Tracing the origin of SARS-CoV-2 Omicron-like Spike sequences detected in wastewater
Importance The origin of highly divergent “cryptic” SARS-CoV-2 Spike sequences, which appear in wastewater but not clinical samples, is unknown. These wastewater sequences have harbored many of the same mutations that later emerged in Omicron variants. If these enigmatic sequences are human-derived and transmissible, they could both be a source of future variants and a valuable tool for forecasting sequences that should be incorporated into vaccines and therapeutics.
Objective To determine whether enigmatic SARS-CoV-2 lineages detected in wastewater have a human or non-human (i.e., animal) source.
Design On January 11, 2022, an unusual Spike sequence was detected in municipal wastewater from a metropolitan area. Over the next four months, more focused wastewater sampling resolved the source of this variant.
Setting This study was performed in Wisconsin, United States, which has a comprehensive program for detecting SARS-CoV-2 in wastewater.
Participants Composite wastewater samples were used for this study; therefore, no individuals participated.
Main Outcome(s) and Measure(s) The primary outcome was to determine the host(s) responsible for shedding this variant in wastewater. Both human and non-human hosts were plausible candidates at the study’s outset.
Results The presence of the cryptic virus was narrowed from a municipal wastewater sample (catchment area 100,000 people) to an indoor wastewater sample from a single facility (catchment area ∼30 people), indicating the human origin of this virus. Extraordinarily high concentrations of viral RNA (∼520,000,000 genome copies / L and ∼1,600,000,000 genome copies / L in June and August 2022, respectively) were detected in the indoor wastewater sample. The virus sequence harbored a combination of fixed nucleotide substitutions previously observed only in Pango lineage B.1.234, a variant that circulated at low levels in Wisconsin from October 2020 to February 2021.
Conclusions and Relevance High levels of persistent SARS-CoV-2 shedding from the gastrointestinal tract of an infected individual likely explain the presence of evolutionarily advanced “cryptic variants” observed in some wastewater samples.
Question What is the source of unusual SARS-CoV-2 Omicron-like Spike variants detected in wastewater but not in clinical samples?
Findings We identified a cryptic SARS-CoV-2 lineage in wastewater collected at a central wastewater treatment facility and traced its source to a single wastewater outlet serving six restrooms. The virus in this sample resembled a 2020-2021 lineage except for the Spike protein, in which Omicron-like variants were observed.
Meaning Prolonged shedding from the human gastrointestinal tract is the most likely source for evolutionarily advanced SARS-CoV-2 variant sequences found in wastewater.
### Competing Interest Statement
The authors have declared no competing interest.
### Funding Statement
This study was made possible by the generous support of the Rockefeller Foundation's Regional Accelerators for Genomics Surveillance (DHO/TCF), Wisconsin Department of Health Services Epidemiology and Laboratory Capacity funds ([www.dhs.wisconsin.gov][1], 144 AAJ8216) to DHO, CDC contract 75D30121C11060 (DHO/TCF), Wisconsin Department of Health Services ELC Wastewater Surveillance funds ([www.dhs.wisconsin.gov][1], 130:AAI8627) to the UW-Madison Wisconsin State Laboratory of Hygiene (WSLH), and NIDA contract 1U01DA053893-01 (MJ).
### Author Declarations
I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.
Yes
I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.
Yes
I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).
Yes
I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.
Yes
Sequencing data is available in NCBI SRA and Genbank. Additional data is available from https://go.wisc.edu/4134pl.
https://go.wisc.edu/4134pl
[1]: https://www.dhs.wisconsin.gov