The data above clearly show that a previously uncharacterized BTV protein, here referred to as NS4, is a non-structural protein that localises to the nucleolus of infected cells. Next, we generated by reverse genetics BTV NS4 deletion mutants in order to assess the requirement of this protein for viral replication. We generated a set of plasmids necessary for the rescue of BTV-1 and BTV-8 and engineered three mutations in the plasmids containing segment 9 of BTV-1 and BTV-8 such that the NS4 initiation codon was removed along with the introduction of two stop codons in the NS4 coding sequence. All the mutations introduced were designed in order to leave the VP6 amino acid sequence unaltered ( Figure 4A ). As a negative control for BTV rescue, we designed a VP6 deletion mutant with a premature stop codon incorporated into the VP6 coding sequence (position 79). As shown in Figure 4B , viable BTV1-DNS4 and BTV8-DNS4 were rescued with similar efficiency to the respective wild-type (wt) viruses, upon transfection of RNA transcribed in vitro from the appropriate plasmids representing the genomic segments of wt or mutated BTV-1 and BTV-8. As expected, BTV-1DVP6 and BTV8-DVP6 could not be rescued.