Molnupiravir, an antiviral medication that has been widely used against SARS-CoV-2, acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus, and many will be lethal. Molnupiravir-induced elevated mutation rates have been shown to decrease viral load in animal models. However, it is possible that some patients treated with molnupiravir might not fully clear SARS-CoV-2 infections, with the potential for onward transmission of molnupiravir-mutated viruses. We set out to systematically investigate global sequencing databases for a signature of molnupiravir mutagenesis. We find that a specific class of long phylogenetic branches appear almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age-groups with widespread usage of the drug. We calculate a mutational spectrum from the AGILE placebo-controlled clinical trial of molnupiravir and show that its signature, with elevated G-to-A and C-to-T rates, largely corresponds to the mutational spectrum seen in these long branches. Our data suggest a signature of molnupiravir mutagenesis can be seen in global sequencing databases, in some cases with onwards transmission.

### Competing Interest Statement

The authors have declared no competing interest.

### Funding Statement

TS was supported by the Wellcome Trust (210918/Z/18/Z) and the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001043), the UK Medical Research Council (FC001043), and the Wellcome Trust (FC001043). This research was funded in whole, or in part, by the Wellcome Trust [210918/Z/18/Z, FC001043]. For the purpose of Open Access, the authors have applied a CC-BY public copyright licence to any Author Accepted Manuscript resulting from this preprint. ID-B is supported by PhD funding from the National Institute for Health and Care Research (NIHR) Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE) (now UKHSA), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford (award 200907). The views expressed are those of the authors and not necessarily those of the Department of Health and Social Care or NIHR. Neither the funders or trial sponsor were involved in the study design, data collection, analysis, interpretation, nor the preparation of the manuscript. TP was funded by the G2P-UK National Virology Consortium funded by the MRC (MR/W005611/1). CR was supported by a Fondation Botnar Research Award (Programme grant 6063) and UK Cystic Fibrosis Trust (Innovation Hub Award 001).

### Author Declarations

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

No new data were generated for this study. Source global sequencing data were available from GISAID (epicov.org) and the INSDC via GenBank: https://www.ncbi.nlm.nih.gov/genbank/. AGILE2 genomic data was published in https://www.nature.com/articles/s41467-022-34839-9 and deposited at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA854613 from which we analysed it.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.

Yes

No new primary data was generated for this study. We used data from international sequencing databases (GISAID and INSDC), and from the AGILE clinical trial, where genomic data were obtained from BioProject PRJNA854613 at the SRA. Our GitHub repository is available at https://github.com/theosanderson/molnupiravir.

The excess risk of serious adverse events found in our study points to the need for formal harm-benefit analyses, particularly those that are stratified according to risk of serious COVID-19 outcomes. These analyses will require public release of participant level datasets.