FIG 8.

Analyses of virus infection of HAE-ALI cultures. HAE-ALI cultures were prepared in Transwell inserts and infected with HBoV1 WT or its mutants from the apical surface at an MOI of 10 (DRP/cell). (A) Apical virus release. At the indicated days p.i., the apical surface was washed with 100 μl of PBS to collect released virus. Virion particles (DRP) were quantified by qPCR (y axis) and plotted to the days p.i. as shown. Means and standard deviations are shown. (B to D) Western blot analysis of HBoV1-infected HAE-ALI cultures. At 14 days p.i., the cells of the infected HAE-ALI culture were lysed for Western blotting with anti-NS1C (B), anti-NS1NL (C), or anti-VP (D) antibody. The blots were reprobed for β-actin, and the blot in panel C was further reprobed with an anti-NP1 antibody. The identities of detected proteins are shown with arrows at the right of the blot. In panel D, the band indicated by an asterisk is likely a cleaved band of VP1 or a new VP. (E) Southern blot analysis of viral DNA replication. At 14 days p.i., the cells of the infected HAE-ALI cultures were lysed for Hirt DNA preparation. The Hirt DNA samples were analyzed by Southern blotting with an HBoV1 NSCap probe and a mitochondrial DNA (Mito DNA)-specific probe (40). Detected mitochondrial DNA was used for normalization of viral DNA quantification. The identities of detected bands are shown to the right.