The E1/E3-deleted adenovirus vector expressing the Ebola Zaire glycoprotein was created by cloning the open reading frame (ORF) sequence of the glycoprotein in the plasmid pShuttle (Adeno-X Expression system I, BD Clonetech, Palo Alto, CA) for subsequent insertion in the E1 region of the human adenovirus serotype 5 genome. The human cytomegalovirus (CMV) promoter included in the Adeno-X expression system was used to drive the expression of the Ebola Zaire glycoprotein in the final recombinant adenovirus serotype 5 construct. Authenticity of the final product was confirmed by sequencing of the recombinant virus rescued by transfecting the linearized DNA into 293 cells. Virus was sequentially amplified to large-scale infections (5610 8 cells) and purified on an affinity column (Adeno-X virus purification mega kit, BD Clonetech, Mountain View, CA) according to the manufacturer's instructions. Genome structures of vectors were analyzed by restriction digestion of isolated viral DNA and compared with those of the original molecular clones. Particle number and infectivity of vectors were determined by standard optical density reading and immunodetection of the hexon protein, respectively, following infection of 293 cells with limiting dilutions of each vector preparation according to the recommendations by the manufacturer (Adeno-X rapid titer kit, Clontech, Mountain View, CA). Purified virus was administered in sterile phosphate buffered saline (pH 7.4) and had particle to plaque forming unit (pfu) ratios of 100:1 or less.