Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

Abstract

SARS-CoV-2, a β-coronavirus, has rapidly spread across the world, highlighting its high transmissibility, but the underlying morphogenesis and pathogenesis remain poorly understood. Here, we characterize the replication dynamics, cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison, HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion, apoptosis, destruction of epithelium integrity, cilium shrinking and beaded changes are observed in the plaque regions. Taken together, our results provide important insights into SARS-CoV-2 cell tropism, replication and morphogenesis.

Fig. 1. Characterization and cell tropism of SARS-CoV-2 in human airway epithelia (HAE).

a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control (n = 3). b Transepithelial electrical resistance (TEER in Ω cm²) between the apical and basal poles was measured at each time point (n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a, b are the means ± s.d. of three independent biological replicates. Source data a–d are provided as a Source Data file.

Fig. 2. Cytopathic effect of SARS-CoV-2 infection on HAE cells.

Characterization of SARS-CoV-2 infection of the HAE surface a–h. a Plaques (arrow) induced by SARS-CoV-2 infection under a light microscope. b Cell fusion and net-like structure (dashed line square) under a laser scan confocal microscope in the plaque region. c Plaque area featured with less content under a scanning electron microscope (dashed line circle). d Deformation of cilia in the plaque area under a scanning electron microscope. e Cilia polarity disorder and granular formation on cilium with rough surface under a scanning electron microscope. f Normal HAE cell cilium with polarity order. g Cilia polarity disorder under a scanning electron microscope. h Mock HAE cell cilium with smooth surface and polarized order. Ultra-pathology of SARS-CoV-2-infected HAE cells i, j. i Overview of a virus-infected ciliated cell. The black line box indicates double membrane vesicles (DMVs) induced by virus infection in ciliated cells. The dashed line box indicates aggregation of denatured mitochondria (Mt) and enlarged endoplasmic reticulum (ER) on the top area of ciliated cells. Virus particles on cilia (Cl) (arrow) and microvilli (Mv) (empty arrow). j Overview of a virus-infected secretory cell with cell organelles and secretory vesicles (SV) aggregated on the top area of the cell. The black line box indicates double membrane vesicles (DMVs) induced by virus infection in secretory cells. The dashed line box indicates virus particles both in the cytoplasm and on microvilli (Mv) (arrows). k Syncytial cell formation (star) and cell tight junction destruction (white arrow) caused by SARS-CoV-2 infection. l Apoptosis induced by SARS-CoV-2 infection in HAE. Apoptotic cells (green) stained with Apopxin Green (ApGreen) and TdT-mediated dUTP Nick-End Labeling (TUNEL) indicator. Source data a–i are provided as a Source Data file.

Fig. 3. SARS-CoV-2 morphogenesis in HAE cells.

SARS-CoV-2 infected both secretory cells a–h and ciliated cells k–q and presented similar morphogenetic processes (72 h p.i.). a Virus attaching (arrow) on the cell surface. b Virus and cell membrane fusion (arrow). c Virus budding (arrow) into endoplasmic reticulum vesicles (ERV). d Virus-associated inclusion bodies (IB) filled with electron condensed matrix in the cytoplasm. e Strands of the endoplasmic reticulum contained rows of viral particles in the cytoplasm (arrow). f Inclusion full of virus particles (star) with membrane-bound compressed by secretory vesicles (SV). g Virus particles (arrow) released from the cell together with cytoplasmic components (dashed box). h Virus particles (arrow) released with secretory vesicles (SV, dashed line box). i Provirus particles (arrow) generated in membrane-rich areas in the cytoplasm of ciliated cells. j Virus-containing vacuoles (arrow) in the Golgi compartments (Go). k Virus particle aggregation (star) with matrix but without bound membrane enclosed by mitochondria (Mt) in the cytoplasm. l Strands of endoplasmic reticulum containing rows of viral particles in the cytoplasm. m Inclusion body (IB) filled with different sizes of spherical virus particles and condensed granular matrix. n Inclusion body (IB) filled with pleomorphic virus particles. o Inclusion body (IB) filled with spherical mature virus particles of different sizes. p Scattered virus particles in the vesicle (V)-rich area in the cytoplasm. q Virus particle release by exocytosis (arrow). r The negatively stained SARS-CoV-2 are spherical. Particle with distinctive spikes (arrow), without spikes (empty arrow) or with partial spikes (triangle). Scale bar: 100 nm. Source data a–r are provided as a Source Data file.