Fluorescent confocal microscopy confirmed that NS4 is expressed in both BTV and GIV infected cells, and starts to accumulate in the cytoplasm and nucleus (as fine aggregates) as early as 4 hours post-infection. However, at 72 hours postinfection NS4 was associated with the cell membrane. This is consistent with analyses suggesting similarities between NS4 and ER-lipid-or membrane-associated proteins [37] . Cells infected with BTV or GIV or transfected with plasmids expressing NS4, showed interaction of NS4 with lipid droplets within the cytoplasm. This is consistent with bioinformatic analysis that identified similarities between NS4 and lipid-associated domains. Similar data were recently reported for VP2, VP6 and NSP5 of rotavirus [39] . . Colorimetric assay to detect interactions of NS4 with dsRNA. The graph shows colorimetric OD readings plotted against concentrations of dsRNA. Increasing concentrations (from 1 to 640 ng) of a biotinylated dsRNA were bound to wells coated with streptavidin. BTV NS4 or GIV NS4 were added to the wells in triplicate. Wells not containing dsRNA/NS4 were included as negative controls. Wells from which dsRNA was omitted, but in which NS4 (BTV or GIV) alone was incubated were also included as controls. Only wells containing the dsRNA to which GIV NS4 was added reacted with anti-GIV antibodies as indicated by increasing OD readings. The readings were almost linear (reaching a plateau at 320 ng of dsRNA) indicating that dsRNA acted as a target for binding of GIV NS4. doi:10.1371/journal.pone.0025697.g023