Virus and cells
The Hu-1 (BetaCoV/Korea/KCDC03/2020, NCCP43326) and B1.617.2 strains (hCoV-19/Korea/KDCA119861/2021, NCCP43390) were obtained from the Korea Disease Control and Prevention Agency. African green monkey kidney epithelial cells (Vero E6, ATCC CRL-1586) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 100 U/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific). The propagation and titration of SARS-CoV-2 in the Vero E6 cells were calculated using TCID50, as described previously1. Briefly, Vero E6 cells were infected with the B1.617.2 (2.9 × 106 TCID50/mL) or Wuhan strain (1 × 106 TCID50/mL), and the cytopathic effect (CPE) was monitored at 3–5 dpi. The experiments associated with SARS-CoV-2 infection were performed at a biosafety level 3 laboratory with the use of personal protection equipment in accordance with the biosafety manual instructions issued by the Korea Zoonosis Research Institute of Jeonbuk National University.
Heterozygous K18-hACE2 mice [strain: JAX 034,860 B6.Cg-Tg(K18-hACE2)2Prlmn/J] were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Seven-week-old male K18-hACE2 mice were administrated 2.5 × 104 TCID50/mL SARS-CoV-2 via the intranasal route. The K18-hACE2 mice were monitored for changes in weight loss, lethality, and clinical symptoms every day after inoculation. At 6 dpi, SARS-CoV-2–infected K18-hACE2 mice were sacrificed by isoflurane and autopsied to assess the clinical lesions in several organs such as the brain, heart, lungs, spleen, and kidneys. The animal experiments were approved by the Institutional Animal Committee of the Jeonbuk National University (JBNU 2020-11-001) and performed in accordance with the guidelines of the Institutional Biosafety Committee. The study was conducted in compliance with the ARRIVE guidelines.
Measurement of the viral burden and viral protein levels in K18-hACE2 mouse tissues
Different tissues of SARS-CoV-2–infected K18-hACE2 mice were obtained by autopsy and then homogenized in a 2-mL homogenous tube (Bertin Technologies SAS, France) with RIPA lysis buffer or TRIzol (Invitrogen, Carlsbad, CA, USA) after treatment with RNA protect-tissue reagent (Qiagen, Venlo, Netherlands). Total RNA was purified as per a commercial manual, and, subsequently, cDNA was synthesized using an all-in-one master mix (Cellsafe, Yongin, South Korea) for 5 min at 25 °C, for 60 min at 42 °C, and 5 s at 85 °C. The target genes were quantified by qRT-PCR with the IQ SYBR Green (Bio-Rad, Seoul, South Korea) using target-specific primer sets. The supernatants were collected from homogenized tissues in the RIPA lysis buffer. The quantification of total protein was performed by using a BCA protein assay kit (Thermo Fisher Scientific) and subjected to SDS-PAGE, followed by Western blotting with specific antibodies. The protein was detected by developing (Poohung, Kyunggi, South Korea) into X-ray films (AGFA, Mortsel, Belgium) using an ECL kit (ELPIS, Daejeon, South Korea). The western blot images comply with the digital and integrity policy (the full, unprocessed images are included in the supplementary information file S1). The target-specific primer sets and primary antibodies are described in Supplementary Table S2.
Histopathological and immunohistochemical analyses
SARS-CoV-2–infected brain and lung tissues were fixed in 10% formalin solution (Sigma–Aldrich, St. Louis, MO, USA) and then embedded in paraffin wax (Leica Biosystems, Wetzlar, Germany). Formalin-fixed, paraffin-embedded tissue blocks were sectioned at a thickness of 4 μm with an HM 340 electronic rotary microtome (Thermo Fisher Scientific). The tissue sections were then stained with hematoxylin and eosin as per the standard laboratory protocol47, and the pulmonary abnormalities were scored based on the representative microscopic lesions. The severity of each criterion was scored 0–3 as described in Supplementary Table S1. As the lesions were not uniformly distributed and different patterns were detected in the tissues, caution was practiced when scoring. The scores for each criterion were summed, with the higher scores indicating more severe damage. For immunohistochemistry, the sections were mounted onto silane-coated slides and treated with citrate buffer (pH 6.0) at 95 °C for 30 min and room temperature for 20 min. The sections were incubated overnight with SARS-CoV-2 nucleocapsid protein (Sino Biological, China), MAP2 (Invitrogen, Carlsbad, CA, USA), and GFAP antibody (Cell Signaling Technology, CA, USA) at 4 °C. Each slide was washed thrice for 15 min each with the wash buffer (0.145 M NaCl, 0.0027 M KCl, 0.0081 M Na2HPO4, 0.0015 M KH2PO4, pH 7.4 in PBS). The sections were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Vector Laboratories, CA, USA). The antibodies were visualized with 3,3′-diaminobenzidine (Vector Laboratories) in accordance with the manufacturer’s instructions. The histopathological examinations were performed in a double-blinded manner by trained pathologists. To quantify the immunohistochemistry outcomes, the images were randomly captured from each stained tissue and analyzed by the TS Auto 5.1 (Olympus, Tokyo, Japan). The percent immunohistochemistry-positive area was analyzed in a defined magnification field and area (magnification, × 200; field, 0.144 mm2).
SARS-CoV-2–infected K18-hACE2 mouse lungs or brains were homogenized with PBS. The clarified supernatants were collected via centrifugation and then serially diluted with the DMEM without serum. Vero E6 cells (3 × 104 cells/well) were inoculated with four replicates from 1 × 10−8 to 1 × 10−1 diluents. The diluents were then removed, and the medium was sequentially replaced with DMEM supplemented with 2% FBS. At 3–5 dpi, CPE was monitored, and TCID50 of SARS-CoV-2 was calculated by the Reed and Muench method48.
Total RNA was isolated and quantified with the Bioanalyzer 2100 (Agilent Technologies, CA, USA). Redundant ribosomal RNA (rRNA) was eliminated from total RNA using the RiboCop rRNA Depletion Kit (Lexogen, Vienna, Australia). The RNA-seq libraries were prepared using the Next Ultra II Directional RNA kit (NEB, MA, USA) according to the manufacturer’s protocol. The libraries were pooled and analyzed as paired-end sequenced on the NovaSeq 6000 (Illumina, CA, USA) targeting 40 million read pairs and extended. The RNA-seq reads were then aligned to the mouse reference genome (mm10) with the TopHat. All gene counts were preprocessed with the EdgeR to adjust the samples for differences in the library size using the trimmed mean of M values. The results of the differential signature genes were analyzed with the ExDEGA (eBiogen, Seoul, South Korea). Gene Ontology analysis and classification were performed using the data from the Database for Annotation, Visualization, and Integrated Discovery.
RT2-profiler PCR array
Total RNA extracted from SARS-CoV-2–infected K18-hACE2 mouse brains at 3 and 4 dpi was synthesized using the RT2 First Strand Kit (Qiagen, Hilden, Germany). The synthesized cDNA was mixed with RT2 SYBR Green Mastermix, and the RT2 profiler™ PCR array mouse necrosis pathway (Qiagen, PAMM-141ZA/330231). The qPCR array was performed by holding for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The result was then analyzed with GeneGlobe (dataanalysis2.qiagen.com/pcr), and the Ct values were normalized with the supplied internal housekeeping gene.