Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, causing a respiratory disease (coronavirus disease 2019, COVID-19) of varying severity in Wuhan, China, and subsequently leading to a pandemic. The transmissibility and pathogenesis of SARS-CoV-2 remain poorly understood. We evaluate its tissue and cellular tropism in human respiratory tract, conjunctiva, and innate immune responses in comparison with other coronavirus and influenza virus to provide insights into COVID-19 pathogenesis.

Methods: We isolated SARS-CoV-2 from a patient with confirmed COVID-19, and compared virus tropism and replication competence with SARS-CoV, Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and 2009 pandemic influenza H1N1 (H1N1pdm) in ex-vivo cultures of human bronchus (n=5) and lung (n=4). We assessed extrapulmonary infection using ex-vivo cultures of human conjunctiva (n=3) and in-vitro cultures of human colorectal adenocarcinoma cell lines. Innate immune responses and angiotensin-converting enzyme 2 expression were investigated in human alveolar epithelial cells and macrophages. In-vitro studies included the highly pathogenic avian influenza H5N1 virus (H5N1) and mock-infected cells as controls.

Findings: SARS-CoV-2 infected ciliated, mucus-secreting, and club cells of bronchial epithelium, type 1 pneumocytes in the lung, and the conjunctival mucosa. In the bronchus, SARS-CoV-2 replication competence was similar to MERS-CoV, and higher than SARS-CoV, but lower than H1N1pdm. In the lung, SARS-CoV-2 replication was similar to SARS-CoV and H1N1pdm, but was lower than MERS-CoV. In conjunctiva, SARS-CoV-2 replication was greater than SARS-CoV. SARS-CoV-2 was a less potent inducer of proinflammatory cytokines than H5N1, H1N1pdm, or MERS-CoV.

Interpretation: The conjunctival epithelium and conducting airways appear to be potential portals of infection for SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated similarly in the alveolar epithelium; SARS-CoV-2 replicated more extensively in the bronchus than SARS-CoV. These findings provide important insights into the transmissibility and pathogenesis of SARS-CoV-2 infection and differences with other respiratory pathogens.

Funding: US National Institute of Allergy and Infectious Diseases, University Grants Committee of Hong Kong Special Administrative Region, China; Health and Medical Research Fund, Food and Health Bureau, Government of Hong Kong Special Administrative Region, China.

Figure 1

Tissue tropism of SARS-CoV-2 and SARS-CoV viruses in ex-vivo cultures of human respiratory tract and conjunctiva Ex-vivo cultures of human bronchus and lung were infected with mock, SARS-CoV-2, SARS-CoV, MERS-CoV, and H1N1pdm viruses and the tissues were fixed with formalin at 96 hpi. Conjunctiva tissues were infected with mock, SARS-CoV-2, and SARS-CoV, and the tissues were fixed with formalin at 48 hpi. Paraffin-embedded sections were subjected to immunohistochemical staining with a monoclonal antibody against the SARS-CoV nucleoprotein, MCoV nucleoprotein, and influenza nucleoprotein. Positive cells are brown. Inset images are 200x magnification. SARS-CoV=severe acute respiratory syndrome-associated coronavirus. MERS-CoV=Middle East respiratory syndrome-associated coronavirus. H1N1pdm=2009 pandemic influenza H1N1.

Figure 2

Cellular tropism of SARS-CoV-2 in ex-vivo cultures of human bronchus and lung Ex-vivo explant cultures of (A) human bronchus and (B) lung were infected with SARS-CoV-2. At 96 hpi the tissues were fixed in formalin, embedded in paraffin, and immunohistochemically stained (brown) for indicated cell markers: (A) Ac-Tub-positive for ciliated cells, MUC5AC-positive for secretory goblet cells, CC10-positive for club cells, p63a-positive for basal cells; and (B) AE1/3 for epithelial cells (brown) and CD68 for macrophages (red), and a monoclonal antibody against the SARS-CoV-2 nucleoprotein (red). SARS-CoV-2-infected cells are identified with brown arrows, and CD68-positive cells are identified with red arrows. The images are representative of three individual donors. NP=nucleoprotein. Ac-Tub=acetyl-α-tubulin. MUC5AC=mucin 5AC. SCGB1A1=secretoglobin family 1A member 1. CC10=club cell protein 10. p63a=p63-alpha. AE1/3= cytokeratin AE1/AE3. CD68=cluster of differentiation 68.

Figure 3

Viral replication kinetics of SARS-CoV-2 and SARS-CoV viruses in ex-vivo cultures of human respiratory tract, conjunctiva, Caco-2 cells, AECs and macrophages (A and B) Human ex-vivo cultures of bronchus (n=5) and lung (n=4), were infected with 5 × 10⁵ TCID₅₀ per mL at 37°C. (C) Human ex-vivo cultures of conjunctiva (n=3) were infected with 5 × 10⁵ TCID₅₀ per mL at 33°C. (D) Caco-2 cells (n=3), (E) AECs (n=3) and (F) macrophages (n=3) were infected with the indicated viruses at a MOI of 0·1 cultured at 37°C for 72 h. Culture supernatants were harvested at the indicated times and virus titres were measured by TCID₅₀ assay. Bar charts show the mean (SD). The horizontal dotted line denotes the limit of detection in the TCID₅₀ assay. Statistical significances compared with SARS-CoV-2 are shown and statistical significances with exact p values compared among other viruses are listed in the appendix (p 8). TCID₅₀=50% tissue culture infection dose. SARS-CoV=severe acute respiratory syndrome-associated coronavirus. MERS-CoV=Middle East respiratory syndrome-associated coronavirus. H1N1pdm=2009 pandemic influenza H1N1. H5N1=highly pathogenic avian influenza H5N1 virus.

Figure 4

Viral gene, cytokine, chemokine, and ACE2 expression profile of SARS-CoV-2 Expression of the mRNA of viral genes (SARS-CoV-2 and SARS-CoV ORF1b gene; MERS-CoV UpE gene; influenza matrix gene), TNF-α, IP-10 and RANTES, IL-6, and ACE2 in alveolar epithelial cells (n=3), human macrophages (n=3) at 24 hpi, and Caco-2 cells (n=3) at 48 hpi. ACE2 is shown only for AECs and MCP-1 is shown for macrophages and Caco-2 cells. Graphs show mean mRNA copies expressed per 1 × 10⁵ β-actin copies from three independent experiments. Data are mean (SD). Statistical significances with exact p values compared among other viruses are shown. SARS-CoV=severe acute respiratory syndrome-associated coronavirus. MERS-CoV=Middle East respiratory syndrome-associated coronavirus. TNF-α=tumour necrosis factor alpha. IP10=interferon gamma-induced protein 10. RANTES=regulated on activation, normal T cell expressed and secreted. IL-6=interleukin 6. ACE2= angiotensin-converting enzyme-2. AECs=alveolar epithelial cells. Caco-2=human colorectal carcinoma cells. MCP-1=monocyte chemoattractant protein 1. H1N1pdm=2009 pandemic influenza H1N1. H5N1=highly pathogenic avian influenza H5N1 virus.