After HBoV1 infection, ALI membranes were fixed with 3.7% paraformaldehyde in PBS at room temperature for 15 min. The fixed membranes were cut into several small pieces, washed in PBS three times for 5 min, and permeabilized with 0.2% Triton X-100 for 15 min at room temperature. The membranes were then incubated with primary antibody at a dilution of 1:100 in PBS with 2% FCS for 1 h at 37uC. This was followed by incubation with a fluorescein isothiocyanate-or rhodamine-conjugated secondary antibody. Confocal images were taken with an Eclipse C1 Plus confocal microscope (Nikon, Melville, NY) controlled by Nikon EZ-C1 software. Primary antibodies used were anti-(HBoV1) NS1, NP1 and VP1/2 antibodies, as reported previously [82] .