The six patients were all female and between 13 and 78 years of age (median 38 years). Five patients were apparently healthy prior to neurological disease, without any signs of immunodeficiency. One patient (patient 1) was therapeutically immunosuppressed after kidney transplantation .
Initially, four patients developed flu-like symptoms, including fever, followed by neurological signs, which progressed to loss of consciousness. One patient presented with right-sided weakness and unsteady gait. Death of the five previously healthy patients occurred between 2 and 6 weeks after admission to the hospital. The immunosuppressed patient 1 developed a severe axonal motor neuropathy with Guillain–Barré syndrome-like spread on post-transplant day 80. Symptoms progressed to tetraplegia and deficit of all cranial nerves, followed by persistent coma and death 14 weeks after initial symptoms. For detailed clinical presentation, see Table 1.
Findings of the general organ autopsies
General autopsy of patient 1 revealed signs of multiple organ failure. Both autologous kidneys displayed high-grade atrophy with underlying mesangioproliferative glomerulonephritis. The transplanted kidney revealed no clear signs of rejection, but showed numerous nodular lymphocytic infiltrates, containing CD20- and CD3-positive cells, whereof the latter were composed of approximately equal proportions of CD4- and CD8-positive cells. Underlying diseases including atherosclerosis of aorta and coronary vessels plus myocardial hypertrophy and fibrosis as well as chronic pulmonary emphysema but no signs of a clinically suspected pneumonia were observed.
General autopsy of patient 2 revealed massive pulmonary edema and acute pulmonary congestion with consecutive acute biventricular cardiac dilation. Except for signs of shock, the other organs were without pathological findings. Likewise, in case of patient 6, no signs of inflammation were seen in the peripheral organs.
General autopsies were not conducted for the remaining patients, as the families gave consent to brain autopsy only.
Macroscopic examination of the CNS
Macroscopically, the brain of patient 1 showed a considerable increase in volume with softened texture. Transtentorial as well as tonsillar herniation was observed on both sides. Coronal brain dissection revealed livid discoloration of the cerebral cortex with areas of an indefinite cortico-medullary junction. The ventricular system appeared constricted at full-length but without midline shift. Cerebellum, brain stem, and spinal cord were increased in volume, too. The brain of patient 2 appeared macroscopically unremarkable. In the brain of patient 3, the basal nuclei were softened on both sides. Globally, the brain of patient 4 appeared softened with mild edema. Incipient tonsillar herniation was noted. After coronal dissection, the cerebral and cerebellar tissue were of softened texture with accentuation of the cortical areas. The brain of patient 5 appeared edematous with discretely prominent cerebellar tonsils but without distinct tonsillar herniation. After coronal brain dissection, the ventricular system was slightly constricted but without any midline shift. Malacia of the hippocampal region was noted on both sides. The brain of patient 6 showed uncal herniation on the left side, but without considerable compression of the midbrain. Coronal sections of the cerebrum displayed slight edema of the subcortical white matter.
Microscopic examination of the CNS and PNS
Cerebral tissue of all six patients exhibited a non-purulent panencephalomyelitis with edematous and spongy texture as well as perivasculary accentuated infiltration of lymphocytes (Fig. 1a). The number of diffusely infiltrating lymphocytes was highest in patient 1, followed by patients 4 and 5, whereas the remaining patients showed substantially lower numbers. Even though lymphocytic infiltrates were found in all examined samples, the inflammation was accentuated in the basal nuclei of patients 3 and 4 and hippocampal area of patient 5. In the CNS of patient 6, inflammation was mostly pronounced in the upper brain stem and subcortical nuclei. The brains of patients 1 and 2 showed a more global diffuse infiltration with no clear accentuation. Diffusely distributed macrophages, filled with eosinophilic, PAS-positive material, and plasma cells were observed most prominently in patient 1. Beyond formation of microglial nodules, all brains demonstrated distinct astrogliosis with remarkably enlarged reactive astrocytes exhibiting enlarged nuclei with thin and marginal chromatin and opaque-eosinophilic cytoplasm (Fig. 1c). In patient 1, global malacia with tremendous loss of neurons with remaining nuclear debris was observed. In patient 4, severe malacia was noted in the hippocampus and in the cortex of frontal and occipital lobe. The loss of neurons was less pronounced in the brains of the remaining patients. In all patients, neurons and astrocytes showed eosinophilic, spherical, intranuclear inclusions without clear halo, so-called Joest–Degen inclusion bodies (Fig. 1b).
The majority of infiltrating lymphocytes were CD3-positive (Fig. 1d), with more CD4-positive than CD8-positive cells. Furthermore, IHC revealed numerous CD68-positive macrophages and strong microglial (Fig. 1e) and glial activation, demonstrated by Iba1 and GFAP immunostaining.
Brain stems and spinal cords displayed inflammatory changes as well whereas the cerebella were milder affected. Here, a global, almost complete loss of Purkinje cells with Bergmann glia proliferation was observed in patients 1, 2, 4, and 6, which was considerably milder in patients 3 and 5.
Sections of peripheral nerves of patient 1 showed massive necrotizing lymphocytic neuritis (Fig. 1i) with CD4- and predominantly CD8-positive lymphocytes. Small nerves of peripheral organs in patients 1 and 2 appeared inconspicuous, though.
For local distribution of lymphocytic infiltration, microglial activation, edematous, and hypoxic changes, see Table 2 and Fig. 2.
Immunohistochemistry of BoDV-1 nucleoprotein and BoDV-1 RNA in situ hybridization
BoDV-1 nucleoprotein and RNA were detected in all examined CNS tissue samples including the spinal cord of all six patients, with less BoDV-1-positive cells detectable in the brain of patient 3 as compared to all other patients. Immunoreaction and RNA signal were present in neuronal cells, astrocytes, and oligodendrocytes. The neurons showed somatic as well as axonal and dendritic positivity (Figs. 1f, 3a).
Patient 1 showed a strong diffuse BoDV-1 distribution in all examined samples of cerebrum, cerebellum, and brain stem without clear focal accentuation. A uniform distribution of BoDV-1-positive cells was seen in patient 2, but with a smaller number of positive cells. Patients 3 and 4 showed a distinct accentuation of viral presence in the basal nuclei, primarily in neuronal, less in glial cells. In patient 4, samples of frontal, parietal, and occipital cortex and white matter showed a strong positivity in subcortical reactive astrocytes with only a small number of positive neurons and cortical glial cells. In patient 5, an accentuation of BoDV-1-positive neurons was noted in the hippocampal area on both sides. Interestingly, the occipital lobe showed a clearly lower number of BoDV-1-positive neurons and glial cells compared to other cortical and subcortical locations. In patient 6, the strongest immunoreaction was observed in the samples of the mesencephalon, cerebellum, and hippocampal region. Interestingly, the cortex of the occipital lobe showed a strong immunoreaction in the neurons as well as in the glial cells, whereas glial cells of the subcortical white matter were completely BoDV-1-negative. In all patients, the ependymal cells were partially BoDV-1-positive (Fig. 3b). For cerebral virus distribution, see also Fig. 2.
The pituitary gland of patient 2 showed a strong immunoreaction and RNA signal of the posterior pituitary compared to absence of BoDV-1 in the adenohypophysis (Fig. 1h) demonstrating the strong neurotropism of BoDV-1 in accidental dead-end hosts.
Furthermore, a positive immunoreaction and detection of BoDV-1 RNA were observed in all examined peripheral nerves of patient 1 (Figs. 1i, 3c). Diminutive nerves of visceral organs were BoDV-1-positive in both tested patients, including trachea and esophagus of patient 1 (Fig. 1g) and adrenal gland and thyroid gland of patient 2. No viral antigen or RNA was detected in the parenchyma and endothelial cells of visceral organs, though.
Co-staining of BoDV-1 nucleoprotein and RNA with cellular markers
To determine the cell types infected by BoDV-1, co-staining for the BoDV-1 nucleoprotein with either GFAP, synaptophysin, or S-100 was performed. In the CNS, BoDV-1 antigen was detected in synaptophysin-positive neurons and GFAP-positive astrocytes. In the PNS, S-100-positive Schwann cells were strongly positive for BoDV-1 antigen.
These observations were corroborated by BoDV-1 RNA in situ hybridization combined with IHC. NFpan-positive neurons (Fig. 3d), as well as GFAP-positive reactive astrocytes (Fig. 3e) and MBP-positive oligodendrocytes (Fig. 3f) showed strong BoDV-1 RNA signal. No RNA signal was seen in microglial cells (Fig. 3g), macrophages (Fig. 3h) and lymphocytes (Fig. 3i). In the PNS, viral RNA was associated with NFpan-positive axons and S-100-positive Schwann cells.
Transmission electron microscopy (TEM) of neocortical, subcortical, and brainstem specimens of patient 6 showed perivascular accentuated lymphomonocytic inflammation at low magnification. Intranuclear inclusions were predominantly seen in astrocytes and oligodendrocytes (Fig. 4a, c). Higher magnification of these inclusions revealed concentric complexes of membranous structures, the architecture characteristic of viral RNA replication centers (Fig. 4b, d).