Detection and identification of coronaviruses in human tissues using electron microscopy

Abstract

The identification of viral particles within a tissue specimen requires specific knowledge of viral ultrastructure and replication, as well as a thorough familiarity with normal subcellular organelles. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored how challenging the task of identifying coronavirus by electron microscopy (EM) can be. Numerous articles have been published mischaracterizing common subcellular structures, including clathrin- or coatomer- coated vesicles, multivesicular bodies, and rough endoplasmic reticulum, as coronavirus particles in SARS-CoV-2 positive patient tissue specimens. To counter these misinterpretations, we describe the morphological features of coronaviruses that should be used to differentiate coronavirus particles from subcellular structures. Further, as many of the misidentifications of coronavirus particles have stemmed from attempts to attribute tissue damage to direct infection by SARS-CoV-2, we review articles describing ultrastructural changes observed in specimens from SARS-CoV-2-infected individuals that do not necessarily provide EM evidence of direct viral infection. Ultrastructural changes have been observed in respiratory, cardiac, kidney, and intestinal tissues, highlighting the widespread effects that SARS-CoV-2 infection may have on the body, whether through direct viral infection or mediated by SARS-CoV-2 infection-induced inflammatory and immune processes. HIGHLIGHTS: The identification of coronavirus particles in SARS-CoV-2 positive tissues continues to be a challenging task. This review provides examples of coronavirus ultrastructure to aid in the differentiation of the virus from common cellular structures.

Keywords: COVID-19; SARS; SARS-CoV-2; coronavirus; electron microscopy; ultrastructure.

FIGURE 1

Electron microscopic images of three isolates of different members of the family Coronaviridae that have caused serious disease in recent years. (a) Severe acute respiratory syndrome coronavirus 1 (SARS‐CoV‐1) from 2002–2003. Intracellular viral particles are held within membrane‐bound vacuoles (arrow), and extracellular viral particles line the cell surface (arrowhead). Scale bar: 1 μm. Inset: Higher magnification of SARS‐CoV‐1 particles. Scale bar: 100 nm. (b) Intracellular (arrow) and extracellular (closed arrowhead) accumulations of Middle East respiratory syndrome coronavirus (MERS‐CoV). A clathrin‐coated vesicle, often misidentified as coronavirus, is also visible free in the cytoplasm (open arrowhead). Scale bar: 100 nm. (c) Severe acute respiratory syndrome coronavirus −2 (SARS‐CoV‐2) from 2020. Vacuole containing SARS‐CoV‐2 particles fused to the plasma membrane of an infected cell. Scale bar: 100 nm

FIGURE 2

Coronaviruses detected in autopsy tissues. (a) Intracellular SARS‐CoV‐2 particles within a type II pneumocyte from well preserved autopsy tissue. The vacuolar membrane is clearly visible (arrowhead) as are the cross sections through the viral nucleocapsids (arrow). Scale bar: 100 nm. (b) Intracellular SARS‐CoV‐1 particles within a pneumocyte from formalin‐fixed, paraffin‐embedded (FFPE) autopsy tissue. Overall ultrastructure is deteriorated with viral particles appearing smaller than normal and more electron dense. The vacuolar membrane is visible (arrowhead) but appears less contiguous. Scale bar: 100 nm. Image reproduced from Shieh et al. (2005). (c) MERS‐CoV particles in a deteriorating cell within the lung from FFPE autopsy tissue. Portions of the vacuolar membrane are visible (arrowhead) as are cross sections through the viral nucleocapsid on some viral particles (arrow). Image courtesy of Maureen Metcalfe, CDC. Scale bar: 100 nm

FIGURE 3

Subcellular structures commonly misidentified as coronavirus. (a) Clathrin‐coated vesicles (CCVs; arrows). Scale bar: 500 nm. (b) Circular cross sections through rough endoplasmic reticulum (RER; arrow). Scale bar: 500 nm. (c) Multivesicular body (MVB). Scale bar: 250 nm

FIGURE 4

Immuno‐electron microscopy (IEM) for SARS‐CoV‐2 using hyperimmune mouse ascites fluid raised against SARS‐CoV‐1. (a) IEM of SARS‐CoV‐2 infected cell culture embedded in LR white resin without osmification. Colloidal gold (electron dense, 12 nm black dots) identifies areas immunoreactive with the SARS‐CoV‐1 antibody, including vacuolar accumulations of presumed viral particles (arrow) and nucleocapsid inclusions (arrowhead). Scale bar: 200 nm. (b) SARS‐CoV‐2 infected cell culture osmicated and embedded using epoxy resin. The ultrastructure of the infected cell, vacuolar accumulation of coronavirus (arrows), and viral nucleocapsid inclusions (arrowheads) are more clearly defined than in the LR white embedded sample. Scale bar: 200 nm