Innovative methods for evaluating viral risk and spread, independent of test-seeking behavior, are needed to improve routine public health surveillance, outbreak response, and pandemic preparedness. Throughout the COVID-19 pandemic, environmental surveillance strategies, including wastewater and air sampling, have been utilized alongside widespread individual-based SARS-CoV-2 testing programs to provide population-wide data. To date, environmental surveillance strategies have mainly relied on pathogen-specific detection methods to monitor viruses through space and time. However, this provides a limited picture of the virome present in a sample, leaving us blind to most circulating viruses. In this study, we explore whether virus-agnostic deep sequencing can improve the utility of air sampling to detect human viruses captured in air samples. We show that sequence-independent single-primer amplification sequencing of nucleic acids from air samples can detect common and unexpected human respiratory and enteric viruses, including influenza virus type A and C, respiratory syncytial virus, human coronaviruses, rhinovirus, SARS-CoV-2, rotavirus, mamastrovirus, and astrovirus.

The authors have declared no competing interest.

This work was made possible by financial support through the National Institutes of Health grant (AAL4371). M.D.R. is supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number T32AI55397.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

The air sample sequencing data generated in this study have been deposited in the Sequence Read Archive (SRA) under bioproject PRJNA950127. The accession numbers for influenza C virus samples used in the phylogenetic analysis are provided in Supplementary Data 1.