Aliquots (100 ml apical or 50 ml basolateral) of the samples were incubated with 25 units of Benzonase (Sigma, St Louis, MO) for 2 h at 37uC, and then digested with 20 ml of proteinase K (15 mg/ ml) at 56uC for 10 min. Viral DNA was extracted using QIAamp blood mini kit (Qiagen), and eluted in 100 ml or 50 ml of deionized H 2 O. The extracted DNA was quantified with respect to the number of HBoV1 gc, by a qPCR method that has been used previously [84] . Briefly, the pskHBoV1 plasmid [82] , which contains the HBoV1 sequence (nt 1-5299), was used as a control (1 gc = 5.4610 212 mg) to establish a standard curve for absolute quantification with an Applied Biosystems 7500 Fast system (Foster City, CA). The amplicon primers and the PrimeTime duallabeled probe were designed by Primer Express (version 2.0.0; Applied Biosystems/Life Technologies) and synthesized at IDT Inc. (Coralville, Iowa). Their sequences are as follows (GenBank: JQ411251): forward primers, 59-GCA CAG CCA CGT GAC GAA-39 (nt 2391 to 2408); reverse primer, 59-TGG ACT CCC TTT TCT TTT GTA GGA-39 (nt 2466 to 2443); and PrimeTime probe, 59 6FAM-TGA GCT CAG GGA ATA TGA AAG ACA AGC ATC G-39 Iowa Black FQ (nt 2411 to 2441). Premix Ex Taq (Takara Bio USA, Madison, WI) was used for qPCR following a standard protocol. 2.5 ml of extracted DNA was used in a reaction volume of 25 ml.