Recombinant BTVs were rescued by reverse genetics as previously described [26] . Briefly, plasmids containing the genomic segments of BTV-1 or BTV-8 or resulting mutants were linearized with the appropriate restriction enzymes and then purified by phenol-chloroform extraction. Digested plasmids were used as a template for in vitro transcription using the mMESSAGE mMA-CHINE T7 Ultra Kit (Ambion), according to the manufacturer's instructions. ssRNAs were purified sequentially by phenol/chloroform extraction and through Illustra Microspin G25 columns (GE Healthcare Life Sciences), following the manufacturer's protocol. Monolayers of 95% confluent BSR cells grown in 12 well plates were transfected twice with BTV RNAs using Lipofectamine 2000 (Invitrogen). Firstly, 0.5610 11 (BTV-1) or 1610 11 (BTV-8) molecules of each of the BTV segments encoding VP1, VP3, VP4, NS1, VP6 and NS2 were diluted in Opti-MEM I Reduced Serum Medium containing 0.5 U/mL of RNAsin plus (Promega) and then mixed with Lipofectamine 2000 diluted in Opti-MEM I Reduced Serum Medium. After 25 min of incubation at room temperature, the mixture was added to the cells. 16 to 18 h after the first transfection, the cells were transfected as before but with all 10 BTV segments. 3 to 4 h after the second transfection the cells were overlaid with 2 ml of minimal essential media containing 1.5% agarose type VII and 2% FBS, and monitored for development of plaques. Finally, individual BTV rescued clones were picked through the agarose overlay and used to infect fresh BSR cells in order to obtain a virus stock. Where necessary, BTV dsRNA was extracted from infected cells using Trizol (Invitrogen). The ssRNA fraction was precipitated using lithium chloride, and the harvested dsRNA fraction was precipitated using isopropanol in the presence of sodium acetate.