Abstract
In the current epidemiological context, rapid characterization of high-pathogenicity avian influenza viruses (HPAI) phenotypes is critical to implement risk-based measures to control the spread of viruses in poultry, in particular in areas where multiple genotypes circulate. Existing AIV characterization methods rely primarily on labour-intensive pathogenesis studies often restricted to few poultry species (e.g. chickens and turkeys).
To address these limitations, we developed a biobank of cryopreserved tissues from different avian species to timely conduct in-vitro phenotyping characterization during epidemics. This biobank includes tracheal organ cultures (TOC), precision-cut lung slices (PCLS), and enteroids derived from chickens, turkeys, and ducks. Expression profiles and viability assessments demonstrated that cryopreserved TOC and PCLS retained functional comparability to fresh tissues, with 20% reduction in cellular viability, while cryopreserved and fresh enteroids exhibited equivalent viability. Both fresh and cryopreserved TOC, PCLS, and enteroids were tested for infection susceptibility with LPAI and HPAI viruses, including the HPAI H5N1 strains associated with recent outbreaks in wild and domestic avian populations. Results indicate that in cryopreserved TOC and PCLS viruses exhibit on average 10-50 times lower mean infectious doses. Furthermore, replication kinetics and cell-specific tropism in the cryopreserved tissues reflected differences between viruses with extremely divergent phenotypes in-vivo. This biobanking platform represents a promising tool for efficient and timely AIV phenotyping, nonetheless, further development will be necessary to assess the predictive value of assays conducted in these substrates in relation to pathogenesis studies.
To address these limitations, we developed a biobank of cryopreserved tissues from different avian species to timely conduct in-vitro phenotyping characterization during epidemics. This biobank includes tracheal organ cultures (TOC), precision-cut lung slices (PCLS), and enteroids derived from chickens, turkeys, and ducks. Expression profiles and viability assessments demonstrated that cryopreserved TOC and PCLS retained functional comparability to fresh tissues, with 20% reduction in cellular viability, while cryopreserved and fresh enteroids exhibited equivalent viability. Both fresh and cryopreserved TOC, PCLS, and enteroids were tested for infection susceptibility with LPAI and HPAI viruses, including the HPAI H5N1 strains associated with recent outbreaks in wild and domestic avian populations. Results indicate that in cryopreserved TOC and PCLS viruses exhibit on average 10-50 times lower mean infectious doses. Furthermore, replication kinetics and cell-specific tropism in the cryopreserved tissues reflected differences between viruses with extremely divergent phenotypes in-vivo. This biobanking platform represents a promising tool for efficient and timely AIV phenotyping, nonetheless, further development will be necessary to assess the predictive value of assays conducted in these substrates in relation to pathogenesis studies.
Co-Author(s)
Alessandra Napolitan* (a,b), Eva Mazzetto* (b), Elisa Mazzacan (b), Lucrezia Vianello (b), Sami Ramzi (b,c), Terregino Calogero (b), Francesco Bonfante (b)
(a) Department of Comparative Biomedicine and Food Science, University of Padua, 35020 Legnaro (Padua), Italy
(b) EU/WOAH/National Reference Laboratory for Avian Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro (Padua), Italy
(c) Department of Animal Medicine, Production and Health, University of Padua, 35020 Legnaro (Padua), Italy
Abstract Category
Transmission pathways, pathobiology, immune responses