Development of a large-volume concentration method to recover infectious avian influenza virus from the aquatic environment

Since late 2021, outbreaks of highly pathogenic avian influenza virus have caused a record number of mortalities in wild birds, domestic poultry, and mammals in North America. Wetlands are plausible environmental reservoirs of avian influenza virus; however, the transmission and persistence of the virus in the aquatic environment are poorly understood. To explore environmental contamination with the avian influenza virus, a large-volume concentration method for detecting infectious avian influenza virus in waterbodies was developed. A variety of filtering, elution, and concentration methods were explored, in addition to testing filtering speeds using artificially amended 20 L water matrices (deionized water with sterile dust, autoclaved wetland water, and wetland water). The optimal protocol was dead-end ultrafiltration coupled with salt solution elution and centrifugation concentration. Using this method, infectious virus was recovered at 1 × 10⁻¹ 50% egg infectious dose per milliliter (EID₅₀/mL), whereas viral RNA was detected inconsistently down to 1 × 10⁰ EID₅₀/mL. This method will aid in furthering our understanding of the avian influenza virus in the environment and may be applicable to the environmental detection of other enveloped viruses.