key: cord-330597-nftwj0d5
authors: Hopfer, Helmut; Herzig, Martin C.; Gosert, Rainer; Menter, Thomas; Hench, Jürgen; Tzankov, Alexandar; Hirsch, Hans H.; Miller, Sara E.
title: Hunting coronavirus by transmission electron microscopy – a guide to SARS‐CoV‐2‐associated ultrastructural pathology in COVID‐19 tissues
date: 2020-09-27
journal: Histopathology
DOI: 10.1111/his.14264
sha: 
doc_id: 330597
cord_uid: nftwj0d5

Transmission electron microscopy has become a valuable tool to investigate tissues of COVID‐19 patients because it allows visualisation of SARS‐CoV‐2, but the “virus‐like particles” described in several organs have been highly contested. Because most electron microscopists in pathology are not accustomed to analysing viral particles and subcellular structures, our review aims to discuss the ultrastructural changes associated with SARS‐CoV‐2 infection and COVID‐19 with respect to pathology, virology, and electron microscopy. Using micrographs from infected cell cultures and autopsy tissues, we show how coronavirus replication affects ultrastructure and put the morphological findings in the context of viral replication, which induces extensive remodelling of the intracellular membrane systems. Virions assemble by budding into the endoplasmic reticulum‐Golgi intermediate complex and are characterized by electron dense dots of cross‐sections of the nucleocapsid inside the viral particles. Physiological mimickers such as multivesicular bodies or coated vesicles serve as perfect decoys. Compared to other in‐situ techniques, transmission electron microscopy is the only method to visualize assembled virions in tissues and will be required to prove SARS‐CoV‐2 replication outside the respiratory tract. In practice, documenting in tissues the characteristic features seen in infected cell cultures, seems to be much more difficult than anticipated. In our view, the hunt for coronavirus by transmission electron microscopy is still on.

SARS-CoV-2 currently dominates all headlines as a highly contagious pandemic with considerable mortality [1] [2] [3] [4] . While hygiene precautions and lockdown measures have changed our personal and professional daily lives during the last months, many investigators are eager to understand the biological basis of SARS-CoV-2's contagiousness and pathogenesis leading to respiratory and multi-organ failure. Autopsy studies have established the morphological changes associated with COVID-19 and have tried to visualise the virus in tissues [5] [6] [7] [8] [9] . Transmission electron microscopy (TEM) seems a logical tool to look for SARS-CoV-2 infection, but some of the published results are highly contested (kidney [8, [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] , endothelium [8, 9, [23] [24] [25] [26] [27] [28] , intestine [8] , liver [29] [30] [31] [32] , placenta [33] [34] [35] [36] [37] ). Because most of us are not virologists or electron microscopists dedicated to viral diseases, this review aims at combining multidisciplinary expertise of SARS-CoV-2 pathology, virology, and electron microscopy.

Pathologists are good at detecting some viral infections -at least in identifying unusual inclusions on an H&E-stained slide. However, unless there is a knowledge of virus morphology (what they look like) and morphogenesis (how and where in the cell they are assembled), it is difficult to identify them. Depending on the virus, we use immunohistochemistry targeting viral proteins or in-situ hybridization to highlight their DNA or RNA. Molecular pathology techniques allow us to test for viruses in tissues when in situ-techniques are not yielding results. All of these techniques have been applied successfully in the context of SARS-CoV-2 (figure 1 and [5, 38, 39] ). It is important to note that any of these tests require an a priori notion of what is present; otherwise, it is difficult to choose the right reagent (e.g. if a herpesvirus is suspected, and an anti-herpesvirus antibody is used, but the infection is caused by an adenovirus, then the test is negative, and the diagnosis is no closer to being made).

Virus detection by TEM is rarely deployed in the routine setting (outside of the viral EM diagnostic laboratories that examine tissues and fluids) because it is expensive, time-consuming, covers only minute portions of the tissue, and is not available in most laboratories.

Nephropathology is one of few areas in pathology that routinely performs TEM. Therefore, it is not surprising that renal pathologists were among the first to search for SARS-CoV-2 infection in the kidneys by TEM [10] [11] [12] 40, 41] . Most changes they routinely look for in kidney biopsies are visible at magnifications between 1,000x and 10,000x (which can be considered as "low power" in the context of TEM). Thus, the magnifications required to identify viral structures are way out of their normal "comfort zone". As a consequence, "hunting coronavirus by electron microscopy"

This article is protected by copyright. All rights reserved takes us to subcellular structures that we usually do not study because they are not important in the context of the standard pathology diagnostic work-up. Complicating things further, ultrastructural preservation is limited in autopsy samples with delayed fixation obscuring subtle changes of the intracellular membrane systems associated with replication of enveloped viruses.

To better understand the ultrastructural morphology of SARS-CoV-2 infection and COVID-19, we will first briefly discuss the pathogenesis of COVID-19 and coronavirus replication in general and then examine the TEM findings in more detail.

Transmission of community-acquired respiratory viruses including SARS-CoV-2 usually results from close-range contacts through respiratory droplets or aerosols making contact with mucus membranes of the upper respiratory tract [42] [43] [44] [45] . SARS-CoV-2 infection of host cells involves specific binding of the viral spike glycoprotein S (S protein) to its receptor ACE2. The S protein is then cleaved by host cell proteases to allow for conformational changes mediating fusion between the viral envelope and the host cell membrane. The viral RNA is uncoated and released into the cytoplasm. Host cells are ciliated epithelial cells of the upper and lower airways and pneumocytes type II [46] [47] [48] [49] [50] . The level of innate immune response appears to increase as the infection progresses to the lower respiratory tract and the patients increasingly show symptoms [51] . This gradual progression may explain the range of the clinical manifestations from asymptomatic to severe disease.

In the alveoli, SARS-CoV-2 infects pneumocytes type II as well as pneumocytes type I via local cell-to-cell transmission according to preclinical primate models [52] . SARS-CoV-2 infection impairs production of surfactant and fluid resorption leading to increased transmural microcapillary pressure and microvascular leakage, finally resulting in adult respiratory distress syndrome clinically and diffuse alveolar damage histologically [2, 5, 6, 8, [53] [54] [55] [56] [57] [58] . While direct infection of the cells in the upper and lower respiratory tract drives these initial phases, the innate immune response is mostly responsible for the hyperinflammatory phase ("cytokine storm") characteristic of severe COVID-19 [51, 59, 60] .

Some evidence suggests that SARS-CoV-2 infection may not be restricted to the respiratory tract but can spread to other organs. Viral RNA has been detected by quantitative nucleic acid amplification technique (QNAT) initially in blood of severely ill patients, in faeces, and rarely in urine samples [61] [62] [63] [64] [65] [66] [67] . It can also be amplified from multiple tissues obtained at autopsies

This article is protected by copyright. All rights reserved including heart, liver, kidneys, intestine, skin, and brain [5, 38, 39, 68] . In-situ hybridization and immunohistochemistry studies support the idea of viral spread throughout the body [8, 12, 33, 38, [69] [70] [71] . However, except for lower respiratory fluids such as sputum or bronchoalveolar lavage [61, 64, [72] [73] [74] and very rarely faeces [65] , other viral RNA-containing samples have not been reported to allow for a productive infection in cell cultures [64, 75, 76] .

These data suggest limited sensitivity of current culture assays and/or low infectiousness. The time point of viral dissemination is unclear but -from a virological point-of-view -would require significant local replication and access to blood, blood cells, and release at new and distant sites. A better knowledge of these events may help to predict the clinical symptoms and their relevance to the disease course.

Currently, our knowledge of morphological changes in COVID-19 is mostly based on autopsy tissue obtained from severely affected individuals in the pulmonary or hyperinflammatory phase making it difficult to differentiate between changes driven by local viral replication, changes due to the systemic inflammatory response and repair, or possibly therapy effects. Indisputable detection of SARS-CoV-2 by TEM would confirm viral replication outside the upper respiratory tract and the lungs and firmly establish a role of direct viral infection to some of the organs mentioned above.

Interpretation of TEM findings in tissues of COVID-19 patients benefits from a good understanding of coronavirus replication in cells [77] [78] [79] . Like all members of this family, SARS-CoV-2 virions are enveloped infectious particles with a diameter of 60-140 nm [80] . 

This article is protected by copyright. All rights reserved MERS-CoV [81] [82] [83] [84] [85] [86] [87] . Recent data show that SARS-CoV-2 replication induces very similar ultrastructural changes [88] .

SARS-CoV-2 infection and replication can be arbitrarily divided into three phases (figure 2), which may occur simultaneously.

1. After binding to its receptor ACE2, SARS-CoV-2 is shuttled into the endosomal pathway, likely by clathrin-coated vesicles [89] . Depending on the presence of furin, a serine protease, cleavage of the S protein triggers early fusion of the viral and the endosomal membranes and causes the release of viral genomes into the cytoplasm [46, 47] . Cleavage may also occur by other proteases after fusion of the late endosome with a lysosome [90] .

Ribosomes recognize the positive-sense genomic RNA strand (+gRNA) as mRNA and translate the viral proteins making up the replication-transcription complex (RTC) at the ER. This initiates an extensive remodelling of intracellular membranes forming a three-dimensional structure referred to as the replication membranous web (RMW) [83, 84, 91, 92] . Virus replication within the RMW has several advantages: all factors necessary are concentrated in close proximity to each other making the process very efficient and, additionally, the RMW may hide viral RNA from innate immune sensors within the cytoplasm. Morphologically, the RMW is a fascinating and confusing structure containing multiple interconnected vesicles with single or double membranes (termed double-membrane vesicles and convoluted membranes). One has to assume that the RMW is a dynamic structure with multiple fission and scission events, which, unfortunately, cannot be 

This article is protected by copyright. All rights reserved Coronaviruses including SARS-CoV-2 and the morphological changes associated with replication can be visualised by TEM in infected cell lines (figure 3A-G) [81] [82] [83] [84] [85] 87, 88] or organoids [96, 97] .

Non-infected cultures serve as controls (figure 3H-K). For comparison with tissues from COVID-19 patients, understanding the morphology of the assembled viruses and the replication membranous web is most important.

Budding of the nucleocapsid into membranes containing the structural proteins, forms the viruses with a circular shape (figure 3C). Thus, assembled SARS-CoV-2 virions reside within vacuoles and cannot be seen free in the cytoplasm (figure 3B, D-E) [82] . Cross-sections of viruses measure 60-140 nm [80] , and show the membrane of the envelope with the helical electron dense nucleocapsid inside as several small granules of approximately 12 nm. Frequently, the centre of the viral cross section is electron lucent. Unless a negative staining procedure for viewing viruses in fluids or tannic acid staining of tissue for thin sections is used, the S proteins forming the "corona" are not readily discernible [98] . Convoluted membranes (CM) are less frequent structures that develop early after infection ( figure   3F ) [83, 84] . They associate with the DMV and the ER, forming an unorganized reticular structure measuring 200-600 nm with a single limiting membrane. CM may be involved in the formation of the DMV [83] . Another hypothesis suggests that these structures may serve as a storage for or are generated by an excess of non-structural proteins not incorporated into the DMV [84] .

Although rare, cubic membrane structures (CMS) are the most eye-catching membrane rearrangement in coronavirus-infected cells because the membranes are highly organized (figure

This article is protected by copyright. All rights reserved 3G) [84] . In cell cultures, they emerge late after infection. CMS consist of highly curved membranes arranged in a recurrent (three-dimensional) pattern. Their function is unknown; one hypothesis proposes that they are formed from membranes with an excess in S protein [82, 84, 99] .

CMS are not specific for coronavirus infection [100] . For example, CMS resulting from chloroquine therapy may be seen in kidney biopsies of patients with lupus nephritis (termed "curvilinear bodies" in this context) [101] .

Late after infection, cell cultures frequently contain large virion-containing vacuoles (LVCV, figure 3B ) [81] [82] [83] . These are large circular vesicles belonging to the secretory pathway containing multiple cross sections of assembled virus and evidence of additional virus budding.

Marker studies suggest that LVCV are ERGIC/Golgi-derived cisternae [84] .

Based on the cell culture findings outlined above, we expect to find the same SARS-CoV-2 morphology and distribution in vesicles of autopsy and biopsy tissues of COVID-19 patients. There are few reports on upper airway and lung tissue demonstrating assembled SARS-CoV-2 virions that morphologically replicate the cell culture findings [58, 80] . Others, including some of us, have reported on "virus-like particles" by TEM not completely matching the cell culture findings in a variety of organs, including lung [5, 8, 26, 56, 102, 104] , kidneys [5, 8, [10] [11] [12] 23, 104] , liver [29] , heart [104] , intestine [8, 103] , skin [71] , and placenta [33, 36, 69, 70] . Colleagues have rightfully raised their concerns that these particles are not consistent with SARS-CoV-2 [14] [15] [16] [17] [18] 24, 27, 30, 31, 34] , but depict other subcellular structures, making identification of SARS-CoV-2 by TEM much more challenging than expected.

This article is protected by copyright. All rights reserved Physiological structures including coated vesicles, multivesicular bodies and cross-sections of the rough ER are morphological look-alikes of genuine corona viruses [105] .

Coated vesicles (CV) are single-membrane-bound vesicles of variable size (typically 50-150 nm) characterized by "spiny adornments on their limiting membrane" (F. Ghadially) (figure 4E) [106] .

They are involved in endocytosis and membrane trafficking (reviewed in [107] ).

In clathrin-coated vesicles, the best-studied example, the CV bud off so-called coated pits on the cell surface during micropinocytosis. Clathrin and other quantitatively minor proteins provide a three-dimensional structural lattice, which is readily seen in electron micrographs.

Morphologically identical structures with coats provided by the main proteins COPI or COPII are involved in transport processes of the trans-Golgi network.

Internalization of SARS-CoV-2, after binding to its receptor ACE2 involves this mechanism [46, 47] . While CV may transport viral proteins ,as shown for vesicular stomatitis virus [108] , and may be used for replication of poliovirus [109] , and, further, have a similar size to that of coronavirus, they are not the assembled virus itself. However, although the projections appear as a perfect "corona" in cross-sections, CV lack the nucleocapsid present inside of coronavirus cross sections, and they are located within the cytoplasm and not within vacuoles. The process is used to sort and target membrane-associated proteins to lysosomal degradation, which happens after fusion of a MVB with a lysosome. A divergent pathway for MVB is the release of the ILV as exosomes after fusion with the cell membrane. Interestingly, the molecular machinery involved in these processes is also used for the budding of enveloped viruses such as HIV (reviewed in [111] ).

MVB are the perfect "decoy" for electron microscopists searching for viral particles. Some of us have fallen for them in our COVID-19 autopsy series [5] because the ILV have a similar size as SARS-CoV-2 and are located within vesicles. The key difference is that ILVs do not show the electron dense granules of the nucleocapsid.

Pathologists regularly performing TEM are familiar with the ER. Two types of this largest closed and interconnected membrane structure in eukaryotic cells are recognized: the smooth ER, the site

This article is protected by copyright. All rights reserved of lipid synthesis and metabolism, and the rough ER, where ribosomes, attached to the outside of the membrane, translate proteins for membranes, organelles, and secretion [112] . Dilatation of the ER occurs in the context of cell stress of various aetiologies. In COVID-19 tissues, the ER is frequently swollen, and the accumulation of membrane structures further adds to the confusing ultrastructure ( figure 4C) . A cross-section through rough ER can easily be mistaken for a "viruslike particle", but these are located within the cytoplasm and not in vesicles and lack the nucleocapsid structures inside.

In kidneys of COVID-19 autopsies, we encountered a peculiar subcellular structure closely mimicking SARS-CoV-2 but likely related to the ER (figure 4C, G-I) [5, 13] . Larger vesicles with a smooth outside membrane contained several round to oval small vesicles with prominent electron dense granules on the inside. These granules were bigger than SARS-CoV-2's nucleocapsid seen in our infected cell cultures and had the same size as the ribosomes visible in areas containing rough ER (ribosomes: 20-21 nm (range: 17-23 nm) vs. nucleocapsid: 12 nm (range: 9-16 nm). These vesicles with "outside-in" ribosomes are possibly derived from the rough ER by membrane invaginations as suggested in some of the TEM pictures ( figure 4I ). Because the particles inside are larger than nucleocapsid cross sections, we believe that they likely do not represent assembled virions. However, it is theoretically possible that nucleocapsids may have deteriorated and swollen to approach the appearance of ribosomes due to autolysis. 

This article is protected by copyright. All rights reserved (too late: the virus has already been cleared by the immune system) and sensitivity (too insensitive: there are too few infected cells or too few virions such that detection by TEM becomes very unlikely; the low viral loads found by quantitative nucleic acid amplification techniques (QNAT) in tissues other than lung would support this argument). We think that the extensive intracellular membrane remodelling seen could be a result of direct infection, but this is difficult to prove in the absence of newly formed viral particles. Ideally, TEM morphology will be backed by other in-situ techniques in the same case.

Another important concept to keep in mind is that viral components (RNA and proteins) are not produced in balanced amounts (as suggested in figure 1 and [113] ). Therefore, surplus viral RNA and proteins may be encountered at the site of infection, in the circulation, and at distant sites.

Detection of viral RNA and proteins does not necessarily reflect the presence of intact and infectious particles. It is also conceivable that pathology in non-respiratory organs could be the result of viral disease distantly, due to transport of viral components, and not a direct result of infection. Therefore, TEM investigation is essential to verify assembled virions in SARS-CoV-2 infection and COVID-19.

In our view, the hunt by TEM for coronavirus in tissues of COVID-19 patients outside the upper airways and the lungs is still open.

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved The structure next to the MVB is a lysosome. Original magnification 44,000x. 

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