2.1. Ethical Statement
The University-Town Hospital of Chongqing Medical University Ethics Committee approved this study.
2.2. Viral Strains and Cell Lines
The BoDV-1 strain, Hu- H1, was isolated from a patient with bipolar depression and was kindly provided by Professor Hanns Ludwig (Free University, Berlin, Germany) and Professor Liv Bode (Robert Koch Institute, Berlin, Germany).
Cell-free virus stock was prepared to infect the oligodendroglioma (OL) cells (34). Virus stock was tested and determined free from mycoplasma and other rodent pathogens.
2.3. Measurement of Virus Titer by RT‑qPCR
The virus titers of BoDV-1 were determined according to the method described previously (35). The plasmid DNA containing P40 amplified sequences was used to draw the standard curve of absolute quantification of BoDV-1. Regression analysis was performed to correlate CT values with the log number of copies of P40 plasmid input in the qPCR reaction. The line of best fit was generated for CT, and the log amount of P40 DNA revealed a reportable range of 109 to 102 copies/reactions. The values of the slope, intercept (Y-inter), R2, and efficiency (Eff%) were used to validate the mentioned test. The TRIZOL reagent (Takara) was used to extract total RNA. According to the manufacturer’s protocol, the RNA was reverse-transcribed into cDNA by the PrimeScript RT reagent kit (TaKaRa, Japan). Quantitative Real-time PCR was performed with the LightCycler 96 system (Roche, Switzerland) and GoTaq qPCR Master Mix (Promega). The equation generated for the line of best fit was used to calculate the absolute amount of the virus in each sample.
2.4. Immunofluorescence Assays
The OL cells grown in six-well plates were fixed in 4% paraformaldehyde for 15 minutes at room temperature. After being permeabilized with 1%Triton X-100 for 20 minutes, the cells were rinsed three times with PBS and blocked with 5% BSA for 30 minutes. Cells were incubated with anti-BoDV-1 nucleoprotein (P40) antibody (GenScript, rabbit polyclonal antibody generated against recombinant P40 protein) overnight at 4°C. The cells were then incubated with the Alexa Fluor 488-conjugated anti-rabbit antibody for 2 hours at room temperature. The cells were counterstained with DAPI before detecting immunofluorescence using an inverted fluorescence microscope.
2.5. Cholesterol Depletion Experiments
The experiment is divided into two parts. In the first part, various concentrations (0-10mM) of MβCD (Sigma-Aldrich) were added before BoDV-1 infection, incubated for 1 hour, and washed twice with PBS. In the second part, 10mM MβCD was added before or after BoDV-1 infection, incubated for 1 hour, and washed twice with PBS. The cells were then cultured for 48h. Finally, the cells were fixed with 4% paraformaldehyde and assessed through immunofluorescence assays as described above.
2.6. Measurement of Cell Viability by CCK-8
The cytotoxicity of MβCD was assessed by Cell counting kit-8 (CCK-8) (Beyotime, China). The OL cells were seeded in a 96-well culture plate and cultured at 37℃ with 5% CO2. After incubating with MβCD, CCK-8 detection reagent was added to the cell culture medium, and the absorbance at 450nm was measured using an enzyme-linked immunosorbent assay reader after 2 hours. The routine culture was continued, and five time points (1, 2, 3, 4, and 5 days) were detected for the OL cells treated with the MβCD.
2.7. Infection Inhibition Assays
Monolayers of OL cells in 6-well plates were pre-treated with increasing concentrations of anti-HSP70 polyclonal antibody (Abcam, UK) and incubated for 2 hours at 37℃. We used normal rabbit IgG as the control. After being treated with anti-HSP70 antibody for 2 hours, the cells were rinsed with PBS three times and then infected with BoDV-1 at a multiplicity of infection (m.o.i.) of 10.
2.8. siRNA Experiments
To determine the responsible member of the HSP70 family, siRNA experiments were conducted. The siRNA targeting HSP70 (5’-CGACGGAGACAAGCCCAAG-3’) (36), HSC70 (5’-GAGAGACCAAAAGCUUCUA-3’) (36) and GRP78(5’-GUGCGUACGUAGCUAGC-3’) (37) were purchased from Invitrogen. The HSP70 siRNA sequence with two mismatches (5’-CGACCGAGACAAGCGCAAG-3’) was used as a control (36). After being transfected with siRNA for 72 hours, subsequent experiments were conducted. Knockdown efficiencies were evaluated by western blotting.
2.9. Cell culture and lentivirus infection
The cells cultured in high-sugar Dulbecco’s modified Eagle's medium (DMEM) (HyClone), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and penicillin/streptomycin (Sigma-Aldrich), at 37℃ with 5% CO2. The lentiviral overexpression vector (LV-HSP70) was purchased from Obio Technology Corp., Ltd.
2.10. Lipid-Raft Isolation
According to Invent Biotechnologies's protocol, the collected cells were centrifuged at 500g for 5 minutes. After washing with pre-cooled PBS, 500μl buffer A was added for re-suspension, transferred into the centrifugal column, and centrifuged at 16000g for 30 seconds. The centrifugal columns were discarded, and the vortex was oscillated to re-suspend the precipitation. The suspension was centrifuged at 1900g for 5 minutes. All supernatants were transferred to new 1.5ml EP tubes. The centrifuge was performed at 3000g, 4℃, for 15 minutes, and all supernatants were carefully discarded. Then 400μl precooling buffer B was added and incubated at 4℃ for 30 minutes. 400μl buffer C was added into the EP tubes and swirled well. EP tubes were centrifuged for 10000g for 5 minutes. After centrifugation, a pipette was inserted with a thin suction tip into the bottom of the tube, and the water phase was removed entirely. The EP tubes were centrifuged at 16000g for 2 minutes. The LR was centrifuged to the bottom of the tube. The residual liquid was removed entirely, and the obtained LR was used for subsequent experiments.
2.11. Western Blotting and Co-immunoprecipitation
The cell collection and homogenization were performed as described previously. Then, the homogenates were centrifuged at 14000g for 15 minutes at 4ºC to obtain the supernatants. The BCA assay kit determined the protein content (Jiangsu Kaigen Biotech). After SDS electrophoresis and transfer, the PVDF membranes were blocked with 5% skim milk powder in TBS at room temperature for 30 minutes. Subsequently, the primary antibodies were incubated overnight at 4ºC. After being incubated with horseradish peroxidase-conjugated IgG (1:10000), blots were washed, and immunoreactive proteins were visualized using chemiluminescent detection. The following antibodies were used in western blotting: P40 (GenScript, USA), HSP70 (Abcam, UK), HSC70 (Invitrogen, USA), GRP78(Abcam, UK), Caspase-3 (Cell Signaling, USA), and Flotillin-1 (Sigma-Aldrich, USA).
To investigate protein interactions, we performed co-immunoprecipitation (Co-IP). For this, we incubated a portion of lysate with either an anti-Caspase-3 antibody bound to Protein G-Sepharose (Sigma, USA) or an anti-HSP70 antibody bound to Protein G-Sepharose for 18 hours. Following washing, the samples were boiled for 10 minutes in SDS-sample buffer and then subjected to Western blotting as described above.
2.12. Experimental Animals and Treatment Regimens
Pregnant SD rats at 16-18 days of gestation were purchased from the Animal Experimental Center of the Chongqing Medical University and maintained under specific pathogen-free conditions. Animal research was approved by an appropriate protocol and conducted following the institutional animal welfare guidelines of Chongqing Medical University. The experimental process followed the National Research Council's Guide for the Care and Use of Laboratory Animals and was approved by the University-Town Hospital of Chongqing Medical University Ethics Committee. After natural delivery, neonatal rats were randomly divided into control group, BoDV-1 group and ‘MβCD + BoDV-1’ group. MβCD (300mg/kg) were injected into the left lateral ventricle of ‘MβCD + BoDV-1’ group within 24h of birth. The other groups injected the same dose of saline were used as controls. Repeat the above procedure once after two days. Three days later, 2µl BoDV-1 solution (BoDV-1 and ‘MβCD + BoDV-1’ groups) were injected into the left lateral ventricle of rats. The titer of the virus was 1×107 f.f.u./ml. Routine feeding for six weeks.
2.13. Hematoxylin and Eosin Staining
Brains were harvested at a specific time point. The brains were post-fixed overnight at 4℃ in 4% paraformaldehyde and followed by dehydration. Brain samples were sectioned at 7μm thickness (Leica, Germany). Sections were deparaffinized in xylene (twice, 10 minutes each), followed by gradual rehydration using 100%, 90% and 70% ethanol and PBS (5 minutes each). The sections stained with hematoxylin for 5 minutes and eosin for 1 minutes at room temperature and followed by dehydration using 95% ethanol (twice, 5 minutes each) and 100% ethanol (twice, 5 minutes each). Treat twice with xylene for 10 minutes each time. Images were taken with a conventional microscope.
2.14. Morris water maze and Y-Maze Tests
Morris water maze (MWM) was used to measure the hippocampus-dependent spatial memory. All behavioral tests were conducted during an active period of animals’ light cycle. All parameters were recorded by ANY- maze software. The pool was filled with water (21 ± 1°C). An escape platform (11cm in diameter) was placed in the pool (180cm in diameter) and the top of the platform was 1 cm below the water surface. The trials were 60 s long, with a 15 min interval between each trial. If the rats could not find the platform within 60s, they were placed on the platform for 10s. Animals were trained in four quadrants daily for 5 consecutive days. On the sixth day, a probe test was administered. During the probe test, the platform was removed from the pool, and the mouse was allowed to swim freely for 1 min.
Working memory and exploratory activity were measured using a Y-maze apparatus (arm length: 40 cm, arm bottom width: 3 cm, arm upper width: 13 cm, height of wall: 15 cm). Each rat was placed in the central area. The number of entries into the arms and alterations were recorded for 10 min. The percentage was calculated as number of alterations/numbers of total arm entry to assess the working memory.
2.15. Statistical Analysis
All data were analyzed by the SPSS software package (version 28.0, SPSS IBM, USA). All data are presented as mean±standard error (SE) in at least three independent experiments. Student’s t-test evaluated the differences between groups and repeated measures one-way analysis of variance (ANOVA) was used to determine differences in the Morris water maze. The values of p<0.05 were considered statistically significant.