Virus isolation and titration.

The GP78 strain of JEV was propagated in suckling BALB/c mice (postnatal day 3 or 4) of either sex until the appearance of symptoms of sickness (limb paralysis, poor pain response, and whole-body tremor). Following the onset of symptoms, the mice were sacrificed to collect the infected brains. The brains were homogenized in minimum essential medium (MEM) and centrifuged at 10,000 × g to remove cellular debris. The resulting suspension was filtered through a 0.22-μm sterile filter, and aliquots of the filtered virus suspension were stored at −80°C until further use (21).

Virus was titrated by plaque formation assay using the porcine stable kidney (PS) cell line as described previously (22). Briefly, a monolayer of PS cells was incubated with JEV (a 10-fold dilution prepared in MEM containing 1% fetal bovine serum [FBS]) at 37°C for 1 h. The viral inocula were removed from the monolayers, and MEM containing 4% FBS, 1% low-melting-point agarose, and a cocktail of antibiotic-antimycotic solutions was added. Then, the culture plates were incubated at 37°C for 72 to 96 h until the appearance of visible plaques. The plaques were fixed and stained with 0.1% crystal violet (23).

JEV was inactivated by exposure to short-wavelength (254-nm) UV radiation (UVC) at a distance of 5 cm for 10 min on ice, using a UV cross-linker (UVC 500; Hoefer Scientific, USA), as described previously (24).

BV-2 cell culture.

The BV-2 cell line (a mouse microglia cell line) was provided by Steve Levison, University of Medicine and Dentistry, New Jersey, USA. BV-2 cells were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% sodium bicarbonate (NaHCO₃), 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, as described previously (25).

Primary microglial culture.

Primary microglia cells were isolated from BALB/c mouse pups of either sex (postnatal days 0 to 2), as reported previously (25). Briefly, the whole brain cortex was dissected from the mouse brain, and the meninges were removed under a dissecting microscope. Tissue was digested using trypsin-DNase solution with a brief mechanical dissociation to obtain a cell suspension. The cell suspension was passed through 100-mm cell strainers and centrifuged at 400 × g for 8 min to obtain a cell pellet. After a viable-cell count, cells were plated on 75-cm² tissue culture flasks at a density of 2 × 10⁵ viable cells/cm² in complete MEM (supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.6% glucose, and 2 mM glutamine). The complete MEM was changed every 2 days until the mixed glial culture became confluent. On day 12, microglia cells were dislodged by shaking the mixed glial flasks on an orbital shaker (Excella E25; New Brunswick Scientific, NJ, USA) at 250 rpm for 60 to 75 min at 37°C. The media containing the nonadherent cells were removed and plated in bacteriological petri dishes. After complete adherence (60 to 90 min), the microglia cells were treated with Accutase, scraped, and centrifuged. Subsequently, the cells were plated on 2-well chamber slides (Nunc, Denmark) at 8 × 10⁴ viable cells/cm² and incubated at 37°C for further experiments.

Infection of BV-2 and primary microglia cells with JEV.

BV-2 cells were plated at a density of 1.5 × 10⁶ cells in DMEM containing 10% FBS. After 15 to 18 h, the cells were incubated with serum-free medium for 4 to 5 h. The cells were infected with JEV or UV-treated JEV at a multiplicity of infection (MOI) of 5 for 1.5 h. The unbound virus was removed by washing gently with 1× phosphate-buffered saline (PBS), and the cells were further incubated in serum-free medium for different time interval studies. Primary microglia cells plated on chamber slides were treated similarly with JEV for 12 or 24 h.

Reverse transcription real-time PCR and miRNA assay.

After isolation of total RNA from treated cells using Tri Reagent (Sigma-Aldrich), cDNA was prepared using an Advantage RT-for-PCR kit (Clontech Laboratories). Amplification of SHIP1 (forward, 5′-CCA GGG CAA GAT GAG GGA GA-3′; reverse, 5′-GGA CCT CGG TTG GCA ATG TA-3′) and beta interferon (IFN-β) (forward, 5′-TTG CCA TCC AAG AGA TGC TC-3′; reverse, 5′-TCA GAA ACA CTG TCT GCT GG-3′) mRNA was carried out by quantitative PCR (qPCR) using power SYBR green PCR master mix (Applied Biosystems, Foster City, CA). The conditions used for the PCR were as follows: 95°C for 3 min (1 cycle) and 94°C for 20 s, 55°C for 30 s, and 72°C for 40 s (40 cycles). The relative mRNA abundance was determined by normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA using the 2^−ΔΔCt method (C_t refers to the threshold value).

miRNA was isolated using a miR-Neasy kit (Qiagen), and cDNA was prepared using a miScript II RT Kit (Qiagen) according to the manufacturer's instructions. qPCR was performed using specific miR-155 primer, 5′ UUA AUG CUA AUU GUG AUA GGG GU 3′ (Qiagen), and the miScript SYBR green PCR kit (containing the miScript Universal reverse primer). The conditions used for qPCR were as follows: 95°C for 15 min (1 cycle), 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s (40 cycles). The thermal cycler ViiA 7 Real-Time PCR (Applied Biosystems) was used for qPCR, and the data were analyzed using iCycler Thermal Cycler software (Applied Biosystems). Relative miRNA abundance was determined by normalizing to U6 or SNORD 68 small nuclear RNA using the 2^−ΔΔCt method.

miRNA overexpression and inhibition in BV-2 cells.

miR-155 was overexpressed in BV-2 cells by transfecting the cells with 100 pmol of miScript miR-155 mimic (Qiagen) or miRNA mimic negative control (Ambion) using commercial medium (Opti-MEM; Invitrogen) and Lipofectamine 2000 (Invitrogen). After 24 h of transfection, cells were harvested and qPCR was performed for miR-155 expression. In another set of experiments, miR-155 was overexpressed, and SHIP1 expression was analyzed by immunoblotting.

For specific knockdown of miR-155 expression, BV-2 cells were transfected with 100 pmol of miScript anti-miR-155 inhibitor (Qiagen). After 24 h of transfection, the cells were infected with JEV for 24 h and used to isolate protein and RNA. Cy3-labeled control anti-miR was used to assess transfection efficiency.

SHIP1 siRNA transfection.

BV-2 cells were transfected with 0.5 μM small interfering RNA (siRNA) targeting SHIP1 (forward, 5′-GCTAAGTGCTTTACGAACA-3′; reverse, 5′-TGTTCGTAAAGCACTTAGC-3′) or nontargeting control siRNA (Sigma) using Lipofectamine 2000 (Invitrogen) for 24 h. After 24 h of transfection, the cells were infected with JEV and used for further studies.

MicroRNA target prediction.

The bioinformatics prediction tools PicTar (http://pictar.mdc-berlin.de/) and Target Scan (version 5.2) (http://www.targetscan.org/) were used to identify the potential targets of miR-155. The miR-155 target binding sites in the 3′ UTRs of human and mouse SHIP1 transcripts were identified with Target Scan software.

Luciferase reporter assay.

BV-2 cells were transfected either with pMIR-REPORT SHIP1 3′ UTR luciferase reporter (Add Gene), pMIR-REPORT plasmid, or pMIR-REPORT-β-galactosidase (Ambion) and cotransfected with 100 pmol of miR-155 or miR-29b for 24 h. Assays were performed using a luciferase assay system (Promega, Madison, WI, USA). In another experiment, BV-2 cells were transfected with SHIP1 construct and were exposed to JEV and UV-treated JEV for 24 h. Briefly, the cells were washed with 1× PBS and lysed in reporter lysis buffer provided by the manufacturer. After a brief centrifugation (12,000 × g for 5 min at 4°C), the supernatant was collected. Luciferase assay reagent (100 μl) was dispensed into three sets of luminometer tubes, to which 20 μl of the collected supernatant was added; the readings were taken using a Sirius single-tube luminometer (Berthold Detection Systems GmBH, Germany). The luciferase units were measured as relative luciferase units (RLU) and normalized to the total protein.

Effect of LNA anti-miR-155 treatment in JEV-infected BALB/c mice.

Adult BALB/c mice of either sex were randomly assigned to three groups: group 1, the control group (CON); group 2, the JEV-infected group (JEV); and group 3, the JEV-infected and LNA anti-miR-155-treated group (JEV + LNA). Mice in groups 2 and 3 received JEV strain GP78 (3 × 10⁵ PFU) via the tail vein, while group 1 received an equal volume of PBS. Twenty-four hours after JEV infection, mice in groups 2 and 3 received anti-miR-155 LNA (5′-TCACAATTAGCATTA-3′) or LNA scrambled anti-miR (Exiqon) (20 mg/kg body weight intravenously [i.v.]) for three consecutive days. After 4 days, mice in the JEV group developed signs of JEV infection, and on the 7th day, the animals developed restriction of movement, limb paralysis, poor pain response, whole-body tremor, piloerection, and hind limb paralysis. All the mice were euthanized, and brain samples were collected for mRNA, miRNA, protein, and immunohistochemistry studies.

Isolation of miRNA from human brain tissue sections and qPCR.

Human brain tissue sections were obtained from the Human Brain Tissue Repository, National Institute of Mental Health and Neurosciences, Bangalore, India, in accordance with the institutional scientific ethics, protecting the confidentiality of the subjects. Formalin-fixed, paraffin-embedded (FFPE) sections from the basal ganglia/frontal cortex were obtained postmortem from confirmed cases of JE. All cases had JEV IgM antibodies in the CSF, microglial nodules, and necrolytic lesions characteristic of JE. Brain tissues from individuals who succumbed to traffic accidents and had no known prior neurological disease were used as non-JEV controls. The miRNA extraction was performed using an miRNeasy FFPE Kit (Qiagen) according to the manufacturer's instructions. The 20-μm-thick FFPE sections were deparaffinized, treated with proteinase K, and processed for RNA isolation. cDNA was synthesized using an miScript II RT Kit (Qiagen) as described above. qPCR analysis was performed using primers specific for human miR-155: 5′-UUAAUGCUAAUCGUGAUAGGGGU-3′ (Qiagen) and the universal SYBR green Power Master Mix (Applied Biosystems). The relative miRNA abundance was determined by normalizing to Hs-RNU6 (Qiagen). The real-time PCR results were analyzed and expressed as fold changes over non-JEV samples using the 2^−ΔΔCt method.

Immunoblotting.

Immunoblotting was carried out at the end of the treatment period as previously described (21). Briefly, control and treated BV-2 cells or mouse brain was lysed in complete lysis buffer containing 1% Triton X-100, 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Nonidet P-40 (NP-40), 1 mM EDTA, 0.2% EGTA, 0.2% sodium orthovanadate, and protease inhibitor cocktail (Sigma-Aldrich). Fifty to 100 μg of each protein sample was electrophoresed on SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then blocked using 5% skim milk in 1× PBST (1× PBS-Tween 20) and incubated with the following primary antibodies: SHIP1, phospho- and total interferon-regulatory factor 3 (IRF3), phospho-IRF7, phospho- and total NF-κB (Cell Signaling), phospho-TBK (Millipore), total TBK, and total IRF7 (Santa Cruz Biotechnology, CA, USA) overnight at 4°C. All primary antibodies were prepared at a dilution of 1:1,000 in 1% bovine serum albumin (BSA) in 1× PBST. The blots were labeled with goat anti-rabbit horseradish peroxidase (1:5,000; Vector Laboratories, CA, USA) in 1× PBST and developed by exposure in a Chemigenius Bio-Imaging System (Syngene; Cambridge, United Kingdom) using Gene Snap software. The images were analyzed using Gene Tools software from Syngene. The blots were then stripped and probed with anti-β-actin antibody (1:10,000; Sigma-Aldrich).

CBA.

The cytokine levels in the brains of control and JEV-infected mice were quantified using a mouse cytokine bead array (CBA) kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. The beads, coated with interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and monocyte chemotactic protein 1 (MCP-1), were mixed with test lysates and standards, to which fluorescent dye was added. The mixtures were incubated for 2 h at room temperature (RT) in the dark, following which the beads were washed and resuspended in 300 μl of wash buffer and acquired using Cell Quest Pro software in a FACSCalibur (Becton, Dickinson, San Diego, CA). The data were analyzed using BD CBA software (Becton, Dickinson, San Diego, CA, USA) (26), and the concentrations of various cytokines were expressed as pg/ml.

Immunohistochemistry.

For immunohistochemical staining, brains from sacrificed mice were excised following repeated transcardial perfusion with ice-cold saline and fixed with 4% paraformaldehyde. Twenty-micrometer-thick cryosections were prepared using a Leica CM3050S cryostat and processed for immunohistochemical staining. Sections were incubated overnight with anti-SHIP1 (1:100; Cell Signaling) and anti-Iba and Neu-N (1:250; Millipore) at 4°C. After washes, the slides were incubated with fluorescein isothiocyanate (FITC) (1:200; Vector Laboratories)- or Alexa Fluor 594 (1:1,000; Molecular Probes, Eugene, OR)-conjugated secondary antibodies. After final washes, the sections were mounted with 4′-6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc.). The sections were observed and photographed using a Zeiss Apotome microscope (Zeiss, Gottingen, Germany) at the indicated magnification.

In situ hybridization (ISH).

Fresh-frozen (FF) brain sections 20 μm thick were obtained using a cryostat, as mentioned above. FFPE sections of uninfected (control) and JEV-infected human brains were deparaffinized with xylene and hydrated using series of alcohol. The FF sections were fixed in fresh 4% neutral buffered formalin overnight. Following washes with PBS, the sections were treated with 20 mg/ml proteinase K at 37°C for 10 min. Then, the sections were thoroughly washed with PBS and incubated with hybridization buffer containing 40 nM double 5′-digoxigenin (DIG)- and -3′-DIG-labeled LNA miR-155 probe (5′-TATCACAATTAGCATTAA-3′) (Exiqon) for 60 min at 48°C in a humidified chamber. Following hybridization, the sections were washed with 5×, 1×, and 0.2× SSC 2 times each for 5 min in that order. After treatment with blocking solution, the sections were incubated with anti-DIG reagent (sheep anti-digoxigenin alkaline phosphatase [anti-DIG-AP] antibody at 1:800 dilution) for 60 min at RT in a humidified chamber and washed with PBST. Then, the sections were incubated with freshly prepared AP substrates for 2 h at 30°C in the humidifying chamber (under dark conditions). The reaction was terminated by adding KTBT buffer (an AP stop solution composed of 50 mM Tris-HCl, 150 mM NaCl, and 10 mM KCl), washed with water, dehydrated in series of alcohol, and mounted using DPX. The slides were observed under a Leica DMRXA2 microscope, and photographs were taken under the ideal magnification and conditions for each slide.

SHIP1 is involved during in vitro and in vivo JEV infection.

Second, we searched for a potential target of miR-155 by using Pictar and Target Scan bioinformatics tools. SHIP1 is one of the potential targets of miR-155 showing an 8-mer binding site in the SHIP1 3′ UTR. To establish the involvement of SHIP1 in JEV infection, BV-2 cells were infected with JEV at different time points (0, 3, 6, 12, or 24 h) and evaluated for SHIP1 expression. JEV-infected BV-2 cells showed a time-dependent decrease in SHIP1 mRNA and protein levels (maximal at 12 and 24 h) (Fig. 2A). Interestingly, in vitro knockdown of miR-155 using specific antisense oligonucleotides rescued the JEV infection-induced decrease in SHIP1 protein expression (Fig. 2B). Treatment of anti-miR-155 in the absence of JEV infection also recorded a similar pattern of SHIP1 protein expression (Fig. 2B). Further, to evaluate the changes in expression of SHIP1 in vivo, BALB/c mice were infected with JEV, and brain samples were collected at different time points (1, 3, 5, or 7 days p.i.). A significant decrease in expression of SHIP1 mRNA was observed that was comparable to in vitro findings (Fig. 2C). Minimal expression of SHIP1 mRNA was observed on the 7th day, when the animals developed all the major signs of infection. To further verify the involvement of SHIP1 in JEV infection, we knocked down the expression of miR-155 in BALB/c mice by administrating anti-miR-155 LNA for 3 days after JEV infection. We observed a significant reduction in the brain SHIP1 mRNA (Fig. 2C) and protein expression 7 days after infection (data not shown), while anti-miR-155 LNA treatment significantly recovered the JEV infection-induced decrease in SHIP1 expression (Fig. 2D). The results were verified with immunohistochemistry staining of SHIP1 in the mouse brain cortex and subventricular zone (SVZ) regions (Fig. 2E). Data on the quality of in vitro and in vivo miR-155 inhibition/overexpression are shown in Fig. S1 in the supplemental material. Further, treatment of anti-miR-155/miR-155 mimic or LNA anti-miR-155 did not alter the viral load in JEV-infected BV2 cells or BALB/c mice (data not shown), indicating that manipulation of miR-155 expression does not alter viral replication. Further, we evaluated the effect of control anti-miR transfection on the expression of SHIP1, its immediate downstream TBK-1, and proinflammatory cytokines in JEV-infected and control BV-2 cells. Transfection of control miRNA to uninfected and JEV-infected BV-2 cells did not alter the protein levels of SHIP1 and pTBK-1 and the mRNA level of IFN-β compared to nontransfected BV-2 cells (see Fig. S2 in the supplemental material). This observation clearly indicates that the results observed here are specific to anti-miR-155 transfection.

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FIG 2

SHIP1 is involved during JEV infection in vitro and in vivo. (A) Time-dependent expression of SHIP1 in JEV-infected BV-2 cells. BV-2 cells were infected with JEV for 0, 3, 6, 12, or 24 h, and expression of SHIP1 was evaluated using qPCR and immunoblotting. The graph represents densitometric quantification of SHIP1 normalized to β-actin. *, P < 0.05 compared to uninfected (0-h) BV-2 cells. (B) Effect of miR-155 knockdown on SHIP1 protein expression in JEV-infected BV-2 cells. BV-2 cells were transfected with anti-miR-155 for 24 h, followed by infection with JEV for 24 h. At the end of that time, the cell pellet was used for isolation of protein for SHIP1 expression. Cy3-labeled control anti-miR 155 was used to evaluate the quality of transfection. The graph represents densitometric quantification of SHIP1 normalized to β-actin. *, P < 0.05 compared to uninfected BV-2 cells, and #, P < 0.05 compared to JEV-infected BV-2 cells. CON, uninfected BV-2 cells. (C) Time-dependent expression of SHIP1 mRNA in brains of JEV infected BALB/c mice. Brain samples were collected from BALB/c mice 1, 3, 5, or 7 days after inducing JEV infection, and SHIP1 mRNA expression was evaluated by qPCR. *, P < 0.05 compared to an uninfected (day 0) mouse brain sample. (D) Effect of anti-miR-155 LNA on expression of SHIP1. BALB/c mice were infected with JEV and treated with anti-miR-155 LNA (LNA+JEV) or scrambled anti-miR (JEV). Their brains were collected on day 7. SHIP1 protein expression was evaluated by immunoblotting. β-Actin was used as a loading control, and the graph represents the densitometric quantification of SHIP1. *, P < 0.05 compared to uninfected mouse brains, and #, P < 0.05 compared to JEV-infected mouse brains. CON, brain samples collected from PBS-treated mice. (E) SHIP1 protein expression was evaluated from cortex and SVZ regions of JEV-infected mice treated with anti-miR-155 LNA (JEV+LNA mice) or scrambled anti-miR (JEV mice). BALB/c mice were infected with JEV and treated with anti-miR-155 LNA (LNA+JEV) or scrambled anti-miR (JEV), and brain samples were collected on day 7 for evaluation of SHIP1 protein expression by immunohistochemistry (magnification, ×20). CON, brain samples collected from PBS-treated mice. The data represent means ± SEM from 5 mice per group or 3 independent experiments for in vitro study.

SHIP1 is a target of miR-155 during JEV infection.

We evaluated the role of SHIP1 in modulating miR-155-dependent changes in JEV-induced neuroinflammation. SHIP1 knockdown by siRNA in BV-2 cells resulted in higher expression of inflammatory cytokines than in untreated cells. Further, cotransfection of BV-2 cells with SHIP1 siRNA and anti-miR-155 resulted in higher expression of proinflammatory cytokines than with their individual transfections (see Fig. S3 in the supplemental material). After JEV infection of BV-2 cells, SHIP1 knockdown blunted the beneficial effects of anti-miR-155 transfection (see Fig. S3 in the supplemental material). These sets of observations provide evidence for an antineuroinflammatory role of SHIP1 (see Fig. S3 in the supplemental material). As shown in Fig. 3A, we observed a significant decrease in the SHIP1 protein level by transfection with a miR-155 mimic. This observation, along with the previously mentioned inverse relationship between miR-155 and SHIP1 expression post-JEV infection, suggests that the expression level of SHIP1 is negatively regulated by miR-155. Previous reports have identified SHIP1 as a potential target of miR-155 (20). To determine whether miR-155 binds to the 3′ UTR of SHIP1, we performed a luciferase reporter assay. A pMIR-REPORT construct encompassing a firefly luciferase reporter followed by the full-length 3′ UTR of SHIP1, pMIR-REPORT SHIP1, was used. miR-155 and miR-29b mimic (as a negative/nonspecific miRNA control) oligonucleotides and an empty vector (pMIR-REPORT) were cotransfected with pMIR-REPORT SHIP1. Luciferase activity was diminished in the reporter containing the 3′ UTR of SHIP1 treated with miR-155 compared with pMIR-REPORT SHIP1 alone. In addition, when pMIR-REPORT SHIP1was cotransfected with an irrelevant miR-29b mimic, the luciferase activity did not show any significant changes (Fig. 3C). To further validate interaction between miR-155 and SHIP1 during JEV infection, BV-2 cells were transfected with a pMIR-REPORT SHIP1 construct, followed by infection with either JEV or UV-treated JEV. The luciferase activity in the JEV-treated group was significantly decreased compared with the pMIR-REPORT SHIP1 construct alone, while the UV-treated group did not show any significant changes in luciferase activity. These results clearly imply that miR-155 targets SHIP1 during JEV infection (Fig. 3D).

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FIG 3

SHIP1 is a target of miR-155 during JEV infection. (A) Expression of SHIP1 in BV-2 cells transfected with miR-155 mimic. miR-155 was overexpressed in BV-2 cells by transfection with miR-155 mimic for 24 h. SHIP1 expression was analyzed by immunoblotting. β-Actin was used as a loading control, and the graph represents densitometric quantification of SHIP1 normalized to β-actin. The data represent means and SEM from 3 independent experiments. *, P < 0.05 compared to uninfected BV-2 cells. CON, sample uninfected BV-2 cells. (B) Seed sequence in the miR-155 mRNA coding sequences (CDS) of human and mouse brains with complementary sequence in the SHIP1 mRNA coding sequence and 3′ UTR of human and mouse showing 8-mer binding in the SHIP1 3′ UTR (highlighted). (C) BV-2 cells were transfected with empty luciferase reporter construct (pMIR-REPORT) or the construct containing the 3′ UTR of SHIP1 (pMIR-REPORT SHIP1) and/or cotransfected with miR-155 mimic (pMIR-REPORT + miR-155 mimic/pMIR-REPORT SHIP1+ miR-155 mimic) or miR-29b mimic (pMIR-REPORT SHIP1+ miR-29 mimic) for 24 h. The luciferase reporter assay showed that SHIP1 was a direct target of miR-155; the luciferase activity of the reporter containing the 3′ UTR of SHIP1 treated with miR-155 mimic decreased significantly compared with that with SHIP1 alone (*, P < 0.001). Transfection with miR-29b did not show any changes. (D) BV-2 cells were transfected with empty luciferase reporter construct (pMIR-REPORT) or the construct containing the 3′ UTR of SHIP1 (pMIR-REPORT SHIP1) for 24 h and infected with JEV (pMIR-REPORT SHIP1 + JEV) or UV-treated JEV (pMIR-REPORT SHIP1 + UV treated JEV) for 24 h. In another set of experiments, BV-2 cells were cotransfected with the 3′ UTR of SHIP1 anti-miR-155 mimic for 24 h and infected with either JEV (pMIR-REPORT SHIP1 + JEV + Anti-miR155) or UV-treated JEV (pMIR-REPORT SHIP1 + UV treated JEV + Anti-miR155). The luciferase activity of pMIR-REPORT SHIP1 + JEV decreased significantly compared with pMIR-REPORT SHIP1 (*, P < 0.001), while the luciferase activity of pMIR-REPORT SHIP1 + UV treated JEV did not show any changes. The luciferase value of β-galactosidase was used to normalize the luciferase values. The data represent means ± SEM from 3 independent experiments performed in duplicate. *, P < 0.05 compared to pMIR-REPORT SHIP1.

miR-155 regulates JEV infection of microglia and mouse brain.

Phosphorylation of TBK-1, IRF3/7, and NF-κB determines induction of inflammatory cytokines and IFN-β secretion. Therefore, we evaluated the effects of in vitro and in vivo knockdown of miR-155 on phosphorylation of TBK-1, IRF3/7, and NF-κB. In BV-2 cells and mouse brains, JEV infection increased TBK-1, IRF3/7, and NF-κB phosphorylation, whereas inhibition of miR-155 led to decreased TBK-1, IRF3/7, and NF-κB phosphorylation (Fig. 4 and ​and55).

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FIG 4

miR-155 regulates JEV-induced infection in microglia. Expression of total and phosphorylated TBK-1, IRF3, IRF7, and NF-κB proteins in JEV-infected BV-2 cells. BV-2 cells were transfected with anti-miR-155 for 24 h, followed by JEV infection for 24 h. Subsequently, the cells were lysed, protein was extracted, and the levels of total and phosphorylated TBK-1, IRF3, IRF7, and NF-κB were determined by immunoblotting. The graphs represent the densitometric analysis of the immunoblots. The light parts of the graphs denote the phosphorylated fractions, and the dark parts represent the total protein. β-Tubulin was used as a loading control. CON, noninfected BV-2 cells. The data represent means ± SEM from 3 independent experiments. *, P < 0.05 compared to uninfected BV-2 cells, and #, P < 0.05 compared to JEV-infected BV-2 cells.

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FIG 5

miR-155 regulates JEV-induced infection of mouse brain. Expression of total and phosphorylated TBK-1, IRF3, IRF7, and NF-κB proteins in uninfected, JEV-infected, or anti-miR-155 LNA-treated JEV-infected BALB/c mouse brain. BALB/c mice were treated with PBS (CON) or infected with JEV and treated with anti-miR-155 LNA (JEV+LNA) or scrambled LNA (JEV). At 7 days postinfection, brain samples were collected for extraction of total proteins, and the levels of total and phosphorylated forms of TBK-1, IRF3, IRF7, and NF-κB proteins were determined by immunoblotting. The graphs represent the densitometric analysis of the immunoblots. The light parts denote the phosphorylated fraction, and the dark parts represent the total protein. β-Tubulin was used as a loading control. CON, brain samples collected from PBS-treated mice. The data represent means ± SEM from 5 mice per group. *, P < 0.05 compared to an uninfected (CON) mouse brain sample, and #, P < 0.05 compared to a JEV-infected mouse brain sample (JEV).

Anti-miR-155 treatment reduces JEV-induced proinflammatory cytokines.

To check if the IFN-β-mediated antiviral response induced during JEV infection can be modulated by miR-155 expression, we evaluated the time kinetics expression of IFN-β in the JEV-infected mouse brain. We observed significantly increased expression of IFN-β mRNA, especially at 5 and 7 days after infection (Fig. 6A). Knockdown of miR-155 in JEV-infected mice (LNA + JEV) significantly reduced IFN-β production compared to the control group (Fig. 6B). We evaluated the JEV-induced proinflammatory response in the mouse brain by measuring protein levels of TNF-α, MCP-1, and IL-6. Time-dependent JEV infection resulted in higher induction of TNF-α, MCP-1, and IL-6 in the mouse brain, with maximum responses at 5 and 7 days (Fig. 6A). Further, in mouse brains, LNA treatment reduced TNF-α, MCP-1, and IL-6 protein expression compared to the JEV group (Fig. 6B).

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FIG 6

Anti-miR-155 treatment decreases expression of proinflammatory cytokines. (A) Time-dependent expression of TNF-α, MCP-1, IL-6, and IFN-β. BALB/c mice were infected with JEV, and brain samples were collected 1, 3, 5, or 7 days p.i. for evaluation of IFN-β mRNA and protein expression by cytokine bead array and qPCR, respectively. *, P < 0.05 compared to an uninfected (day 0) mouse brain sample. (B) Effects of anti-miR-155 LNA on expression of TNF-α, MCP-1, IL-6, and IFN-β. BALB/c mice were treated with PBS (CON) or infected with JEV and treated with anti-miR-155 LNA (JEV+LNA) or scrambled LNA (JEV), and brain samples were collected on day 7 for evaluation of TNF-α, MCP-1, IL-6, and IFN-β expression by cytokine bead array and qPCR, respectively. CON, brain samples collected from PBS-treated mice. The data represent means ± SEM from 5 mice per group. *, P < 0.05 compared to an uninfected (CON) mouse brain sample, and #, P < 0.05 compared to a JEV-infected mouse brain sample.

In vivo anti-miR-155 treatment decreases microglia activation and rescues neurons from death.

Pathological changes, such as elevated levels of proinflammatory cytokines, microglia activation, and neuronal death, are key features of in vivo JEV infection. As shown in Fig. 6A, JEV infection of BALB/c mice resulted in higher levels of brain TNF-α, MCP-1, and IL-6 than in control mouse brain. Inhibition of miR-155 in JEV-infected mice reduced the levels of proinflammatory cytokines. Ionized calcium binding adaptor molecule 1 (Iba1) is commonly used as a marker for microglia activation, while neuronal nuclear antigen (NeuN) is a marker of mature neurons. In the present study, we employed double-immunofluorescence staining using Iba1 and NeuN antibodies to demonstrate microglia activation and associated neuronal death. As shown in Fig. 7A, JEV-infected mouse brain sections had increased numbers of activated microglia and reduced numbers of neurons. Inhibition of miR-155 reduced microglia activation and neuronal death (Fig. 7A). Further, immunoblotting revealed that JEV-infected mouse brains had increased protein levels of Iba1 and NeuN compared to control mice. Additionally, LNA miR-155-treated mouse brains had reduced levels of Iba1 and NeuN proteins compared to JEV-infected mice (Fig. 7B). In anti-miR-155-treated mice, high SHIP1 levels correlated with low inflammatory cytokine levels, and regulation of inflammation by SHIP1 could be a mechanism for the decreased microglia activation and neuronal death observed here.

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FIG 7

Anti-miR-155 treatment decreases microglia activation and rescues neurons from death in vivo. Shown are the effects of anti-miR-155 LNA on expression of Iba1 and NeuN. BALB/c mice were treated with PBS (CON) or infected with JEV and treated with anti-miR-155 LNA (JEV+LNA) or scrambled LNA (JEV), and brains were collected on day 7 for evaluation of Iba1 and NeuN protein expression by immunofluorescence staining (magnification, 20×; scale bar, 20 μm) (A) and immunoblotting (B), respectively. The graphs represent densitometric quantification of Iba1 and NeuN normalized to β-actin. CON, brain samples collected from PBS-treated mice. The data represent means and SEM from 5 mice per group. *, P < 0.05 compared to an uninfected (CON) mouse brain sample, and #, P < 0.05 compared to a JEV-infected mouse brain sample.

This work was supported by a core grant from the Department of Biotechnology to the National Brain Research Centre. Menaka Chanu Thounaojam is the recipient of a research associateship, and Shalini Swaroop is the recipient of a Senior Research Fellowship from the Council of Scientific and Industrial Research, Government of India.

We acknowledge Soumya Iyengar for critically analyzing the in situ hybridization data. We also acknowledge Kanhaiya Lal Kumawat and Manish Dogra for technical assistance and Sourish Ghosh for his valuable suggestions.