title: Characterization and purification of recombinant bovine viral diarrhea virus particles with epitope-tagged envelope proteins.
abstract: 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 E(rns), 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 E(rns) or E2 proteins, respectively. The expression of the epitope-tagged E(rns) 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 10(8.75) TCID(50) 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 E(rns) and E2 proteins at the virus membrane.
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Wegelt
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Ilona
authors.initials: I
authors.lastname: Reimann
authors.affiliation: Institute of Infectology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Granzow
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D019065:Virus Assembly; D014776:Virus Cultivation
chemical_list: D009842:Oligopeptides; D010455:Peptides; D014759:Viral Envelope Proteins; C076551:gp53, bovine viral diarrhea virus; C085706:FLAG peptide