key: cord-0969102-ol1hjg28
authors: Bullock, Hannah A.; Goldsmith, Cynthia S.; Miller, Sara E.
title: Detection and identification of coronaviruses in human tissues using electron microscopy
date: 2022-04-04
journal: Microsc Res Tech
DOI: 10.1002/jemt.24115
sha: b9dc7ad20ee112babb8c801e7d5f0d78cbccaab6
doc_id: 969102
cord_uid: ol1hjg28

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.

The coronavirus disease pandemic has resulted in a renewed focus on transmission electron microscopy (EM) as a means of detecting viral particles within clinical and autopsy specimens. While the use of EM as a first-line diagnostic method in infectious disease has waned due to the advent of molecular techniques, EM continues to be an important tool for diagnosis and research into the ultrastructural basis of disease. It is particularly useful when the infectious agent is unknown and has not been detected by molecular or immunological techniques due to test specificity and sensitivity or reagent choice. For example, EM played a key role in the identification of a coronavirus as the causative agent of the 2002-2003 severe acute respiratory syndrome outbreak . During the COVID-19 pandemic, EM has proven valuable in establishing the extent of direct viral infection in clinical specimens Martines et al., 2020; Meinhardt et al., 2020) as well as providing insights into the effects of SARS-CoV-2 infection on tissues throughout the body (Kudose et al., 2020; Sharma et al., 2020) .

Since early 2020, EM has been used to attempt to find SARS-CoV-2 particles in patient tissue specimens. Unfortunately, many of these studies have misidentified common subcellular structures as coronavirus particles, leading to confusion and the publication of inaccurate information. This is most problematic when EM is the only method used to detect a virus in a particular tissue or when all other diagnostic methods have yielded negative results. Numerous articles and letters to the editor have been written to address this evergrowing problem (Akilesh, Nicosia, et al., 2021; Bullock, Goldsmith, Zaki, et al., 2021; Calomeni et al., 2020; Dittmayer et al., 2020; Goldsmith et al., 2020; Kniss, 2020; Miller & Brealey, 2020; Neil et al., 2020; Roufosse et al., 2020) . Additionally, more clinical and research articles are now providing examples of structures observed by EM in SARS-CoV-2 positive tissues that simply mimic viral particles (e.g., coated vesicles and multivesicular bodies) Sharma et al., 2020) .

With the aim of increasing accurate identification of coronavirus particles in COVID-19 cases, we provide herein examples of coronavirus particles as well as common subcellular structures that may be mistaken for viral particles. We also discuss complementary methods for virus identification to support traditional EM data and review studies reporting the ultrastructural pathology of SARS-CoV-2 infection.

Members of the family Coronaviridae all have the same morphology ( Figure 1 ) (Almeida & Tyrrell, 1967; Goldsmith et al., 2004; Oshiro et al., 1971) . The following morphologic characteristics must be present to conclusively identify suspected viral particles as coronavirus:

1) Coronaviruses are enveloped viruses that range in size from 60 nm to 140 nm in diameter. 2) Coronaviruses have surface peplomers (spikes) that may be visible on extracellular viral particles in thin section preparations (tissue specimens embedded in epoxy resin).

Intracellular viral particles rarely have visible surface peplomers. The surface peplomers are most clearly visible in negative stain preparations (liquid samples stained with a heavy metal salt solution) (Hayat, 2000) . 3) Intracellular viral particles are always contained within cytoplasmic vacuoles. 4) Coronaviruses have a helical nucleocapsid that is visible in cross section as small electron-dense dots that are 6-12 nm in diameter ( Figure 1 ).

In addition to viral morphologic appearance, knowledge of the viral replicative process is also essential in correctly identifying a coronavirus. All coronaviruses mature by budding through the membranes (Goldsmith et al., 2004; Oshiro et al., 1971) . Virions are then released from the cell by exocytosis once the viral vacuole fuses with the host cell plasma membrane (Figure 1c ). Virus particles may stay attached to the cell surface. The attachment of the mature particles to the plasma membrane is a hallmark characteristic of coronaviruses ( Figure 1a) ; however, this does not preclude the release of viral particles away from the infected cell. Coronavirus replication may also result in the formation of modified host cell membranes such as double-membrane vesicles and convoluted membranes, though these structures are not always observed in an infected cell (Knoops et al., 2008; Snijder et al., 2020) . Because of the mechanism of coronavirus replication, coronavirus particles will not be found free in the cytoplasm, but instead will be held within membrane-bound vacuoles.

Details of coronavirus morphology and replication should be used as a guide while attempting to identify the virus within an infected cell.

When analyzing autopsy tissues or formalin-fixed, paraffinembedded (FFPE) tissues, the morphologic characteristics of coronaviruses may be difficult to perceive due to tissue autolysis and/or the additional processing that FFPE tissues undergo prior to being prepared for EM ( Figure 2 ). In these cases, it is advisable to use addi- (Table 1) , leading to possible confusion as to whether the virus itself is causing tissue damage throughout the body or if tissue damage is a result of other factors, like the cytokine storm or downstream F I G U R E 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) . Kidney 12

Heart 9

Placenta 9

Intestine 5

Liver 5

Skin 3 (Figure 1b-CCVs, Figure 3 ). By following the coronavirus identification criteria described above, one can easily differentiate a coronavirus particle from these more common cellular organelles Bullock, Goldsmith, Zaki, et al., 2021) . CCVs, MVBs, and RER all lack the internal electron-dense black dots that signify cross-sections through the helical nucleocapsid curled up within the virus particle. While CCVs have a generally spherical shape and a fringe of spike-like clathrin or coatomer protein surrounding the vesicle, CCVs are found free in the cytoplasm, unlike coronaviruses which are found within membrane-bound vacuoles ( Figure 1b, Figure 3a ). The ribosomes along circular cross-sections through the RER may also have a spike-like appearance, but again, these structures are found free within the cytoplasm and vary more greatly in size than coronavirus particles (Figure 3b ). MVBs are membrane-bound but do not contain the dense dots created by cross sections through the spiral-shaped, filamentous nucleocapsid that would be characteristic of a coronavirus (Figure 3c) Hatfield, PA), which is somewhat more porous than the routine EM epoxy . A SARS-coronavirus specific antibody is applied to a thin section, followed by a secondary antibody conjugated to colloidal gold particles. The gold particles appear as electron dense black dots in areas where coronavirus is present or where the protein that the primary antibody targets is present, such as nucleocapsid inclusions (Figure 4a , cell culture). Due to the embedding technique used for IEM (the lack of osmification, as osmium would inactivate the immunological recognition of the antigen), the ultrastructure is less well defined than in a traditionally embedded EM sample. In Figure 4a , spherical structures consistent in size with coronavirus and in membrane-bound vacuoles are labeled with colloidal gold as are more electron dense areas of the cytoplasm that are likely nucleocapsid inclusions. For comparison, Figure 4b shows an area of a traditionally embedded and osmicated EM specimen that contains many of the same viral features as Figure 4a , that is, vacuolar (Grootemaat et al., 2022) . Despite these limitations, IEM can still be a valuable tool to confirm the presence of a virus in cells.

An increasing number of articles have included descriptions of ultrastructural changes in SARS-CoV-2 positive autopsy tissues (Ackermann et al., 2020; Akilesh, Nast, et al., 2021; Deinhardt-Emmer, Böttcher, et al., 2021; Deinhardt-Emmer, Wittschieber, et al., 2021; Duarte-Neto et al., 2021; Lüke et al., 2020; Rizzo et al., 2021; Santana et al., 2021; Saraiva et al., 2021) . These articles do not necessarily include EM images of viral particles but describe possible ultrastructural alterations due to infection. Bear in mind that some ultrastructural changes may be due to or impacted by co-morbidities and/or tissue autolysis, rather than exclusively a result of viral infection. Comparison of infected and healthy tissues is necessary, and correlation with results of other diagnostic techniques is always recommended.

Respiratory system changes due to SARS-CoV-2 infection have been most thoroughly described (Ackermann et al., 2020; Martines et al., 2020; Santana et al., 2021; Saraiva et al., 2021) . Type I pneumocyte degeneration, type II pneumocyte hyperplasia, and hyaline membrane formation were common ultrastructural features of SARS-CoV-2 infection Santana et al., 2021) . as well as in cell culture systems designed to model the alveolarcapillary barrier (Ackermann et al., 2020; Deinhardt-Emmer, Böttcher, et al., 2021; Deinhardt-Emmer, Wittschieber, et al., 2021; Santana et al., 2021) . A study by Ackerman et al., comparing lung tissues from patients who died from COVID-19 to those that had died from influenza, demonstrated that more microthrombi were observed in SARS-CoV-2 infected lungs. These researchers also observed intussusceptive angiogenesis both in early and in prolonged SARS-CoV-2 lung infection, and EM showed the formation of intussusceptive pillars and distorted vessels with ruptured endothelial cells in the alveolar septum (Ackermann et al., 2020; Duarte-Neto et al., 2021 (Ghadially, 1997; Haguenau & Dalton, 1973; Maunsbach & Afzelius, 1998) . In the case of coronaviruses, intracellular viral particles will be held within membranebound vacuoles, and extracellular particles will likely cluster along the outside of the infected cell and may or may not have visible spikes.

The coronavirus particles will be 60 nm to 140 nm in diameter and will show multiple electron dense cross sections through the helical viral nucleocapsid. Successful identification of a coronavirus in infected tissue requires all these morphologic criteria to be present.

Employing multiple methods of viral detection, including IHC, ISH, PCR, and IEM, is always advisable for a more robust argument for the presence of a virus within a tissue specimen.

Combining ultrastructural study with molecular, chemical, histologic, and immunohistochemical data has enabled the building of a more complete picture of the scope of SARS-CoV-2 infection. Observations of ultrastructural damage have emphasized the extent to which COVID-19 is a systemic disease that can have manifestations throughout the body, affecting even organs and tissues not directly infected by SARS-CoV-2. By using EM as part of a multifaceted approach, we can provide accurate and valuable information concerning SARS-CoV-2 infection.

We thank Maureen Metcalfe for providing the image of MERS-CoV from autopsy tissue.

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Hannah A. Bullock https://orcid.org/0000-0002-4811-1274

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