The above experiments required using a relatively high BTV-1 concentration (13 mg/ml), to allow virus detection during entry. This raises the possibility that the virus may have been forced to enter the cells via an endocytic pathway that is not normally used, or is non-productive for infection. Therefore we also investigated the effect of AP180C expression on BTV-1 infection at a low m.o.i., to encourage virus uptake by the most efficient route. At 12 h post-transfection, the cells were incubated with BTV-1 (m.o.i. = 1) for 1 h at 37uC. The cells were then washed to remove excess virus and infection continued for a further 11 h when the cells were fixed and processed for confocal microscopy. Cells expressing AP180C were identified using expression of the c-myc tag as described above. Infected cells were identified using a rabbit antibody (Orab1) that binds the viral NS2 protein (a marker of virus replication) and an Alexa-568 conjugated secondary antibody. Intense areas of NS2 labelling were detected within infected cells (Fig. 1M and N) . Cells expressing AP180C were scored for infection (indicated by labelling for NS2) and the results normalised to the results obtained for the non-expressing cell population. In the cells expressing AP180C the frequency of infection was similar to that of the non-expressing cell population, indicating that AP180C does not inhibit infection (Fig. 1O) . These results show that virus uptake by cells expressing AP180C leads to infection and support the conclusion that CME is not the major pathway used by BTV-1 for entry into BHK cells.