Cryopreservation of chicken and duck tracheal rings and precision-cut lung slices: A promising tool for the rapid characterization of avian influenza viruses

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. 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. Finally, we used duck and chicken TOCs to assess the ability of cryopreserved explants to discriminate viruses based on their divergent fitness and host preference. These findings underscore the potential of cryopreserved TOCs and PCLS as additional tools for the phenotypic characterisation of emerging AIVs.