Immuno-EM analysis of DIII-pseudotyped VLPs. Extracellular VLPs released from Sf9 cells co-expressing Pr55GagHIV and DIII-RIgE fusion protein, were isolated by ultracentrifugation through sucrose cushion. Aliquots of VLP samples were adsorbed on EM grids and reacted with monoclonal anti-His6 tag antibody, and secondary 10-nm colloidal gold-labeled anti-mouse IgG. (a), Control VLPs devoid of DIII (VLP-0); (b-d), VLP-displayed DIII-DENV1-RIgE; (e, f), VLP-displayed DIII-WNVKun-RIgE. Specimens were negatively stained with sodium phosphotungstate. BV, baculovirus vector. Note that all visible gold grains are found to be associated with membrane-enveloped VLPs of 120-130 nm in diameter . Scale bar represents 100 nm in (a-c) and (e-f), and 20 nm in (d).