Sera collected both prior to challenge and at euthanasia were analysed for binding to HeV sG protein using a Luminex microsphere assay. Assays were performed on a Bio-Plex Protein Array System (Bio-RadLaboratories, Inc., CA, USA) as previously described [22] . Bio-Plex Manager Software (v 4.1) (Bio-RadLaboratories, Inc., CA, USA) was used for data acquisition and analysis. All samples were assayed simultaneously for each mouse strain. A strong positive control was assayed alongside all samples as was a negative control. A positive result was defined as samples occasionally seen in microglia, identified with labelling for Iba1 (green) in olfactory bulb at day 9 PI. HeV antigen labeling (arrow) was discrete and circumscribed, consistent with its presence within a cellular compartment such as a lysosome. (E) Section of olfactory bulb showing a capillary amongst HeV infected cells at day 10 PI. HeV antigen (red) and GFAP (green) are not colocalised. The endothelial cell cytoplasm (arrow) is negative for HeV antigen. Scale bars = A) 50 mm, B) 10 mm, C) 15 mm, D) 10 mm and E) 10 mm. doi:10.1371/journal.pone.0040308.g003 with a median fluorescence intensity (MFI) greater than the mean of all pre-challenge samples plus 3 x the standard deviation, MFI .406.