A mouse model for SARS-CoV-2 infection by exogenous delivery of hACE2 using alphavirus replicon particles

transduced with VEEV-VRP-hACE2. These results suggested that VEEV-VRP-hACE2-transduced


Ethics statement
All the mice were cared in accordance with the recommendations of National Institutes

Cells, virus and antibodies
Baby hamster kidney

Production and titration of VRP expressing hACE2
The RNAs used for packaging VRP were in vitro transcribed using a mMESSAGE mMACHINE TM T7 Transcription Kit (Invitrogen) according to the manufacturer's protocols. To generate the VRP expressing hACE2, 8×10 6 BHK-21 cells in 0.8 mL icecold PBS were electroporated with 5 μg of each VEEV-hACE2, VEEV-del nsP4-C and VEEV-del nsP4-E RNAs in a 0.4 cm cuvette with a GenePulser apparatus (Bio-Rad) at 0.85 kV and 25 μF, pulsing three times at 3s intervals. The electroporated cells were seeded in a T75 flask and incubated at 37 °C in 5% CO2. The supernatants of the cells were harvested at 24 h after transfection. The titer of VRP, which was expressed as infectious units (IU) per mL, was determined by IFA assay. The supernatants were serially diluted 10-fold and were used to infect BHK-21 cell monolayers in 24-well plates. The infected cells were fixed with cold (-20 °C) 5% acetic acid in methanol, and subjected to immunofluorescence assay using the anti-S tag antibody as primary antibody.

Western blotting
BHK-21 cells were infected with VRP-hACE2 or VRP-empty at a multiplicity of infection (MOI) of 1. At 24 hpi, the cells were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and supplemented with a phosphatase inhibitor cocktail). The samples were analyzed by SDS-PAGE and transferred onto PVDF membrane. The membranes were sequentially incubated with the primary antibodies against hACE2, S tag and β-actin, respectively and secondary horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG. The signals were detected with a chemiluminescence system (ChemiDoc; Bio-Rad) using Pierce ECL Western Blotting Substrate kit.

Immunofluorescence assay (IFA)
BHK-21 cells and MLE-12 cells were infected with VRP-hACE2 or VRP-empty at an MOI of 1 for 12 h, followed by infection with SARS-CoV-2 (MOI=1). At 24 h after SARS-CoV-2 infection, the cells were fixed with 4% paraformaldehyde and treated with 0.1% Triton-X 100 in PBS for 15 min at room temperature. For the detection of hACE2 expression, anti-S tag mouse antibody and Alexa Fluor 568-anti mouse IgG were used as primary and secondary antibodies, respectively. Viral replication was detected using anti-NP antibody and FITC-conjugated goat anti-rabbit IgG as primary and secondary antibodies, respectively. The nuclei were stained with DAPI.
Fluorescence images were obtained with a Nikon confocal microscope.

Plaque assay
1 × 10 5 Vero-E6 cells per well were seeded into 24-well plates one day before plaque assay. A series of 1:10 dilutions were made by mixing 15 μL of virus sample with 135 μL of DMEM. Then, 100 μL of each dilution was added to individual wells of 24-well plates containing confluent Vero-E6 cells. The plates were incubated at 37℃ with 5% CO2 for 1 h before the layer of 2% methyl cellulose was added. After 4 days of incubation at 37℃ with 5% CO2, the cells were fixed with 3.7% formaldehyde and then stained with 1% crystal violet. Plaque morphology and numbers were recorded after washing the plates with tap water.

Transduction and infection of mice
Two kinds of mouse models were used in this study: six to eight weeks-old female BALB/c and C57BL/6 mice were provided by the Animal Centre of Wuhan Institute of Virology. For animal model establishment, mice were anaesthetized with Avertin (250 mg/kg) and intranasally infected with 1.6×10 6 FFU VRP-hACE2 in 80 μL DMEM per mouse one day before SARS-CoV-2 infection. The second day, mice intranasally infected 1×10 5 PFU SARS-CoV-2 in a total volume of 50 μL DMEM following weighed and observed for clinical signs daily throughout the study. And at the indicated times after SARS-CoV-2 infection, lungs or other organs including heart, liver, spleen, brain, kidney, eyes and small intestines were collected.

RNA extraction and Real-Time Quantitative RT-PCR.
Mouse organs were homogenized in DMEM, and viral RNA was extracted using QIAamp viral RNA mini kit(52906，Qiagen)following the manufacturer's protocol.

Enzyme linked immunosorbent assay (ELISA)
SARS-CoV-2 antibody titer of serum samples collected from immunized mice was determined by indirect ELISA assay. 96-well microtiter plates were coated with inactivated SARS-CoV-2 at 2-8C overnight, and blocked with 5% skim milk for 1 h at room temperature. Diluted sera were applied to each well for 2 h at 37C, followed by incubation with goat anti-mouse antibodies conjugated with HRP for 1 h at 37C after 3 times PBS wash. The plate was developed using TMB, following 1M H2SO4 addition to stop the reaction, and read at 450 nm by ELISA plate reader for final data.

Plaque reduction neutralization test (PRNT)
The neutralizing activities of serum samples from inactivated SARS-CoV-2 immunized mice were performed by plaque reduction neutralization test (PRNT). Briefly, approximately 50 PFU of SARS-CoV-2 was pre-incubated with 2-fold serial dilutions of heat-inactivated mouse sera (starting at 1:10 dilution) for 1 h at 37℃ and then the mixture was added to Vero-E6 cell monolayers in 24-well plates and removed after 1 h of incubation followed quantifying virus by plaque assay as described above.
Neutralizing antibody titers (PRNT50) were determined to be the highest serial dilutions for which the virus plaque count was reduced by 50% compared with the control.

Histological Analysis
Lung samples from mice were fixed with 4% paraformaldehyde, embedded in paraffin followed by sagittal sections of 4-μm thickness on a microtome, and mounted on APScoated slides. For H&E stain, sagittal sections directly stained with H&E. For detection of SARS-CoV-2 antigen in fixed lungs, indirect immunofluorescence assay (IFA) was conducted. Briefly, the slides were deparaffinized, rehydrated and experienced heatinduced antigen retrieval with EDTA (pH 8.0) in a microwave oven. Then tissues were uniformly covered with 5% BSA for incubation at room temperature for 1 h followed by further incubation with the primary antibody (anti-RP3-CoV N protein polyclonal antibody, 1:500) and then washed in PBS. After the slices were slightly dried, tissues were covered with 488s-conjugated goat-anti-rabbit IgG (Abcam, GB25301) at 1:200 dilution. After washing in PBS, slides were stained with DAPI (Beyotime) at 1:100 dilution. The image information was collected using a Pannoramic MIDI system (3DHISTECH, Budapest) and FV1200 confocal microscopy (Olympus).

Blood sampling and biochemistry analysis
Blood was collected retro-orbitally and transferred to a blood collection tube containing EDTA to prevent clotting. Cell classification was analyzed by a ProCyte Dx Hematology Analyzer (IDEXX).

Statistical Analysis
All data were analyzed using GraphPadPrism 8.0.2 software and expressed as mean ± standard deviation (SD). The statistical significance was assigned when P values were < 0.05. Student's T-test was used to analyze the differences between two groups, and significant differences between groups were determined using a two-way analysis of variance (ANOVA).