<p>(<b>A</b>–<b>B</b>) <i>Kinetics of DVR2A</i><sup><i>ΔNS1</i></sup><i>TCPs secretion from helper cell lines</i>. (A) Schematic representation of the experimental setting. DVR2A<sup>ΔNS1</sup> TCPs produced in VeroE6_NS1<sup>WT</sup> cells (ΔNS1-WT<sup>TCP</sup>) were used to infect (MOI = 1) control or helper cell lines expressing different forms of NS1. Culture supernatants were collected 24, 48 and 72 h later, and virus titers were determined by Focus forming unit (FFU) assay on VeroE6_NS1<sup>WT</sup> cells, using an E-specific mouse monoclonal antibody. Cell lysates and culture supernatants were analyzed by western blot. (B) FFU titers were determined as specified in panel (A). As reference, naïve VeroE6 cells were infected with DVR2A. The dashed line indicates the limit of detection of the assay. The lower panel shows representative images of foci morphologies of each trans-complemented NS1 variant. (<b>C</b>) <i>Expression levels of intra- and extra-cellular NS1 in DVR2A</i><sup><i>ΔNS1</i></sup><i>TCP infected cells</i>. NS1 expression and secretion were evaluated by western-blotting using cell lysates (Intra-) or clarified supernatants (Extra-) from (B) and NS1-, NS5- or GAPDH specific antibodies. (<b>D</b>, <b>E</b>) <i>Ultrastructural characterization of cells infected with DVR2A</i><sup><i>ΔNS1</i></sup><i>TCPs</i>. VeroE6_NS1<sup>HA</sup> cells were infected with 1 MOI of DVR2A<sup>ΔNS1</sup> TCPs. Forty-eight hours later, cells were fixed, processed and analyzed by transmission electron microscopy as described in materials and methods. Representative images of the perinuclear area (D) or plasma membrane (E) of infected cells are shown. Black arrowheads indicate Vesicle packets (VPs), red arrowheads indicate virion bags. Electron-dense virus particles in proximity to or at the plasma membrane are indicated with black arrows. Boxed areas on the left panels are shown at higher magnification on the right. Black or white scale bars represent 500 or 200 μm, respectively.</p