‘One Health’ Genomic Surveillance of Avian and Human Influenza A Viruses Through Environmental Wastewater Monitoring

Background Influenza A viruses (IAV) are significant pathogens of humans and other animals. Although endemic in humans and birds, novel IAV strains can emerge, jump species, and cause epidemics, like the latest variant of H5N1. Wastewater-based epidemiology (WBE) has very recently been shown to detect human IAV but whether it can detect avian-origin IAV, and if whole genome sequencing (WGS) can be used to discriminate circulating strains of IAV in wastewater remains unknown. Methods Using a pan-IAV RT-qPCR assay, six wastewater treatment works (WWTWs) across Northern Ireland (NI), were screened from August to December 2022. A WGS approach using Oxford Nanopore technology was employed to sequence positive samples. Phylogenetic analysis of sequences relative to currently circulating human and avian IAVs was performed. Findings We detected a dynamic IAV signal in wastewater from September 2022 onwards across NI. “Meta” whole genome sequences were generated displaying homology to both human and avian IAV strains. The relative proportion of human versus avian-origin IAV reads differed across time and sample site. A diversity in subtypes and lineages was detected (e.g. H1N1, H3N2, and several avian). Avian segment 8 related to those found in recent H5N1 clade 2.3.4.4b was identified. Interpretation WBE affords a means to monitor circulating human and avian IAV strains and provide crucial genetic information. As such WBE can provide rapid, cost-effective, year-round “one-health” IAV surveillance to help control epidemic and pandemic threats. Funding This study was funded by the Department of Health for Northern Ireland as part of the Northern Ireland Wastewater Surveillance Programme. Highlights Author Summary Influenza A virus (IAV) is a major pathogen of humans and other animals and causes regular epidemics and devastating pandemics. Recently, a novel variant of highly-pathogenic H5N1 avian influenza has emerged spreading across the world killing millions of birds and infecting mammals, enhancing its pandemic potential. Strengthening global surveillance systems for human and animal IAV is thus a major priority. Wastewater-based epidemiology (WBE) has been applied to track SARS-CoV-2 and IAV in humans but whether this approach could work for avian IAV is not known. Here, we develop a “one-health” method to survey pan-IAV levels and genetically characterise the viruses. Through this we highlight co-detection of human and avian IAVs in wastewater, with homology to recent H5N1 isolates. Our work demonstrates the potential for WBE to help defend against not only human infections but emerging, zoonotic IAVs of pandemic potential. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Department of Health for Northern Ireland as part of the Northern Ireland Wastewater Surveillance Programme. ### Author Declarations 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 All data produced in the present study are available upon reasonable request to the authors. [1]: pending:yes