We confirmed, by western blotting and confocal microscopy, that the DNS4 mutants do not express NS4 but express levels of VP7 and NS2 comparable to the parental wild type viruses. It was also evident that in BSR cells BTV-8 expresses lower amounts of NS4 relative to BTV-1 ( Figure 4D and not shown). However, BTV-1 replicates better than BTV-8 in these cells and differences in the steady-state levels of VP7 between these two viruses were also observed ( Figure 4D ). In cells infected by BTV1-DNS4 or BTV8-DNS4, we found by electron microscopy all the ultrastructural features of BTV-infected cells (e.g. viral inclusion bodies, NS1 tubules, viral particles) ( Figure 4E ).