First generation recombinant adenovirus expressing Ebola Zaire glycoprotein was prepared and purified as described above. The protein content of the virus preparation was determined using BioRad DC Protein Assay reagents (BioRad, Hercules, CA) and bovine serum albumin as a standard in a microplate format. According to established protocols, 10 mg of monomethoxypoly(ethylene) glycol, activated by tresyl chloride (Sigma Aldrich, St. Louis, MO), was added for each microgram of protein present [38] . The coupling reaction was performed at 25uC with gentle agitation. The reaction was stopped by the addition of L-lysine, in a 10-fold excess with respect to the amount of PEG added. Unreacted PEG, excess L-lysine, and reaction byproducts were removed by buffer exchange over a second Econo-Pac 10DG disposable chromatography column equilibrated with 100 mM potassium phosphate-buffered saline (pH 7.4). PEGylated preparations were administered in sterile potassium phosphate buffered saline (pH 7.4) and had particle to plaque forming unit (pfu) ratios of 100:1 or less as determined by the Adeno-X rapid titer kit. Characterization of these preparations revealed significant changes in biophysical properties of the virus once the reaction was complete such as the PEG-Dextran partition coefficient and peak elution times during capillary electrophoresis (as described previously [30, 38] ). Approximately 13,000 PEG molecules were associated with each virus particle in the studies outlined here as determined by a PEG-biotin assay [30] .