Structural analyses of WNV antigens secreted by HeLa3-WNV cells.

<p>(A) WNV proteins released to the culture medium of HeLa3-WNV cells were purified by centrifugation through a 20% sucrose cushion and loaded onto a 20–60% linear sucrose gradient. The amount of E glycoprotein in each fraction of the gradient was determined by enzyme-linked immunodot assay using a monoclonal antibody against the WNV E protein. (B) Quantification of the integrated density in each dot corresponding to the gradient fractions shown in panel (A). (C) Negative stained TEM image of purified WNV RSPs contained in peak fraction 11 of the gradient in panel (A). Scale bar: 50 nm. (D) Histogram showing the diameter of a total of 259 RSPs visualized by transmission electron microscopy and negative staining as in panel (C). (E) Purified RSPs were immunolabelled with either a monoclonal anti-E (left panel) or a pooled sera from WNV-infected mice (right panel), incubated with protein A or anti-mouse IgG coupled to 5 nm diameter colloidal gold. Samples were negatively stained and observed by transmission electron microscopy. Scale bars: 50 nm.