Water reuse has many applications and has now been recognized as a sustainable and predictable water resource as part of a diverse water supply portfolio. To assess the performance of wastewater treatment and water reuse processes, enteric viruses in wastewater have been monitored. However, cell culture for enteric viruses is arduous and takes several weeks to obtain the results. Some viruses of concern including noroviruses are not readily cultured and detection limits of analytical methods remain challenging. Water and wastewater utilities need rapid, reliable and inexpensive assessment tools of treatment performance for water reuse systems to ensure a reclaimed water quality that is suitable for the desired use at all times.

The project goal is to develop a better understanding of indigenous viruses in different wastewater systems to design improved viral surrogate approaches that address challenges and shortcomings of current methodologies, including low concentrations of viruses in wastewater, quantitative detection and a lack of specificity for addressing human health risk. Our hypotheses focus on (i) indigenous viruses are present in wastewater in high concentrations which provide a major advantage compared to the currently used viral indicators for wastewater treatment such as coliphages; (ii) viruses in wastewater vary geographically, and (iii) viruses in various forms (free, particle-associated and vesicle-cloaked) are one of the reasons for apparent more recalcitrant populations during disinfection. Project objectives are to: (1) characterize the composition of indigenous viruses in wastewater (including viruses with three different forms: free, particle-associated, and vesicle-cloaked viruses) using a metagenomic approach, with the purpose of identification of viral surrogates for assessing treatment performance; (2) design and develop individual based quantitative assays for new viral surrogates using new instrumentation and molecular viability methods; and (3) validate new surrogates and new quantitative detection assays (from Objective 1 & 2) for estimating reduction of viruses through full-scale advanced water treatment processes (with some bench-scale work). Approach: With strategic partnership with water reclamation facilities in California, Florida and Ohio, a comprehensive characterization of indigenous viruses (including free, particle-associated, and vesicle-cloaked viruses) in wastewater will be conducted using metagenomics to identify improved surrogates for assessing treatment performance. This will then lead to the design and development of new analytical methods to provide not only rapid and precise quantification of viruses in wastewater but also able to assess viability. Finally, new quantitative detection assays for estimating reduction of viruses will be validated using both full-scale advanced water reclamation treatment facilities and bench-scale disinfection experiments. Expected Results: The project will develop new knowledge that provides a better understanding of virus attenuation in wastewater and water treatment systems. The development of new quantitative detection tools with novel surrogates will improve sensitivity, reduce analysis turnaround time and reduce cost of measuring viruses in wastewater for validating treatment performance. Knowledge on abundance, diversity, persistence, and fate of viruses and viral vesicles in wastewater will be advanced and thus strategies for risk assessment and control will be improved.