Fig 1: Development of angiotensin-converting enzyme 2-humanized (hACE2) mice sensitized to SARS-CoV-2 mimic challenge. (A) Western blot assay of hACE2 expression using antibodies against GAPDH, ACE2 and the DYKDDDDK (FLAG) tag. Five days post virus transduction, mice were challenged with SARS-CoV-2 mimic via three different deliver methods: nasal (I.N.), intratracheal (I.T.), and intravenous (I.V) injection. Inflammation-related cytokines in bronchoalveolar lavage fluid (BAL); α-FLAG and α-GAPDH cropped from different parts of the same gel, but different exposures, α-ACE2 cropped from different gels (B) and neutrophil infiltration (C) in hACE2 mice. (D) Western blot assay in HEK293T cells transduced with adenovirus 5 (Ad5)-hACE2 or Ad5-EGFP at a multiplicity of infection (MOI) of 25 or 50, or transiently transfected with PCDNA3.1 carrying hACE2 or mouse ACE2 (mACE2), using antibodies against β-actin (control), ACE2, and the FLAG tag; α-ACE2 and α-actin cropped from different parts of the same gel, but different exposures, α-FLAG cropped from different gels. (E) Recombinant SARS-CoV-2 spike-extracellular domain protein (rSpike-ECD) was incubated with HEK293T cells transiently transfected with hACE2 or mACE2 for 1 h at room temperature and detected by flow cytometry using allophycocyanin (APC)-labeled anti-DYKDDDDK antibody. Neutrophil infiltration (F) measured by flow cytometry and inflammatory-related cytokine profile (G) after challenge with SARS-CoV-2 mimic (poly[I:C] + SP), poly(I:C) alone, S alone, saline control (each n ≥ 4), via nasal injection at 5 days post virus transduction). *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA with a post hoc Bonferroni test.