Risk Assessment for Highly Pathogenic Avian Influenza A(H5N6/H5N8) Clade 2.3.4.4 Viruses

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

(Sigma-Aldrich), sheared with plastic pipettes, and strained with 100 μm and 70 μm filters. The collected filtrate was centrifuged at 600 × g for 5 min at 4 o C. Supernatant was removed. The cell pellet was resuspended in red blood cell lysis buffer (Roche) and incubated at room temperature for 5 min, followed by the addition of AdDF+++ basic medium and centrifugation at 600 × g for 5 min at 4 o C. 10 mg/ml cold Cultrex growth factor reduced basement membrane extract type 2 Matrigel (Trevigen) was added to the cell pellet and 40 μl droplets of the cell-in-Matrigel suspension was aliquoted to prewarmed 24-well suspension culture plates. The plates were incubated at 37 o C for 15-30 min. 1 ml organoid medium was added to each well as previously described (1,2). The plates were incubated in a humidified incubator at 37 o C and 5% CO2.
Medium was changed every 4 days and organoids were passaged every 2 weeks. To passage, organoids-in-Matrigel droplets were resuspended in cold AdDF+++, sheared with flamed glass Pasteur pipettes, and centrifuged at 400 × g for 5 min at 4 o C. Organoid fragments were reembedded in new cold Matrigel with the usual expansion of 6 times the original Matrigel droplet number. After 14 days in culture at 37 o C and 5% CO2, human airway organoids were ready for infection.

Human airway organoids -Infection
Human airway organoids of approximately 100 μm in diameter were removed from Matrigel droplets and slightly broken open by shearing with flamed glass Pasteur pipettes.
Around 100-200 organoids were infected with each virus at 6 log TCID50/ml for 1 h at 37 o C.
Organoids were washed 3 times: twice with AdDF+++ basic medium and once with organoid medium. They were re-embedded in Matrigel and cultured in organoid medium in a humidified incubator at 37 o C and 5% CO2. Culture supernatant was collected at 1, 24, 48 and 72 hpi for virus titration by TCID50 assay. Organoids were fixed in 4% paraformaldehyde at 24 and 48 hpi for immunohistochemical double staining. Organoid lysates were collected at 24 hpi by adding Buffer RLT (Qiagen) supplemented with 1% β-mercaptoethanol. Experiments were performed with organoids isolated from at least 4 different donors.

Primary human alveolar epithelial cells -Isolation and Culture
Human lung tissues were minced and washed 3 times using HBSS (Gibco) supplemented with 0.7 mM sodium bicarbonate (Gibco) at pH 7.4. Tissues were digested using 0.5% trypsin (Gibco) and 4 U/ml elastase (Worthington Biochemical Corporation) in a 37 o C water bath for 40 min. DMEM/F12 medium (Gibco) supplemented with 40% FBS and 350 U/ml DNase I (Sigma-Aldrich) were added to stop the digestion. The tissue suspension was pipetted up and down with 10 ml plastic pipettes for 10 min and strained through 50 μm filters. The filtrate was centrifuged at 1500 rpm for 5 min. Supernatant was removed and the cell pellet was resuspended in a 1:1 mixture of DMEM/F12 medium and small airway growth medium (SAGM) (Lonza) supplemented with 5% FBS and 350 U/ml DNase I. The cell suspension was seeded to tissue culture flasks (Corning) and incubated in a humidified incubator at 37 o C and 5% CO2 for 90 min.
Cells not adhered to the flasks were collected and centrifuged at 1500 rpm for 5 min. Supernatant was removed. The cell pellet resuspended in SAGM supplemented with 100 U/ml Penicillin-Streptomycin (Gibco) was seeded to new tissue culture flasks. Cells were kept in a humidified incubator at 37 o C and 5% CO2. After 60 h, culture medium was changed every day until reaching 75% confluence. Cells were then trypsinized and seeded for infection.

Immunohistochemistry double staining
The staining procedures were as previously described with slight modifications (1,2).
Infected organoids were washed with cold AdDF+++ and removed from Matrigel droplets pior to being fixed in 4% paraformaldehyde. Fixed tissues were embedded in paraffin. After sectioning and deparaffinization, the sections were incubated with 0.05% Pronase (Roche) at 37 o C for 16 min and 3% H2O2 at room temperature for 10 min. Subsequently, ImmPRESS® mRNA expression of all genes was normalized to that of β-actin.

Desialylation-haemagglutination assay
The effect of desialylation on IAV haemagglutination of Turkey red blood cells (TRBCs) was studied as previously described (2,3).  1 Table) were used to build a phylogenetic tree based on the nucleotide sequences coding for the mature HA1 protein in this study. Multiple sequence alignment was performed using the MUSCLE algorithm. Phylogenetic analysis was inferred by using the maximum likelihood method and the Tamura-Nei model in MEGA version X. Stability of the phylogenetic tree branch topology was tested using 1000 bootstrap replicates.

Statistical and sequencing analysis
Area Under Curve (AUC) and statistical analyses were performed using GraphPad Prism, version 8.4.3. AUC values were calculated as the areas under the replication kinetic curves from 1 or 24 to 72 hpi above the TCID50 assay detection limit (1.5 log TCID50/ml) using the trapezoid rule (5). AUC values and mRNA levels were compared by one-way analysis of variance (one-