It has been reported that BTV-10 infects Hela and Vero cells using CME for entry [45] . Here we have investigated the involvement of the clathrin pathway in BTV-1 entry and infection of BHK cells using deletion mutants of AP180 (AP180C) and Eps15 (Eps15-Ed95/295; from here known as DN-Esp15) which act as DN inhibitors of CME and block uptake of transferrin [5, 6, 51] . BHK cells were transiently transfected to express AP180C-c-myc, GFP-DN-Eps15, or a control GFP-Eps15 (D3D2; from here known as 'Esp15 control') that does not interfere with the clathrin pathway [5, 51] . At 12 h posttransfection, the cells were incubated with Alexa-568 labelled transferrin for 15 minutes or with BTV-1 for 0.5 h. The cells were then fixed and processed for confocal microscopy (Fig. 1) . Virus was detected using PM10 (anti-VP5) and an Alexa-568 conjugated secondary antibody. Cells expressing Eps15 proteins or AP180C were identified by the GFP or c-myc fusion tags respectively (see methods) and are shown in green on figure 1. Cells were scored for transferrin or BTV uptake as described in the methods. For each DN protein, uptake of transferrin or BTV was normalised to the results obtained for the controls (see methods). In cells expressing GFP-DN-Eps15 the frequency of transferrin uptake was reduced by 53% when compared to cells expressing the Eps15 control (Fig. 1A , B, E, F and O) indicating a block to clathrin-mediated endocytosis. Similarly, the frequency of transferrin uptake was reduced by 82% in cells expressing AP180C when compared to cells of the non-expressing population (Fig. 1I, J and O) . In contrast, expression of DN-Eps15 (Fig. 1C , D, G, H and O) or AP180C (Fig. 1K , L and O) did not significantly inhibit virus uptake. In virtually all cells expressing the DN proteins the pattern of virus labelling was indistinguishable from cells expressing the Esp15 control or cells that were not expressing a transgene. These results show that under conditions where clathrin-mediated uptake of transferrin is inhibited BTV-1 is taken up normally by BHK cells.