Fig. 4

Groups of Balb/c mice were challenged with EEEV Pe-6 by the aerosol route and culled at pre-determined time-points, culled on welfare grounds, or were found dead. Histopathology (a) and immuno-histochemical demonstration of EEEV virus (b) images were obtained from multiple animals, represented here by two mice that illustrate key histopathological features of disease. Mice exposed to CM alone (mock infected) were found to have neuronal tissues within normal limits, with distinct nuclei in well-defined neuronal layers, as well as clear white matter tracts and nerves that were strongly eosinophilic in the olfactory bulb. Neurons were not vacuolated and perivascular spaces were also within normal limits for these tissues 1 day post-challenge. Mice exposed to EEEV presented with severe meningoencephalitis with cell death and degenerative changes in neurons and leukocytes in the encephalon, with marked rarefaction and/or oedema of the tissue by day 3 post-challenge. In the olfactory bulb, there was an abundance of shrunken neurons and cell debris (arrows), and rarefaction of the neural tissue (*). In the piriform cortex, there was abundant pyknotic and karyorrhectic debris in the vessel wall (*) and in the CNS (arrows), as well as considerable changes in the perivascular spaces of the meninges (M). Contralateral piriform cortex sections demonstrate EEEV-specific labelling in neurons (*). The hippocampus had marked vacuolation (*) and clear expansion of perivascular spaces (arrows), with diffuse EEEV-specific labelling of neuronal populations (*). Other tissues with diffuse EEEV-specific labelling included the cortex, hippocampus and thalamus. In the pons region of the brain there was a focal area of spongiosis/vacuolation (*), as well as neuronal degeneration, necrosis and abundant pyknotic and karyorrhectic debris (arrows). The mandibular lymph node (LN) displayed numerous tingible body macrophages (arrows) and had a marked increase in apoptopic bodies