INTRODUCTION
Japanese encephalitis (JE) is an acute zoonotic, mosquito-borne infectious disease caused by JE virus (JEV) infection. JEV is a single-stranded, positive-sense RNA virus, belonging to the genus
Flavivirus of the family
Flaviviridae (
1,
2). JEV is a neurotropic virus and infection causes an acute encephalopathy. JE commonly affects children in the South Pacific regions of Asia (
3,
4). Of nearly 70,000 cases of JE reported each year, ca. 20 to 30% of cases are fatal, and a high proportion of patients that survive have serious neurological and psychiatric sequelae (
5). Pathologically, JE is positively associated with severe neuroinflammation in the central nervous system (CNS) and the disruption of the BBB (
6). However, it is not clear whether blood-brain barrier (BBB) disruption is a prerequisite for or a consequence of JEV infection in the CNS.
The BBB is a physical and physiological barrier composed of cerebral microvascular endothelium together with astrocytes, pericytes, neurons, and the extracellular matrix (
7). Brain microvascular endothelial cells (BMECs) comprise the major component of the BBB, and the tight junctions (TJ) between BMECs determine and limit the paracellular permeability. The cytoplasmic TJ proteins, zonula occludens (ZO), interact with each other and connect the transmembrane TJ proteins occludin and claudins to the actin cytoskeleton. Occludin plays a key role in the formation of TJ complex and is sensitive to the modification in inflammation associated with oxidative stress, as recently reviewed (
8). Claudins are another important family of transmembrane proteins to form the TJ backbone and maintain the integrity of the BBB. Brain endothelial cells predominantly express claudin-3 and claudin-5, and the depletion of claudin-5 induces the disruption of the BBB in mice (
8). Together, these proteins and cells form an elaborate network that selectively regulates the transport of the compounds into and out of the brain (
7,
9–11). Cell adhesion molecules (AMs) are cell surface molecules that facilitate intercellular binding and communication (
12,
13). Intercellular cell adhesion molecule 1 (ICAM-1), vascular endothelial cell adhesion molecule 1 (VCAM-1), and platelet endothelial cell adhesion molecule 1 (PECAM-1) are responsible for recruiting leukocytes onto the vascular endothelium before extravasating to the injured tissues.
Many CNS diseases alter the function of the BBB (
14,
15). Most neurotropic pathogens can cause changes to BBB permeability, such as Nipah virus, JEV, rabies virus (RABV), West Nile virus (WNV), and mouse adenovirus type 1 (MAV-1) (
16–20). Some of these viruses (for example, Nipah virus and MAV-1) enhance BBB permeability by disrupting the TJ complex during infection of BMECs (
16,
21), while others (such as HIV) disrupt the TJ complex and enhance BBB permeability via induction of inflammatory cytokines or chemokines such as gamma interferon (IFN-γ), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), and IL-1β, which indirectly contribute to BBB breakdown (
20,
22). Human T-cell lymphotropic virus can infect endothelial cells and release proinflammatory mediators, facilitating the entry of virus into the CNS (
23). WNV, which also belongs to the genus
Flavivirus of the family
Flaviviridae and shares some common biological feature with JEV, may degrade the TJ proteins and increase multiple matrix metalloproteinases (MMPs), contributing to BBB disruption (
19).
In JEV-infected mice, clinical symptoms and mortality are associated with high virus titers in the brain, elevated production of inflammatory mediators, and BBB disruption (
24,
25). However, it is not known whether BBB disruption is a prerequisite or a consequence of JEV infection. In the present study, the C57BL/6 mice were infected with JEV via the tail vein. It was found that the virus actively propagated in the brain before BBB permeability was found to be enhanced. BBB permeability was accompanied by a dramatic increase in the level of inflammatory cytokines and chemokines. Brain extracts from mice showing clinical signs of JE could enhance BBB permeability
in vitro and administration of IFN-γ-neutralizing antibody ameliorated the enhancement of BBB permeability in JEV-infected mice. These data suggest that inflammatory cytokines and chemokines produced subsequent to CNS infection contribute to BBB disruption during JE pathogenesis.
MATERIALS AND METHODS
Virus and cells.
JEV P3 strain was used for both
in vitro and
in vivo studies. Virus was propagated in the brain of 1-day-old suckling mice intracerebrally with 15 μl containing 5 × 10
4 PFU of viral inoculum. When moribund, mice were euthanized and brains removed. A 10% (wt/vol) suspension was prepared by homogenizing the brain in Dulbecco modified Eagle medium (DMEM). The homogenate was centrifuged to remove debris, and the supernatant was collected and stored at −80°C (
26). A baby hamster kidney fibroblast cell line (BHK-21) was used for viral titration. Mouse BMEC (bEnd.3) was obtained from American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with fetal bovine serum (Gibco, Grand Island, NY).
Inoculation of mice with JEV.
Adult C57BL/6 mice (female, 6 to 8 weeks old) were obtained from the animal housing facility of the Chinese Academy of Sciences, Changsha, China, and maintained according to Committee for Protection, Supervision, and Control of Experiments on Animals guidelines, Huazhong Agricultural University. Mice were injected intravenously with 100 μl containing 105 PFU of JEV (strain P3) diluted with phosphate-buffered saline (PBS; pH 7.4). Control animals received sterile PBS by the same route. The time before intravenous injection is referred to as day 0. JEV-infected mice (six to seven mice) were sacrificed every day after JEV infection and continued for 7 days either for tissue sectioning or protein/RNA extraction.
Virus titration.
At the indicated times, mice were anesthetized and perfused with cold PBS through the left ventricle of the heart. The brain was then homogenized in 2 ml of cold DMEM, and virus in the supernatant was titrated by plaque assay on BHK-21 cells. BHK-21 cell monolayers were grown in 12-well plates and inoculated with serial dilutions of the virus preparation. After absorption for 60 min, virus inoculum was aspirated, and the cells were washed twice with PBS. An overlay consisting of DMEM containing 1.5% carboxy-methylcellulose (CMC; Wako) and 2% fetal calf serum (CMC-DMEM) was added to the cells, and the plates were incubated at 37°C in a CO2 incubator. After incubation for 5 days, the CMC-DMEM was removed, the cells were washed twice with PBS and then fixed and stained with a solution of 0.1% crystal violet and 10% formalin in PBS under UV light. After staining for 2 h, the cells were washed with water, and the plaques were counted. The virus titer was calculated from a virus dilution that produced 10 to 100 plaques per well and is expressed as PFU/ml. Virus titer in the serum was also determined by the same plaque procedure.
Measurement of BBB permeability.
BBB permeability was assessed either with Evans blue dye (EBD; 961 Da) or with sodium fluorescein dye (NaF; 376 Da). The EBD binds to serum albumin (69,000 Da) to become a high-molecular-weight protein tracer when injected into the circulation, whereas the NaF remains an unbound small molecule in the circulation. Mice were injected with 100 μl of EBD solution (2% in PBS) intravenously (
27). After 1 h, all injected mice were sacrificed and transcardially perfused with 20 ml of normal saline. Whole brains were then removed and photographed.
NaF is also utilized as a tracer molecule to evaluate BBB permeability with a modified technique (
28). The animals were injected with 10 mg of NaF in 0.1 ml of sterile saline, administered i.p. under anesthesia. After 10 min to allow circulation of the NaF, peripheral blood was collected. Serum (50 μl) was recovered and mixed with an equal volume of 15% trichloroacetic acid (TCA). After centrifugation for 10 min at 10,000 ×
g, the supernatant was recovered and made up to 150 μl by adding 30 μl of 5 M NaOH and 7.5% TCA. At the indicated times, mice were anesthetized and perfused with cold PBS through the left ventricle of the heart to flush out intravascular fluorescein. The brain tissues were homogenized in cold 7.5% TCA and centrifuged for 10 min at 10,000 ×
g to remove insoluble precipitates. After the addition of 30 μl of 5 M NaOH to 120 μl of supernatant, the fluorescence was determined using a BioTek Spectrophotometers (Bio-Tek Instruments, Wonooski, VT) with excitation at 485 nm and emission at 530 nm. Standards (125 to 4,000 μg/ml) were used to calculate the NaF content of the samples. NaF uptake into tissue is expressed as (μg of fluorescence spinal cord/mg of tissue)/(μg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection.
Quantitative real-time PCR analysis.
The left hemisphere of each mouse was collected and homogenized with DMEM. Total RNA from left hemisphere or cells was extracted with TRIzol (Invitrogen, Grand Island, NY). cDNA was synthesized by using ReverTra Ace qPCR RT kit (Toyobo, Japan) according to the manufacturer's instructions. Quantitative real-time PCR was performed using SYBR green (Invitrogen) on a StepOne Plus using StepOne software v2.2.2 (Applied Biosystems, Foster City, CA). The expression of mRNA was normalized with β-actin. A pair of primers, 5′-GGCTCTTATCACGTTCTTCAAGTTT-3′ and 5′-TGCTTTCCATCGGCCYAAAA-3′, was used for the JEV-C gene. To quantify the viral copy numbers, a standard curve was generated using the pcDNA3.0-HA/JEV-C gene plasmid as a template (the copy number ranged from 6.8 copies/μl to 6.8 × 107 copies/μl).
Luminex assay.
Blood was collected into serum separator tubes (Sarstedt, Nümbrecht, Germany) by retro-orbital bleeding. The serum was stored at −70°C until processing. The right hemisphere of each mouse was lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate) and protease inhibitor cocktail (Roche, South San Francisco, CA) and then centrifuged for 20 min at 12,000 rpm to remove debris.
Extracts from homogenized brains were analyzed simultaneously for the levels of preselected 13 mouse cytokines and chemokines using the Milliplex mouse cytokine magnetic kit (Millipore, Darmstadt, Germany) according to the manufacturer's instructions (
29). The 13 cytokines and chemokines analyzed included TNF-α, IL-1β, IL-4, IL-6, IL-10, IL-12p70, IL-17, IFN-γ, C-C motif ligand 2 (CCL2), CCL3, CCL4, CCL5, and C-X-C motif ligand 10 (CXCL10). Premixed magnetic beads conjugated to antibodies for all 13 analytes were mixed with equal volumes of brain extracts in 96-well plates. Plates were protected from light and were incubated on an orbital shaker overnight at 4°C, followed by washing of magnetic beads with 200 μl of wash buffer three times. Detection antibodies were then added to each well, and the mixtures were incubated at room temperature for 1 h. Streptavidin phycoerythrin was added to each well, and the mixtures were incubated at room temperature for 30 min. The magnetic beads were resuspended in sheath fluid, and plates were assayed on a Luminex-3D system. The data of median fluorescence intensity were analyzed using a five-parameter logistic method for calculating cytokine and chemokine concentrations in brain homogenates.
Transendothelial permeability assay.
A transendothelial permeability assay was carried out as previously described with modifications (
30). Mouse BMECs (b.End3) were cultured on 0.4-μm-pore-size Transwell filters until reaching 100% confluence. After treatments, FITC-dextran-10000 (10 kDa; Sigma-Aldrich, St. Louis, MO) was applied apically at 1 mg/ml for 30 min. Samples were removed from the lower chamber for fluorescence measurements with a fluorometer (excitation, 492 nm; emission, 520 nm).
Histopathology, IHC, and immunofluorescence.
Mice with signs of disease after infection were anesthetized with ketamine-xylazine (0.1 ml/10 g [body weight]) and perfused with PBS, followed by 4% paraformaldehyde as described previously (
31). Brain tissues were removed and paraffin embedded for coronal sections (4 μm). The sections were stained with hematoxylin and eosin (H&E) for histopathology. For immunohistochemistry (IHC), the sections were deparaffinized and rehydrated in xylene and ethanol. Endogenous peroxidase was quenched by incubation in 3% hydrogen peroxide, and antigen retrieval was performed in 0.01 M citrate buffer. Sections were then blocked and incubated with primary rabbit anti-occludin polycolonal antibody (pAb; Santa Cruz, Santa Cruz, CA), rabbit anti-claudin-5 pAb (Invitrogen), and rabbit anti-ZO-1 pAb (Sigma) overnight at 4°C. After a washing step, biotinylated secondary antibodies were applied. The avidin-biotin-peroxidase complex (VectaStain standard ABC kit; Vector Laboratories, Burlingame, CA) was used to localize the biotinylated antibody. Diaminobenzidine (DAB; Vector Laboratories) was utilized for color development. Negative control was performed by substituting primary antibodies with PBS. For antigen quantification, sections were photographed and analyzed using an Olympus BX41 microscope (Olympus, Tokyo, Japan). The integrated optical density of DAB signals was determined by using an Image-Pro Plus (Media Cybernetics, Bethesda, MD).
In immunofluorescence, the following primary antibodies were used: rabbit anti-MAP2 pAb (1:1,000; Proteintech, China), rabbit anti-IBA1 pAb (1:500; Wako, Japan), and rabbit anti-glial fibrillary acidic protein (GFAP) pAb (1:800; Dako, Carpinteria, CA). Sections were incubated with either one primary antibody, followed by incubation with secondary antibody conjugated with either Alexa Fluor 488 or Alexa Fluor 555. The same sections were then incubated with another primary antibody, followed by incubation with the appropriate secondary antibody. Sections were imaged by using a laser confocal microscope (Leica, Germany). The data were obtained and processed using Adobe Photoshop 7.0 software (Adobe Systems, Pasadena, CA).
Western blotting.
BEnd.3 cells and mouse brains were lysed in RIPA buffer and protease inhibitor cocktail, homogenized, and centrifuged at 10,000 × g for 10 min at 4°C. After centrifugation, the insoluble material was removed, and the total protein concentration in supernatant was measured with a BCA protein assay kit (Beyotime, China). Brain extracts or cell lysates were electrophoretically separated by using sodium dodecyl sulfate–8 to 15% polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Richmond, CA) and blocked for 1 h at room temperature in 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST). Membranes were then incubated overnight with either one of rabbit anti-ZO-1 (Sigma), rabbit anti-claudin-5 (Invitrogen), rabbit anti-occludin, rabbit anti-VCAM-1, rabbit anti-PECAM-1, rabbit anti-ICAM-1 (Santa Cruz), and mouse anti-β-actin (as loading control) antibodies. After extensive washing with TBST, the membranes were incubated with species-specific horseradish peroxidase-conjugated antibodies. Antibody binding was visualized using enhanced chemiluminescence reagents (Beyotime). All Western blots were quantified by using ImageJ, and the values represent the relative immunoreactivity of each protein normalized to the respective loading control. The data are presented as means ± the standard errors of the mean (SEM).
Neutralization of IFN-γ.
The time of intravenous injection with 105 PFU of JEV-P3 virus is referred to as day 0. Mice were injected intraperitoneally with 50 μg of neutralizing antibody (BioLegend, San Diego, CA) specific for IFN-γ or a rat IgG1 isotype control antibody (BioLegend) diluted in 200 μl of PBS at 0, 1, 2, and 3 days postinfection (dpi) with JEV. At 5 dpi, BBB permeability was determined based on the uptake of NaF. After animal euthanasia, brains were harvested, and the expression of TJ proteins in the brain was monitored by Western blotting.
Statistical analysis.
Data are expressed as means ± SEM, and the significance of differences between groups was evaluated by using a two-tailed Student t test or one-way analysis of variance, followed by Tukey's post hoc tests. Graphs were plotted and analyzed using GraphPad Prism (v5.0; GraphPad, La Jolla, CA).
DISCUSSION
Understanding the mechanisms of JEV pathogenesis in the CNS is critical to JE prevention and treatment. In the present study, we demonstrate that JEV entry into the CNS occurs prior to BBB dysfunction in a mouse model of JE. It is not the virus itself, but the inflammatory cytokines/chemokines that caused reduction of the expression of TJ proteins, resulting in the enhancement of BBB permeability. IFN-γ was found to be a key cytokine in this process. Indeed, neutralizing IFN-γ can ameliorate the disruption of BBB integrity in JE.
Some viruses require disruption of the BBB to enter the CNS while others can enter the CNS and then cause BBB disruption. WNV can infect endothelial cells, facilitating the entry of cell-free virus into the CNS without disturbing the BBB, although BBB disruption can be observed later, accompanied by the degradation of TJ proteins and an increase in MMPs (
38). In tick-borne encephalitis virus (TBEV) infection, the permeability of the BBB increased at later stages when high virus load was present in the brain, which means BBB breakdown was not necessary for TBEV entry into the brain (
39). Conversely, infection of human microvascular endothelial cells (HMEC-1) with dengue virus (DENV) can induce cell apoptosis, suggesting that DENV might directly cause BBB breakdown in order to gain access to the CNS (
40). HIV-1 and HIV-1 Tat protein have been shown to disrupt BBB integrity via modification of claudin-5, thereby allowing HIV-1 to enter the brain (
41). Thus, depending on the virus, loss of BBB integrity can be the gateway for viral entry into the CNS (HIV-1 and DENV) or it can be the consequence of CNS infection (WNV and TBEV). To determine whether BBB disruption is a prerequisite for or a consequence of CNS infection in JE, BBB permeability and JEV viral loads in the CNS were monitored in mice after intravenous infection. Our data show that JEV-infected mice do not exhibit signs of BBB leakiness until 4 dpi, whereas virus is detectable in the brain as early as 2 dpi, indicating that CNS infection occurs prior to BBB dysfunction, similar to the other two flaviviruses, WNV and TBEV (
39,
42,
43).
Different flaviviruses maneuver different pathways for neuroinvasion. Some viruses such as Murray Valley encephalitis virus and St. Louis encephalitis virus are speculated to enter the CNS via the olfactory pathway, which is independent of BBB dysfunction (
44). Several routes were proposed for WNV entry into CNS. Hematogenous route via crossing the BBB was suggested to be one of the major routes by which WNV enters the CNS since high viremia correlated with early WNV entry into the CNS, although the associated mechanisms remain unclear (
45). Retrograde axonal transport via peripheral nervous system was another route of WNV entering the CNS (
19). It was also suggested that WNV virus enters the CNS by infected monocytes, dendritic cells, or macrophages (
46). Moreover, the infection of endothelial cell
in vitro gives another possibility that WNV might cross the BBB via brain endothelial cells (
38). Similarly, the exact routes of JEV gaining entry into the CNS remain unclear. JEV may enter the CNS via a hematogenous route since the infection is diffuse throughout the brain (
47). Both macrophages and dendritic cells are capable of being infected by JEV; thus, it remains possible that they contribute to the transmission of virus into the CNS (
48–50). However, monocytes appear to have the ability to effectively control flavivirus infection, and
in vivo experiments have failed to identify infected microglia or infected infiltrating macrophages in animal models of flavivirus encephalitis (
25,
51). JEV could cross the BBB via endothelial transcytosis and/or endothelial infection, as reported previously (
24). However, there is little to no evidence
in vivo for endothelial infection by flaviviruses (
52). In our
in vitro model, JEV infection of the endothelial cell line b.End3 was extremely low, even at an MOI as high as 10. There was no significant change in the expression of proteins associated with tight-junction integrity (occludin, claudin-5, and ZO-1; data not shown). These observations are consistent with previous work showing that unequivocal endothelial infection was seen in only 1 of 167 mice following intraperitoneal infection with flavivirus (
52). Therefore, it seems unlikely that the primary route of CNS entry is through JEV-infected microvascular endothelial cells.
In our study, it was found that inflammation induced by JEV infection triggered BBB disruption in the CNS. Large amounts of chemokines and cytokines were produced in the CNS, including CXCL10, CCL2, CCL3, CCL4, CCL5, TNF-α, IL-6, and IFN-γ in mice infected with JEV. The inflammatory mediators observed during the course of infection were more of a Th1-type immune response. Indeed, the Th2 cytokines IL-4 and IL-10 were unchanged in the brain of JEV-infected mice. Importantly, the upregulation of these inflammatory mediators reached a peak just before BBB breakdown. Furthermore, the level of CXCL10 was dramatically elevated in the CNS almost immediately after infection, similar to the observations in mice during WNV infection (
53). Since neurons are the major target for JEV infection, it is possible that infected neurons are the early source of CXCL10 production as seen in other neurotropic virus infections (
53,
54). Coincidently, IFN-γ, a protein that induces the expression of CXCL10, was also upregulated early after infection and could further promote CXCL10 production. We observed that IFN-γ increased in both mixed glial culture and BV-2 cells (a microglial cell line) after JEV infection. Both glia and neurons could be the source of IFN-γ early in infection (
55). Although the ability of glial cells to be infected by flavivirus remains debatable, the studies by us and others clearly show that they are well activated during JE (
25,
56). Activation of glial cells results in the production of multiple inflammatory chemokines and cytokines (
25,
57–60). JEV-activated astrocytes have been shown to be a source of CCL5 and CXCL10 and important for the migration of NK cells and monocytes into the CNS (
56,
57). The local immune responses initiated by activated microglial cells may provide protection against JEV infection of the CNS; however, they are also the major source of TNF-α, which can be deleterious to neurons (
56). The elevated levels of the proinflammatory cytokines IL-6 and TNF-α and the chemokines CCL2 and CCL5 in the CNS could induce irreversible neuronal damage and correlates with an increased mortality rate (
25,
56).
The functions of chemokines and cytokines in the CNS extend beyond their role as mediators of neuroinflammation. Our data showing that brain extracts from mice with JE, but not virus alone, can increase the permeability of an
in vitro BBB model strongly suggest that cytokines and/or chemokines contribute to the BBB disruption
in vivo. Indeed, we went on to show that blocking IFN-γ
in vivo abolishes the increased BBB permeability associated with JEV infection. There are two pathways by which inflammatory cytokines can regulate BBB permeability. First, inflammatory cytokines can upregulate the endothelial AMs, which in concert with chemotactic chemokines, facilitate rolling and adhesion of leukocytes on the endothelial wall, and migration into the affected site (
61,
62). In the brains of JEV-infected mice, the expression levels of ICAM-1, VCAM-1, and PECAM-1 are significantly elevated, indicating the potential for increased recruitment of mononuclear leukocytes from the periphery to the CNS. Second, inflammatory cytokines or chemokines (IL-6, IFN-γ, CXCL10, and CCL2 to CCL5) can deteriorate BBB permeability via downregulation of the TJ proteins claudin-5, occludin, and ZO-1 (
19). Brain extracts from JEV-infected mice contain high levels of chemokines, cytokines, viruses, and soluble cellular proteins, and these extracts significantly enhanced BBB permeability
in vitro even when the virus was inactivated with UV. It has been reported that a recombinant RABV expressing a chemokine (CXCL10, CCL5, or CCL3) could enhance BBB permeability in mice (
63) and reduce the expression of TJ protein claudin-5, occludin, and ZO-1 in BMECs (
37). This is consistent with our mouse model of JE, where we show increased expression of CXCL10, CCL3, and CCL5, along with decreased expression of claudin-5, occludin, and ZO-1 in the CNS
in vivo. Furthermore, the enhancement of BBB permeability is chemokine and cytokine dependent in many other CNS diseases (
64). For example, CCL2 has been demonstrated to reduce the expression of ZO-1, ZO-2, occludin, and claudin-5 in HIV-1-infected BMECs through Rho and Rho kinase signaling (
65). In EAE, IL-17-induced reactive oxygen species activate myosin light-chain kinase and reduce expression levels of ZO-1 and occludin in BMECs (
66). For some reason, experiments
in vitro with transendothelial monolayers cannot represent the expression changes in claudin-5 that are observed
in vivo. It should be understandable that the
in vitro simplified system fails to replicate the precise cellular condition
in vivo. It has been reported that IFN-γ, but not TNF-α, is associated with enhanced BBB permeability through peroxynitrite-dependent radicals in RABV infection (
28). Consistent with these observations, neutralizing IFN-γ with antibodies ameliorated the enhancement of BBB permeability and partially restored the expression of TJ proteins in JEV-infected mice. Taken together, our data show that inflammatory mediators, particularly IFN-γ, play a central role in enhancing BBB permeability in JEV infection by downregulating TJ protein expression.
In summary, the evidence presented here demonstrates that inflammatory chemokines and/or cytokines in the CNS, but not JEV itself, mediate BBB breakdown during JE. As depicted in
Fig. 9, JEV invades the CNS without prior disruption of the BBB permeability where it infects neurons. Infected neurons may produce chemokines that can also induce the activation of glial cells, which in turn produce a preponderance of inflammatory chemokines and cytokines. These inflammatory mediators can break down the BBB by reducing the expression of TJ proteins and thereby damage the integrity of the tight junctions between microvascular endothelial cells. Inflammatory mediators can further compromise the BBB barrier by inducing increased expression of adhesion molecules on BBB endothelial cells, allowing for increased infiltration of inflammatory cells from the periphery to the CNS. Increased inflammatory infiltrates can lead to further neuroinflammation and neuronal injury. Nevertheless, advanced investigations are warranted to determine the pathways by which JEV invades the CNS and induces inflammation. Understanding the mechanisms of inflammation-induced BBB disruption in JVE infection may provide a potential target for therapeutic intervention in CNS diseases that are driven or exacerbated by a compromised BBB.