Confirmed pGEX-BTVNS4 or pGEX-GIVNS4 plasmids were used to transform BL21 or C41 bacteria. A single colony of each plasmid was grown overnight (ON) in TSC/ampicillin, then used to seed 200 ml of fresh TSC/ampicillin. The bacteria were grown until OD600 0.5, then 0.5 mM IPTG was added for induction, for 4 hours at 37uC, or for 8 hours at 28uC. The bacterial cells were pelleted and processed using Bugbuster protein purification (Novagen) as previously described [26] . The soluble fraction of the fusion protein was purified by glutathione affinity chromatography using glutathione sepharose, as directed by the manufacturer (GE Healthcare). Proteins were analysed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% polyacrylamide separating gel (Miniprotean III) with a 3% stacking gel and stained with Coomassie brilliant blue, as described Table 2 . Percentage amino acid identity values between NS4 of BTV, EHDV, AHSV, GIV, PHSV and YUOV. Amino acid identity ranged from 5% to50%. The highest identity exists between BTV and EHDV (50%) followed by PHSV and YUOV-1 (30%). Amino acid identity in NS4 between the tick-borne and insect-borne viruses ranged between 5% and 18%. doi:10.1371/journal.pone.0025697.t002 previously [21] . The purified fusion protein was used to immunize rabbits (Harlan) with an initial injection, followed by 4 boosts at 2 weeks interval in the presence of Montanide ISA50 (Seppic) as an adjuvant.