Targeted Enrichment Sequencing Utilizing a Respiratory Pathogen Panel for Genomic Wastewater-Based Viral Epidemiology in Uruguay

Human respiratory and enteric viruses are responsible for substantial morbidity and mortality worldwide. Wastewater-based epidemiology utilizing next-generation sequencing serves as an effective tool for monitoring viral circulation dynamics at the community level. However, these complex environmental samples are often laden with other microorganisms and host genomic material, which can hinder the sensitivity of viral detection. To address this limitation, targeted enrichment sequencing is emerging as a preferred strategy, facilitating the acquisition of a more comprehensive understanding of specific pathogens. In this study, we evaluated the performance of a targeted enrichment sequencing panel for 42 excreted respiratory viruses (including Picornaviridae, Adenoviridae, Coronaviridae, Paramyxoviridae, Orthomyxoviridae, Orthoherpesviridae, Pneumoviridae, and Parvoviridae families), known as the Respiratory Pathogen ID/AMR enrichment panel (RPIP), coupled with Explify bioinformatics analysis in 3 sewage samples from Uruguay. RPIP panel successfully identified sequences from frequently circulating viruses, along with some that had not been documented previously. We identified and characterized various viruses, including human Enterovirus (Coxsackievirus A1 and A19), Influenza A-H1N1, and full-length sequences of SARS-CoV-2. Additionally, several other viral pathogens were detected, such as human Bocavirus, human Parechovirus, Enterovirus A71, and Enterovirus D68; however, for these viruses further analysis was limited due to the small genomic regions or low-read coverage obtained. While the RPIP panel necessitates substantial sequencing depth and may introduce bias towards the more predominant strains present in the samples, this approach suggests its viability as a genomic epidemiological tool for assessing respiratory and enteric viruses in wastewater.