Application of Pan-Viral Metagenomic Sequencing on United States Air Force Academy Wastewater to Uncover Potential Causes of Acute Gastroenteritis
Authors: Mechikoff Michael A , Collins John P , Golder Philip , Ingersoll Cullen M , McGarry Riley E , Lu Xiang-Jun , Gokden Alper , Cuff Anastasia , Webber Bryant J , Wallace Andrew B , Steel J Jordan , Wickiser J Kenneth , Balboni Armand L 
Published: November 2024

ABSTRACT

Introduction
Wastewater surveillance is an important technique to monitor public health and is being studied extensively for pandemic prevention, force health protection and readiness, and as a potential early warning system for chem-bio defense. Wastewater surveillance has traditionally relied on techniques such as quantitative PCR or targeted sequencing, both of which are microbe- or disease-specific tools. Newer pan-viral metagenomics strategies may provide higher specificity for pathogens of interest, resulting in a lower false negative rate and reduced sequencing of undesired background nucleic acids. One such system, VirCapSeq-VERT, has been developed to target all vertebrate virus pathogens; until recently, its application has been limited to clinical samples. The objective of this study was to use VirCapSeq-VERT to interrogate wastewater samples from the U.S. Air Force Academy (USAFA), Colorado Springs, Colorado, to determine its utility in assessing complex samples and its potential application in public health surveillance.

Materials and Methods
Biweekly samples were analyzed from February 2022 through May 2023. Samples were collected from the wastewater treatment facility at USAFA before treatment and stored at −20 °C until total nucleic acid (tNA: DNA and RNA) extraction. tNA was then subject to the probe-based capture system, VirCapSeq, and run through a collection of public bioinformatics pipelines to identify captured viral pathogens and perform phylogenetic analysis. It was determined by the USAFA IRB that the study was non-human subject research and was deemed exempt.

Results
In total, 68 families of viruses were identified, comprising thousands of individual strains. This study focused on viruses responsible for gastrointestinal dysfunction as a test of the use of the VirCapSeq-VERT to identify human pathogenic viruses within a complex and highly enriched biological sample. Four enteric viruses dominated the wastewater samples, with Adenoviridae most prevalent before the cadet winter recess (December 17, 2022-January 4, 2023) and Astroviridae most abundant thereafter.

Conclusion
Although gastroenteritis outbreaks at USAFA are commonly attributed to norovirus because of clinical presentation and the acute nature of the illness—often diagnosed and treated without confirmatory stool testing—this virus was not found in high prevalence in these wastewater samples. Among adenoviruses, F serotype 41 predominated, suggesting a role in gastrointestinal infections among the cadet population. Phylogenetic investigation of adenovirus and norovirus exposed common variants with seasonal distributions. These findings may prompt correlational studies to assess the clinical predictive capability of VirCapSeq-VERT and to determine the utility of wastewater monitoring as an outbreak early warning system.