The production of progeny virus following HBoV1 infection was monitored daily by collecting samples from both the apical and basolateral chambers of the HAE culture and carrying out (2) been infected with Ad as indicated. Lanes 1-8 in panel B were analyzed on the same gel, and the gels shown in panels B&C were transferred and blotted together. Ten ng of the HBoV1 dsDNA genome (,5.6-kb), excised from pIHBoV1 using the SalI and XhoI sites, was used as a control (Ctrl) for DpnI digestion in panels A-C. (D) HEK293 cells were transfected with pIHBoV1 and its various mutants as indicated. At 48 h post-transfection, Hirt DNA was extracted and digested with (+) or without (2) DpnI, followed by Southern blotting using the HBoV1 dsDNA genome as a probe. dRF DNA, double replicative form DNA; mRF DNA, monomer replicative form DNA. (E) Negative staining electron micrograph. Purified HBoV1 particles were negatively stained and examined by a transmission electron microscopy. Bar indicates 100-nm. doi:10.1371/journal.ppat.1002899.g002