Human origin ascertained for SARS-CoV-2 Omicron-like spike sequences detected in wastewater: a targeted surveillance study of a cryptic lineage in an urban sewershed

Background The origin of novel SARS-CoV-2 spike sequences found in wastewater, without corresponding detection in clinical specimens, remains unclear. We sought to determine the origin of one such “cryptic” wastewater lineage by tracking and characterizing its persistence and genomic evolution over time. Methods We first detected a cryptic lineage in Wisconsin municipal wastewater in January 2022. By systematically sampling wastewater from targeted sub-sewershed lines and maintenance holes using compositing autosamplers, we traced this lineage (labeled WI-CL-001) to its source at a single commercial building. There we detected WI-CL-001 at concentrations as high as 2.7 × 109 genome copies per liter (gc/L) via RT-dPCR. In addition to using metagenomic 12s rRNA sequencing to determine the virus’s host species, we also sequenced SARS-CoV-2 spike receptor binding domains (RBDs), and where possible, whole viral genomes to identify and characterize the evolution of this lineage over the 13 consecutive months that it was detectable. Findings The vast majority of 12s rRNAs sequenced from wastewater leaving the identified source building were human. Additionally, we generated over 100 viral RBD and whole genome sequences from wastewater samples containing the cryptic lineage collected between January 2022 and January 2023. These sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in humans in Wisconsin at low levels from October 2020 to February 2021. Despite this, mutations in the spike gene, and elsewhere, resembled those subsequently found in Omicron variants. Interpretation We propose that prolonged detection of WI-CL-001 in wastewater represents persistent shedding of SARS-CoV-2 from a single human initially infected by an ancestral B.1.234 virus. The accumulation of convergent “Omicron-like” mutations in WI-CL-001’s ancestral B.1.234 genome likely reflects persistent infection and extensive within-host evolution. Funding The Rockefeller Foundation, Wisconsin Department of Health Services, Centers for Disease Control and Prevention (CDC), National Institute on Drug Abuse (NIDA), and the Center for Research on Influenza Pathogenesis and Transmission. Evidence before this study To identify other studies that characterized unusual wastewater-specific SARS-CoV-2 lineages, we conducted a PubMed search using the keywords “cryptic SARS-CoV-2 lineages” or “novel SARS-CoV-2 lineages” in addition to “wastewater” on May 9, 2023. From the 18 papers retrieved, only two reported wastewater-specific cryptic lineages. These lineages were identified by members of our author team in wastewater from California, Missouri, and New York City. None of these could be definitively traced to a specific source. A third study in Nevada identified a unique recombinant variant (designated Pango lineage XL) in wastewater, which was also discovered in two clinical specimens from the same community. However, it was unclear whether the clinical specimens collected were from the same individual(s) responsible for the virus detected in the wastewater. To our knowledge, no prior study has successfully traced novel SARS-CoV-2 lineages detected in wastewater back to a specific location. How and where cryptic lineages are introduced into wastewater is not known. The added value of this study This study documents the presence and likely source of a novel and highly divergent cryptic SARS-CoV-2 lineage detected in Wisconsin wastewater for 13 months. In contrast to previously reported cryptic lineages, we successfully traced the lineage (WI-CL-001) to a single commercial building with approximately 30 employees. The exceptionally high viral RNA concentrations at the source building facilitated the tracing effort and allowed for the sequencing of WI-CL-001’s whole genome, expanding our view of the lineage’s mutational landscape beyond the spike gene. Implications of all the available evidence WI-CL-001’s persistence in wastewater, its heavily mutated Omicron-like genotype, and its identified point source at a human-occupied commercial building all support the hypothesis that cryptic wastewater lineages can arise from persistently infected humans. Because cryptic wastewater lineages have some amino acid changes that subsequently emerge in circulating viruses, increased global monitoring of such lineages could help forecast variants that may arise in the future. ### Competing Interest Statement YK has received unrelated funding support from Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Shionogi, Otsuka Pharmaceutical, KM Biologics, Kyoritsu Seiyaku, Shinya Corporation and Fuji Rebio ### 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 The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All human subjects work was conducted by the CDC and the Wisconsin Department of Health Services and was consistent with applicable federal law and CDC policy (see ethics statement). 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 [1]: http://www.dhs.wisconsin.gov