Rapid and sensitive detection of high pathogenicity Eurasian clade 2.3.4.4b avian influenza viruses in wild birds and poultry.

Avian influenza virus (AIV) is classified as high or low pathogenicity AIV (HPAIV/LPAIV) based on intravenous pathogenicity in chickens and/or the presence or absence of multiple basic residues at the heamagglutinin (HA) cleavage site (CS). Since 2014, Europe has experienced waves of incursions of H5Nx HPAIV. Between November 2020 and March 2021, these included HPAIV H5N8, with sporadic of H5N1 and H5N5 (all clade 2.3.4.4b), detected in more than 300 "found dead" wild birds submitted through a passive surveillance programme in the United Kingdom. Currently, H5Nx HPAIV detection relies on identification of AIV RNA and H5 subtyping using real-time reverse transcription PCR (rRT-PCR) assays. The pathotype is subsequently determined by Sanger sequencing of the HA CS. Here, we report the validation and application of a rapid, more cost-effective HP H5-detection rRT-PCR assay. The HP H5 rRT-PCR assay specifically, sensitively and reproducibly detected RNA from contemporary clade 2.3.4.4b H5 HPAIVs with comparable sensitivity to the diagnostic H5-specific rRT-PCR; LPAIV H5 RNA and non-AIV RNA were not detected. On material from "found-dead" wild birds, and for statutory disease diagnosis on poultry, the HP H5 rRT-PCR results provided 100% discrimination when compared to conventional CS sequencing, significantly reducing time-to-pathotype determination and cost, enhancing the diagnostic workflow.