Virus

A strain of BEFV was obtained from the Kimron Veterinary Institute, Israel, which had been isolated from a cow (Bos taurus) in 2006 in Israel, and its genome fully sequenced [33]. On receipt of this isolate, designated (bovine/Israel/2005-6a), it was passaged once in T175 tissue culture flasks (Greiner Bio-One, Stonehouse, UK) on 80% confluent African green monkey kidney (Vero) cells with Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich, Gillingham, UK) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich), with either 5% heat-inactivated fetal calf serum (+ FCS) or without FCS (− FCS) (Thermo Fisher Scientific, Loughborough, UK). Infected cells were incubated at 37 °C, 5% CO₂ until a 100% cytopathic effect was observed. The cells were then harvested by centrifugation at 1000 g at 8°C for 10 min and the supernatant collected in 1.5- to 3-ml volumes and stored at − 80 °C. Cell pellets were also collected, re-suspended in 5–7.5 ml DMEM and stored at − 80 °C. The infectious titer of the strain (cultured both with FCS and without FCS) was determined by a tenfold endpoint titration run in 96-well flat-bottom microplates (Thermo Fisher Scientific), in quadruplet, on a monolayer of Vero cells in 100 µl DMEM supplemented with 1% penicillin/streptomycin and 10% FCS; this was expressed as the 50% tissue culture-infective dose (TCID₅₀) calculated by the Spearman–Karber method [36]. The infectious titer recorded in Vero cell culture for BEFV + FCS and BEFV − FCS was 7.5 log₁₀ TCID₅₀/ml and 6.6 log₁₀ TCID₅₀/ml, respectively.