A Tale of Two Lenses: Emergency department indoor-air hybrid-capture metagenomics complements wastewater by adding a human-focused respiratory virus perspective

BackgroundContinuous, non-invasive viral surveillance is essential to monitor emerging pathogens and guide public health responses. Most environmental surveillance studies use targeted qPCR approaches, and comparisons between wastewater and indoor air surveillance remain limited. We aimed to compare the utility of emergency department indoor air and urban wastewater for tracking circulating viruses and resolving genomic information. MethodsWe conducted a matched-pair study comparing 19 weekly indoor air samples from the central ventilation exhaust shaft of an emergency department and 19 24-hour composite municipal wastewater samples in Leuven, Belgium, from December 2024 to April 2025. Both sample sets were processed using probe-based hybrid-capture viral metagenomics targeting over 3000 viral species, using influenza A as a clinically relevant test case. FindingsWastewater captured higher overall viral diversity (233 versus 106 species) and more complete genomes compared to indoor air, showing a relatively stable composition, mainly of enteric and animal-associated viruses. Indoor air demonstrated lower overall diversity but was enriched for respiratory viruses, including influenza A, coronaviruses, metapneumovirus, and respiratory syncytial virus, and more frequently achieved high genome coverage for these pathogens. Although both sample types permitted influenza A subtype characterization, influenza A genomes from wastewater were often less well covered. When coverage thresholds were met, indoor air supported targeted antiviral resistance-site screening for influenza A and RSV-A. InterpretationWastewater and indoor air generate distinct but complementary viromes. Wastewater acts as a diverse, population-level monitor for One-Health applications, whereas indoor air serves as a targeted, human-centric sentinel system facilitating further genomic characterization for respiratory viruses. FundingMustafa Karatas is supported by a Research Foundation Flanders (FWO) fundamental research scholarship (number: 11P7I24N). C.G., L.C., E.H., S.G. and E.A. acknowledge support from the DURABLE project. The DURABLE project has been co-funded by the European Union, under the EU4Health Programme (EU4H), project no. 101102733. Research in context Evidence before this studyWe searched PubMed for studies published between Jan 2000 and March 2024 using the terms "wastewater surveillance", "metagenomics", "indoor air", and "viral metagenomics". Previous studies have shown that wastewater surveillance can detect population-level viral circulation, and more recent work has explored indoor air sampling as a method for monitoring respiratory virus transmission. However, environmental metagenomic studies have largely examined these two sample types separately. Furthermore, most studies relied on untargeted sequencing approaches, which often yield fragmented genomes in these environments. To date, no study has systematically compared indoor air and wastewater using a comprehensive hybrid-capture viral metagenomics approach for virus surveillance. Added value of this studyWe conducted a matched comparison of indoor air from a hospital emergency department and municipal wastewater collected during the same weeks in Leuven, Belgium. We analyzed both sample types using an identical hybrid-capture viral metagenomics workflow targeting more than 3000 viral species. This design enabled a direct evaluation of how the two environmental surveillance lenses differ in viral diversity, genomic recovery, and epidemiological relevance. Wastewater captured broader viral diversity and a stable background dominated by enteric and animal-associated viruses, whereas indoor air captured more respiratory viruses and more frequently yielded high genome completeness for these pathogens. When genome coverage thresholds were met, indoor air data enabled influenza subtype identification and screening for antiviral resistance markers. Implications of all the available evidenceOur findings support a layered environmental surveillance strategy in which different environmental samples provide complementary information. Wastewater offers a stable, population-level view of viral circulation and captures broad viral diversity, including human and animal-associated viruses. Indoor air sampling in human-dominated settings provides a more direct signal of respiratory virus circulation and can yield genomes suitable for subtype and mutation-level characterization. Combining these approaches could strengthen metagenomic surveillance frameworks by improving the interpretation of environmental viral signals, supporting early detection of emerging pathogens, and helping distinguish human virus circulation from environmental or animal-derived detections.