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.

During the recent avian influenza epizootics that occurred in France in 2020/21 and 2021/22, the virus was so contagiousness that it was impossible to control its spread between farms. The preventive slaughter of millions of birds consequently was the only solution available. In an effort to better understand the spread of avian influenza viruses (AIVs) in a rapid and innovative manner, we established an amplicon-based MinION sequencing workflow for the rapid genetic typing of circulating AIV strains. An amplicon-based MinION sequencing workflow based on a set of PCR primers targeting primarily the hemagglutinin gene but also the entire influenza virus genome was developed. Thirty field samples from H5 HPAIV outbreaks in France, including environmental samples, were sequenced using the MinION MK1C. A real-time alignment of the sequences with MinKNOW software allowed the sequencing run to be stopped as soon as enough data were generated. The consensus sequences then were generated and a phylogenetic analysis was conducted to establish links between the outbreaks. The whole sequence of the hemagglutinin gene was obtained for the 30 clinical samples of H5Nx HPAIV belonging to clade 2.3.4.4b. The consensus sequences comparison and the phylogenetic analysis demonstrated links between some outbreaks. While several studies have shown the advantages of MinION for avian influenza virus sequencing, this workflow has been applied exclusively to clinical field samples, without any amplification step on cell cultures or embryonated eggs. As this type of testing pipeline requires only a short amount of time to link outbreaks or demonstrate a new introduction, it could be applied to the real-time management of viral epizootics.

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.

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