The Detection and Fate of Enveloped Viruses in Water Environments

Abstract

Removing and inactivating infectious viruses in water is critical in controlling waterborne diseases. Studies on the presence of viruses in wastewater and their fate through wastewater treatment plants have focused primarily on enteric viruses, which transmit gastrointestinal diseases via water. Most enteric viruses are nonenveloped, consisting only of proteins and nucleic acids. Enveloped viruses contain an outer lipid membrane in addition to proteins and nucleic acids. Certain enveloped viruses are responsible for high-profile diseases, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and influenza. Enveloped viruses have often been assumed to be absent from wastewater and rapidly inactivated when they are released to water. However, recent studies suggest that certain enveloped viruses can enter wastewater, and may survive in water for long periods of time. Our current state of knowledge on enveloped viruses in aquatic environments has been limited due to a lack of appropriate methods for capturing and detecting infectious enveloped viruses in water. To address the knowledge gaps, this dissertation research aims to 1) evaluate the survival, partitioning, and recovery of model enveloped viruses in wastewater, 2) characterize the reactivity of enveloped viruses with common disinfectants, and 3) develop a new method for monitoring infectious human viruses in water samples.

To evaluate virus survival and partitioning, we applied four model viruses, two enveloped and two nonenveloped, and used plaque assays to track the infectivity and partitioning of the model viruses in untreated wastewater. We simulated our experimental data with virus sorption and inactivation models to quantitatively characterize the fate of model enveloped viruses and model nonenveloped viruses. Our results suggest that model enveloped viruses can survive in wastewater, especially at cooler temperatures. We also demonstrated that a larger fraction of model enveloped viruses partitioned to the wastewater solids than nonenveloped viruses. As a result, we expect that enveloped viruses are removed to a greater extent than nonenveloped viruses during primary wastewater treatment. With the knowledge gained from the survival and partitioning experiments, we optimized an ultrafiltration method for recovering infectious enveloped viruses from wastewater. The second portion of this dissertation research characterized the reactivity of enveloped viruses in the disinfection process. The reactions in a model virus lipids, proteins, and genome were tracked as a model enveloped virus was treated with disinfectants using quantitative lipid and protein mass spectrometry, and molecular PCR techniques. We found that protein reactions drive the inactivation of the model enveloped virus by free chlorine, and genome reactions drive the inactivation of the model enveloped virus by UV254. Furthermore, our results suggest that the model enveloped virus proteins were more susceptible to oxidant attack than the proteins of a model nonenveloped virus. The final portion of this dissertation research focused on the development of an integrated cell culture-mass spectrometry (ICC-MS) method for detecting infectious human viruses in wastewater. In proof of concept experiments, reoviruses were detected in samples collected throughout a wastewater treatment plant by applying the ultrafiltration concentration method developed in the first study and the ICC-MS detection method. These results suggest that ICC-MS is a promising tool for monitoring infectious enveloped or nonenveloped viruses in water samples.