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 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.

SARS-CoV-2 and other respiratory pathogens are detected in continuous air samples from congregate settings.

Two years after the emergence of SARS-CoV-2, there is still a need for better ways to assess the risk of transmission in congregate spaces. We deployed active air samplers to monitor the presence of SARS-CoV-2 in real-world settings across communities in the Upper Midwestern states of Wisconsin and Minnesota. Over 29 weeks, we collected 527 air samples from 15 congregate settings. We detected 106 samples that were positive for SARS-CoV-2 viral RNA, demonstrating that SARS-CoV-2 can be detected in continuous air samples collected from a variety of real-world settings. We expanded the utility of air surveillance to test for 40 other respiratory pathogens. Surveillance data revealed differences in timing and location of SARS-CoV-2 and influenza A virus detection. In addition, we obtained SARS-CoV-2 genome sequences from air samples to identify variant lineages. Collectively, this shows air sampling is a scalable, high throughput surveillance tool that could be used in conjunction with other methods for detecting respiratory pathogens in congregate settings.

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 deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID's EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

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

Sequencing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from wastewater has become a useful tool in monitoring the spread of viral variants. Approaches to this task have been varied, relying on differing sequencing methods and computational analyses. We used a novel computation workflow based on amplicon sequencing of SARS-CoV-2 spike domains in order to track viral populations in wastewater. As part of this workflow, we developed a program, SAM Refiner, that has a variety of outputs, including novel variant reporting as well as functions designed to remove polymerase chain reaction (PCR) generated chimeric sequences. With these methods, we were able to track viral population dynamics over time. We report here on the emergence of two variants of concern, B.1.1.7 (Alpha) and P.1 (Gamma), and their displacement of the D614G B.1 variant in a Missouri sewershed.