Beginning in early 2021, unique and highly divergent lineages of SARS-CoV-2 were sporadically found in wastewater sewersheds using a sequencing strategy focused on the most mutagenic region of SARS-CoV-2, the receptor binding domain (RBD). Because these RBD sequences did not match known circulating strains and their source was not known, we termed them “cryptic lineages”. To date, more than 20 cryptic lineages have been identified using the RBD-focused sequencing strategy. Here, we identified and characterized additional cryptic lineages from SARS-CoV-2 wastewater sequences submitted to NCBI’s Sequence Read Archives (SRA). Wastewater sequence datasets were screened for individual sequence reads that contained combinations of mutations frequently found in cryptic lineages but not contemporary circulating lineages. Using this method, we identified 18 cryptic lineages that appeared in multiple samples from the same sewershed, including 12 that were not previously reported. Partial consensus sequences were generated for each cryptic lineage by extracting and mapping sequences containing cryptic-specific mutations. Surprisingly, seven of the mutations that appeared convergently in cryptic lineages were reversions to sequences that were highly conserved in SARS-CoV-2-related bat Sarbecoviruses. The apparent reversion to bat Sarbecovirus sequences suggests that SARS-CoV-2 adaptation to replicate efficiently in respiratory tissues preceded the COVID-19 pandemic.

The authors have declared no competing interest.

This project has been funded in part by the National Institutes of Health (NIH) contract 75N93021C00045 for the Johns Hopkins Center of Excellence in Influenza Research to MCJ. This project has been funded by a gift from Heart of Racing and a grant from Inkfish (MCJ and DHO).

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