The BVDV-1 NADL (courtesy of Dr. Ferrari, Istituto Zooprofilattico Sperimentale di Lombardia ed. Emilia Romagna, Brescia, Italy), BVDV-2232/02 [17 ], and HoBi-like Pestivirus Italy-83/10cp [21 (link)] cytopathogenic strains were used for immunization procedures (as inactivated preparations) and for the virus neutralization (VN) assays to assess the VN titer in immunized animals. All viruses were propagated in pestivirus-free Madin-Darby bovine kidney cell lines (MDBK; ATCC CCL-22), cultured in Dulbecco’s modified essential medium (DMEM) (Sigma-Aldrich, Schnelldorf, Germany) containing 10% fetal bovine serum (FBS, Gibco, Thermo Scientific, Rodano, Italy, tested negative for pestivirus or anti-BVDV antibody), 2 mM of L-glutamine, 100 IU/ml of penicillin (Sigma-Aldrich), 100 mg/ml of streptomycin (Sigma-Aldrich), and 2.5 mg/ml of amphotericin B. Cells were incubated at 37 °C with 5% CO2. Human embryo kidney cell lines (HEK293T; ATCC, CRL-1573) were cultured following the same procedure and used for transfection experiments.
For immunization, viral suspensions were inactivated with 0.05% β-propiolactone and emulsified initially with Tween-80 (4.1% v/v) and then with a mixture (50% v/v) of Montanide ISA 563 (Seppic Inc., Paris, France), Marcol 52 (Esso Italiana S.r.l., Rome, Italy), and Montane 80 (Seppic Inc.) in a 30:63:7 ratio, as previously described [22 (link)].
For immunization, viral suspensions were inactivated with 0.05% β-propiolactone and emulsified initially with Tween-80 (4.1% v/v) and then with a mixture (50% v/v) of Montanide ISA 563 (Seppic Inc., Paris, France), Marcol 52 (Esso Italiana S.r.l., Rome, Italy), and Montane 80 (Seppic Inc.) in a 30:63:7 ratio, as previously described [22 (link)].
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