Cell tropism and viral clearance during SARS-CoV-2 lung infection

Abstract

Pulmonary capillary microthrombosis has been proposed as a major pathogenetic factor driving severe COVID-19. Autopsy studies reported endothelialitis but it is under debate if it is caused by SARS-CoV-2 infection of endothelial cells. In this study, RNA in situ hybridization was used to detect viral RNA and to identify the infected cell types in lung tissue of 40 patients with fatal COVID-19. SARS-CoV-2 Spike protein-coding RNA showed a steadily decreasing signal abundance over a period of three weeks. Besides the original virus strain the variants of concern Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529) could also be detected by the assay. Viral RNA was mainly detected in alveolar macrophages and pulmonary epithelial cells, while only single virus-positive endothelial cells were observed even in cases with high viral load suggesting that viral infection of endothelial cells is not a key factor for the development of pulmonary capillary microthrombosis.

Keywords: COVID-19; Cellular target; Endothelialitis; SARS-CoV-2 infection; Time-course.

Fig. 1

Temporal course of SARS-CoV-2 lung infection, A) Early COVID-19 pneumonia showing alveolar edema and hyaline membrane formation. B) Intermediate proliferative stage of COVID-19 pneumonia with type II pneumocyte hyperplasia and squamous metaplasia. C) Late stage disease with coexistence of alveolar macrophages accumulation, type II pneumocyte hyperplasia and septal broadening due to interstitial fibrosis. D) Same patient as in A, diffuse SARS-CoV2-Spike RNA signals (red) in alveolar cells and hyaline membrane. E) Same patient as in B, focal piling of SARS-CoV2-Spike RNA signals (red) in alveolar and interstitial cells. F) Same patient as in C, focal detection of single hybridization signals (red) in this case. G) CD68 RNA positive macrophages (red) are packed with SARS-CoV-2 Spike RNA (green). H) Co-hybridization with a KRT18 probe (red) demonstrates presence of viral RNA (green) in pneumocytes (arrows). I) Very few Spike RNA signals (green) are seen in MCAM RNA (red) positive endothelial cells (arrows). I) Each bar represents 50 µm.

Fig. 2

Quantification of viral RNA in lung tissue A) Clearance of viral RNA from lung tissue over time. B) Comparison of SARS-CoV-2 RNA signals in deceased COVID-19 patients with and without prior anti-SARS-CoV-2 vaccination. C) Viral RNA signals in different stages of COVID-19 pneumonia. D) Quantification of viral RNA in different cell types. CD68 marks alveolar macrophages. KRT 18 was used to detect lung epithelial cells and MCAM was used to identify SARS-CoV-2 in endothelial cells. *p < 0.05; **p < 0.01; ns, not significant.