The infectivity of the HBoV1 virions purified from pIHBoV1transfected HEK293 cells was examined in polarized primary HAE, the in vitro culture model known to be permissive to HBoV1 infection [31] . Three sets (different donors, culture lots #B29-11, B31-11 and B33-11) of B-HAE were generated, and these were Figure 1 . Sequencing the terminal hairpins of the HBoV1 Salvador1 isolate. Sequence and predicted structure of the left-end, LEH (A&B), and right-end, REH (C), hairpins are shown and diagramed, with PCR primers indicated by arrowed lines. PCR products were analyzed by electrophoresis on 2% agarose gels; the expected DNA bands are indicated by arrowheads (D). In both the LEH and REH, nucleotides in red represent new sequences identified in this study, nucleotides in blue represent sequences identified from the head-tail junction of an HBoV1 episome [28, 29] , and nucleotides in black are the 59end and 39end sequences of the incomplete HBoV1 genome (GenBank: JQ411251). doi:10.1371/journal.ppat.1002899.g001 infected with HBoV1 from the apical side. Initially the B-HAE cultures were infected with various amounts of virus, and when a multiplicity of infection (MOI) of ,750 gc/cell was used, most of the cells (,80%) were positive for anti-NS1 staining (indicating that the viral genome had replicated and that genes encoded by it had been expressed) at 5 days post-infection (p.i.). This MOI was subsequently used for apical infection. Notably, B29-11, B31-11 and B33-11 HAE each supported productive HBoV1 infection ( Figures 3 and S3 ). Immunofluorescence (IF) analysis of infected B31-11 HAE at 12 days p.i. showed that virtually all the cells expressed NS1 and NP1 ( Figures 3A and 3B ), and that a good portion of the infected cells expressed capsid proteins (VP1/2; Figure 3C ).