On the distributions of restriction sites in human and pangolin sarbecoviruses

Abstract

Since early 2020, several theories have suggested that a distribution of restriction endonuclease recognition sites in the SARS-CoV-2 genome indicates a synthetic origin. The most influential of these, a 2022 preprint by Bruttel et al. claimed: "The BsaI/BsmBI restriction map of SARS-CoV-2 is unlike any wild-type coronavirus, and it is unlikely to evolve from its closest relatives." To test this, I reanalyzed the same 11 contested sites using an expanded set of sarbecovirus genomes, including bat coronaviruses published after the Bruttel et al. preprint. For each site, I identified the bat coronaviruses most closely matching SARS-CoV-2 in the surrounding sequences, excluding the sites themselves. The Bruttel et al. hypothesis predicts that these closely related viruses should differ from SARS-CoV-2 at many of the contested sites if restriction sites had been artificially introduced or removed. Contrary to this prediction, one or more of the most closely related bat coronaviruses are identical to SARS-CoV-2 at all 11 sites. Equivalent "synthetic fingerprints" were identified in natural pangolin sarbecoviruses. Finally, I conducted a re-analysis of the dataset that Bruttel et al. used to test where the SARS-CoV-2 BsaI/BsmBI restriction map was significantly more "evenly spaced" than expected in a natural genome. I found technical and conceptual errors that resulted in Bruttel et al. reporting that their chosen metric was 0.07% likely to occur by chance rather than 4.2%, reducing the apparent rarity 60-fold. Using a more informative metric, I tested whether restriction sites in SARS-CoV-2 or two pangolin sarbecoviruses were significantly more evenly spaced than expected and found they were not. These results show that the restriction maps of SARS-CoV-2 and related pangolin viruses are unremarkable in the context of related bat coronaviruses.