Wastewater surveillance of SARS-CoV-2 genomic populations on a country-wide scale through targeted sequencing.

SARS-CoV-2 surveillance of viral populations in wastewater samples is recognized as a useful tool for monitoring epidemic waves and boosting health preparedness. Next generation sequencing of viral RNA isolated from wastewater is a convenient and cost-effective strategy to understand the molecular epidemiology of SARS-CoV-2 and provide insights on the population dynamics of viral variants at the community level. However, in low- and middle-income countries, isolated groups have performed wastewater monitoring and data has not been extensively shared in the scientific community. Here we report the results of monitoring the co-circulation and abundance of variants of concern (VOCs) of SARS-CoV-2 in Uruguay, a small country in Latin America, between November 2020-July 2021 using wastewater surveillance. RNA isolated from wastewater was characterized by targeted sequencing of the Receptor Binding Domain region within the spike gene. Two computational approaches were used to track the viral variants. The results of the wastewater analysis showed the transition in the overall predominance of viral variants in wastewater from No-VOCs to successive VOCs, in agreement with clinical surveillance from sequencing of nasal swabs. The mutations K417T, E484K and N501Y, that characterize the Gamma VOC, were detected as early as December 2020, several weeks before the first clinical case was reported. Interestingly, a non-synonymous mutation described in the Delta VOC, L452R, was detected at a very low frequency since April 2021 when using a recently described sequence analysis tool (SAM Refiner). Wastewater NGS-based surveillance of SARS-CoV-2 is a reliable and complementary tool for monitoring the introduction and prevalence of VOCs at a community level allowing early public health decisions. This approach allows the tracking of symptomatic and asymptomatic individuals, who are generally under-reported in countries with limited clinical testing capacity. Our results suggests that wastewater-based epidemiology can contribute to improving public health responses in low- and middle-income countries.

Humidity Reduces Rapid and Distant Airborne Dispersal of Viable Viral Particles in Classroom Settings.

The transmission of airborne pathogens is considered to be the main route through which a number of known and emerging respiratory diseases infect their hosts. While physical distancing and mask wearing may help mitigate short-range transmission, the extent of long-range transmission in closed spaces where a pathogen remains suspended in the air remains unknown. We have developed a method to detect viable virus particles by using an aerosolized bacteriophage Phi6 in combination with its host Pseudomonas phaseolicola, which when seeded on agar plates acts as a virus detector that can be placed at a range of distances away from an aerosol-generating source. By applying this method, we consistently detected viable phage particles at distances of up to 18 feet away from the source within 15 min of exposure in a classroom equipped with a state of the art HVAC system and determined that increasing the relative humidity beyond 40% significantly reduces dispersal. Our method, which can be further modified for use with other virus/host combinations, quantifies airborne transmission in the built environment and can thus be used to set safety standards for room capacity and to ascertain the efficacy of interventions in closed spaces of specified sizes and intended uses.

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.

Protocol for safe, affordable, and reproducible isolation and quantitation 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. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.