Advancing Wastewater-Based Epidemiology Through a Mechanistic Understanding of Viruses in Wastewater

Abstract

Some of the world’s most virulent diseases are caused by viruses, including COVID-19, smallpox, Ebola, HIV/AIDS, and influenza. In modern times, we track viral diseases in an effort to prevent infections. In the U.S., there are a number of clinical surveillance networks for viral diseases at both the federal and state levels; however, these systems are limited to a small number of infectious diseases and rely on the availability of approved testing protocols and materials, reliable reporting infrastructure, and that infected persons seek testing. Wastewater-based epidemiology (WBE) has recently increased in popularity due to its ability to test one composite wastewater sample for virus targets in a sewershed. This is a relatively low-cost, high-throughput method that accounts for asymptomatic carriers and does not rely on robust clinical testing or an individual seeking care. Despite the vast number of WBE studies that have been carried out since the emergence of COVID-19, fundamental questions remain about the presence and behavior of viruses in wastewater. This dissertation sheds light on advancing WBE to new targets, analyzing viral signals and their decay in wastewater, and better defining the composite wastewater samples used to measure virus trends. The first chapter of this dissertation demonstrates that WBE can be expanded to new respiratory virus targets, namely respiratory human adenovirus (HAdV). Human adenovirus types 40/41, which typically cause gastrointestinal illness, were most abundant in wastewater. Nonetheless, specific assays targeting respiratory adenoviruses in wastewater revealed an outbreak in the local community. The findings are valuable for public health to identify and type respiratory HAdV outbreaks earlier than when clinical specimens are sent for sequencing. The second chapter of this dissertation answers important remaining questions about the stability of viral RNA and DNA in municipal wastewater. Specifically, the persistence of the nucleic acids was quantified both when the nucleic acids were within the virus capsid (i.e. encapsidated) and when the nucleic acids were outside of the virus capsid (i.e. extraviral). The results demonstrate that encapsidated viral genomes are relatively stable in wastewater and persist for days. Extraviral genomes, however, decay over 99% after minutes (RNA) or hours (DNA). Ultimately, the decay kinetics of the encapsidated genome was compared to the virus inactivation kinetics and the results suggest the nucleic acid signal in wastewater disappeared shortly after the viruses lost infectivity. The findings have important implications for interpreting WBE measurements, and also for when molecular measurements are used to predict the presence and concentration of infectious viruses in wastewater (i.e. HuNoV measurements for potable reuse). The third and final research chapter focuses on the implications of the type of samples used for WBE. Wastewater surveillance samples are commonly collected as either composite influent samples or as primary settled solids samples collected from the underflow of primary clarifiers. Whereas the representativeness of 24-hour composite influent samples is well-defined, this is not the case for samples collected from primary clarifiers. This study characterized the mean age of primary settled solids in two wastewater treatment plants over two seasons. The findings suggest that there is wide variation in solids ages across clarifiers, seasons, and treatment plants on the order of several hours to several days.