A vian influenza is a viral disease caused by influenza A viruses, segmented, negative, single-stranded RNA viruses belonging to the Orthomyxoviridae family. Wild aquatic birds are the virus reservoir and generate occasional worldwide panzootic outbreaks during seasonal migrations (1). Highly pathogenic avian influenza (HPAI) virus subtypes can cause panzootic outbreaks associated with high mortality in wild and domestic birds, as well as substantial economic losses for the poultry industry, and are a major threat to public health because of their zoonotic potential.During winter 2020-21, the HPAI H5N8 virus belonging to the A/goose/Guangdong/1/1996 clade 2.3.4.4b lineage caused hundreds of outbreaks among wild and domestic flocks across Europe (2,3). France was severely affected; 492 poultry farms, primarily duck farms, were infected during December 5, 2020-May 3, 2021. Despite reinforced surveillance activities, the virus spread rapidly, posing major challenges for surveillance and control. Officially recognized surveillance methods involve tracheal or cloacal swab-based sampling (4,5). However, these methods are laborious and have technical requirements that make application on such a massive scale difficult; thus, newer surveillance methods are needed.Epidemiologic modeling of this outbreak suggested within-farm viral transmission was extremely fast, and the environment was a major source of contamination for neighboring farms (6). HPAI viruses disperse in aerosols, in fomites carried by human and animal vectors, and via feathers, fecal particles, and to a great extent, dust (7-9). Poultry farms are known to heavily generate dust particles that spread from feed, litter, feces, and animal skin and feathers (9,10). These particles can act as vehicles for bacteria and viruses and are classified, depending on their size, as inhalable (<100 µm), thoracic (<10 µm), or respirable (<4 µm) (10). In poultry houses, most dust consists of nonrespirable particles >4 µm (10). We evaluated the role of dust as a vehicle of H5N8 clade 2.3.4.4b virus and assessed whether dust or aerosol sampling is a viable alternative to bird swab sampling for HPAI virus surveillance.

Highly pathogenic avian influenza viruses (HPAIV) are a major threat to the global poultry industry and public health due to their zoonotic potential. Since 2016, Europe and France have faced major epizootics caused by clade 2.3.4.4b H5 HPAIV. To reduce sample-to-result times, point-of-care testing is urgently needed to help prevent further outbreaks and the propagation of the virus. This study presents the design of a novel real-time colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of clade 2.3.4.4b H5 HPAIV. A clinical validation of this RT-LAMP assay was performed on 198 pools of clinical swabs sampled in 52 poultry flocks during the H5 HPAI 2020-2022 epizootics in France. This RT-LAMP assay allowed the specific detection of HPAIV H5Nx clade 2.3.4.4b within 30 minutes with a sensitivity of 86.11%. This rapid, easy-to-perform, inexpensive, molecular detection assay could be included in the HPAIV surveillance toolbox.