ABSTRACT Diverse influenza A viruses (IAVs) circulate in wild birds, including dangerous strains that infect poultry and humans. Consequently, surveillance of IAVs in wild birds is a cornerstone of outbreak prevention and pandemic preparedness. Surveillance is traditionally done by testing birds, but dangerous IAVs are rarely detected before outbreaks begin. Testing environmental specimens from wild bird habitats has been proposed as an alternative. These specimens are thought to contain diverse IAVs deposited by broad range of avian hosts, including species that are not typically sampled by surveillance programs. We developed a targeted genomic sequencing method for recovering IAV genome fragments from these challenging environmental specimens, including purpose-built bioinformatic analysis tools for counting, subtyping, and characterizing each distinct fragment recovered. We demonstrated our method on 90 sediment specimens from wetlands around Vancouver, Canada. We recovered 2,312 IAV genome fragments originating from all 8 IAV genome segments. 11 haemagglutinin (HA) subtypes and 9 neuraminidase subtypes were detected, including H5, the current global surveillance priority. Recovered fragments originated predominantly from IAV lineages that circulate in North American resident wild birds. Our results demonstrate that targeted genomic sequencing of environmental specimens from wild bird habitats can be a valuable complement to avian influenza surveillance programs.

Targeted genomic sequencing of avian influenza viruses in wetlands sediment from wild bird habitats