1D), as well as the neural cell lineages were further confirmed by the expression of specific NPC markers, including N-cadherin (Fig. 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 A-1165442 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. Introduction 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]. Patients infected with Japanese encephalitis virus (JEV) who do not obtain the proper vaccination or treatment may develop acute encephalitis and have a high mortality rate [2]. Neuropathological features found in JEV-infected brains include multiple foci of acellular necrotic plaques in gray matter areas, such as the cerebral cortex, hippocampus, thalamus, substantia nigra and medulla oblongata [3], [4], [5], [6]. 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 also be detected in astrocytes, microglia, vascular endothelial cells and ependymal cells [7]. Although immunohistochemical studies indicate the correlation of Japanese encephalitis and severe neuron loss, direct evidence is still lacking as to whether primary A-1165442 viral infection or a secondary immunological cytokine storm causes the death of neurons. In addition, the cell identification of the most reported JEV tropism in autopsied brains or primary cultures relies on the morphological features of infected cells [6], [7], [8], [9]. Further confirmation is required by using human neural cells for JEV primary infection and applying immunological double-staining with both viral antigens and cell lineage-specific markers. Here we used human embryonic A-1165442 stem cell (hESC)-derived neuroepithelial precursor cells (NPCs) and functional mature neural cells to investigate the cell tropism of JEV infection. The hESC-derived specific NPCs and mature neural cells can be sequentially generated as embryonic developmental stages, and the derived neurons faithfully recapitulate the same neurophysiological properties as ex vivo neuronal cells [10], [11], [12]. Here, we carefully evaluate the infectivity and cell tropism of JEV in early-stage NPCs and late-stage mature neural cells by using a Taiwan-isolated neurovirulent JEV strain [13]. Our results show that NPCs and glial cells, but not mature neurons, are the primary targeted cells for JEV infection in humans. Materials and Methods Viruses and titer determination A plaque-purified neurovirulent RP-9 JEV strain [13] was a gift from Dr. Yi-Ling Lin at Academia Sinica Taiwan and amplified in mosquito C6/36 cells, which were cultured in RPMI 1640 A-1165442 medium with 5% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA). Baby hamster kidney fibroblast cells (BHK-21) were grown in RPMI 1640 medium containing 5% RB FBS and 2 mM L-glutamine and used for the viral plaque assay. JEV titer was determined by the number of JEV plaques in infected BHK-21 cells on fourth day post-infection (4 d.p.i.), revealed by crystal violet staining. hESC cultures The TW1 hESC lines (XY, passages 80C90) were grown in mTeSR1 media (Stem Cell Technologies, Vancouver, BC, Canada) on 1% Matrigel (Becton Dickinson, BD, Franklin Lakes, NJ, USA) coated 6 cm dishes (Corning, Corning, NY, USA). The TW1 cells have been previously described [14] and were obtained from Lee Women’s Hospital in Taiwan. Following the Policy Instructions on the Ethics of Human Embryo and Embryonic Stem Cell Research, the Institutional Review Board of the Industrial Technology Research Institute, Hsinchu, Taiwan approved the establishment of hESC lines from surplus blastocysts donated by Taiwanese infertile couples undergoing IVF treatment at Lee Women’s Hospital, Taichung, Taiwan. Both parties of each couple signed an informed- consent form after receiving oral and written information about the research [14]. The culture medium for the hESCs was refreshed daily. The cells were passaged using dispase II (0.5 mg/ml, Invitrogen) and replated at a cell dilution of 15 to 18. Neural induction After being treated with dispase II for 5 min,.