Japanese encephalitis is characterized by profound neuronal destruction/dysfunction and concomitant microgliosis/astrogliosis. Although substantial activation of glia is observed in Japanese encephalitis virus (JEV)-induced Japanese encephalitis, the inflammatory responses and consequences of astrocytes and microglial activation after JEV infection are not fully understood. In this study, infection of cultured neurons/glia with JEV caused neuronal death and glial activation, as evidenced by morphological transformation, increased cell proliferation and elevated tumour necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6 and RANTES (regulated upon activation, normal T-cell expressed and secreted) production. Replication-competent JEV caused all glial responses and neurotoxicity. However, replication-incompetent JEV lost these abilities, except for the ability to change microglial morphology. The bystander damage caused by activated glia also contributed to JEV-associated neurotoxicity. Microglia underwent morphological changes, increased cell proliferation and elevated TNF-a, IL-1b, IL-6 and RANTES expression in response to JEV infection. In contrast, IL-6 and RANTES expression, but no apparent morphological changes, proliferation or TNF-a/IL-1b expression, was demonstrated in JEV-infected astrocytes. Supernatants of JEV-infected microglia, but not JEV-infected astrocytes, induced glial activation and triggered neuronal death. Antibody neutralization studies revealed that TNF-a and IL-1b, but not RANTES or IL-6, released by activated microglia appeared to play roles in JEV-associated neurotoxicity. In conclusion, following JEV infection, neuronal death was accompanied by concomitant microgliosis and astrogliosis, and neurotoxic mediators released by JEV-activated microglia, rather than by JEV-activated astrocytes, had the ability to amplify the microglial response and cause neuronal death.

h i g h l i g h t sMixing state significantly slows down the reactivity of levoglucosan toward OH. Low RH and high temperature are favorable to degradation of levoglucosan. Degradation of levoglucosan should be prominent in the atmosphere. To understand the atmospheric stability of levoglucosan, which is a major molecular tracer used for source apportionment of biomass burning aerosols, degradation kinetics of levoglucosan by hydroxyl radical (OH) have been investigated using a flow reactor under different conditions. The second-order rate constant (k 2 ) for the degradation of pure levoglucosan by OH is (9.17 AE 1.16) Â 10 À12 cm 3 molecules À1 s À1 at 25 C and 40% relative humidity (RH), while it depends on environmental conditions such as temperature, RH, and mixing state. At 25 C, k 2 of pure levoglucosan linearly decreases with increasing RH (k 2 ¼ (1.50 AE 0.04) Â 10 À11 À (1.31 AE 0.11) Â 10 À11 RH), while it increases with increasing temperature and follows the Arrhenius equation k 2 ¼ (6.2 AE 5.6) Â 10 À9 exp[(e1922.5 AE 268.2)/T] when the RH is 40%. At 25 C and 40% RH, compared to pure levoglucosan, levoglucosan coated on (NH 4 ) 2 SO 4 or NaCl (levoglucosan@(NH 4 ) 2 SO 4 and levoglucosan@NaCl) shows larger k 2 to OH with (9.53 AE 0.39) Â 10 À12 and (10.3 AE 0.45) Â 10 À12 cm 3 molecules À1 s À1 , respectively, whereas levoglucosan coated on soot (levoglucosan@soot) shows the smaller k 2 of (4.04 AE 0.29) Â 10 À12 cm 3 molecules À1 s À1 . Either (NH 4 ) 2 SO 4 or NaCl internally mixed with levoglucosan ((NH 4 ) 2 SO 4 @levoglucosan and NaCl@levoglucosan) prominently inhibits the degradation of levoglucosan. Based on the rate constants, atmospheric lifetimes of levoglucosan were estimated to be 1.2e3.9 days under different conditions. All the results indicate that the degradation of levoglucosan by OH is prominent during air mass aging, and it should have an important influence on the uncertainty of source apportionment if the contribution of degradation to levoglucosan concentration is not considered in source apportionment models. a r t i c l e i n f o

h Though the compromised blood-brain barrier (BBB) is a pathological hallmark of Japanese encephalitis-associated neurological sequelae, the underlying mechanisms and the specific cell types involved are not understood. BBB characteristics are induced and maintained by cross talk between brain microvascular endothelial cells and neighboring elements of the neurovascular unit. In this study, we show a potential mechanism of disruption of endothelial barrier integrity during the course of Japanese encephalitis virus (JEV) infection through the activation of neighboring pericytes. We found that cultured brain pericytes were susceptible to JEV infection but were without signs of remarkable cytotoxicity. JEV-infected pericytes were found to release biologically active molecules which activated ubiquitin proteasome, degraded zonula occludens-1 (ZO-1), and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. Infection of pericytes with JEV was found to elicit elevated production of interleukin-6 (IL-6), which contributed to the aforementioned endothelial changes. We further demonstrated that ubiquitin-protein ligase E3 component n-recognin-1 (Ubr 1) was a key upstream regulator which caused proteasomal degradation of ZO-1 downstream of IL-6 signaling. During JEV central nervous system trafficking, endothelial cells rather than pericytes are directly exposed to cell-free viruses in the peripheral bloodstream. Therefore, the results of this study suggest that subsequent to primary infection of endothelial cells, JEV infection of pericytes might contribute to the initiation and/or augmentation of Japanese encephalitis-associated BBB breakdown in concerted action with other unidentified barrier disrupting factors.

Blood-brain barrier (BBB) characteristics are induced and maintained by crosstalk between brain microvascular endothelial cells and neighboring cells. Using in vitro cell models, we previously found that a bystander effect was a cause for Japanese encephalitis-associated endothelial barrier disruption. Brain astrocytes, which neighbor BBB endothelial cells, play roles in the maintenance of BBB integrity. By extending the scope of relevant studies, a potential mechanism has been shown that the activation of neighboring astrocytes could be a cause of disruption of endothelial barrier integrity during the course of Japanese encephalitis viral (JEV) infection. JEV-infected astrocytes were found to release biologically active molecules that activated ubiquitin proteasome, degraded zonula occludens-1 (ZO-1) and claudin-5, and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. JEV infection caused astrocytes to release vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and matrix metalloproteinases (MMP-2/MMP-9). Our data demonstrated that VEGF and IL-6 released by JEV-infected astrocytes were critical for the proteasomal degradation of ZO-1 and the accompanying disruption of endothelial barrier integrity through the activation of Janus kinase-2 (Jak2)/signal transducer and activator of transcription-3 (STAT3) signaling as well as the induction of ubiquitin-protein ligase E3 component, n-recognin-1 (Ubr 1) in endothelial cells. MMP-induced endothelial barrier disruption was accompanied by MMP-mediated proteolytic degradation of claudin-5 and ubiquitin proteasome-mediated degradation of ZO-1 via extracellular VEGF release. Collectively, these data suggest that JEV infection could activate astrocytes and cause release of VEGF, IL-6, and MMP-2/MMP-9, thereby contributing, in a concerted action, to the induction of Japanese encephalitis-associated BBB breakdown. GLIA 2015;63:1915-1932.