Abstract
Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.
Highlights
Japanese encephalitis virus (JEV), which belongs to the flavivirus family and contains a positive-sense, single-stranded RNA genome, is a severe public-health threat in Asia [1]
On D10, over 90% of differentiating human embryonic stem cells (hESCs) consistently displayed a classical neural rosette conformation after plating on culture plates (Fig. 1D), and the neural cell lineages were further confirmed by the expression of specific neuroepithelial precursor cells (NPCs) markers, including N-cadherin (Fig. 1E), nestin (Fig. 1F), Pax6 and Sox1 transcription factors (Fig. 1G) [11]
We discovered that glial fibrillary acid protein (GFAP)+ glial cells (53¡14% of GFAP+ cells, n5742) (Fig. 4A) were much more susceptible than TuJ1+ neurons (1.4¡0.6%, n5711) (Fig. 4B) to JEV infection at multiplicity of infection (MOI) 1
Summary
Japanese encephalitis virus (JEV), which belongs to the flavivirus family and contains a positive-sense, single-stranded RNA genome, is a severe public-health threat in Asia [1]. Reactivated astrocytes and microglia nodules aggregate in the surrounding damaged inflammatory regions, which are accompanied by edema, hemorrhage and extensive perivascular inflammatory infiltrates [3, 7]. Neuronal cells, such as the pyramidal neurons in the hippocampus and spinal cord, have been reported as the primary target cells of JEV [2, 7]. Immunohistological observations reveal that the viral antigens can be detected in astrocytes, microglia, vascular endothelial cells and ependymal cells [7]
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