Abstract: medRxiv preprint doi: https://doi.org/10.1101/2022.12.21.22283811; this version posted December 22, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license . 1 1 2 Antiviral treatments lead to the rapid accrual of hundreds of SARS-CoV-2 mutations in immunocompromised patients 3 4 Authors: Nicholas M. Fountain-Jones1,2*, Robert Vanhaeften1, Jan Williamson1, Janelle Maskell1, I-Ly J Chua1, Michael Charleston2 & Louise Cooley1,3 5 Affiliations: 1 6 7 2 Royal Hobart Hospital, Pathology Department, Hobart Australia 7001. School of Natural Sciences, University of Tasmania, Hobart Australia 7001. 3 8 School of Medicine, University of Tasmania, Hobart Australia 700.1 9 10 11 12 13 14 Keywords: Paxlovid, Molnupiravir, Lageviro, virus evolution, resistance Abstract The antiviral Molnupiravir (Lageviro) is widely used across the world to treat SARS-CoV-2 15 infection. Molnupiravir reduces viral replication by inducing mutations throughout the 16 genome, yet in patients that do not clear the infection, the longer-term impact of the drug on 17 virus evolution is unclear. Here, we used a case-control approach to monitor SARS-CoV-2 18 genomes through time in nine immunocompromised -patients with five treated with 19 Molnupiravir. Within days of treatment, we detected a large number of low-frequency 20 mutations in patients and that these new mutations could persist and, in some cases, were 21 fixed in the virus population. All patients treated with the drug accrued new mutations in the 22 spike protein of the virus, including non-synonymous mutations that altered the amino acid 23 sequence. Our study demonstrates that this commonly used antiviral can ‘supercharge’ viral 24 evolution in immunocompromised patients, potentially generating new variants and 25 prolonging the pandemic. 26 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2022.12.21.22283811; this version posted December 22, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license . 27 2 Main text: 28 29 Persistent SARS-CoV-2 infection in immunocompromised patients is recognized as an 30 important source of genomic variation and is linked to the evolution of novel variants 1–3. 31 How commonly used antivirals such as Molnupiravir shape viral evolution is an important 32 knowledge gap. As of December 2022, Molnupiravir is routinely used globally to treat 33 COVID patients in and outside of hospital settings, including treatment of 34 immunocompromised patients 4. Molnupiravir and other similar direct-acting SARS-CoV-2 35 antivirals promote mutagenesis by incorporating the prodrug NHC (β-D-N4-hydroxycytidine) 36 into the virus-dependent RNA polymerase (RdRp)5. When the RdRp uses the NHC modified 37 RNA as a template it promotes an ‘error catastrophe’ 5,6 that inhibits the replication of SARS- 38 CoV-2. For immunocompromised patients that do not clear infection after antiviral treatment, 39 the impact that these drugs have on..