Detection of viruses of public health importance in wastewater samples using conventional PCR techniques and a targeted enrichment whole genome sequencing panel

Abstract Wastewater-based epidemiology (WBE) is an effective surveillance approach for monitoring viruses of public health relevance at the community level, complementing clinical surveillance systems. Molecular methods such as PCR/qPCR are widely used for targeted detection, while next-generation sequencing (NGS) with targeted enrichment panels has emerged as a complementary strategy for broader viral detection and genomic characterization. This study comparatively evaluated conventional PCR/qPCR and a targeted enrichment whole-genome sequencing Viral Surveillance Panel (VSP, Illumina) for virus detection in wastewater. Fifty-six wastewater samples collected between 2017 and 2023 from a wastewater treatment plant in Córdoba, Argentina, were concentrated by polyethylene glycol precipitation and pooled by season and year, reaching a total of 14 pools. Each pool was analyzed in parallel by PCR/qPCR for eight human viruses of public health importance and by the VSP, targeting 66 viral species, sequenced on a NovaSeq 6000 platform, and analyzed with the DRAGEN pipeline. Detection frequencies for each virus using PCR/qPCR and VSP were: RoV A 100%/14.3%; NoV 100%/14.3%; AiV 50%/42.9%; SARS-CoV-2 14.3%/0%; HAV 42.9%/0%; HEV 14.3%/0%; JCPyV 35.7%/85.7%; BKPyV 28.6%/71.4%, respectively. In addition, VSP detected the genomes of Astrovirus (71.4%), Salivirus (21.4%), Coxsackie A (14.3%), Rotavirus C (14.3%), and Merkel Cell virus (7.1%), and enable the recovery of 16 near complete genomes (coverage > 92.5%) of AiV, JCPyV, BKPyV, Salivirus and Astrovirus. PCR/qPCR and targeted enrichment NGS provide complementary information wastewater viral surveillance. Their combined application improves virus detection and genomic characterization, reinforcing the value of integrated approaches in environmental virology and public health monitoring.