Ethical statement

All animal experiments were reviewed and approved by the Animal Care and Use Committee of the Laboratory Animal Center, Air Force Medical University. The number of Animal Experimental Ethical Inspection is 20220109. And all experiments were carried out in accordance with the recommendations set out in the Guide for the Care and Use of Laboratory Animals.

Cells and virus

The JEV-P3 strain was propagated in the brains of 3-day-old inbred BALB/C suckling mice and titrated by conventional plaque assay. The neuroblast cell line Neuro2a, baby hamster kidney (BHK) cells, brain microvascular endothelial cell line bEnd.3, and HEK 293T cells (purchased from American Type Culture Collection (ATCC)) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, United States) containing 10% fetal bovine serum (FBS, Gemini, United States) and 1% penicillin streptomycin combination (Solarbio, China).

Cell culture and JEV infection

Neuro2a cells were seeded in 24 wells with the density of 5 × 103 overnight. The cells were washed by phosphate-buffered saline (PBS) and then infected with JEV (MOI = 1) at 37 °C for 1 h. After adsorption, virus-containing suspension was replaced with fresh DMEM for subsequent cell culture.

Mice

AXL/Mertk double knockout mice were kindly donated by Professor Dai Shu Han (Peking Union Medical College, Chinese Academy of Medical Sciences) and were maintained in a specific pathogen-free (SPF) facility. AXL/Mertk double knockout mice were backcrossed with wild-type C57BL/6J mice, and toe DNA from newborn mice was extracted and amplified through PCR with PrimeStar (Takara, Japan) for genotyping. Then, the amplified products were analyzed by agarose gel electrophoresis to screen WT, Mertk+/−, Mertk−/− descendants (sequences of primers are listed in Supplementary Table S1). The sizes of PCR products are 519 bp for WT mice, 519 and 650 bp for Mertk+/− mice, and 650 bp for Mertk−/− mice.

Mice infection

WT and Mertk−/− mice (6–8 weeks) were inoculated with 1 × 106 plaque-forming units (PFUs) of JEV-P3 in 20 µl of PBS via footpad injection. Body weight, behavior score, and death cases of each group were recorded twice a day at 8:00–9:00 and 16:00–17:00 for 20 days until all the groups were totally stable. The scoring criteria were referred as Bian et al. [18] described. The scoring criteria were as follows: 0: no significant abnormal behaviors, piloerection, restriction of movement, body stiffening, or hind limb paralysis; 1: piloerection, no restriction of movement, body stiffening, or hind limb paralysis; 2: piloerection, restriction of movement, no body stiffening or hind limb paralysis; 3: piloerection, restriction of movement, body stiffening, no hind limb paralysis; 4: piloerection, restriction of movement, body stiffening, and hind limb paralysis; 5: piloerection, restriction of movement, body stiffening, hind limb paralysis, sometimes tremor and even death.

Hematoxylin–eosin (HE) staining

Brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections (5 μm) were then dewaxed and hydrated. Subsequently, the sections were sequentially stained with hematoxylin and eosin using a hematoxylin–eosin (HE) staining kit (Servicebio). Finally, the sections were examined under an optical microscope.

Brain sectioning and immunofluorescence staining

Brain tissues used for sectioning and Immunofluorescence staining were obtained from JEV-infected WT and Mertk−/− mice. Mice were subjected to cardiac perfusion under anesthesia, then the brain tissues were collected in 4% PFA. The primary and secondary antibodies used in this study were listed in Supplementary Table S2.

DNA construction

Two shRNA targeting Mertk (sequences in Supplementary Table S3) with the restriction endonucleases AgeI and EcoRI were annealed and inserted into the pLK0.1-puro plasmid. The plasmids from positive clones were extracted and sequenced to obtain the correct recombinant interfering plasmid.

Preparation of recombinant lentiviral particles

Lentiviral pseudoparticles were generated by cotransfecting 293T cells in 6-well plates with the plasmids pLenti-shRNAi-Mertk-puro proviral DNA (3 μg); envelope plasmid (pMD2.G, 1 μg); and packing plasmid (psPAX2, 2 μg). The cell culture medium was changed to 1.7 ml DMEM before transfection. For each transfection, 18 µl transfection regent LipoFectMAX (ABP Biosciences, United States) was mixed with 6 µg total DNA in 0.3 ml DMEM for 30 min and then added to the 6-well plates. The cells were maintained at 37 °C for 6 h, after which the medium was changed with 5 ml DMEM with 10% FBS. The supernatants were harvested at 48 and 72 h. The cell debris was removed by centrifugation at 1000×g for 10 min and then 10,000×g for 30 min.

Lentivirus infection

bEnd.3 cells were seeded into 6-well plates at 2 × 105 overnight. The supernatant was removed, and 2 ml Mertk-shRNA lentiviral particles mixed with polybrene (8 µg/ml) was added. Lentiviral supernatants were replaced every 2 h until the viral supernatant was used up. Forty eight hours after the infection, fresh DMEM containing puromycin (5 µg/ml) was added to screen the positive cells.

Blood–brain barrier permeability measurements

WT and Mertk−/− mice were injected intraperitoneally with sodium fluorescein solution (0.1 g/ml, 200 μl/mice). After 30 min, eyeball blood was collected into the EP tube pretreated with EDTA-Na2. The peripheral blood cells were removed by cardiac perfusion before brain tissue dissection. The eyeball blood was centrifuged at 6000g for 10 min, then 100 μl serum mixed with 100 μl 15% trichloroacetic acid was centrifuged at 10000g for 30 min. After weighing the brain tissues, 7.5% trichloroacetic acid was added according to 150 μl/mg and ground with high flux tissue grinder (QIAGEN), then centrifuged at 10000g for 30 min. 120 μl centrifugal supernatants obtained from blood and brain tissue respectively were mixed with 30 μl 5M NaOH. 100 μl of the mixture was added to the 96-well blackboard. Fluorescence emission at 485 and 535 nm was determined using multimode microplate reader SPARK (TECAN). The value of fluorescein sodium uptake in the brain tissue was considered as the ratio of brain tissue fluorescence value/plasma fluorescence value.

In vitro BBB model establishment and TEER measurement

The Transwell chamber (Thermo, Waltham, MA, United States) was inverted inserted into 24-well plate, and then C8-Dia cell suspension (25,000/well) was seeded at the bottom side of the chamber and incubated at 37 °C for 6 h. Then Neuro2a cell suspension (5000/well) was inoculated in the 24-well plate, and the chamber was inserted into the corresponding 24-well plate. BEnd.3 or bEnd.3-KD Mertk cell suspension (50,000/well) were then seeded at the upper side of the chamber. The cells were cultured in 10%FBS DMEM medium containing penicillin. Before the daily electrical impedance test, cell culture medium should be replaced with fresh medium. The electrical impedance values were detected by the electrical impedance detector and recorded daily. After about 4 days, the electrical impedance was increased steadily, and a stable liquid column difference was formed compared with the blank chamber, indicating that the in vitro BBB model was successfully constructed. Then the virus was infected and the culture medium was replaced with serum-free DMEM. The virus (MOI = 1) was inoculated onto the Neuro2a cells in the 24-well plate, and the upper chamber was subsequently placed into the 24-well plate for further cultivation. the resistance was detected and recorded every day. The impedance value calculates the TEER = resistance value Ω/0.33 cm.

qRT-PCR

Total RNA from mouse tissues and cells were extracted with Total RNA Kit I (Omega, America). cDNA was prepared by the PrimeScript RT reagent Kit (TaKaRa, Japan). qRT-PCR experiments used SYBR Green Real-Time PCR Master Mix (TaKaRa, Japan) (the primers used were listed in the Supplementary Table S4). The mRNA expression was normalized to β-actin expression, and the data are shown as the relative change to the corresponding reference for each group.

Western blotting

Total protein from the brain of each mouse was extracted with radio-immunoprecipitation assay (RIPA) buffer containing phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitors and then quantified by protein reagent assay BCA kit (Thermo, Waltham, MA, United States). Thirty micrograms of protein from each sample were loaded and electrophoresed using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE Gel Quick Preparation Kit, Beyotime, China) and transferred protein onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, United States). The membranes were incubated with primary antibodies (antibodies in Supplementary Table S2) overnight at 4 °C, followed by DyLight 800/700-labeled secondary antibodies for 1 h at RT. The blots were visualized using an infrared imaging system (Odyssey, LI-COR, NE, United States).

Plaque assay

BHK cells were cultured overnight in 12-well plates at a density of 2 × 105 per well. The supernatants of the cells were removed, and the cells were washed twice with 1 × PBS solution. Then, the serial 5-fold diluted viral suspension samples with DMEM were added and incubated with BHK cells for 2 h at 37 °C. The viral supernatant was replaced with 4 ml overlay media (25 ml 4 × DMEM, 50 ml 4% methylcellulose, 2 ml FBS, 23 ml ddH2O) and incubated for 5 days. The overlay medium was washed off with 1×PBS several times until the upper overlay was rinsed, and then cells in each well were immobilized with 2 ml Crystal violet dye for 15 min. Finally, 12-well plate was washed with flowing tap water and plaques were counted.

Enzyme-linked immunosorbent assay

The enzyme-linked immunosorbent assay (ELISA) kit (Proteintech, China) was used to detect the concentration of inflammatory cytokines in serum of WT and Mertk−/− mice, such as tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and IL-6. Twenty-five microliters of serum were added to the enzyme-labeled plate coated with TNF-α antibody, and incubated at 37 °C for 2 h. Then, the serum was removed, the plate was washed with wash buffer for four times, and TNF-α antibody was added for another 60 min. Subsequently, the working solution of the enzyme conjugate was added to the plate, and the plate was incubated for 40 min. After washing the plate, the color-developing substrate was added, and the plate was incubated for 15 min. The optical density (OD) at 450 nm was measured using a microplate reader after the termination liquid was added. The contents of IL-10 and IL-6 were detected using the same procedure as that for TNF-α.

Statistical analysis

All statistical analysis were performed using GraphPad Prism version 8 software. Statistical differences were determined using Student’s t-test or two-way analysis of variance (ANOVA). P-values < 0.05 were considered significant.