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.
Replication dynamics of SARS-CoV-2 in human airway epithelium
To confirm the replication dynamics of SARS-CoV-2 in HAE, fully differentiated HAE cultures derived from three different donors (1210, XK35, ZR05) were inoculated with SARS-CoV-2 or human coronavirus NL63 (HCoV-NL63) (Amsterdam, ATCC) at a multiplicity of infection (MOI) of 0.1, which was consistent with the infection of HAE by other coronaviruses, including HCoV-NL63 SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43 and HCoV-HKU1 to assess the viral growth kinetics. Additionally, the replication efficiency of the two kinds of viruses could be compared by using the same MOI to inoculate HAE cells. As shown in Fig. 1a, HAE cells were highly susceptible to SARS-CoV-2 infection with peak virus production from apical wash at 48–72 h post infection (h pi) and remained at a high level from 3 to 6 days. In contrast, HCoV-NL63 reached peak virus load at 72–96 h pi, similar to previously reported SARS-CoV replication kinetics in HAE14. SARS-CoV-2 progeny viruses were released into the basolateral medium as the infection progressed, yet nearly no progeny viruses were released into the basolateral medium post infection with HCoV-NL63 (Fig. 1a). We further monitored transepithelial electrical resistance (TEER), considered a surrogate of epithelium integrity, during infection with SARS-CoV-2 or HCoV-NL63, and we found that at 96 h pi, the TEER of HAE inoculated with SARS-CoV-2 was reduced nearly 40% but not in HCoV-NL63-infected cells (Fig. 1b). Notably, the decrease in TEER in SARS-CoV-2-infected HAE was accompanied by an increase in SARS-CoV-2 progeny virus detected in the basolateral medium
Cell tropism of SARS-CoV-2 in human airway epithelium
Previous studies showed that SARS-CoV-2 utilized ACE2 as its cell surface receptor8,9, suggesting that SARS-CoV-2 may share a similar cell tropism (ciliated cells in HAE) with SARS-CoV and HCoV-NL63 by using the same receptor. To further confirm the tropism of SARS-CoV-2 in HAE, transmission electron microscopy (TEM) and laser scanning confocal microscopy analyses were performed. Surprisingly, we found that SARS-CoV-2 infects both ciliated cells and secretory cells. As shown in Fig. 1c, virus particles were found on the apical surface of both ciliated cells and secretory cells; inclusion bodies formed by viral components were observed in the cytoplasm, which confirmed the infection of both cell types. Immunofluorescent staining of SARS-CoV-2 N protein colocalized with ciliated cells (marker: β-tubulin IV) and secretory cells, including goblet cells (marker: Muc5AC) and club cells (marker: CCSP) (Fig. 1d). This was dramatically different from six other human coronaviruses. It had been demonstrated that HCoV-HKU1, HCoV-OC43, HCoV-NL63, and SARS-CoV infect ciliated cells while HCoV-229E and MERS-CoV infect secretory cells. Additionally, the presence of SARS-CoV-2-infected ACE2-positive cells further confirmed ACE2 as a surface receptor of SARS-CoV-2
Cytopathic effects (CPE) and ultrastructural pathology induced by SARS-CoV-2 in human airway epithelium
In our previous study, unique CPE were observed in HAE induced by SARS-CoV-2 infection1. To investigate the CPE in more detail, SARS-CoV-2-infected HAE were analyzed by laser scanning confocal microscopy, scanning electron microscopy (SEM) and immunofluorescence staining. Plaque-like CPE was consistently observed in different propagations of SARS-CoV-2-infected HAE (Fig. 2a). The size and number of plaques increased with the extension of the incubation time. As shown in Fig. 2b, multinucleated syncytial cells arranged in a net-like structure were observed in the plaque regions. Immunofluorescence staining using a specific SARS-CoV-2 N protein antibody and cell tight junction antibody showed giant syncytial cell formation and destruction of cell tight junctions (Fig. 2k). Cilium shrinking (Fig. 2d) in the plaque region (Fig. 2c) and beaded changes (Fig. 2e) in the periphery of plaques were detected compared with mock-infected cells
Discussion
The organotypic HAE system forms a pseudostratified epithelial layer that morphologically and functionally resembles the human airway. The cultures of basal, ciliated, and secretory cells produced protective mucus and beating cilia that are visible under a light microscope27. ACE2 is mainly expressed on ciliated epithelial cells of the human lungs8 and is thought to be the cell surface receptor for SARS-CoV-210. Interestingly, SARS-CoV and HCoV-NL63 share the same receptor as SARS-CoV-2 and a tropism for ciliated cells. Here, we reported SARS-CoV-2 infection of both ciliated cells and secretory cells in HAE, which consequently suggested the possibility of more receptors for SARS-CoV-2 attachment in addition to the ACE2 receptor10. We also described parts of the morphogenesis process and cytopathic effect of SARS-CoV-2 infection in both cell types in detail.
Epidemiological investigations have suggested that SARS-CoV-2 is highly infectious and transmissible among humans. In comparison to SARS-CoV or MERS-CoV, SARS-CoV-2 replicated more efficiently in primary HAE than in standard immortalized cells. This might be because there were more susceptible cells to SARS-CoV-2 infection than to other coronavirus infections. SARS-CoV-2 replicates more efficiently than HCoV-NL63 in HAE. This observation itself indicated that SARS-CoV-2 is more transmissible. The efficient transmissibility may be due to there being one more permissible cell type for SARS-CoV-2 transmission. Furthermore, the secretory cells released vesicles with large amounts of virus particles dispersed in the cytoplasm by exocytosis, which underlies the high detection rate of SARS-CoV-2 in sputum28 and its transmission by droplets. Additionally, few pathologies have been reported due to the limited access to autopsy or biopsy specimens, but all the reports5,6 have referred to the observation of a large amount of foam or gelatinous mucus in the trachea at autopsy. This may be induced by infection of secretory cells and dysregulation of mucus secretion balance, indicating that clinical treatment should be considered to restore the balance in mucus secretion.
Respiratory ciliary function abnormalities have been associated with various diseases, such as cystic fibrosis, chronic obstructive pulmonary disease, and sinusitis29. In this study, large areas of disordered cilia were visually confirmed by SEM analysis, indicating that SARS-CoV-2 infection disrupted cilia synchronicity. Cilium shrinking in the center of CPE plaque was first observed as no ciliary beating under the light microscope could be found. The beaded changes in cilia in the peripheral region of plaques were considered characteristic of severe pathological changes. The abnormal ciliary beating and disruption of cilia synchronicity lead to poor mucociliary clearance (MCC), which can result in secondary infections30. Aggregation of organelles close to the apical surface featuring a large number of mitochondria with abnormal morphology was also identified as another striking ultrastructural change. The extensive cell death observed in the CPE region was shown to be apoptosis.
In addition, unique plaque-like CPE on the apical surface of HAE were observed consistently across different propagations. The size and numbers of these plaques expanded and increased over the incubation period as well as the formation of syncytial cells and destruction of cell tight junctions. Collectively, our findings also suggested that SARS-CoV-2 might either be released via the apical surface of infected cells or transmit through direct cell-to-cell contact.
Taken together, our data support the notion that SARS-CoV-2 is fully adapted to the human airway, which is distinct from other coronaviruses that were reported to have interspecies transmission. Our study shows SARS-CoV-2 multicellular tropism and severe ultrapathological changes and malfunction of cilia, which could provide clues for clinical treatment and drug screening strategies. Recent publications7,31 indicated a high expression level of the SARS-CoV-2 receptor ACE2 gene in both goblet cells and ciliated cells by scRNA sequencing, which is consistent with our study. One of the limitations in our work is the lack of host response analyses after HAE are infected with SARS-CoV-2 as has been performed in a recent study32. Nevertheless, the results reported here should improve the understanding of SARS-CoV-2 transmission and pathogenesis.
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