Characterization and purification of recombinant bovine viral diarrhea virus particles with epitope-tagged envelope proteins

Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. The lipid membrane of the virions is supposed to contain the three glycosylated envelope proteins Erns, E1 and E2, but detailed studies of virus assembly are complicated because no efficient purification method for pestiviruses has been described so far. In this study, we generated infectious BVDV with N-terminally FLAG-tagged Erns or E2 proteins, respectively. The expression of the epitope-tagged Erns and E2 proteins could be shown by immunofluorescence and Western blot experiments. Furthermore, an affinity tag purification protocol for the isolation and concentration of infectious BVDV was established. In the preparation with a titre of 108,75 TCID50 ml-1, spherical particles with a diameter of 43-58 nm (mean diameter: 48 nm) could be detected by negative staining electron microscopy, and immunogold labelling located both Erns and E2 proteins at the virus membrane.