andrea Antinori collected the clinical data. Fabio Giovanni Tucci, Emanuela Giombini, and Cesare Ernesto Maria Gruber did the bioinformatic analyses of the data. Cesare Ernesto Maria Gruber, Emanuela Giombini, and Fabio Giovanni Tucci wrote the original draft of the manuscript. Fabrizio Maggi, Andrea Antinori, and Daniele Focosi critically reviewed the manuscript. All authors had access to the data in the study and were ultimately responsible for deciding to submit it for publication.
CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest.
ETHICS STATEMENT The study was conducted in accordance with the Declaration of
References
Abbate, Vlassi, Rozera, Detection of quasispecies variants predicted to use CXCR4 by ultra-deep pyrosequencing during early HIV infection, AIDS,
doi:10.1097/QAD.0b013e328343489eAlteri, Fox, Scutari, A proof-of-concept study on the genomic evolution of Sars-Cov-2 in molnupiravir-treated, paxlovid-treated and drug-naïve patients, Commun Biol
Bernal, Da Silva, Musungaie, Molnupiravir for oral treatment of Covid-19 in nonhospitalized patients, N Engl J Med,
doi:10.1056/NEJMoa2116044Caraco, Crofoot, Moncada, Phase 2/3 trial of molnupiravir for treatment of Covid-19 in nonhospitalized adults, NEJM Evid,
doi:10.1056/EVIDoa2100043Curtis, Merck presentation about molnupiravir for the U.S. Food & Drug Administration Antimicrobial Drugs Advisory Committee
Donovan-Banfield, Penrice-Randal, Goldswain, Characterisation of SARS-CoV-2 genomic variation in response to molnupiravir treatment in the AGILE phase IIa clinical trial, Nat Commun,
doi:10.1038/s41467-022-34839-9Focosi, Casadevall, A critical analysis of the use of cilgavimab plus tixagevimab monoclonal antibody cocktail (Evusheld™) for COVID-19 prophylaxis and treatment, Viruses,
doi:10.3390/v14091999Fountain-Jones, Vanhaeften, Williamson, Effect of molnupiravir on SARS-CoV-2 evolution in immunocompromised patients: a retrospective observational study, Lancet Microbe,
doi:10.1016/S2666-5247(23)00393-2Giorgio, Martignano, Torcia, Mattiuz, Conticello, Evidence for host-dependent RNA editing in the transcriptome of SARS-CoV-2, Sci Adv,
doi:10.1126/sciadv.abb5813Hakmaoui, Khan, Liacini, Relevant SARS-CoV-2 genome variation through six months of worldwide monitoring, BioMed Res Int,
doi:10.1155/2021/5553173Koboldt, Chen, Wylie, VarScan: variant detection in massively parallel sequencing of individual and pooled samples, Bioinformatics,
doi:10.1093/bioinformatics/btp373Nakamura, Fujimoto, Hasegawa, A phase I, randomized, placebo-controlled study of molnupiravir in healthy Japanese to support special approval in Japan to treat COVID-19, Clin Transl Sci,
doi:10.1111/cts.13395Penrice-Randal, Bentley, Sharma, The effect of molnupiravir and nirmatrelvir on SARS-CoV-2 genome diversity in infected and immune suppressed mice. bioRxiv, J Med Virol
Roy, Mandal, Mondal, Trends of mutation accumulation across global SARS-CoV-2 genomes: implications for the evolution of the novel coronavirus, Genomics,
doi:10.1016/j.ygeno.2020.11.003Rueca, Bartolini, Gruber, Compartmentalized replication of SARS-Cov-2 in upper vs. lower respiratory tract assessed by whole genome quasispecies analysis, Microorganisms,
doi:10.3390/microorganisms8091302Rueca, Giombini, Messina, The easy-to-use SARS-CoV-2 assembler for genome sequencing: development study, JMIR Bioinform Biotech
Sanderson, Hisner, Donovan-Banfield, A molnupiravirassociated mutational signature in global SARS-CoV-2 genomes, Nature,
doi:10.1038/s41586-023-06649-6Sanderson, Hisner, Donovan-Banfield, A molnupiravirassociated mutational signature in global SARS-CoV-2 genomes, Nature,
doi:10.1038/s41586-023-06649-6Tian, Tong, Sun, Mutation N501Y in RBD of spike protein strengthens the interaction between COVID-19 and its receptor ACE2. bioRxiv,
doi:10.1101/2021.02.14.431117Wang, Wang, Zhang, Intra-host variation and evolutionary dynamics of SARS-CoV-2 populations in COVID-19 patients, Genome Med,
doi:10.1186/s13073-021-00847-5{ 'indexed': {'date-parts': [[2024, 5, 7]], 'date-time': '2024-05-07T00:31:43Z', 'timestamp': 1715041903785},
'reference-count': 33,
'publisher': 'Wiley',
'issue': '5',
'license': [ { 'start': { 'date-parts': [[2024, 5, 6]],
'date-time': '2024-05-06T00:00:00Z',
'timestamp': 1714953600000},
'content-version': 'vor',
'delay-in-days': 5,
'URL': 'http://creativecommons.org/licenses/by-nc/4.0/'}],
'funder': [ {'DOI': '10.13039/501100000780', 'name': 'European Commission', 'doi-asserted-by': 'publisher'},
{ 'DOI': '10.13039/501100003196',
'name': 'Ministero della Salute',
'doi-asserted-by': 'publisher'}],
'content-domain': {'domain': ['onlinelibrary.wiley.com'], 'crossmark-restriction': True},
'published-print': {'date-parts': [[2024, 5]]},
'abstract': '<jats:title>Abstract</jats:title><jats:p>Molnupiravir, an oral direct‐acting antiviral '
'effective in vitro against SARS‐CoV‐2, has been largely employed during the COVID‐19 '
'pandemic, since December 2021. After marketing and widespread usage, a progressive increase '
'in SARS‐CoV‐2 lineages characterized by a higher transition/transversion ratio, a '
'characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing '
'All Influenza Data\xa0(GISAID) and International Nucleotide Sequence Database '
'Collaboration\xa0(INSDC) databases. Here, we assessed the drug effects by SARS‐CoV‐2 '
'whole‐genome sequencing on 38 molnupiravir‐treated persistently positive COVID‐19 outpatients '
'tested before and after treatment. Seventeen tixagevimab/cilgavimab‐treated outpatients '
'served as controls. Mutational analyses confirmed that SARS‐CoV‐2 exhibits an increased '
'transition/transversion ratio seven days after initiation of molnupiravir. Moreover we '
'observed an increased G‐>A ratio compared to controls, which was not related to '
'apolipoprotein B mRNAediting enzyme, catalytic polypeptide‐like (APOBEC) activity. In '
'addition, we demonstrated for the first time an increased diversity and complexity of the '
'viral quasispecies.</jats:p>',
'DOI': '10.1002/jmv.29642',
'type': 'journal-article',
'created': {'date-parts': [[2024, 5, 6]], 'date-time': '2024-05-06T10:40:21Z', 'timestamp': 1714992021000},
'update-policy': 'http://dx.doi.org/10.1002/crossmark_policy',
'source': 'Crossref',
'is-referenced-by-count': 0,
'title': 'Molnupiravir increases SARS‐CoV‐2 genome diversity and complexity: A case‐control cohort study',
'prefix': '10.1002',
'volume': '96',
'author': [ { 'ORCID': 'http://orcid.org/0000-0002-8831-3380',
'authenticated-orcid': False,
'given': 'Cesare Ernesto Maria',
'family': 'Gruber',
'sequence': 'first',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'given': 'Fabio Giovanni',
'family': 'Tucci',
'sequence': 'additional',
'affiliation': [{'name': 'Department of Biology University of Rome Tor Vergata Rome Italy'}]},
{ 'given': 'Emanuela',
'family': 'Giombini',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'ORCID': 'http://orcid.org/0000-0002-0240-7504',
'authenticated-orcid': False,
'given': 'Valentina',
'family': 'Mazzotta',
'sequence': 'additional',
'affiliation': [ { 'name': 'Clinical and Research Infectious Diseases Department National '
'Institute for Infectious Diseases Lazzaro Spallanzani IRCCS Rome '
'Italy'}]},
{ 'given': 'Pietro Giorgio',
'family': 'Spezia',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'ORCID': 'http://orcid.org/0000-0001-5162-046X',
'authenticated-orcid': False,
'given': 'Martina',
'family': 'Rueca',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'given': 'Ilaria',
'family': 'Mastrorosa',
'sequence': 'additional',
'affiliation': [ { 'name': 'Clinical and Research Infectious Diseases Department National '
'Institute for Infectious Diseases Lazzaro Spallanzani IRCCS Rome '
'Italy'}]},
{ 'given': 'Lavinia',
'family': 'Fabeni',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'given': 'Giulia',
'family': 'Berno',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'given': 'Ornella',
'family': 'Butera',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Microbiology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'given': 'Silvia',
'family': 'Rosati',
'sequence': 'additional',
'affiliation': [ { 'name': 'Clinical and Research Infectious Diseases Department National '
'Institute for Infectious Diseases Lazzaro Spallanzani IRCCS Rome '
'Italy'}]},
{ 'given': 'Eliana',
'family': 'Specchiarello',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'given': 'Fabrizio',
'family': 'Carletti',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]},
{ 'ORCID': 'http://orcid.org/0000-0001-8811-195X',
'authenticated-orcid': False,
'given': 'Daniele',
'family': 'Focosi',
'sequence': 'additional',
'affiliation': [ { 'name': 'North‐Western Tuscany Blood Bank Pisa University Hospital Pisa '
'Italy'}]},
{ 'given': 'Emanuele',
'family': 'Nicastri',
'sequence': 'additional',
'affiliation': [ { 'name': 'Clinical and Research Infectious Diseases Department National '
'Institute for Infectious Diseases Lazzaro Spallanzani IRCCS Rome '
'Italy'}]},
{ 'given': 'Enrico',
'family': 'Girardi',
'sequence': 'additional',
'affiliation': [ { 'name': 'Scientific Direction National Institute for Infectious Diseases '
'“L. Spallanzani”—IRCCS Rome Italy'}]},
{ 'given': 'Andrea',
'family': 'Antinori',
'sequence': 'additional',
'affiliation': [ { 'name': 'Clinical and Research Infectious Diseases Department National '
'Institute for Infectious Diseases Lazzaro Spallanzani IRCCS Rome '
'Italy'}]},
{ 'given': 'Fabrizio',
'family': 'Maggi',
'sequence': 'additional',
'affiliation': [ { 'name': 'Laboratory of Virology National Institute for Infectious '
'Diseases Lazzaro Spallanzani IRCCS Rome Italy'}]}],
'member': '311',
'published-online': {'date-parts': [[2024, 5, 6]]},
'reference': [ { 'key': 'e_1_2_10_2_1',
'unstructured': 'Fact sheet for healthcare providers: emergency use authorization for '
'Lagevrio™ (molnupiravir) capsules. Accessed January 12 '
'2024.https://www.fda.gov/media/155054/download'},
{'key': 'e_1_2_10_3_1', 'doi-asserted-by': 'publisher', 'DOI': '10.1007/s40265-022-01684-5'},
{ 'key': 'e_1_2_10_4_1',
'unstructured': 'EMA Assessment Report. Use of molnupiravir for the treatment of '
'COVID‐19. Accessed\xa0January 12 '
'2024.https://www.ema.europa.eu/en/documents/referral/lagevrio-also-known-molnupiravir-or-mk-4482-covid-19-article-53-procedure-assessment-report_en.pdf'},
{'key': 'e_1_2_10_5_1', 'doi-asserted-by': 'publisher', 'DOI': '10.1038/s41594-021-00651-0'},
{ 'key': 'e_1_2_10_6_1',
'doi-asserted-by': 'crossref',
'unstructured': 'TianF TongB SunL et al. Mutation N501Y in RBD of spike protein '
'strengthens the interaction between COVID‐19 and its receptor '
'ACE2.bioRxiv. Preprint posted online February 18 '
'2021.doi:10.1101/2021.02.14.431117',
'DOI': '10.1101/2021.02.14.431117'},
{'key': 'e_1_2_10_7_1', 'doi-asserted-by': 'publisher', 'DOI': '10.1111/cts.13395'},
{'key': 'e_1_2_10_8_1', 'doi-asserted-by': 'publisher', 'DOI': '10.1056/EVIDoa2100043'},
{'key': 'e_1_2_10_9_1', 'doi-asserted-by': 'publisher', 'DOI': '10.1056/NEJMoa2116044'},
{ 'key': 'e_1_2_10_10_1',
'unstructured': 'European Medicine Agency. Lagevrio.2023. Accessed May 26 '
'2023.https://www.ema.europa.eu/en/medicines/human/summaries-opinion/lagevrio'},
{ 'key': 'e_1_2_10_11_1',
'doi-asserted-by': 'publisher',
'DOI': '10.1038/s41586-023-06649-6'},
{'key': 'e_1_2_10_12_1', 'doi-asserted-by': 'publisher', 'DOI': '10.1126/science.abn0048'},
{ 'issue': '4',
'key': 'e_1_2_10_13_1',
'doi-asserted-by': 'crossref',
'DOI': '10.1093/molbev/msad085',
'article-title': 'Evolution of the SARS‐CoV‐2 Mutational Spectrum',
'volume': '40',
'author': 'Bloom JD',
'year': '2023',
'journal-title': 'Mol Biol Evol'},
{ 'key': 'e_1_2_10_14_1',
'doi-asserted-by': 'crossref',
'DOI': '10.1093/molbev/msad085',
'article-title': 'Evolution of the SARS‐CoV‐2 mutational spectrum',
'volume': '40',
'author': 'Bloom JD',
'year': '2023',
'journal-title': 'Mol Biol Evol'},
{ 'key': 'e_1_2_10_15_1',
'unstructured': 'Sean Curtis. Merck presentation about molnupiravir for the U.S. Food & '
'Drug Administration Antimicrobial Drugs Advisory Committee. November 30 '
'2021. Accessed\xa0October 6 2022.\xa0'
'https://www.fda.gov/media/154472/download'},
{'key': 'e_1_2_10_16_1', 'doi-asserted-by': 'publisher', 'DOI': '10.1126/sciadv.abb5813'},
{'key': 'e_1_2_10_17_1', 'doi-asserted-by': 'publisher', 'DOI': '10.4049/jimmunol.1501504'},
{ 'key': 'e_1_2_10_18_1',
'doi-asserted-by': 'publisher',
'DOI': '10.1097/QAD.0b013e328343489e'},
{ 'key': 'e_1_2_10_19_1',
'doi-asserted-by': 'publisher',
'DOI': '10.3390/microorganisms8091302'},
{ 'key': 'e_1_2_10_20_1',
'doi-asserted-by': 'publisher',
'DOI': '10.1186/s13073-021-00847-5'},
{ 'issue': '1',
'key': 'e_1_2_10_21_1',
'doi-asserted-by': 'crossref',
'DOI': '10.1038/s42003-022-04322-8',
'article-title': 'A proof‐of‐concept study on the genomic evolution of\xa0Sars‐Cov‐2 in '
'molnupiravir‐treated, paxlovid‐treated and drug‐naïve patients',
'volume': '5',
'author': 'Alteri C',
'year': '2022',
'journal-title': 'Commun Biol'},
{ 'key': 'e_1_2_10_22_1',
'doi-asserted-by': 'crossref',
'first-page': '7284',
'DOI': '10.1038/s41467-022-34839-9',
'article-title': 'Characterisation of SARS‐CoV‐2 genomic variation in response to '
'molnupiravir treatment in the AGILE phase IIa clinical trial',
'volume': '13',
'author': 'Donovan‐Banfield I',
'year': '2022',
'journal-title': 'Nat Commun'},
{ 'key': 'e_1_2_10_23_1',
'doi-asserted-by': 'crossref',
'unstructured': 'Fountain‐Jones\xa0NM \xa0Vanhaeften\xa0R \xa0Williamson\xa0J et al. '
'Effect of molnupiravir on SARS‐CoV‐2 evolution in immunocompromised '
'patients: a retrospective observational study.\xa0Lancet Microbe. '
'Published online March 22 2024.\xa0doi:10.1016/S2666-5247(23)00393-2',
'DOI': '10.1016/S2666-5247(23)00393-2'},
{ 'key': 'e_1_2_10_24_1',
'doi-asserted-by': 'publisher',
'DOI': '10.1016/j.virol.2016.03.017'},
{'key': 'e_1_2_10_25_1', 'doi-asserted-by': 'publisher', 'DOI': '10.3390/v14091999'},
{ 'key': 'e_1_2_10_26_1',
'doi-asserted-by': 'publisher',
'DOI': '10.1093/bioinformatics/btu170'},
{'key': 'e_1_2_10_27_1', 'doi-asserted-by': 'publisher', 'DOI': '10.2196/31536'},
{ 'key': 'e_1_2_10_28_1',
'doi-asserted-by': 'publisher',
'DOI': '10.1093/bioinformatics/btp373'},
{ 'key': 'e_1_2_10_29_1',
'unstructured': 'UK Health Security Agency. England COVID19 (Coronavirus) '
'Dashboard-Potential MOV sequences. Accessed January 12 '
'2024.https://ukcovid.xyz/molnupiravir/'},
{ 'key': 'e_1_2_10_30_1',
'doi-asserted-by': 'publisher',
'DOI': '10.1038/s41586-023-06649-6'},
{ 'key': 'e_1_2_10_31_1',
'doi-asserted-by': 'crossref',
'unstructured': 'SheahanTP StevensLJ NarowskiTM et al. The antiviral mechanism of action '
'of molnupiravir in humans with COVID‐19.medRxiv. Preprint posted online '
'November 27 2023.doi:10.1101/2023.11.21.23298766',
'DOI': '10.1101/2023.11.21.23298766'},
{ 'key': 'e_1_2_10_32_1',
'doi-asserted-by': 'publisher',
'DOI': '10.1016/j.ygeno.2020.11.003'},
{'key': 'e_1_2_10_33_1', 'doi-asserted-by': 'publisher', 'DOI': '10.1155/2021/5553173'},
{ 'key': 'e_1_2_10_34_1',
'doi-asserted-by': 'crossref',
'unstructured': 'Penrice‐RandalR BentleyEG SharmaP et al. The effect of molnupiravir and '
'nirmatrelvir on SARS‐CoV‐2 genome diversity in infected and immune '
'suppressed mice.\xa0bioRxiv.\xa0Preprint posted online February 28 '
'2024.doi:10.1101/2024.02.27.582110',
'DOI': '10.1101/2024.02.27.582110'}],
'container-title': 'Journal of Medical Virology',
'original-title': [],
'language': 'en',
'link': [ { 'URL': 'https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmv.29642',
'content-type': 'unspecified',
'content-version': 'vor',
'intended-application': 'similarity-checking'}],
'deposited': { 'date-parts': [[2024, 5, 6]],
'date-time': '2024-05-06T10:40:30Z',
'timestamp': 1714992030000},
'score': 1,
'resource': {'primary': {'URL': 'https://onlinelibrary.wiley.com/doi/10.1002/jmv.29642'}},
'subtitle': [],
'short-title': [],
'issued': {'date-parts': [[2024, 5]]},
'references-count': 33,
'journal-issue': {'issue': '5', 'published-print': {'date-parts': [[2024, 5]]}},
'alternative-id': ['10.1002/jmv.29642'],
'URL': 'http://dx.doi.org/10.1002/jmv.29642',
'relation': {},
'ISSN': ['0146-6615', '1096-9071'],
'subject': [],
'container-title-short': 'Journal of Medical Virology',
'published': {'date-parts': [[2024, 5]]},
'assertion': [ { 'value': '2024-03-05',
'order': 0,
'name': 'received',
'label': 'Received',
'group': {'name': 'publication_history', 'label': 'Publication History'}},
{ 'value': '2024-04-22',
'order': 1,
'name': 'accepted',
'label': 'Accepted',
'group': {'name': 'publication_history', 'label': 'Publication History'}},
{ 'value': '2024-05-06',
'order': 2,
'name': 'published',
'label': 'Published',
'group': {'name': 'publication_history', 'label': 'Publication History'}}]}
