Protocol for Safe, Affordable, and Reproducible 1 Isolation and Quantitation 2 of SARS-CoV-2 RNA from Wastewater

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Furthermore, the RNA obtained using this protocol, minus the pasteurization step, can be sequenced both using a targeted approach sequencing specific regions or the whole genome. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to support their efforts in monitoring SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. Owing to a pasteurization step, it is safe for use in a BSL1+ facility. This step also increases the genetic signal of the virus while making the protocol safe for the personnel involved. This protocol could be used to isolate a variety of other clinically relevant viruses from wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens

Detection of Mutations Associated with Variants of Concern Via High Throughput 2 Sequencing of SARS-CoV-2 Isolated from NYC Wastewater

Monitoring 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 date, most data on SARS-CoV-2 genetic diversity has come from the sequencing of clinical samples, but such studies may suffer limitations due to costs and throughput. Wastewater-based epidemiology may provide an alternative and complementary approach for monitoring communities for novel variants. Given that SARS-CoV-2 can infect the cells of the human gut and is found in high concentrations in feces, wastewater may be a valuable source of SARS-CoV-2 RNA, which can be deep sequenced to provide information on the circulating variants in a community. Here we describe a safe, affordable protocol for the sequencing of SARS CoV-2 RNA using high-throughput Illumina sequencing technology. Our targeted sequencing approach revealed the presence of mutations associated with several Variants of Concern at appreciable frequencies. Our work demonstrates that wastewater-based SARS-CoV-2 sequencing can inform surveillance efforts monitoring the community spread of SARS-CoV-2 Variants of Concern and detect the appearance of novel emerging variants more cheaply, safely, and efficiently than the sequencing of individual clinical samples

Live imaging of center of calcification formation during septum development in primary polyps of Acropora digitifera

Recent studies have revealed that stony corals create their extracellular skeletons via biologically controlled calcification, in which amorphous calcium carbonate (ACC), regarded as precursors of aragonite crystals, have been observed at nanoscale using electron microscopy. However, the exact mechanism by which ACC is generated, and how it contributes to skeletal growth in coral calcifying tissue, remains enigmatic. The septal skeleton of an individual polyp is composed of radially aligned plates extending upward from the aboral calcifying tissue. This structure includes microstructure known as the centers of calcification (CoC). However, despite its importance, direct in vivo observation of septal growth has not been reported. Observations under transmitted illumination using polarized light microscopy on calcifying tissue of young Acropora digitifera revealed small crystals, a few micrometers in size, that accompany subtle movements and that emerge exclusively on the inner wall of the pocket in extracellular calcifying fluid (ECF). Crystal growth initiated from small, scattered crystals on a glass plate resembles this phenomenon observed in coral skeletons. Time-lapse photographs of 12 individuals in early primary polyp settlement revealed this process in three individuals, documenting 13 of these crystal events. This phenomenon occurred solely at the bases of subsequently formed septa. These crystals differ notably from fusiform crystals and from dumbbell-like or rod-like crystals growing individually. Upright two-photon microscopy captured movement of sub-micron-sized fluorescent calcein-accumulating particles, emphasizing their presence on the surface of the growing fronts of septa. Methodological advances that facilitate comprehensive in vivo observation of sub-micron-sized structures, calcein-accumulating particles to the skeleton, are needed to develop a more detailed understanding of coral skeletal growth

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

L. pneumophila resists its self-harming metabolite HGA via secreted factors and collective peroxide scavenging

ABSTRACT Many pathogenic bacteria, including Legionella pneumophila, infect humans from environmental reservoirs. To survive in these reservoirs, bacteria must withstand microbe-on-microbe competition. We previously discovered that L. pneumophila can compete with neighboring bacteria via an antimicrobial metabolite called homogentisic acid (HGA). Curiously, L. pneumophila strains that secrete HGA are not wholly immune to its effects: low-density bacteria are strongly inhibited by HGA, whereas high-density cells are tolerant. How do these bacteria tolerate HGA and avoid self-harm during interbacterial competition? Here, we find that HGA toxicity occurs via the production of toxic hydroperoxides, and multiple factors facilitate high-density tolerance. First, HGA becomes fully toxic only after >1 h of oxidation. While this manifests as a delay in killing within well-mixed liquid cultures, in a biofilm environment, this could provide time for HGA to diffuse away before becoming toxic. Second, HGA generates quantities of hydroperoxides that can be collectively scavenged by high-density, but not low-density, cells. Third, high-density cells produce one or more secreted factors that are transiently protective from HGA. In combination, we propose that the bacteria are able to deploy HGA to generate a pool of reactive oxygen species surrounding their own biofilms while maintaining non-toxic conditions within them. Overall, these findings help to explain how broadly toxic molecules can be used as inter-bacterial weapons. They also provide insights about why some of our current decontamination methods to control L. pneumophila are ineffective, leading to recurrent disease outbreaks. IMPORTANCE Before environmental opportunistic pathogens can infect humans, they must first successfully grow and compete with other microbes in nature, often via secreted antimicrobials. We previously discovered that the bacterium Legionella pneumophila, the causative agent of Legionnaires’ disease, can compete with other microbes via a secreted molecule called HGA. Curiously, L. pneumophila strains that produce HGA is not wholly immune to its toxicity, making it a mystery how these bacteria can withstand the “friendly fire” of potentially self-targeting antimicrobials during inter-bacterial battles. Here, we identify several strategies that allow the high-density bacterial populations that secrete HGA to tolerate its effects. Our study clarifies how HGA works. It also points to some explanations of why it is difficult to disinfect L. pneumophila from the built environment and prevent disease outbreaks