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 is an effective approach for monitoring viruses of public health relevance in the community. PCR/qPCR are widely used for targeted detection, while NGS with targeted enrichment panels has emerged as a complementary strategy for broader viral detection and genomic characterization. This study comparatively evaluated PCR/qPCR and a targeted enrichment whole-genome sequencing Viral Surveillance Panel (VSP, Illumina) for virus detection in wastewater. Fifty-two wastewater samples collected between 2017 and 2023 from a wastewater treatment plant in Córdoba, Argentina, were concentrated by PEG precipitation and pooled by season and year, reaching 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 Rotavirus A 100%/14.3%; Norovirus 100%/14.3%; Aichivirus 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%), Coxsackievirus A19 (14.3%), Rotavirus C (14.3%), and Merkel Cell virus (7.1%), and enable the recovery of 16 near complete genomes of Aichivirus, JCPyV, BKPyV, Salivirus and Astrovirus. PCR provided the sensitivity and speed necessary for viral screening of the main human pathogens of public health concern, while the VSP generated valuable genomic information on understudied viruses. According to these results, PCR/qPCR panels would represent a more practical, sensitive and cost-effective alternative for wastewater key public health viruses’ surveillance purposes in our region.