Wastewater-based epidemiology (WBE) — identifying pathogens in wastewater — has been employed for decades, notably in the 1940s when WBE was used for the detection of poliovirus in sewage of large US cities including New York and Chicago. WBE again rose to prominence during the pandemic; surveilling community infection trends of SARS-CoV-2.
However, scientists have long recognized that WBE results often underrepresent disease prevalence due to a limited understanding of RNA stability in wastewater. It was just this issue that Associate Professor Benoit Van Aken in George Mason University’s Chemistry and Biochemistry Department wanted to investigate. Van Aken had researched viral pathogens detection during the pandemic, monitoring said SARS-CoV-2 RNA in wastewater. This experience led him to the idea that large macromolecules present in wastewater could help stabilize viral RNA, making its lifetime longer than usually suspected. In collaboration with his colleague Zhiwu (Drew) Wang at Virginia Tech, who also has a background in wastewater contamination, they proposed the research to 4-VA and were awarded a grant to look closer at this question.
Following a year of more than 300 analyses to characterize the decay of SARS-CoV-2 RNA in the presence of macromolecules (proteins, lipids, and carbohydrates) — — the team’s research yielded robust, quantitative evidence on the reaction rates of SARS-CoV-2 RNA degradation in macromolecule-rich solutions that mimic wastewater. The research was conducted in large part by George Mason graduate student Brandi Williams under Van Aken’s direction. Further complementary experiments with different lengths of the marker to quantify RNA – using a technique known as reverse-transcriptase quantitative PCR — revealed that measured decay rates depend strongly on target length, with shorter amplicons exhibiting greater apparent persistence.

Williams pictured with the team’s thermocycler, which is described as a “high-tech copy machine” that reproduces a single DNA fragment into millions of identical copies, allowing for further detection and precise quantification.
“Our research provides new insights about the stability of RNA in complex environments by showing that macromolecules in wastewater can stabilize free RNA molecules—a finding that directly challenges current beliefs about the stability of RNA in environmental sample,” explains Van Aken.
Initially, the team planned a secondary aim to measure RNA decay in real wastewater samples from local wastewater treatment plants, but based on the observation of the dependance of RNA degradation rate on amplicon length, they redirected the research for the systematic evaluation of this effect using newly developed primer/probe sets targeting markers of different lengths. “Our revised findings produced innovative and field-relevant results as it clearly established that amplicon length is a critical parameter governing observed RNA degradation kinetics in WBE assays and, consequently, the comparability and interpretability of viral RNA signals across studies,” added Van Aken.

George Mason University PhD Candidate Brandi Williams pictured above right with
(now) VA Governor Abigail Spanberger and Benoit Van Aken.
Concludes Van Aken, “The novelty of this study provides a mechanistic framework that may partially explain the persistence of free RNA molecules in complex environments, such as wastewater. This work will be of interest — bridging environmental chemistry and public health and demonstrating how the fundamental understanding of nucleic acid stability in complex matrices can enhance community-level pathogen monitoring and inform future biosurveillance systems.”


























