Development of innovative substrates and phenotyping assays aimed at characterizing in vitro the fitness of emerging avian influenza viruses in both poultry and wild bird species

Since its emergence in 1996, highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/96 lineage have diversified into multiple clades, culminating in the 2020–2021 global panzootic caused by H5N1 viruses of the clade 2.3.4.4b. Further reassortment events have significantly diversified the phenotypes of these viruses, underscoring the need for continuous monitoring and strain characterization to better adjust control measures and mitigate the impact of the disease in wild birds and poultry. Standardized, ready-to-use ex vivo tissue platforms for rapid phenotyping of avian influenza viruses (AIVs) offer a valid alternative to in vivo models that are financially, ethically and logistically demanding. We optimized explant production and cryopreservation protocols for chicken and duck tracheal organ cultures (cTOCs and dTOCs) and precision-cut lung slices (cPCLS and dPCLS), assessing post-thaw viability, histological integrity, and susceptibility to AIV infection . Adult-derived tissues were used instead of embryonic tissues to better replicate the cellular architecture, receptor distribution, and protease expression relevant to natural infection. Trehalose supplementation of cryopreservation solutions based on dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) significantly improved tissue viability. Although cryopreserved tissues were less viable than the fresh explants, viral replication was similar and only a modest reduction in susceptibility to infection was observed . The platform was validated using LPAI and HPAI strains with different tissue tropisms and host origins to discriminate viruses based on their divergent fitness and host preference. H9N2 showed superior replication in chicken TOCs, whereas H5N1 replicated more efficiently in duck TOCs, reflecting in vivo replication patterns. In addition, enteroids derived from adult duck crypts supported replication of duck-adapted H5N1 strains but not H9N2 or human-origin H1N1, further confirming tissue-specificity. The strong concordance with in vivo data supports their utility as scalable phenotyping tools during outbreaks. These findings underscore the potential of cryopreserved explants as additional tools for the phenotypic characterisation of emerging AIVs .