SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract

Abstract

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

Keywords: ACE2; COVID-19; SARS-CoV-2; infectious clone; nasal infection; neutralization assay; primary cells; reporter virus; respiratory tropism; reverse genetics.

Graphical abstract

Figure 1

Design and recovery of SARS-CoV-2 recombinant viruses (A) Full-length cDNA clone constructs and genomes of recombinant viruses. Restriction sites, cohesive ends, and the genetic marker T15102A (^∗) are indicated in the schematic diagram. GFP or GFP-fused nLuc genes were introduced into the ORF7 (replacing aas 14–104) of SARS-CoV-2 genome. (B) Plaques of recombinant viruses. (C) CPE and GFP signals were observed in Vero-E6 cells electroporated with sub-genomic RNA (sgRNA)-N alone (mock) or sgRNA-N mixed with full-length RNA transcripts (recombinant viruses) at two days after transfection. Scale bar, 100 μm. (D and E) SacI digestion (D) and Sanger sequencing (E) of a 1.5-kb region covering the genetic marker in vial genomes. (F) Northern blot analysis of genomic and sgRNAs isolated from the virus-infected cells. Abbreviations are as follows: Isolate, clinical isolate strain WA1; WT, icSARS-CoV-2-WT; GFP, icSARS-CoV-2-GFP, nLuc-GFP: icSARS-CoV-2-nLuc-GFP. See also Figure S1.

Figure S1

Additional information for the SARS-CoV-2 infectious cDNA clone, related to Figure 1 (A) Electrophoresis of seven restriction enzyme-digested infectious cDNA clone plasmids. Plasmid A was digested with NotI and BsaI; plasmids B, C, and D were digested with BsaI; plasmids E and F were digested with BsmBI; plasmid G was digested with SalI and BsaI. (B) Amplification SARS-CoV-2 sgRNAs using primers targeting sgRNA-5 (M) and −9 (N). Cellular RNA samples were collected from Vero-E6 cells electroporated with viral RNA transcripts at 20 h. Mock cells were electroporated with SARS-CoV-2 sgRNA-9 alone. (C) Alignment of sequences containing the T #15102 in nsp12 gene among 9 different group 2b CoVs.

Figure 2

Growth curves and the role of proteases in SARS-CoV-2 replication (A and B) One-step (A) and multi-step (B) growth curves of clinical isolate and recombinant viruses in Vero E6 cells, with MOI of 5 and 0.05, respectively. (C and D) Fluorescent images (C) and viral titers (D) of the SARS-CoV-2-GFP replicates in Vero cells supplemented with different concentrations of trypsin. (E and F) Fluorescent images (E) and viral titers (F) of the SARS-CoV-2-GFP replicates in normal Vero or Vero-furin cells. (G and H) Fluorescent images (G) and viral titers (H) of the SARS-CoV-2-GFP replicates in normal LLC-MK or LLC-MK-TMPRSS2 cells. All scale bars, 200 μm. Data are presented in mean ± SD. See also Figure S2.

Figure S2

Cytopathic Effect of Cells Infected with icSARS-CoV-GFP Virus, related to Figure 2 (A) Infected Vero cells supplemented with different concentrations of trypsin. (B) Infected Vero or Vero-furin cells. (C) Infected LLC-MK or LLC-MK-TMPRSS2 cells. All scale bars, 200 μm.

Figure 3

Neutralization assays using luciferase reporter coronaviruses (A and B) mAbs (A) and COVID-19 sera (B) against icMERS-CoV-nLuc. (C and D) mAbs (C) and SARS and COVID-19 sera (D) against icSARS-CoV-nLuc. (E–G) mAbs (E), SARS and COVID-19 sera (F), and vaccinated mouse serum (G) against icSARS-CoV-2-nLuc-GFP. (H) ID₅₀ values of SARS and COVID-19 sera cross-neutralizing SARS-CoV and SARS-CoV-2. The same sera samples are indicated with arrows. The MERS-CoV neutralizing mAbs were the following: MERS-27 and m336; the SARS-CoV neutralizing mAbs were the following: S230, S230.15, and S227.9; the Dengue virus mAb was the following: EDE1-C10. SARS patient serum samples are labeled as “A” to “E”; COVID-19 patient serum samples are labeled as “1” to “10”. Mouse serum was produced by immunized BALB/c mice with SARS-CoV-2 spike.

Figure 4

Intraregional ACE2 and TMPRSS2 mRNA expression in normal human airways (A) Representative RNA-ISH images demonstrating regional distribution of ACE2 and TMPRSS2 mRNA localization (red signal) in normal human airway surface epithelium. Scale bars, 20 μm. (B) Comparison of ACE2 and TMPRSS2 mRNA expression between matched nasal and bronchial brushed tissues obtained from seven healthy subjects. (C) Relative expression of ACE2 and TMPRSS2 mRNA in different airway regions enriched for epithelial cells, including tracheas, bronchi, bronchiole, and alveoli, obtained from matched seven normal lungs. (D) Frequency of ACE2⁺ and TMPRSS2⁺ cells among total cells identified in distinct anatomical airway regions in a re-analysis of scRNA-seq data (Deprez et al., 2019). (E) RNA-ISH images depicting mRNA expression of ACE2 and cell type markers, including FOXJ1 (ciliated) (i, ii, and iv), MUC5B (secretory) (iii and v), SFTPC (alveolar type 2) (vi), and HOPX (alveolar type 1 or 2) (vii) on cytospins of nasal versus bronchial superficial epithelial and purified alveolar cells. Scale bars, 10 μm. (F) Frequency of ACE2⁺ cells among nasal and bronchial preparations. A total of 1,000 cells were analyzed for ACE2 expression per donor (N = 3). G. Frequency of ACE2⁺ cells among FOXJ1⁺ or MUC5B⁺ cells in nasal or bronchial preparations. A total of 200 FOXJ1⁺ or MUC5B⁺ cells were analyzed for ACE2 expression per donor (N = 3). (H and I) Histograms depicting number of dot signals of ACE2 expression in FOXJ1 or MUC5B⁺ cells in nasal (H) or bronchial (I) preparations identified by scRNA-ISH. ACE2⁺ dot signals were counted in 200 FOXJ1 or MUC5B⁺ cells per donor (N = 3). Statistics for (B), (C), (F), and (G) used linear mixed-effect model with the donor as random-effect factor for comparison between groups, and pairwise comparisons of groups with more than two levels were performed using Tukey post hoc tests. (H) and (I) used generalized linear mixed-effect models with Poisson distribution to compare the difference in cell counts at varying ACE2 expression amounts between FOXJ1⁺ and MUC5B⁺ cells. Histobars and error bars represent mean ± SD. Different symbol colors indicate results from different individual donors. See also Figure S3.

Figure S3

ACE2 and TMPRSS2 expression in human tonsillar epithelium and nasal surface epithelium and submucosal glands, related to Figure 4 (A) Tonsillar surface squamous epithelium stained with (i) H&E staining and (ii) dual-color-fluorescence RNA-ISH showing TMPRSS2 (green) and ACE2 (red) along with nuclear staining (blue). Scale bars, 50 um. (iii) Enlarged images of (ii) showing surface (iii) and basal (iv) expression; scale bars, 20um. Images are representative from N = 3 tonsils, N = 4-8 regions per tonsil. (v) Signal dots for ACE2 and TMPRSS2 mRNAs were counted and normalized to the number of cells in surface and basal layer of tonsillar surface epithelium as described in the STAR Methods. Each bar represents the average of N = 4-8 regions for each tonsil studied. Data are presented in mean ± SD. (B) Frequency of FOXJ1- or MUC5B-positive cells identified by RNA-ISH among total nasal surface epithelial cells isolated. A total of 1,000 cells were analyzed for FOXJ1 or MUC5B expression per donor. N = 3. (C) Cytospins of nasal submucosal glands cells probed by dual-color-immunofluorescent RNA-ISH. (i) shows lack of ACE2 in MUC5B-positve nasal gland cells, while (ii) depicts occasional co-expression of TMPRSS2 in a subset of MUC5B-positive cells. Scale bars, 20 μm. (iii) Frequency of detection of ACE2 or TMPRSS2 positive cells in MUC5B positive cells from nasal glands. N = 1 gland preparation, a total of 200 MUC5B positive cells were counted.

Figure S4

Additional data of SARS-CoV and SARS-CoV-2 infected primary human cells, related to Figure 6 (A) Representative whole-mount extended focus views of icSARS-CoV-2-GFP-infected (i) HNE and LAE cell cultures. Red = filamentous actin (phalloidin), White = α-tubulin (multiciliated cells), Blue = nuclei (Hoechst 33342). Green = GFP (left). Green = SARS-CoV-2 Nucleocapsid (right). Yellow = MUC5AC (left). Yellow = MUC5B (right); (ii) LAE and SAE cell cultures. Yellow = filamentous actin (phalloidin), White = α-tubulin (multiciliated cells), Blue = nuclei (Hoechst 33342). Green = GFP (virus). Red = CCSP. Scale bars, 50 μm. (B) Merged of GFP and bright field mages taken from AT1 and AT2 cells infected with icSARS-CoV-2-GFP at 48 h. The AT-1 cells are present inside the enclosed areas. Bar = 100 μm. (C) GFP signals of icSARS2-GFP-infected HNEs collected from five different donors at 72 hpi, MOI = 3. (D) (i) Fluorescent signals of the two viruses in LAE (ii) Growth curves of three SARS-CoV-2 viruses in LAE from the same donor. Scale bar, 200 μm. (iii) Growth curves of two SARS-Urbani viruses in LAE. Data are presented in mean ± SD. All the infections in this figure were in MOI = 0.5.

Figure 5

Inflammatory cytokines alter ACE2 and TMPRSS2 expression (A) RNA-ISH images demonstrating regional distribution of ACE2 and TMPRSS2 mRNA localization in normal and CF human airways. Scale bars, 20 μm. Images were obtained from four different airway regions from one normal or CF subject as representative of N = 6 normal or CF subjects studied. (B) mRNA expression of ACE2 and TMPRSS2 measured by Taqman assay after inflammatory cytokine challenge in primary human large airway epithelial cells. Shown in (i) is IL-1β (10 ng/mL, 7 days, N = 8), in (ii) is IFN-β (10 ng/mL, 3 days, N = 4 donors, 2–3 cultures per donor), and in iii is IL-13 (10 ng/mL, 7 days, N = 8). Wilcoxon matched pairs signed rank test was used for comparison between control and cytokine treatment groups. Histobars and error bars represent mean ± SD. Different symbol colors indicate results from different individual donors.

Figure 6

Replication of SARS-CoV-2 in primary human respiratory cells (A) Representative GFP signals in icSARS-CoV-2-GFP-infected HNE, LAE, SAE, AT2-like, and AT1-like cultures at 48 h. Scale bar, 80. (B) Growth curves of icSARS-CoV-2-GFP in (i) HNE, n = 9 donors; (ii) LAE, n = 7 donors; (iii) SAE, n = 3 donors; (iv) AT1-like (empty symbols) and AT2-like (filled symbols) cells, n = 3 donors per cell type. Cells from female and male donors are labeled in pink and blue, respectively. Triplicated viral infections under MOI of 3 or 0.5 are shown in solid and dotted lines, respectively. In (v) is a comparison of the highest titers of individual culture among cell types and in (vi) is a comparison of individual titers in HNE and LAE at different time points. (C) Representative whole-mount extended focus views of icSARS-CoV-2-GFP-infected HNE and LAE cell cultures. Color coding is as follows: red, filamentous actin (phalloidin); white, α-tubulin (multiciliated cells); green, GFP (virus); blue, nuclei (Hoechst 33342); yellow, MUC5B (left) and MUC5AC (right). An arrow represents viral-infected α-tubulin⁺ (ciliated) and MUC5B⁺ (secretory) transitional HNEs. Scale bars, 50 μm. (D) Shown in (i) is the variability of GFP and cilia signals in icSARS-CoV-2-GFP-infected LAE cultures collected from five different donors at 72 hours after infection, scale bar, 200 μm. Shown in (ii) is the quantification of ciliated area in the LAE cultures. (E) Growth curves of icSARS-CoV-2-GFP infected in LAE and SAE collected form the same donor. Cultures were infected with SARS-CoV-2 clinical isolate (i), WT (ii), and GFP (iii) with MOI of 0.5. Data are presented in mean ± SD. See also Figure S4.

Figure 7

Characterization of cell types for SARS-CoV-2 infection in SARS-CoV-2 autopsy lungs (A) Sections from an autopsy lung with SARS-CoV-2 infection were stained by hematoxylin and eosin (i) and probed for SARS-CoV-2 by RNA-ISH (ii–iv). A SARS-CoV-2 sense probe (ii) was used. Scale bars, 1 mm. (B) The trachea from a SARS-CoV-2 autopsy was probed for SARS-CoV-2 by RNA ISH. Shown in (i) is a colorimetric detection of SARS-CoV-2 (red) showing infection of surface epithelium. Shown in (ii–iv) is the co-localization of SARS-CoV-2 (red) with cell-type-specific markers (green) determined by dual-immunofluorescent staining (ii, acetylated α-tubulin cilia marker; iii, MUC5B secretory cell marker; and iv, MUC5AC mucous (goblet)-cell marker). Scale bars, 10 μm. (C) Co-localization of SARS-CoV-2 with alveolar cell-type-specific markers in the alveolar space from a SARS-CoV-2 autopsy. Shown in (i) is the dual color-fluorescent RNA-ISH co-localization of SARS-CoV-2 (green) with alveolar type II cell marker SFTPC (red). Shown in (ii) is the dual-immunofluorescent co-localization of SARS-CoV-2 (green) with alveolar type I cell marker AGER (magenta). Scale bars, 20 μm. (D) Mucin expression in SARS-CoV-2 autopsy lung. Shown in (i) is the AB-PAS (blue to purple) stain for complex carbohydrate (mucin), in (ii) is MUC5B immunohistochemistry, in (iii–v) is the dual-immunofluorescent staining for MUC5B (green) and MUC5AC (red) in the large airway (iv) and the alveoli (v). Abbreviation is as follows: SM, submucosal grand. Scale bars, 2mm (i–iii) and 200 μm (iv and v). See also Figure S5.

Figure S5

SARS-CoV-2 infection in SARS-CoV-2 autopsy lungs, related to Figure 7 (A) Sections from of a second region of an autopsy lung with SARS-CoV-2 infection were stained by hematoxylin and eosin (H&E) (i) and probed for SARS-CoV-2 by RNA in situ hybridization (ISH) (ii, iii, and iv). Related to Figure 7A. (B) Frequency of acetylated alpha tubulin, MUC5AC, or MUC5B colocalization with SARS-CoV-2 positive cells in the trachea from a SARS-CoV-2 autopsy. A total of 200 randomly selected SARS-CoV-2 positive cells were analyzed for each dual staining condition. Related to Figure 7B ii, iii, iv. (C) Absence of SARS-CoV-2 infection in submucosal glands (SMG). (i-ii) H&E staining (i) and RNA-ISH (ii) for SARS-CoV-2 (red) in a large cartilaginous airway of one autopsy lung. SARS-CoV-2 is only present in the surface epithelium near the lumen, not in SMG. (iii-iv) H&E (iii) and dual-immunofluorescence staining using acetylated alpha tubulin (red) and anti-SARS-CoV-2 rabbit polyclonal antibody (green) (iv) from the trachea of a separate autopsy. Related to Figure 7B and S5Di. (D) Regional distribution of SARS-CoV-2 RNA from trachea to alveoli identified by RNA-ISH in one SARS-2-CoV autopsy lung (in i and ii, viral staining is red; in iii, viral staining is turquoise). RNA-ISH dual color images demonstrate SARS-CoV-2 RNA and SFTPC mRNA (alveolar type 2 cell marker) localization in alveoli of a SARS-CoV-2 autopsy lung. SARS-CoV-2 (turquoise) was identified in a SFTPC (red)-positive (iii, arrow) and a SFTPC-negative cell (iv, arrowhead); Scale bars, 2mm (A); 100 μm (C); 20 μm (D).