Characterizing Viral Infection by Electron Microscopy: Lessons from the Coronavirus Disease 2019 Pandemic

Abstract

The severe acute respiratory syndrome coronavirus 2 pandemic has infected millions of individuals in the United States and caused hundreds of thousands of deaths. Direct infection of extrapulmonary tissues has been postulated, and using sensitive techniques, viral RNA has been detected in multiple organs in the body, including the kidney. However, direct infection of tissues outside of the lung has been more challenging to demonstrate. This has been in part due to misinterpretation of electron microscopy studies. In this perspective, we will discuss what is known about coronavirus infection, some of the basic ultrastructural cell biology that has been confused for coronavirus infection of cells, and rigorous criteria that should be used when identifying pathogens by electron microscopy.

Figure 1

Subcellular mimics of coronaviruses. A: Diagram of a cell with some of the intracellular structures that have been mistaken for coronavirus. Coatomer-coated vesicles (yellow) are involved in the anterograde and retrograde transport of vesicles between the endoplasmic reticulum (ER) and Golgi. Clathrin-coated vesicles (blue) are involved in endocytosis. Multivesicular bodies (MVBs) are derived from early endosomes and contain cargo (ie, destined to be degraded through fusion with lysosomes or expulsion of exosomes). B: Glomerular endothelial cell with coated pit (arrow) and vesicle (arrowhead), consistent with clathrin-coated pit and vesicle. C: Tubular epithelial cell with coated vesicles (arrowheads) adjacent to ER/Golgi; note vesicle budding (arrow; inset) from ER/Golgi. D: Glomerular endothelial cell with coated vesicles in cytoplasm that have club-shaped spikes (arrow). E: Podocyte with multivesicular bodies. F: Podocyte with microvilli inside an invagination of the plasma membrane, resembling a cytoplasmic vesicle. B–F: Insets: Higher magnifications of the dashed boxed areas. Scale bars: 100 nm (B–D, main images, and B–F, insets); 500 nm (E and F).

Figure 2

Coronavirus infection in cells. A: Diagram of cell demonstrating structures that are associated with coronavirus infection. Double-membrane vesicles (DMVs) are found near the nucleus and represent the site of viral genome replication. Coronavirus particles bud into the cisternae of the endoplasmic reticulum (ER)/Golgi and accumulate in cytoplasmic vesicles that fuse with the plasma membrane and release virus particles into the extracellular space. B: A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–infected HBEC3-KT cell showing perinuclear DMV (arrow) and enlarged vesicles (arrowhead) filled with viral particles. C: Higher-magnification image of viral particles in cytoplasmic vesicles (arrowhead). D: Viral particles (arrowheads) within cisternae of ER/Golgi; particles have characteristic electron-dense dots corresponding to the helical nucleocapsid within the envelope. E: Viral particles (arrowhead) at the cell surface. The particles have an average envelope diameter of 64 nm and a range of 56 to 75 nm. The spikes are vague and not a prominent morphologic feature in transmission electron microscopy images. Scale bars: 500 nm (B); 100 nm (C–E).

Figure 3

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of cells with immunohistochemistry (IHC) and in situ hybridization (ISH). The figure shows tissues and cells that have been stained with anti-spike antibody (SARS-CoV/SARS-CoV-2 spike antibody; chimeric monoclonal antibody; 40150-D001; Sino Biological, Wayne, PA; left panels), and probed with anti-spike gene probe that recognizes intact virions (RNAscope Probe - V-nCoV2019-S; ACD Biosciences, Newark, CA; middle panels) or replicating virus (RNAscope Probe - V-nCoV2019-S-sense; ACD Biosciences; right panels). The rows from top to bottom are lung tissue from a patient who died of coronavirus disease 2019 (COVID-19), HBEC3-KT cells infected with SARS-CoV-2 (HBEC3), HSAEC1-KT cells infected with SARS-CoV-2 (HSAEC1), HEK 293 angiotensin-converting enzyme 2 (ACE2) cells infected with SARS-CoV-2 (HEK293-CoV2), and mock-infected HEK 293 ACE2 cells (HEK293-Mock). The HBEC3-KT and HSAEC1-KT cells are immortalized human bronchial epithelial and small airway and cell lines, respectively (ATCC, Manassas, VA); the HEK 293 ACE2 cells are HEK293T cells that are stably transformed with the human ACE2 gene. The anti-spike IHC and the intact virion ISH show similar staining patterns in all of the samples. The replicating virus ISH was undetectable in autopsy lung tissue, and positive in a small fraction of cells in the other samples. All images are the same magnification. Scale bar = 100 μm.