The full-length VP2 genes of HBoV1, 2, 3, and 4 were cloned into the baculovirus expression vector pFastbac1 and expressed using Bac-to-BacH Baculovirus Expression System (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. VLPs were obtained as previously described [17, 37] . High Five cells (Invitrogen) were infected with recombinant baculoviruses at a multiplicity of infection (MOI) of five and harvested after three days. Cells were suspended in 25 mM NaHCO 3 solution at 2610 7 cells/mL and kept on ice for 30 min. After centrifugation at 18,000 rpm for 10 min at 4uC, (NH4) 2 SO 4 was added to the supernatants at a final concentration of 20% (w/v). Precipitants were harvested by centrifugation and dissolved in CsCl solutions with densities of 1.4 g/mL in Tris-EDTA buffer (10 mmol/L Tris, pH 8.7, 1 mmol/L EDTA, and 0.5% Triton X-100). After centrifugation at 35,000 rpm and 18uC for 39 h in a SW41 rotor centrifuge (Beckman Coulter, Fullerton, CA), the fractions were analyzed by SDS-PAGE and Western blot using mouse sera against HBoV VP2 proteins. A Tecnai12 transmission electron microscope (FEI, Hillsboro, OR) at 80 kV was used to verify the morphology of the VLPs. SDS-PAGE and Western blot analysis were used to identify the VLPs [18] .