Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing

PLoS Pathog. 2022 Oct 14;18(10):e1010636. doi: 10.1371/journal.ppat.1010636. eCollection 2022 Oct.


Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • COVID-19* / epidemiology
  • Genetic Variation
  • Humans
  • SARS-CoV-2* / genetics
  • Wastewater


  • Waste Water

Supplementary concepts

  • SARS-CoV-2 variants

Grant support

This project has been funded in part with federal funds from the NIDA/NIH ( under contract numbers 1U01DA053893-01 to JW and MCJ and by the New York City Department of Environmental Protection ( under contract number 1484-RDOP to JJD. This work was supported by financial support through Rockefeller Regional Accelerator for Genomic Surveillance (,133 AAJ4558), Wisconsin Department of Health Services Epidemiology and Laboratory Capacity funds (, 144 AAJ8216) to DHO. The work was supported by funds from the California Department of Health ( to RSK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The DEP played no role in study design, data collection, analysis or preparation of the manuscript. However, they did require that they review the manuscript and approve its publication.