Figure 1.
In vivo BLM imaging of infection progress using WEEV.McM.FLUC.
A: 24 hpi B: 48 hpi C: 72 hpi D: Survival analysis of WEEV.McM.FLUC and wild-type virus (WEEV.McM) from subcutaneous and intranasal virus challenge experiments. Note uniform lethality resulting from intranasal exposure by WEEV.McM.FLUC. E: FLUC activity was quantitatively measured in each animal at 1, 2, and 3 d.p.i. time points. Results from BLM analysis demonstrate robust FLUC activity as infection progressed with the greatest increase observed between days 2 and 3 post-infection. F: Brains of animals infected with WEEV.McM attain a higher viral titer more rapidly when compared with WEEV.McM.FLUC. WEEV.McM.FLUC titers approach that of McM by 72 h.p.i. G: Regression analysis of viral titer versus FLUC activity. Linear regression line appears curved due to log10 scaling of axis as required to clearly depict all data points (R2 = 0.9807).
Figure 2.
Schematic depiction of anatomical organization of mouse brain in medial sagittal view.
AON: anterior olfactory nucleus, BF: basal forebrain, CC: cerebral cortex (isocortex), CP: caudate putamen, F: fornix, HIP: hippocampus, OB: olfactory bulb, VS: ventral striatum. Progress of infection with WEEV after intranasal inoculation (A–D). Whole heads were bisected along sagittal midline and imaged at 24 hpi (A), 48 hpi (B), 60 hpi (C) and 72 hpi (D). Luciferase activity pattern is consistent with dissemination along olfactory pathways. Regions consistent with initial infection of the nasal turbinates show pronounced FLUC activity at 24 hpi. However, nasal turbinate BLM activity is exceeded by signal from areas consistent with infection proceeding through olfactory information processing within the CNS, including the lateral olfactory tract, anterior olfactory nucleus, basal ganglia, thalamus, and cerebrum. Immunological markers of disease (MCP-1 and IP-10), resulting from WEEV.MCM.FLUC, are strongly induced and comparable to WEEV.McM at 3 d.p.i. (E–F).
Figure 3.
I. Extraneural lesions 24 hpi.
A) Focal erosion/necrosis of the olfactory mucosa with deciliation of the flanking epithelium and neutrophil infiltration into the mucosa and submucosa (Bar = 100 µm). B) IHC positive staining for FLUC is highlighted in a few neuropeithelial cells subjacent to a focal loss of olfactory mucosa (Bar = 200 µm). II- Neuroinvasion from olfactory nerve 48–72 hpi. C) Terminal end of olfactory nerve shows a digestion chamber (arrow) with occasional lymphocytes infiltrating vacuolated branches (Bar = 100 µm). D) Early immunoreactivity (anti-FLUC) in the main olfactory bulb involving scattered neurons in the external plexiform and granular layers (Bar = 100 µm). E) Sagittal section H&E showing the connection between olfactory nerve (ON) and main olfactory bulb layers affected by multifocal necrotizing lesions with associated status spongiosis and infiltration of neutrophils. Glom = glomerular layer; EPI = external plexiform layer; IPL = internal plexiform layer; and ONL = olfactory nerve layer at the ventrum of the olfactory bulb (Bar = 400 µm). F) Neuropil of the olfactory nerve layer shows a large vacuolar lesion (demyelination) with individual neuronal necrosis and infiltration of small numbers of neutrophils (Bar = 100 µm). G) Perivascular cuffs and multifocal gliosis in the glomerular layer 72 hpi (Bar = 200 µm). H) Close-up view of the congested glomerular vessels with pleocellular perivascular cuffs comprising moderate numbers of neutrophils, lymphocytes and glial cells (Bar = 100 µm).
Figure 4.
Later stage dissemination throughout the brain 72 hpi.
A) Expanded view of the olfactory bulb showing progression of virus into caudal regions of the brain with multifocal necrosis and secondary demyelination (arrows) (Bar = 1000 µm). B) Multifocal demyelination and positive immunoreactivity in the anterior olfactory nucleus (AON), ventral striatum (VS), basal forebrain (BF) thalamus, hypothalamus, midbrain, hippocampus (HIP), and cerebral cortex (CC). Fornix (F) and optic tract (circled in blue) do not show any immunoreactivity (Bar = 1000 µm). C) Neuronal immunoreactivity in caudal olfactory bulb that shows multifocal demyelinating lesions (Bar = 100 µm). D) Multifocal immunoreactivity in caudal olfactory bulb and anterior olfactory nucleus (Bar = 200 µm). E) Strong immunoreactivity in hippocampus (Bar = 100 µm). F) Hippocampal H&E showing focal loss of pyramidal neurons with mild gliosis (Bar = 100 µm). G) Multifocal areas of necrosis and demylelination in the cerebral cortex (Bar = 200 µm). H) Strong immunoreactivity in cerebral neurons and their dendrites revealing interneuronal spread (Bar = 100 µm). All IHC images within this figure are from anti-FLUC staining. Comparison images using anti-WEEV staining may be found in the supplemental information.
Figure 5.
Neuroinvasion from trigeminal nerve.
A) Cranial nerves including a branch of trigeminal nerve show neutrophilic perineuritis with a large glial nodule extending to the meninges of the overlying brain section (Bar = 200 µm). B) Close up of epineurium of cranial nerves infiltrated by neutrophils and lymphocytes (Bar = 100 µm). C & D) Strong immunoreactivity (anti-FLUC) of maxillary nerve including Schwann cells. Note strong and diffuse immunoreactivity of olfactory neuropeithelium, variable staining of surrounding skeletal muscles and bone marrow elements (Bar = 100 µm). E) Trigeminal ganglion IHC positivity (Bar = 100 µm). F) Trigeminal positivity is associated with immunoreactivity of the overlying meninges and brain tissue (Bar = 100 µm). G) Brainstem demyelinating lesion (potential consequence of trigeminal invasion) (Bar = 400 µm). H) IHC positivity (anti-FLUC) in the brain stem with interneuronal spread and rare immunoreactivity of glial cells (astrocytes) (Bar = 100 µm). All IHC images within this figure are from anti-FLUC staining. Comparison images using anti-WEEV staining may be found in the supplemental information.
Figure 6.
In vivo imaging assessment of liposome-antigen-nucleic acid-complex vaccination strategy.
A) Average radiance in vaccinated, untreated, and uninfected mice at 48 hpi. Treated animals show significantly reduced luciferase activity (p<0.01) compared to untreated animals. Error bars indicate standard deviation. B) Corresponding images used for the quantitative analysis. Scale was normalized for all images and is shown on the left-hand side. Images presented are from 48 hours post intranasal challenge with WEEV.McM.FLUC.