Fixed sections of optic nerve, brain, and eyes from mice at 3 or 6 days pi were stained by the previously described immunohistochemical methods using antiviral nucleocapsid antiserum. To visualize viral antigen by EGFP expression directly, frozen sections from infected mice were examined by fluorescence microscopy. Viral antigen was also assayed by Western blotting using antiYhepatitis virus nonstructural protein 9 monoclonal antibody (Rockland, Inc., Gilbertsville, PA), according to the manufacturer's instructions. Briefly, protein was extracted from the optic nerve and retina using RIPA buffer (Sigma, St. Louis, MO) in the presence of a complete protease inhibitor cocktail (Pierce, Rockford, IL) at 4-C. Protein concentration was determined with a Micro BCA Protein Assay Kit (Pierce). Protein (30 Kg) was electrophoresed on 4% to 15% SDS-PAGE, transferred to cellulose membrane, blocked, and probed with 1:1000 dilution of primary antibody. Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined as a loading control using anti-GAPDH (Sigma) diluted 1:10,000. After incubation with horseradish peroxidaseYconjugated secondary antibodies, signals were developed with enhanced chemiluminescence agent (GE Healthcare, Buckinghamshire, UK), and intensity was determined using the NIH Image J program.