Enteric viruses pose a significant health risk to the public through exposure to contaminated environmental waters, costing society billions of dollars to treat millions of excess illnesses annually. Current available methods to monitor human fecal pollution in impacted waters either do not correlate with viral pathogen presence and risk, cross-react with other animal sources, or are not abundant enough in environmental waters to be reliably detected. This dissertation investigates a novel target for a human-specific indicator of fecal pollution, the bacteriophage “crAssphage”. First, a metagenomic evaluation was conducted as an initial step to evaluate the potential of the crAssphage genome for assay development. Results indicated that crAssphage is enriched in sewage and more abundant than other viruses and bacteriophages in sewage, suggesting its high potential as a target for human-specific marker development. Next, end-point PCR primers were designed along the length of the crAssphage genome and screened against human sewage samples and non-human animal fecal samples to assess which genomic regions may be the most useful as source tracking markers. The two best performing primer pairs were adapted to TaqMan qPCR assays. These assays were evaluated head-to-head against two bacterial qPCR human source tracking markers to evaluate their performance. The assays were widespread in geographically diverse human sources and as abundant or more abundant in sewage and impaired environmental water than the bacterial-based assays. Moreover, the assays displayed high human-association. Finally, an initial environmental evaluation was conducted to correlate the crAssphage-based assays to pollution events, culturable bacterial and phage indicators, and molecular bacterial and viral indicators. This study demonstrated the usefulness of the crAssphage assays in a real-world system, validating their high abundance and correlation with pollution events and other indicators of fecal pollution. Ultimately, the research in this dissertation contributes two novel viral-based technologies for detection of human fecal pollution that will enhance management of environmental waters and protect public health through the development of an abundant viral fecal source-tracking marker
