Extended Data Fig. 5: Determinants of BTN3A3 sensitivity.

a, PR8:Mallard reassortants and Udorn wild type (WT) were used to perform plaque assays in MDCK cells expressing BTN3A3 and empty vector control cells. Note that reassortant PR8 7:1 Mallard seg 1, Mallard 7:1 PR8 seg 2 and Mallard 7:1 PR8 seg 5 failed to rescue. Reassortant PR8 Mallard 3PNP is formed by PR8 (segments 4, 6, 7 and 8) and segments 1, 2, 3 and 5 from Mallard. Conversely, reassortant Mallard PR8 3PNP contain segments 4, 6, 7 and 8 from Mallard and segments 1, 2, 3 and 5 from PR8. Data are mean +/− SEM of 2 technical replicates from 2 independent experiments. Statistical differences between cells expressing BTN3A3 and control cells were calculated using multiple t-tests and corrected for multiple comparisons using the Holm-Šídák method. NS-Non-significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. b, Identity of amino acid residues in positions 100 (left) and 313 (right) of all NP proteins available in our dataset collected from GenBank. c, Identity of combinations between amino acid residues 100 and 313 (left) and 52 and 313 (right). d, Infectious virus titres obtained in A549-Empty and A549-BTN3A3 cells infected with either Mallard or Cal04 residues 100 and 313 mutants. Cells were infected with an MOI of 0.001 for 48h and viruses were titrated by plaque assay. Data are mean +/− SEM of 2 technical replicates from 3 independent experiments. Statistical analysis was carried out as in (a). e, Viral replication assays in avian cells were carried out in chicken fibroblasts (DF1 cells). Cells were infected an MOI of 0.001 for 48 h. Infectious virus titres were determined by plaque assay. Data are mean +/− standard error of the mean (SEM) of 3 independent experiments (each using two technical replicates).