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

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.

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing

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 substitution. 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 immunocompromised patients 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. Author SummaryDuring the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source.

Tracking Cryptic SARS-CoV-2 Lineages Detected in NYC Wastewater

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we amplified regions of the SARS-CoV-2 Spike protein gene from RNA acquired from all 14 NYC wastewater treatment plants (WWTPs) and ascertained the diversity of lineages from these samples using high throughput sequencing. Here we report the detection and increasing frequencies of novel SARS-CoV-2 lineages not recognized in GISAIDs EpiCoV database. These lineages contain mutations rarely observed in clinical samples, including Q493K, Q498Y, H519N and T572N. Many of these mutations were found to expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. In addition, pseudoviruses containing the Spike amino acid sequence of these lineages were found to be resistant to many different classes of receptor binding domain (RBD) binding neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these mutations, including the possibility of a non-human animal reservoir. Although wastewater sampling cannot provide direct inference of SARS-CoV-2 clinical sequences, our research revealed several lineages that could be relevant to public health and they would not have been discovered if not for wastewater surveillance.

Monitoring SARS-CoV-2 Populations in Wastewater by Amplicon Sequencing and Using the Novel Program SAM Refiner

Sequencing SARS-CoV-2 from wastewater has become a useful tool in monitoring the spread of variants. We use a novel computation workflow with SARS-CoV-2 amplicon sequencing in order to track wastewater populations of the virus. As part of this workflow, we developed a program for both variant reporting and removal of PCR generated chimeric sequences. With these methods, we are able to track viral population dynamics over time. We observe the emergence of the variants of concern B.1.1.7 and P.1, and their displacement of the D614G B.1 variant.