Abstract Title
Digital droplet RT-PCR assays for A(H5N1) 2.3.4.4b surveillance in a One-Health perspective
Abstract
Since March 2024, numerous cases of highly pathogenic avian influenza A(H5N1) caused by viruses of clade 2.3.4.4b, genotype B3.13, have been reported in U.S. dairy cattle, followed by spillover to cats, mice, and over avian and mammalian species. The CDC later confirmed human infections linked to professional exposure to A(H5N1)-positive cattle and poultry, though human-to-human transmission hasn't been reported yet. The virus propensity to infect mammals and gain adaptive mutations raise public health concerns requiring enhanced monitoring to prepare against possible pandemics, including wastewater surveillance integrating existing cross-border governance and measurement structures.
To detect A(H5N1), two digital droplet RT-PCR assays targeting conserved regions in the matrix protein (MP) and the hemagglutinin (HA) genes were developed. The MP assay detects influenza A viruses, while the HA assay specifically targets H5 HPAI 2.3.4.4b strains. After in vitro optimisation, validation was performed using control materials and samples from various European animal specimens infected with H5N1 HPAI 2.3.4.4b . An inter-laboratory exercise employing target and non-target isolates demonstrated the assays were easily deployable. They were applied also to nucleic acids extracted from wastewater samples spiked with H5N1 HPAI 2.3.4.4b RNA, confirming high specificity, sensitivity, and reproducibility.
In support to One Health strategies pivotal for zoonotic avian influenza surveillance, we propose the combined use of these assays for rapid and sensitive detection of A(H5N1) 2.3.4.4b in biological and environmental samples drawing upon extensive peer-reviewed evidence to inform both one health policy cycle formation and practice.
To detect A(H5N1), two digital droplet RT-PCR assays targeting conserved regions in the matrix protein (MP) and the hemagglutinin (HA) genes were developed. The MP assay detects influenza A viruses, while the HA assay specifically targets H5 HPAI 2.3.4.4b strains. After in vitro optimisation, validation was performed using control materials and samples from various European animal specimens infected with H5N1 HPAI 2.3.4.4b . An inter-laboratory exercise employing target and non-target isolates demonstrated the assays were easily deployable. They were applied also to nucleic acids extracted from wastewater samples spiked with H5N1 HPAI 2.3.4.4b RNA, confirming high specificity, sensitivity, and reproducibility.
In support to One Health strategies pivotal for zoonotic avian influenza surveillance, we propose the combined use of these assays for rapid and sensitive detection of A(H5N1) 2.3.4.4b in biological and environmental samples drawing upon extensive peer-reviewed evidence to inform both one health policy cycle formation and practice.
Co-Author(s)
Antonio Marchini (1)*, Mauro Petrillo (2), Gerhard Buttinger (1), Viviana Valastro (3), Sabrina Marciano (3), Marika Crimaudo (3), Valeria D’Amico (3), Gabriele Leoni (4), Valentina Paracchini (1), Philippe Corbisier (1), Bénédicte Lambrecht (5), Bernd Gawlik (4), Calogero Terregino (3), Maddalena Querci (4), Sandra Coecke (4), Tobias Wiesenthal (1), Ursula Vincent (1), Valentina Panzarin (3)
1) European Commission, Joint Research Centre (JRC), Geel, Belgium.
2) Seidor Italy S.r.l., Milan, Italy.
3) EU/WOAH/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Legnaro, Italy.
4) European Commission, Joint Research Centre (JRC), Ispra, Italy.
5) Avian Virology and Immunology, Sciensano, Brussels, Belgium.
*Corresponding author: Antonio.MARCHINI@ec.europa.eu
Abstract Category
Avian influenza in mammals, pandemic preparedness, and one health