BTV infection is known to be dependent on the low pH within endosomes [45] . Concanamycin-A is a potent and specific inhibitor of the vacuolar proton ATPase and is commonly used to raise endosomal pH [52] . Pre-treatment of BHK cells with concanamycin-A inhibited BTV-1 infection ( Fig. 2A and 2B ) confirming the requirement for active endosomal acidification. Similarly, when concanamycin-A was added coincident with the start of infection (i.e. at time 0 on figure 2C) a strong inhibitory effect was also seen. When added after the start of infection, concanamycin-A also had an inhibitory effect on infection. When added 2 h after infection was initiated, the frequency of infection was reduced by ,50% when compared to mock treated cells (Fig. 2C) . Near identical results were obtained using ammonium chloride in place of concanamycin-A (data not shown). These results suggest that BTV-1 is delivered to acidic endosomes much slower than expected for uptake by CME [53] .