HEK293 cells were cultured on fifteen 150-mm plates in DMEM-10%FCS, and transfected with 15 mg of pIHBoV1 per dish using LipoD293 (SignaGen, Gaithersburg, MD). After being maintained for 48 h at 5% CO 2 and 37uC, the cells were collected, resuspended in 10 ml of phosphate buffered saline, pH7.4 (PBS), and lysed by subjecting them to four freezing (2196uC) and thawing (37uC) cycles. The cell lysate was then spun at 10,000 rpm for 30 min. The supernatant was collected and assessed on a continuous CsCl gradient. In brief, the density was adjusted to 1.40 g/ml by adding CsCl, and the sample was loaded into an 11ml centrifuge tube and spun in a Sorvall TH641 rotor at 36,000 rpm, for 36 h at 20uC.