Tracing the origin of SARS-CoV-2 Omicron-like Spike sequences detected in wastewater

ABSTRACT

ImportanceThe 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. ObjectiveTo determine whether enigmatic SARS-CoV-2 lineages detected in wastewater have a human or non-human (i.e., animal) source. DesignOn 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. SettingThis study was performed in Wisconsin, United States, which has a comprehensive program for detecting SARS-CoV-2 in wastewater. ParticipantsComposite 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 studys outset. ResultsThe 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 RelevanceHigh 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. Key points QuestionWhat is the source of unusual SARS-CoV-2 Omicron-like Spike variants detected in wastewater but not in clinical samples? FindingsWe 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. MeaningProlonged shedding from the human gastrointestinal tract is the most likely source for evolutionarily advanced SARS-CoV-2 variant sequences found in wastewater.