AAV-hACE2 transduction allows for productive SARS-CoV-2 infection in vivo. (a) Schematic of experimental plans. C57BL/6J mice were transduced intratracheally with an AAV coding for hACE2 (AAV-hACE2) or control (AAV-GFP or PBS) and infected with SARS-CoV-2 2 wk after. Lung and blood samples were collected at days 2, 4, 7, and 14 for analysis. (b) Viral RNA from lung homogenates were measured using quantitative PCR against SARS-CoV-2.(c) Viral titers from lung homogenates were performed by plaque assay on VeroE6 cells (AAV-hACE2 values noted as mean ± SEM from three independent experiments; n = 7 at 2 and 14 DPI; n = 6 at 4 and 7 DPI. Control values noted as mean ± SEM from two independent experiments;n = 4 at 2, 7, and 14 DPI; n = 3 at 4 DPI). (d) Frozen lung tissue was stained for SARS-CoV-2 N protein (red) and epithelial cells (EpCAM, green).(e) Fixed lung tissue was paraffin-embedded and stained with H&E. Magnified panels highlight leukocyte infiltration and perivenular inflammation. (f) Images from panel e were scored by a pulmonary pathologist for perivenular inflammation (n = 2). (g) At 2 DPI, single-cell suspensions of lung were analyzed by flow cytometry. Data are shown as frequency of CD45⁺ cells (monocyte-derived macrophages, Ly6Chi monocytes, and neutrophils), frequency of parent cells (CD44⁺CD69⁺CD4⁺ T cells, CD44⁺CD69⁺CD8⁺ T cells, and CD69⁺ NK cells), or mean fluorescence intensity of CD64 (Ly6Chi monocytes; AAV-hACE2 and control values noted as mean ± SEM from two independent experiments,n = 4). (h) Serum antibodies were measured against spike protein using an ELISA. (i) Day 7 and 14 sera from panel h were used to perform a PRNT on VeroE6 cells incubated with SARS-CoV-2 (AAV-hACE2 noted as mean ± SEM from two independent experiments, n = 4; control value,n = 1). P values were calculated by two-tailed unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Scale bars, 100 µm.