Chemicals, virus and cell lines
Chloroquine and NH4CL were purchased from Sigma-Aldrich (St. Louis., MO, USA). Bafilomycin A1 was purchased from MedChemExpress (New Jersey, USA). The SARS-CoV-2 strain BetaCoV/wuhan/AMMS01/2020 was originally isolated by CFQ’s laboratory at the Academy of Military Medical Sciences . African green monkey kidney (Vero E6) cells were previously preserved in our laboratory. HEK293T-hACE2 stable cells were kindly provided by Zhong Ji Dang Kang Biotechnology Co. (Beijing, China). Human hepatoma cells (Huh-7) were purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U ml−1 of penicillin and 50 µg ml−1 of streptomycin at 37℃ with 5% CO2. All experiments with live SARS-CoV-2 were conducted in a Biosafety Level 3 laboratory in the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences.
Cell viability assays
Cell viability was assayed by the CCK-8 regent (Dojindo Molecular Technologies, Rockville, USA) in accordance with the manufacturer’s instructions. In brief, Vero E6 cells (7 × 103 cells/well) were transferred into a 96-well plate. After incubation for 24 h, treatments with viruses or drugs were applied. Chloroquine or bafilomycin A1 was given 2 h prior to virus treatment, the CCK-8 reagent was added (10 μl per well) after the indicated incubation times, and 450 nm OD values were determined with a multifunction microplate reader after 2 h of incubation.
Crystal violet staining
Cell proliferation ability was examined as previously described using crystal violet staining . In brief, Vero E6 cells (2 × 105 cells/well) were transferred into 6-well plates and incubated for 24 h. Prior to virus administration, Vero E6 cells were pre-treated with chloroquine or bafilomycin A1 for 2 h. After incubation for 48 h, cells were then stained with crystal violet and fixed with 4% paraformaldehyde for 1 h. Cells were then analyzed by microscopy and representative images were captured with a digital camera.
Female hACE2 transgenic mice, aged 6 weeks, were in a C57/B6 background and obtained from the National Institute for Food and Drug Control (Beijing, China). Transgenic mice expressing hACE2 receptor are driven by the mice ACE2 promoter as described previously . All animals were fed under conditions of controlled lighting (12 h light/dark), temperature (23 ± 1 °C), and humidity (45%). Mice were randomly distributed into experimental groups (n = 6) and the following experiments were conducted in a Biosafety Level 3 laboratory. Chloroquine (60 mg kg−1) or bafilomycin A1 (0.1 mg kg−1) was given intraperitoneally 2 h prior to SARS-CoV-2 injection and once daily for a further 5 days. Each mouse was intratracheally inoculated with 6.7 × 103 PFU of SARS-CoV-2 in 30 μl of PBS under anesthesia by intraperitoneal barbiturates. On day 5, mice were sacrificed by cervical dislocation and the primary organs were collected for RNA extraction, in situ hybridization and histopathological examination. All experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
RNA extraction and qPCR
The viral RNA in cell supernatants or tissue homogenates was extracted by the QIAamp Viral RNA Kit (Qiagen, Hilden, Germany). Virus copies were then detected by RT-qPCR methods with the HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme Biotech, Nanjing, China). The protocol for qRT-PCR was as follows: 50 °C for 15 min, 95 °C for 30 s, followed by 45 cycles at 95 °C for 10 s and 63 °C for 35 s. The primers used to detect the SARS-CoV-2N gene are as follows: Forward: GGG GAA CTT CTC CTG CTA GAA T; Reverse: CAG ACA TTT TGC TCT CAA GCTG. The PCR products were then examined using an ABI 7500 real time PCR system (Applied Biosystems, CA, USA).
RNA in situ hybridization assay
Lung tissues were fixed in 4% paraformaldehyde solution containing 0.1% DEPC. Tissues were then dehydrated by a gradient of ethanol concentrations, embedded in paraffin wax, and cut into thin sections. The sequence of the probe used for RNA hybridization is as follows: 5´-DIG-ACTACAGCCATAACCTTTCCACATACCGCAGAC-DIG-3´. The DIG label was detected by an anti-DIG-HRP. After incubation with 3,3′-diaminobenzidine (DAB), the images were captured by light microscopy and the integrated optical density was analyzed by Image pro plus 6.0.
Lung tissues were fixed in 4% paraformaldehyde solution and paraffin-embedded sections were prepared. Hematoxylin and eosin (H&E) staining was then used to identify pathological changes in the lung tissues. Images were then observed and captured by light microscopy.
All data were analyzed by GraphPad Prism, Version 8.0. Data were analyzed with the t-test or by analysis of variance (ANOVA) followed by a two-tailed t-test and expressed as means ± SEM. p values < 0.05 were considered to be statistically significant.