Assessing Viral Pollution in Seawater, Using Passive Samplers, Quantitative PCR and Target Enrichment Sequencing

Abstract Detecting pathogenic viruses in seawater presents significant challenges due to their typically low concentrations and the transient nature of contamination events. To address these challenges, this study evaluates passive sampling (PS) as a semiquantitative methodology for viral surveillance in seawater, focusing on human adenovirus (HAdV), norovirus GI (NoVGI) and GII (NoVGII). To optimize the approach, nylon and nitrocellulose membranes were compared, with nylon demonstrating greater consistency and reliability. In Barcelona, across 14 sampling events, 6 of them during combined sewer overflow (CSO) events, PS ( n = 51) was assessed against grab sampling ( n = 21) coupled with ultrafiltration (UF). Using PS, HAdV, NoVGI, and NoVGII were detected in 100%, 62.5%, and 75% of non-CSO events, respectively, outperforming UF, which detected these viruses in only 37.5%, 25%, and 37.5% of the same events. PS was applied during CSO events at both the CSO discharge point and at a nearby bathing site. As expected, the sampler deployed at the discharge point detected higher viral loads of HAdV, NoVGI, and NoVGII. However, viral genomes were also detected at the bathing site. Target enrichment sequencing of seawater vertebrate-infecting viruses conducted on PS samples, identified vertebrate viral pathogens, including members of the Circoviridae , Parvoviridae and Picornaviridae families, showcasing the extensive viral diversity in seawater due to fecal contamination. This study emphasizes the risk of viral exposure when seawater is impacted by CSO and the potential of PS as a robust tool for seawater viral surveillance, offering enhanced sensitivity and utility for quality management and public health risk assessment. Graphical Abstract