On the last day of infection, the HAE on the Millicell inserts were washed with PBS and fixed in 4% paraformaldehyde for ,30 min. The fixed membranes were cut into several small pieces, and washed with PBS three times. Each membrane fragment was transferred to 20% sucrose in a 15-ml conical tube and allowed to drop to the bottom; it was then embedded vertically in cryoprotectant OCT in an orientation that enabled sectioning of the membrane perpendicular to the blade. Cryostat sections were cut at a thickness of 10 mm, placed onto slides, and stained with hematoxylin and eosin (H&E). Images were taken with a Nikon 80i fluorescence microscope at a magnification of 660. Figure S1 Sequencing of PCR DNA fragments. PCR DNA fragments indicated by arrowheads in Figure 1D were extracted and sequenced. A representative result of sequencing is shown in each chromatogram. The sequences between the arrows in the chromatograms (A-C) show the sequences which are complementary to those sequences between the arrows in the hairpin drawings in Figure 1A -C, respectively. (TIF) Figure S2 Construction of a full-length pIHBoV1 clone. (A) HBoV1 genome. The full-length genome of HBoV1 is diagramed with structures of the left-end hairpin (LEH) and rightend hairpin (REH) in a form of negative ssDNA from 39end to 59end. Restriction enzyme sites of BspEI and BglII in the replicative form (RF) DNA are shown. (B) Cloning of the LEH. PCR-amplified DNA fragments from the LEH, shown in red, were ligated into pBBSmaI or its derivative. (C) Cloning of the REH. PCR-amplified or synthesized HBoV1 DNA fragment from the REH, shown in blue, were ligated into pBBSmaI or its derivatives. (D) Cloning of the pIHBoV1. The pIHBoV1 was constructed by ligating HBoV1 DNA nt 1-517 digested from pBB-LEH and nt 518-4139 amplified from viral DNA extract (HBoV1 Salvador isolate) into the pBB-REH that contains HBoV1 REH (nt 4140-5543). All the numbers are nucleotide numbers of the HBoV1 genome (Genbank JQ923422). (TIF) Figure S3 Kinetics of virus release from HBoV1 infection of primary B31-11 and B29-11 HAE. Primary B-HAE (donor B31-11 or B29-11) was infected with purified HBoV1 at an MOI of ,750 genome copy numbers (gc)/cell. Virus was collected from the apical chamber (A), or from both the apical and basolateral chambers (B) for detection of nuclease-resistant viral gc. Averages and standard deviations are shown. ND, not determined. (TIF) Figure S4 Immunofluorescence analysis of HBoV1infected HAE polarized from NuLi-1 cells (NuLi-HAE). NuLi-1 cells were polarized by growth at an ALI for 4 weeks on Millicell inserts of 0.6 cm 2 , until a transepithelial electrical resistance (TEER) of .500 V.cm 2 was detected. Polarized HAE was infected with purified HBoV1 at an MOI of ,750 gc/cell. At 10 days p.i., infected NuLi-HAE was fixed and stained with an anti-(HBoV1)NS1 antibody. Nuclei were stained with DAPI and cells were visualized by confocal microscopy at a magnification of 640.