The optimized conditions were applied to transfect G, F and M expression plasmids in 293T cells grown in 10 cm dishes. The VLP-containing culture SUPs were harvested 48 hours later, and concentrated and purified as described. Briefly, the clarified SUPs were concentrated by ultracentrifugation through 20% sucrose cushion, and then purified on a discontinuous sucrose gradient. The VLP pellet was resuspended in TN buffer and viewed by EM after negative staining. The result presented in Figure 2A shows a VLP-containing band in the sucrose gradient. Viewing of the negatively stained purified particles by transmission electron microscopy ( Figure 2B) showed numerous virus-like particles. The size variation of these VLPs was consistent with the parental virus: NiV is a pleomorphic virus ranging in size from 40-1900 nm [63, 64] ; the sizes of the VLPs ranged from ,40-500 nm. The particles also resembled authentic NiV morphologically, and this is seen more clearly in the magnified images presented in Figure 2C ; here, the fringe of the glycoproteins is clearly visible on the VLP surface. An occasional VLP had what appeared to be a double fringe (shown with an arrow), a feature more frequently associated with Hendra rather than NiV virus particles [64] . The image in Figure 2D is a cryoelectron micrograph of one of our VLPs; the overall surface appearance is virus-like, which is described as dense, ordered and repetitive [31] , and it shows the surface glycoproteins and their spatial arrangement even more definitively.