Evidence of Hepatitis E Virus Breaking Through the Blood–brain Barrier and Replicating in the Central Nervous System

R. Shi; M. H. Soomro; R. She; Y. Yang; T. Wang; Q. Wu; H. Li; W. Hao

Disclosures

J Viral Hepat. 2016;23(11):930-939. 

In This Article

Results

Evaluation of HEV Antigen and Hepatic Function Indexes in Serum

Using ELISA assay, HEV antigen was detected in the serum samples of inoculated gerbils from 7 dpi to 56 dpi (Table 1). The ALT, AST and T-BIL levels in serum of the experimental groups were significantly higher than the control groups (P < 0.05, or P < 0.01). The peaks of ALT, AST and T-BIL levels of inoculated group occur between 21 dpi and 42 dpi. The detailed data concerning the ALT, AST and T-BIL levels in our research were showed in published literature.[24]

Detection of Positive- and Negative-strand HEV RNA in Gerbil Brain and Spinal Cord

Positive-strand and negative-strand HEV RNA were detected simultaneously in the CNS tissues of inoculated gerbils from 7 dpi to 28 dpi, with test results summarized in Table 1 (Figs 1 & 2). All the brain and spinal cord from gerbils in control group were tested negative for HEV RNA.

Figure 1.

PCR for positive-strand HEV RNA in the CNS: ConB, control brain; B4–6, HEV inoculated brain; ConS, control spinal cord; S4–6, HEV inoculated spinal cord; −, negative control; +, positive control; M, marker (from 100 bp to 2000 bp).

Figure 2.

PCR for negative-strand HEV RNA in the CNS: B, HEV inoculated brain; S1 and S2, HEV inoculated spinal cord; −, negative control; +, positive control; M, marker (from 100 bp to 2000 bp).

Quantitative Real-time PCR Results

The linear correlation (R2) of the standard curve established with cloned amplicons was 0.999, the slope of the standard curve was −3.380, and the intercept was 45.34. The HEV viral load in the brain and spinal cord samples is shown in Table 1. The HEV viral load in the spinal cord samples was higher than it in the brain at each time point. For the liver, HEV RNA was detected from 7 dpi to 42 dpi, and the viral load peaked at 28 dpi (7.73 logs g−1), which was detailed in our published research.[24]

Immunohistochemical Staining for HEV Antigen

With immunohistochemical staining, the localization of brown HEV ORF2-positive signal was observed in brains and spinal cords detected as HEV RNA positive. In the brain, HEV ORF2 protein expression was targeted in the cytoplasm of neurons (Fig. 3d, arrows), ependymal epithelium (Fig. 3d, arrowhead), choroid plexus areas (Fig. 3e), perivascular area (Fig. 3f and g), Purkinje cells (Fig. 3h) and some mononuclear cells in the granular layer of cerebellum (Fig. 3i). In the spinal cord, positive staining was detected in the ependymal epithelium of central canal (Fig. 3j and k, arrows) and cytoplasm of neurons in the grey matter (Fig. 3j and k, arrowhead), perivascular area in the white matter and spinal pia mater (Fig. 3l arrowhead and arrow, respectively). No positive signal was detected in brain or spinal cord of gerbils from control group (Fig. 3a,b,c).

Figure 3.

Immunohistochemical staining for HEV ORF2 antigen. No positive signal was detected in brain or spinal cord of gerbils from control group (a, b, c): hippocampus and ependymal epithelium (a, arrowhead and arrow, respectively); Purkinje cell layer and choroid plexus (b, arrowhead and arrow, respectively); spinal central canal (c, arrow). In brains detected as HEV RNA positive, HEV ORF2 protein expression was targeted in the cytoplasm of neurons and ependymal epithelium (d, arrows and arrowhead, respectively), choroid plexus areas (e, arrow), perivascular area (f and g, arrows), Purkinje cells (h, arrow) and some mononuclear cells in the granular layer of cerebellum (i, arrows). In the HEV RNA-positive spinal cord, positive staining was detected in the ependymal epithelium of central canal (j and k, arrow), cytoplasm of neurons in the grey matter (j and k, arrowhead), perivascular area in the white matter and dorsal median septum (l, arrow and arrowhead, respectively).

Histopathological Changes of Brain and Spinal Cord

In the control group, no obvious histopathological changes were observed (Fig. 4a–d). For the gerbils detected as HEV positive, varying degrees of pathological changes in the brain and spinal cord were observed, including multifocal vacuolar degeneration and necrosis of neurons, neuronophagia (Fig. 4e–h), massive inflammatory cells infiltrating to the ependymal epithelium (Fig. 4k), choroid plexus region (Fig. 4l), perivascular area (Fig. 4m and n) and severe necrosis of Purkinje cells in the cerebellum (Fig. 4o). At 14 dpi, typical 'perivascular cuff' (Fig. 4m) and microglia nodules (Fig. 4i and j) were observed in the brain of gerbils 14B and 14E, and central canal haemorrhage (Fig. 4p) was found in the spinal cord of gerbil 14B.

Figure 4.

Histopathological analysis of brain and spinal cord tissues. (a–d) The normal structure of the CNS tissue from control group gerbils: cerebrum (a); hippocampus (b, *) and ependymal epithelium (b, arrowhead); cerebellum Purkinje cell layer (c, arrow); spinal cord (d). (e–p) Various pathological changes observed in HEV RNA-positive brain and spinal cord sections, brain (e–g, i–o), spinal cord (h and p): neurons degeneration and necrosis (e–h, arrow); neuronophagia (e–h, arrowhead); microglia nodules (i and j, arrow); inflammatory cells infiltrating to the ependymal epithelium (k, arrowhead); choroid plexus region haemorrhage and inflammatory cells infiltration (l, arrow and arrowhead, respectively); 'perivascular cuff' formation (m and n, arrow); Purkinje cells necrosis (o, arrow); haemorrhage in dorsal median septum and central canal of spinal cord (p arrowhead and arrow, respectively).

Ultrastructural Analysis Through Transmission Electron Microscope

Transmission electron microscope (TEM) was used to evaluate the ultrastructure of brain and spinal cord and focused on the components of BBB including vessel endothelium, basal membrane layers and the junctional complexes. No apparent lesions were observed in the control groups. The nucleus of the neurocytes was round and big with majority of euchromatin. Organelles in the cytoplasm were abundant and well organized. The blood vessel wall was smooth with uniform thickness, clearly defined layers and compact junctional complexes between the endothelium (Fig. 5, Fig. 6).

Figure 5.

(a–d) Normal ultrastructure of spinal cord from control group gerbils: nucleus (a, arrowhead), longitudinal section of myelin sheath (b); blood vessel with clearly defined layers and endothelial nuclei (c, arrowhead and arrow, respectively); compact junctional complexes (d, arrow). (e–h) Ultrastructural examination of HEV RNA-positive spinal cord sections: karyopyknosis (e, arrow); degeneration of myelin sheath (f, arrows); rough inner surface of capillary endothelium (g and h, arrows); swelling and degenerative mitochondrion in endothelial cytoplasm (g, arrowhead); loss of endothelial junctional complexes (h, arrowhead).
Correction added on 15 July 2016, after first online publication: Figure 5 was previously incorrect and is now corrected in this version.

Figure 6.

(a–d) No apparent lesions was observed in the brain section from the control group gerbils: round nucleus with majority of euchromatin (A, arrow); mitochondria with abundant cristae (b, arrow); smooth blood vessel wall with uniform thickness (c, d); compact endothelial junctions (d, arrowhead). Various ultrastructural changes were observed in HEV RNA-positive brain section: nucleus distortion manifested as enlarged nucleus with prominent nucleoli and concave karyotheca (e–g, arrows); karyorrhexis (g, h, arrowhead); neuron vacuolation (i, arrow); rarefaction and vacuoles of the mitochondrial structure (j, arrow); endothelial degeneration and necrosis (k, l, arrowhead); nucleus crescent formation as an indication of apoptosis (k, arrow); coarse vessel walls with disorganized layers and convex, fractured endothelial membrane (m, n, arrows); endothelial junction complexes break (o, arrowhead); lymphocytes infiltration formed the perivascular cuff (p, arrows).

Ultrastructural examination of HEV RNA-positive brain and spinal cord from the experimental group revealed degeneration and necrosis of neurocytes, which were characterized by cytoplasmic density reduction, rarefaction and vacuoles of the mitochondrial structure, break and disappearance of the mitochondrial cristae, karyopyknosis and karyorrhexis. It is worth noting that nucleus distortion manifested as enlarged nucleus with prominent nucleoli and concave karyotheca was apparently and commonly seen in HEV-positive brain tissue. Severe myelin sheath degeneration was observed in the spinal cord sections. Moreover, the vessel walls were coarse with swelling endothelium and disorganized cellular and acellular layers, and the tight junctions associated with BBB were defective. Besides, part of the endothelium membrane protruded into the vessel lumen and some of it broke. The electron density in the endothelium cytoplasm was low and the mitochondria in it were swollen with thin and sparse cristae (Fig. 5, Fig. 6).

ZO-1 and GFAP Expression Analysis in the Brain

In the brain of HEV-positive gerbils, the blood–brain barrier-associated protein ZO-1 was determined with immunohistochemistry method and the positive area density decreased significantly compared with control group (P < 0.05). However, using immunohistochemistry staining of GFAP, astrocyte endfeet enveloped the blood vessels was found obviously thickened and the positive area density was significantly increased in comparison with control group (P < 0.05) (Fig. 7).

Figure 7.

ZO-1 and GFAP expression analysis in the brain: IHC staining of GFAP in control group brain sections (a) and HEV RNA-positive brain sections (b); IHC staining of ZO-1 in control group brain sections (c) and HEV RNA-positive brain sections (d); area density analysis of GFAP and ZO-1 expression is demonstrated in (e).

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