Light Sheet Microscopy-Assisted 3D Analysis of SARS-CoV-2 Infection in the Respiratory Tract of the Ferret Model

Abstract

The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host–pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (>150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and suggest clustering of infection foci in close proximity. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of IFN-mediated innate immune defenses

“…While SARS-CoV-2 is known to induce a comparatively weak immune response relative to other respiratory viruses (e.g., IAV) [ 20 , 39 ], we observed localized areas of epithelial infection ( Fig 1A , arrows) that were coincident with elevated levels of ISG expression by 72 h postinfection ( Fig 7F , Mx1). These findings are consistent with reports that have shown localized areas of infection in the respiratory airway of infected ferrets and surface epithelium of organoid cultures [ 29 – 31 , 49 ]. Thus, the proportion of epithelial infection and/or rate of intraepithelial spread may account for the differences observed in immune signature reported between different respiratory pathogens [ 20 , 51 ].…”

“…Infection of respiratory airway cultures with SARS-CoV-2 (England/02/2020; MOI 0.05, 10 4 plaque-forming unit (PFU)/Tissue) at 37°C demonstrated these tissues to support infection and viral replication, with intraepithelial and apical vRNA accumulation readily detected by in situ hybridization (ISH) by 72 h postinfection ( Fig 1A and 1B ). Notably, we observed discrete clusters of vRNA accumulation within respiratory epithelia ( Fig 1A , arrows), indicative of localized areas of intraepithelial infection, propagation, and spread [ 29 , 30 ]. The overall morphology of the respiratory epithelium remained largely intact, with little shedding of ciliated cells from the epithelial surface ( Fig 1A , 120 h).…”

“…phenotype in SARS-CoV-2 replication in respiratory epithelia that occurs independently of the robust induction of IFN-mediated innate immune defenses known to restrict SARS-CoV-2 replication [37,38]. The differentiation of stratified respiratory epithelium has proven to be a valuable research tool to investigate the cellular tropism, replication kinetics, and immune regulation of SARS-CoV-2, as these tissue models mimic many aspects of infection observed in animal models and COVID-19 patients [16][17][18][19][20][21][22][23][24]29,[45][46][47][48][49]. While the use of such 3D models represents an important advancement over traditional two-dimensional cell culture systems, the absence of circulating immune cells (e.g., macrophages, natural killer cells, dendritic cells, and neutrophils), which modulate fever and proinflammatory immune responses to infection is an important limiting factor [9,10,50].…”

Comparative pathogenesis of different phylogroup I bat lyssaviruses in a standardized mouse model

“…Immunostaining and clearing protocols from previous reports [51][52][53] were modified and performed as described previously [54]. All incubation steps were performed on an orbital shaker.…”

Studying SARS-CoV-2 with Fluorescence Microscopy

“…Confocal imaging of the infected human enteroids after co-staining of viral NP and villin, a marker of human enterocytes, demonstrated that the most infected cells in the enteroids were villin-positive, indicating that enterocytes are the major target cell of SARS-CoV-2. Additionally, Zaeck et al combined the light sheet fluorescence microscopy and tissue optical clearing to make the 3D overview of SARS-CoV-2 infection in the ferret model [ 26 ]. Modified iDisco protocol allowed to achieve optically cleared ferret nasal turbinates and lung tissues and then to perform immunostaining with polyclonal serum and monoclonal antibodies against N protein.…”