Review 1: "Identification of a Molnupiravir-associated Mutational Signature in SARS-CoV-2 Sequencing Databases"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

Sanderson and colleagues present strong evidence that molnupiravir treatment sometimes leads to SARS-CoV-2 virus with hyper-mutated genomes, that in some instances have been submitted further. Whether molnupiravir would lead to heavily mutated genomes was debated earlier when molnupiravir was approved. This paper clearly shows that this has happened repeatedly.

Molnupiravir (MTP) is used to treat SARS-CoV-2 infection and acts as a mutagenic nucleoside analog during negative and positive strand synthesis. Presence of MTP at high enough concentrations should introduce a large number of mutations in the viral genome during replication, such that the production of replication competent virus is sufficiently low that viral load is greatly reduced and the infection can be cleared.

The mutations introduced by MNT are overwhelmingly transitions, while the viral RdRp also produces a large fraction of G->T mutations (using T instead of U for simplicity). These differences were known a priori from the clinical study and were now used by Sanderson et al to search for sequences with MNT specific mutation pattern in the large body of publicly available SARS-CoV-2 genomes. Specifically, they used the very large phylogenies shared by the UShER team to identify branches in the tree that have many mutations but almost no G->T mutations (or other transversions). This should be a very sensitive filter for sequences mutated through processes other than viral replication, but does not rule out artifacts in sample processing and sequencing. To further strengthen the case that these sequences are molnupiravir-derived, the authors show that the great majority of these sequences were deposited after molnupiravir was approved and that these sequences tend to come from countries with wide-spread use of the drug. Countries with little use of the drug have very few of such sequences. In addition, the authors show a small number of examples where viruses, likely mutated by MNT, have transmitted further. These are a small number of examples, but are convincing evidence that onward transmission can happen.

The phylogenetic analysis of all global data is complemented by a de-novo analysis of only the Australian data where MNT was used at scale. This analysis confirms the global picture and adds additional, even more extreme, examples that were filtered as outliers in the automated global tree.

The authors make no claims beyond the well supported points above, but recommend these cases are followed up by the relevant authorities to confirm the link to molnupiravir established here.