IBDV was purified following established procedures [11 (link)]. Briefly, supernatants of IBDV-infected QM7 cells were harvested at 72 h post-infection (hpi) and precipitated with 3.5% polyethylene glycol (PEG) 6000, 0.5 M NaCl and incubated (overnight, 4 °C, with mild shaking). The precipitate was isolated by centrifugation (3000  ×  g, 30 min, 4 °C), resuspended in PES buffer (50 mM PIPES pH 6.2, 150 mM NaCl, 20 mM CaCl2) supplemented with protease inhibitors (Complete Mini, Roche), and further purified by ultracentrifugation through a 25% sucrose cushion (170 000  ×  g, 150 min) followed by a 25–50% linear sucrose gradient (200 000  ×  g, 45 min).
To produce HT-VP2-466 VLP, H5 cell monolayers were infected with rBV HT-VP2-466 at a multiplicity of infection (moi) of 5 plaque-forming units per cell (pfu/cell) and harvested at 48 hpi. The cell pellet was lysed in PES buffer supplemented with 1% Igepal CA-630 (Sigma) and protease inhibitors (Complete Mini, Roche) for 20 min. The cell suspension was clarified by centrifugation, the supernatant loaded on a 25% sucrose cushion and centrifuged (170 000  ×  g, 150 min). The pellet was resuspended in PES buffer and centrifuged in a 25–50% linear sucrose gradient (200 000  ×  g, 45 min). Fractions (1 ml) were concentrated 10-fold by ultracentrifugation (240 000  ×  g, 120 min). All purification steps were performed at 4 °C.