Cytochalasin-D treated cells were also infected with BTV-1 for 1 h in the presence of the drug (see methods). Mock-treated cells were infected in parallel. After the 1 h incubation with virus the cells were washed to remove excess unbound virus and inhibitor, and infection continued for a further 11 h. At the end of infection the cells were processed for confocal microscopy using NS2 to detect infected cells. The effects of cytochalasin-D on actin filaments are reversible and it is possible that following drug washout, virus that remained bound to the outsides of the cells (as a result of actin disruption) may cause an infection. Therefore ammonium chloride was added to the culture medium immediately after the virus inoculums and cytochalasin-D was removed. Ammonium chloride is a membrane permeable weak base that rapidly raises endosomal pH [57] and was added to prevent further infection events by virus that have not yet entered the cell and undergone acid-induced membrane penetration. Under these conditions infection can only occur during the period when cytochalasin-D was present. To control for possible effects on intracellular virus replication, cytochalasin-D was also added to other cells immediately after the virus inoculums were removed and co-incident with the addition of ammonium chloride. Under these conditions, cytochalasin-D can only have an inhibitory effect on infection by interfering with post-entry virus replication. Figure 6E shows that pre-treatment of BHK cells with cytochalasin-D inhibited BTV-1 infection. In contrast, when added after the virus internalisation step no inhibitory effect was seen (Fig. 6E) . Similar results were obtained using latrunculin-A in place of cytochalasin-D (Fig. 6E) . These results confirm that BTV-1 infectious entry into BHK cells is actin dependent.