Characterizing Viral Infection by Electron Microscopy: Lessons from the COVID-19 Pandemic

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic causing COVID-19 has presented many challenges and spurred intense investigations into the pathogenesis of this disease. In addition to respiratory disease, many patients with SARS-CoV-2 infection are experiencing systemic illnesses, including kidney failure, heart failure, liver injury, neurological dysfunction and skin manifestations (e.g. “COVID toe”). The etiology and pathogenesis of these sequelae are the current focus of intense research and speculation. A fundamental question is whether the extrapulmonary disease processes encountered in COVID-19 patients result from direct infection of target organs or indirect injury resulting from initially localized infection in the lungs and upper respiratory tract and subsequent systemic responses such as cytokine release/cytokine storm. Viruses come in all shapes and sizes but are invariably very small and require an electron microscope to resolve the morphology of individual particles 1 . With the emergence of SARS-CoV-2 we are witnessing a renaissance in the use of electron microscopy (EM) to help identify virally infected cells and uncover the pathogenesis of this disease. Several papers have used EM to propose direct evidence of infection of the kidney [2], [3], [4], [5] and other tissues 4 , [6], [7], [8], [9], [10], [11], [12], [13], [14] by SARS-CoV-2. These reports have fueled speculation that direct infection of tissues throughout the body contributes to the morbidity and mortality of COVID-19. Unfortunately, many of these studies are fraught with confusion over differentiating virus from normal structures within cells, and hence there has been an explosion of misinformation. Indeed, published manuscripts claiming to provide direct evidence of SARS-CoV-2 virus infecting in kidney cells and endothelial cells have provoked letters to the editor challenging these claims[15], [16], [17], [18], [19], [20]. In this perspective we will discuss what is known about coronavirus infection and some of the basic ultrastructural cell biology that has been confused for coronavirus infection of cells, namely the machinery that controls endocytosis and exocytosis, and membrane transport within cells 21 . Electron Microscopy of Viral Infections Understanding the biology of viruses is essential to accurately identify viral particles by EM since cells have organelles that can mimic the structure of viral particles (Table 1 ) 1 , 22 . The location inside the cell and the type of membrane-bound organelles with which viral particles are associated can be important clues to identifying the virus. Accurate interpretation of electron micrographs requires integration of morphology and biology. This is especially important with studies that may be compromised by low resolution and poor tissue preservation, which is common in autopsy material. Table 1 Subcellular structures that can be confused with viral particles Subcellular structure∗ Virus mimic∗ Perichromatin granules Small icosahedral viruses Improperly fixed chromatin Nucleocapsids Nuclear pores Herpesvirus nucleocapsids Melanosomes Poxvirus Cilia and microvilli Enveloped viruses Microtubules Viruses with helical nucleocapsids Secretory vesicles and granules Enveloped viruses Multivesicular bodies and exosomes Enveloped viruses ER/Golgi and coatomer-coated vesicles† Enveloped viruses Clathrin-coated vesicles† Enveloped viruses Granules and glycogen Small icosahedral viruses ∗ personal observations and references 1 , 22 † protein coats can be misinterpreted as spike proteins Viruses have DNA or RNA genomes that are contained within a protein coat (capsid). The nucleic acid together with the protein coat forms the nucleocapsid. The nucleocapsid can be membrane-bound (enveloped viruses) or without a membrane (naked viruses). The coronavirus is an enveloped RNA virus that infects cells after it binds to cell surface enzymes that serve as receptors, such as ACE2 for SARS-CoV and SARS-CoV-2, and is internalized in endocytic vesicles 23 . The S-protein of the virus is cleaved and activated, the viral envelope fuses with the vesicle membrane, and the nucleocapsid is released into the cytoplasm, where the replicative stage of the viral life cycle begins. Observing viral infection in cultured cells has provided much detail about the steps in coronavirus replication, which include the formation of double-membrane vesicles that constitute the site for synthesis of viral replicase proteins and genomes (the viral replication transcription complex). The viral genomes and structural proteins are assembled into particles that bud into the endoplasmic reticulum (ER)-Golgi intermediate compartment 23 . Within an infected cell, viral particles are identifiable by EM within structures that resemble ER, Golgi, larger vesicles and vacuoles, as well as outside of the cells 24 . An elegant series of electron micrographs from a nasal mucosal biopsy depicts coronavirus infection of epithelial cells during a naturally acquired infection 25 . The putative virions detected in the kidney renal tubular epithelial cells, podocytes and endothelial cells that are described in several recent publications are shown as free particles in the cytoplasm 2 , 3 , 6 , 7 , a location that would not be expected for coronavirus. In vitro studies and the rare examples of in vivo coronavirus infections reported prior to the current pandemic [25], [26], [27], as well as recent reports of in vitro studies and human infections for the current pandemic 12 , 28 all demonstrate coronavirus within membrane-bound organelles, or outside of cells. Similar problems lie with proposed “virus” detected in multiple cells types in the chorionic villi of the placenta 9 , 10 , endothelial cells within the lung 6 , endothelial cells within the skin 11 and cardiomyocytes and interstitial cells in the heart 13 , 14 . These reports do not discuss alternative explanations for the identified structures or why SARS-CoV-2 infection of human tissues would break the existing paradigm for coronavirus infection. This raises important questions about the interpretation of the micrographs. Cellular structures mistaken for virus Cells have many organelles comparable in size to the coronavirus with varying degrees of electron dense material surrounding and inside of these structures. Cells contain numerous small vesicles that are important for moving membranes and cargo between different compartments within the cell, and into and out of cells (Figure 1 A). Notable examples include clathrin-coated and non-clathrin-coated vesicles. Clathrin-coated vesicles help bring cargo into cells via receptor-mediated endocytosis and move cargo between the trans-Golgi network (TGN) and endosomes 29 . Non-clathrin coated vesicles include the coatamer-coated vesicles (COPI and COPII) that sort cargo between the endoplasmic reticulum and Golgi apparatus during retrograde and anterograde transport 30 , AP3-coated vesicles involved in the biogenesis of melanosomes and platelet dense bodies, AP4-coated vesicles involved in sorting cargo between the TGN and endosomes, as well as the basolateral membrane 29 , and caveolin coated vesicles that are involved in endocytosis, transcytosis, regulation of membrane lipids and signaling 31 . Cellular vesicles can be difficult to classify based on morphology alone but can be deduced from their relationship with other membranes in the cell. Vesicles seen budding from the plasma membrane that are about 60-100 nm in diameter, surrounded by an electron dense coat and appear spiculated are likely clathrin-coated (Figure 1B) and Ref. 32 . Vesicles that measure approximately 60-100 nm in diameter, have similar spiculated electron dense coats, are found in the vicinity of ER and Golgi and bud from these organelles are likely coatamer-coated (Figure 1, C and D). Other coated vesicles identified in the cell cytoplasm can be difficult to classify based on ultrastructural morphology alone (Figure 1D). 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 antero- and retrograde transport of vesicles between the ER and Golgi. Clathrin-coated vesicles (blue) are involved in endocytosis. Multivesicular bodies are derived from early endosomes and contain cargo that is 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 (bar 100 nm). C) Tubular epithelial cell with coated vesicles (arrowheads) adjacent to ER/Golgi; note vesicle budding (arrow, inset) from ER/Golgi (bars 100 nm). D) Glomerular endothelial cell with coated vesicles in cytoplasm that have club shaped “spikes” (arrow) (Bars 100 nm). E) Podocyte with multivesicular bodies (bar 500 nm). . F) Podocyte with microvilli inside an invagination of the plasma membrane resembling a cytoplasmic vesicle (bar 500 nm). The insets in B,C,D, E and F show higher magnification of the areas designated with the dashed box (inset bars = 100 nm). Multivesicular bodies (MVBs) are also involved in the endocytic and exocytic functions of cells 33 , 34 . Early endosomes pinch off molecules that are destined for removal or degradation into intraluminal vesicles (ILVs), forming MVBs. The MVBs may fuse with autophagosomes or lysosomes to degrade the contents, or fuse with the plasma membrane to expulse exosomes. The ILVs found within a larger vesicles (Figure 1E) and Ref. 18 , 19 , 32 have been confused with SARS-CoV-2 particles 8 . Microvilli captured in plasma membrane invaginations can also mimic MVBs and be confused for viral particles (Figure 1F). Proposed criteria for identification of viral infection of tissues by electron microscopy in COVID-19 and future pandemics. To ensure the rigor and reproducibility for the identification of viruses in tissues by electron microscopy we propose that the following 4 criteria be met. Structure: morphologic features of the viral particles should conform to prior knowledge of the virus, including size and uniformity, formation of higher order structures (aggregates/arrays/inclusions), the absence or presence of a clearly discernible membrane, and the qualities of internal (e.g. nucleocapsid) and external (e.g. peplomers/spikes) electron densities. If prior knowledge is lacking or incomplete, the structure of the viral particles should be established with an appropriate model system, such as electron microscopy of in vitro infected cells. For coronavirus, Goldsmith and colleagues note that coronavirus spikes are often difficult to visualize in thin sections using transmission electron microscopy (TEM) 19 , and usually less obvious than clathrin coats. In addition, the nucleocapsid within the membrane of the viral particle has characteristic dot-like electron densities that are typically absent from cellular vesicles (Ref 19 and Figure 2 ). The reported diameter of the virus is approximately 80 nm 35 . In our studies, the SARS-CoV2 viral particles had an average diameter of 64 nm (range 56-75 nm) (Figure 2). Tissue preservation is also critical, and poor preservation, as is common for autopsy material, will compromise objective interpretation of electron micrographs and the ability to conclusively identify viral particles. Location: viral particles should be present in sites that conform with the known biology of viral replication; strong supporting evidence is required when attempting to identify viral particles in tissues with suboptimal preservation, necrosis and autolysis in order to differentiate these particles from normal cellular structures. Coronavirus particles are found inside the cisternae of the ER-Golgi and secretory compartment, as well as outside of cells (Figure 2.). Independent evidence to corroborate EM findings: additional validated tests, such as PCR, IHC, ISH and immunoelectron microscopy should be performed independently to confirm viral infection and further support the interpretation of the EM findings (Figure 3 ). Expertise: electron microscopy should be performed and interpreted by experienced individuals and aided by appropriate controls and bona fide images of the virus that is sought. Simply having experience with electron microscopy for diagnosis of kidney diseases is not sufficient to accurately discern subcellular organelles from novel viruses, and appropriate experience should be gained or sought. Figure 2 Coronavirus infection in cells. A) Diagram of cell demonstrating structures that are associated with coronavirus infection. Double-membrane vesicles (DMV) are found near the nucleus and represent the site of viral genome replication. Coronavirus particles bud into the cisternae of the ER/Golgi and accumulate in cytoplasmic vesicles that fuse with the plasma membrane and release virus particles into the extracellular space. B) A SARS-CoV-2 infected HBEC3-KT cell showing perinuclear DMV (arrow) and enlarged vesicles (black arrowhead) filled with viral particles (bar = 500 nm). C) Higher magnification image of viral particles in cytoplasmic vesicles (black arrowhead, bar = 100 nm). D) Viral particles (white arrowhead) within cisternae of ER/Golgi; particles have characteristic electron dense dots corresponding to the helical nucleocapsid within the envelope (bar = 100 nm). E) Viral particles (white arrowhead) at the cell surface (bar = 100 nm). The particles have an average envelope diameter of 64 nm and a range of 56-75 nm. The “spikes” are vague and not a prominent morphologic feature in TEM images. Figure 3 Detection of 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 (left column, SARS-CoV/SARS-CoV-2 Spike antibody,Chimeric Mab, 40150-D001, Sino Biological, Wayne PA), and probed with anti-Spike gene probe that recognizes intact virions (middle column, ACD Biosciences, RNAscope Probe - V-nCoV2019-S, ACD Biosciences, Newark CA) or replicating virus (right column, RNAscope Probe - V-nCoV2019-S-sense, ACD Biosciences). The rows from upper to lower are lung tissue from a patient that died of COVID-19, HBEC3-KT cells infected with SARS-CoV-2 (HBEC3), HSAEC1-KT cells infected with SARS-CoV-2 (HSAEC1), HEK 293 ACE2 cells infected with SARS-CoV-2 (HEK 293 CoV2) and mock infected HEK 293 ACE2 cells (HEK 293 mock). The HBEC3-KT and HSAEC1-KT cells are immortalized human bronchial epithelial and small airway and cell lines, respectively (American Type Culture Collection, 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 very 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 (bar = 100 um). Conclusions Early reports on the identification of novel pathogens during a pandemic leave a lasting impression. If erroneous, they have the potential to misdirect other researchers, clinicians and the general public. Adherence to rigorous criteria for the identification of pathogens by electron microscopy will help to establish with confidence critical information that is needed to better understand the biology of the disease and achieve effective treatments for this and future pandemics.