22Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne 23 zoonotic diseases, thereby posing a serious threat to human health. Although JEV is 24 potentially neurotropic, its pathogenesis and distribution in the host have not been 25 fully elucidated. In this study, an infected mouse model was established using a 26 highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, 27 combined with anatomical imaging of the mouse brain, were used to dynamically 28 localize the virus and construct three-dimensional (3D) images. Consequently, onset 29 of mild clinical symptoms occurred in some mice at 84h post JEV infection, while 30 most mice displayed typical neurological symptoms at 144h post infection. Moreover, 31 brain pathology revealed typical changes associated with non-suppurative 32 encephalitis, which lasted up to 192h. The earliest detection of viral antigen was 33 achieved at 72h post infection, in the thalamus and medulla oblongata. At 144h post 34 infection, the positive viral antigen signals were mainly distributed in the cerebral 35 cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 36 192h post infection, the antigen signals gradually decreased, and the localization of 37JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was 38 detected in the brain at 504h post infection. In this model, the viral antigen was first 39 expressed in the reticular thalamic nucleus (Rt), at a consistent concentration. The 40 expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory 41 nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images 42 showed that viral signals were mostly concentrated in the parietal cortex, occipital 3 / 28 43 lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in 44 mice, a large number of viral antigens were detected in denatured and necrotic 45 neurons, suggesting that JEV directly causes neuronal damage. From the time of its 46 entry, JEV is widely distributed in the central nervous system thereby causing 47 extensive damage. 48Author summary 49 There are many theories regarding the mechanism of entry of the Japanese 50 encephalitis virus (JEV) into the nervous system. The inflammation cascade effect, 51 resulting from the virus entering the central nervous system (CNS), is a major cause 52 of brain injury in JEV patients. In this study, we found that the earliest point at which 53 viral antigen was detected in the brain tissues following peripheral infection of JEV 54 was at 72h. The virus was located in the nerve nuclei of the thalamus and medulla 55 oblongata and, subsequently, viral antigens were found in the anterior olfactory 56 nucleus. At 96h post infection, the virus was extensively distributed in the brain 57 tissue, and at 144h-192h the viral antigen was widely distributed and highly 58 concentrated. The viral concentration detected in the ventromedial thalamic nucleus 59 (VM), d...