Abstract: medRxiv preprint doi: https://doi.org/10.1101/2023.01.26.23284998; this version posted January 27, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . Identification of a molnupiravir-associated mutational signature in SARS-CoV-2 sequencing databases Theo Sanderson 1, , Ryan Hisner 2 , I’ah Donovan-Banfield 3,4 5 , Thomas Peacock , and Christopher Ruis 6,7,8, 1 Francis Crick Institute, London, UK; 2 Independent researcher, USA; 3 Department of Infection Biology and Microbiomes, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, UK; 4 NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool, UK; 5 Department of Infectious Disease, Imperial College London, London, UK; 6 Molecular Immunity Unit, University of Cambridge Department of Medicine, MRC-Laboratory of Molecular Biology, Cambridge, UK; 7 Department of Veterinary Medicine, University of Cambridge, Cambridge, UK; 8 Cambridge Centre for AI in Medicine, University of Cambridge, Cambridge, UK 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. Molnupiravirinduced 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-CoV2 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 agegroups 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. Correspondence: theo.sanderson@crick.ac.uk cr628@cam.ac.uk cation in 24 hours by 880-fold in vitro, and to reduce viral load in animal models (Rosenke et al., 2021). Molnupiravir initially showed some limited efficacy as a treatment for COVID-19 (Jayk Bernal et al., 2022; Extance, 2022), but subsequent larger clinical trials found that molnupiravir did not reduce hospitalisation or death rates in high risk groups (Butler, 2022). As one of the first orally bioavailable antivirals on the market, molnupiravir has been widely adopted by many countries, most recently China (Reuters, 2022). However, recent trial results and the approval of more efficacious antivirals have since led to several countries recommending against molnupiravir usage on the basis of limited effectiveness (NICE Guidance ; NC19CET, 2022). MTP appears to be incorporated into nascent RNA primarily by acting as an analogue of cytosine (C), pairing opposite guanine (G) bases (Fig. 1). However, once incorporated, the molnupiravir..