Seasonality of Respiratory, Enteric and Urinary Viruses Revealed by Wastewater Genomic Surveillance

Wastewater surveillance can reveal population-level infectious disease burden and emergent public health threats can be reliably assessed through wastewater surveillance. While molecular methods for wastewater monitoring of microorganisms have traditionally relied on PCR-based approaches, next-generation sequencing can provide deeper insights via genomic analyses of multiple diverse pathogens. We conducted a year-long sequencing surveillance of 1,408 composite wastewater samples collected from 12 neighborhood-level access points in the Greater Tempe area, Arizona, USA, and show that variation in wastewater viromes is driven by seasonal time and location. Wastewater virome temporal dynamics were influenced in a cyclical manner, with the most dissimilarity between samples 23 weeks apart (i.e., winter vs summer, spring vs fall). We identified diverse urinary and enteric viruses including polyomaviruses, astroviruses and noroviruses, and showed that their genotypes/subtypes shifted across season. We show that while wastewater data of certain respiratory viruses like SARS-CoV-2 strongly correlate with clinical case rates, laboratory-reported case incidences were discordant with surges of high viral load in wastewater for other viruses like human coronavirus 229E. These results demonstrate the utility of wastewater sequencing for informing decision making in public health. IMPORTANCE Wastewater genomic sequencing surveillance can provide insights into the spread of pathogens in communities. Advances in next-generation sequencing methodologies allow for more precise detection of viruses in wastewater. Long-term wastewater genomic sequencing surveillance is an important tool for public health preparedness. This system can act as a public health observatory that gives real-time early warning for infectious disease outbreaks and improved response times. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was funded by Arizona State University, the Centers for Disease Control and Prevention (CDC BAA 75D30121C11084) and Tohono O-Odham Nation (2020-01 ASU). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. * SARS-CoV-2 : Severe acute respiratory syndrome coronavirus 2 COVID-19 : Coronavirus Disease 2019 qRT-PCR : Quantitative reverse transcription polymerase chain reaction RT-dPCR : Reverse transcription digital polymerase chain reation RSV : Respiratory syncytial virus NRVESS : National Respiratory and Enteric Virus Surveillance System PCoA : Principal coordinates analysis DNA : Deoxyribonucleic acid RNA : Ribonucleic acid PMMoV : Pepper mild mottle virus JCPyV : JC polyomavirus BKPyV : BK polyomavirus HDPE : High-density polyethylene PES : Polyethersulfone MWCO : Molecular weight cutoff NGS : Next generation sequencing RPM : Reads per million