Misinterpreting Electron Microscope Images

It should be clear to anyone being intellectually honest that purification (free of foreign material, contaminants, pollutants, etc.) and isolation (separated from everything else) of particles believed to be a “virus” is essential and can not be skipped. Without separating the assumed “viral” particles from everything else in the sample, there is no way for a virologist to know what exactly they are looking for, especially in regards to a “novel virus” no one has supposedly ever seen before. It is easy to see that virologists look at numerous similar particles and mistake them for the preconceived one that they are looking for from the very start.

Below are numerous examples of misinterpretations of Electron Microscope images for “SARS-COV-2.” It is a long read, but you will see that there are multiple issues when trying to identify particles believed to be “SARS-COV-2.” There are many subcellular particles which are either similar or completely identical to “Coronaviruses.” Within an unpurified sample, there are guaranteed to be millions of these particles present. Attempting to identify images of this “virus” is an area fraught with subjective analysis of a predetermined idea of what a “SARS-COV-2” particle is supposed to look like and where it should be located.

Caution in Identifying Coronaviruses by Electron Microscopy

We are concerned about the erroneous identification of coronavirus directly in tissues by authors using electron microscopy. Several recent articles have been published that purport to have identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly in tissue.14 Most describe particles that resemble, but do not have the appearance of, coronaviruses.57

“In the article by Farkash et al.,8 the electron microscopic images in their Figure 3, A–C do not demonstrate coronaviruses. Rather, the structures described as virus are clathrin-coated vesicles (CCVs), normal subcellular organelles involved in intracellular transport.”

“Additionally, Farkash et al.8 document their findings by referring to an article by Su et al.2 that purports to have identified coronavirus in kidney. Likewise, that article shows only normal cell structures that, to the non-electron microscopist virologist, may resemble coronavirus. Their interpretation has been refuted in Letters to the Editor of Kidney International.5,6

Identification of viruses is not always straight forward. Consideration should be given to the mechanism of virus production, including the location inside of cells, as well as the appearance (size, shape, internal pattern of the nucleocapsid, and surface spikes).1416 Care should be taken to prevent mistaking cell organelles for viral particles.17,18

https://jasn.asnjournals.org/content/31/9/2223

Clarinth-Coated Vesicles…or “SARS-COV-2?”

Multivesicular bodies mimicking SARS-CoV-2 in patients without COVID-19

Most of the published images depicting the suspected virus are very similar, if not identical, to multivesicular bodies (MVBs). MVBs have been well-known since the 1960s and their appearance and occurrence is detailed in the classic monograph of Feroze Ghadially;3 however, their exact significance and function is unclear. We suspect that the EM images of SARS-CoV-2 published to date are in fact MVBs.

“MVBs theoretically may represent podocyte endocytosis with subsequent formation of intracytoplasmic microvesicles resembling viruses. Seeing an MVB by EM is just a snapshot and we do not know how and when they evolved or how long they remain. MVBs contain microvesicles. However, microvesicles are commonly “free floating” in the cytoplasm of many cell types, including tubular epithelial cells (frequently representing endocytotic vesicles). Su et al.1 show such cytoplasmic microvesicles in tubular epithelial cells in their Figure 2, but the particles in Figure 2a may have come from an MVB after its membrane dissolved. While these “free floating” cytoplasmic microvesicles could represent viral particles, they are nonspecific. Endocytic vesicles may be coated by proteins, such as clathrin. The presence of coating proteins may cause an electron-dense area around these vesicles giving the appearance of a viral “corona.”

Transmission EM of tissue sections is not a specific or sensitive method for the detection of viral particles; there are numerous structures found by EM that resemble viruses (so-called viral-like particles), such as the well-known endothelial tubuloreticular inclusions (also called myxovirus-like particles). Therefore, caution is suggested when identifying a virus by EM in tissue sections.”

https://www.kidney-international.org/article/S0085-2538(20)30529-9/fulltext

Multivesicular Body…or “SARS-COV-2?”

Electron microscopy of SARS-CoV-2: a challenging task

“We read with interest the Correspondence by Zsuzsanna Varga and colleagues1 on the possible infection of endothelial cells by SARS-CoV-2 using electron microscopic (EM) images as evidence. However, we believe the EM images in the Correspondence do not show coronavirus particles but instead show cross-sections of the rough endoplasmic reticulum (RER). These spherical structures are surrounded by dark dots, which might have been interpreted as spikes on coronavirus particles but are instead ribosomes.”

“Just recently, there have been two additional reports34 in which structures that can normally be found in the cytoplasm of a cell have been misinterpreted as viral particles.5 EM can be a powerful tool to show evidence of infection by a virus, but care must be taken when interpreting cytoplasmic structures to correctly identify virus particles.”

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)31188-0/fulltext

Rough Endoplasmic Reticulum…or “SARS-COV-2?”

Alternative interpretation to the findings reported in visualization of severe acute respiratory syndrome coronavirus 2 invading the human placenta using electron microscopy

“The authors examined the placenta by transmission electron microscopy to identify SARS-CoV-2 particles. They identified circular inclusions in the cytoplasm of several syncytiotrophoblasts that they concluded were SARS-CoV-2 virions.”

The structures identified as SARS-CoV-2 virions look exactly like clathrin-coated pits or vesicles. Clathrin-coated vesicles are spherical structures employed by trophoblasts and other cell types to internalize cargos from the extracellular space.2 Coated vesicles and coated pits derive their name from the external scaffold coat composed of clathrin triskelions that decorate the surface of the structure. In transmission electron micrographs in which tissue-thin sections are stained with uranyl acetate and lead citrate, coated vesicles have an electron-dense studded surface that appears identical to the “corona” comprising the spike protein that decorates all coronaviruses, including SARS-CoV-2 virions.”

I propose that the structures identified by Algarroba et al1 in their journal preproof paper are clathrin-coated vesicles and not SARS-CoV-2 particles. This conclusion is based on the following evidence: (1) the circular structures in the electron micrographs in the paper, identified as virions, have the size and shape of clathrin-coated vesicles found in nearly all eukaryotic cells3; (2) there is no evidence of virions bound to the apical surface of the syncytiotrophoblast (ACE2 or SARS-CoV-2 receptor)4 as would be predicted in virus-infected cells; (3) U-shaped, corona-studded structures are apparent at the surface of the syncytiotrophoblast representing newly forming coated vesicles that have not yet pinched off (ie, endocytosed their cargo) (Figure); and (4) the neonate was determined to be virus-negative using RT-PCR.”

https://www.ajog.org/article/S0002-9378(20)30632-3/fulltext

Clarinth-Coated Vesicles again…or “SARS-COV-2?”

Why misinterpretation of electron micrographs in SARS-CoV-2-infected tissue goes viral

With interest we follow the publications that show the presence of putative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by electron microscopy (EM) in patient tissues and the debate about these results, which should have sufficiently raised attention to their correct interpretation.12

Nevertheless, ultrastructural details in autopsy tissues have been misinterpreted as coronavirus particles in recent papers. Bradley and colleagues3 described “coronavirus-like particles” in autopsy specimens of the “respiratory system, kidney, and gastrointestinal tract”, and in a case report Dolhnikoff and colleagues4 described “viral particles” in “different cell types of cardiac tissue” of a deceased child. However, the images in these publications show putative virus particles that lack sufficient ultrastructure for an unambiguous identification as virus. Some of these particles definitely represent other cellular structures, such as rough endoplasmic reticulum (eg, Dolhnikoff and colleagues,4 figure 3B), multivesicular bodies (Bradley and colleagues,3 figure 5C) and coated vesicles (Bradley and colleagues,3 figure 5B, G). Moreover, it is remarkable that Dolhnikoff and colleagues4 referred to findings, described by Tavazzi and colleagues,5 of “viral particles” in interstitial cells, which are clearly non-viral structures, such as coated vesicles.45 Furthermore, Bradley and colleagues3 quoted publications as a reference for their virus particle identification, which, in our opinion, both identified non-coronavirus structures as coronavirus particles, as already discussed by Goldsmith and colleagues1 and by Miller and Brealey.2

“As diagnostic EM requires both specialised staff and expensive equipment, and has been replaced by other methods (eg, immunohistochemistry) in several fields of application, its use has been in decline in the past decades, resulting in irreversible loss of expertise that now becomes dramatically overt during the SARS-CoV-2 pandemic. This dilemma of diagnostic EM should alarm us all, as misleading information on the presence of SARS-CoV-2 in tissue has already made its way into the scientific literature and seems to be perpetuated.”

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736%2820%2932079-1/fulltext

“SARS-COV-2″…or Multivesicular Bodies, Clarinth-Coated Vesicles, and/or Rough Endoplasmic Reticulum?

Characterizing Viral Infection by Electron Microscopy

“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.

“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 articles have used EM to propose direct evidence of infection of the kidney and other tissues 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, leading to an explosion of misinformation. Indeed, published articles claiming to provide direct evidence of SARS-CoV-2 virus infection in kidney cells and endothelial cells have provoked letters to the editor challenging these claims.”

“Understanding the biology of viruses is essential to accurately identify viral particles by EM because certain cellular 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.”

“The putative virions detected in the kidney renal tubular epithelial cells, podocytes, and endothelial cells described in several recent publications appear as free particles in the cytoplasm,2,3,6,7 a location that would not be expected for coronavirusIn vitro studies and the rare examples of in vivo coronavirus infections reported before the current pandemic,252627 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 cell 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 inside and surrounding them.”

Cellular vesicles can be difficult to classify on the basis of 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 to 100 nm in diameter, surrounded by an electron-dense coat, and appear spiculated, are likely clathrin-coated (Figure 1B).32 Vesicles that measure approximately 60 to 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 on the basis of ultrastructural morphology alone (Figure 1D).

“Multivesicular bodies are also involved in the endocytic and exocytic functions of cells.33,34 Early endosomes pinch off molecules destined for removal or degradation into intraluminal vesicles, forming multivesicular bodies. The multivesicular bodies may fuse with autophagosomes or lysosomes to degrade the contents, or with the plasma membrane to expulse exosomes. The intraluminal vesicles found within larger vesicles (Figure 1E)18,19,32 have been confused with SARS-CoV-2 particles.8 Microvilli captured in plasma membrane invaginations can also mimic multivesicular bodies 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 four 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 (eg, nucleocapsid) and external (eg, 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 Miller19 note that coronavirus spikes are often difficult to visualize in thin sections using transmission EM, and are 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 (Figure 2).19 The reported diameter of the virus is approximately 80 nm.35 However, in our studies, the SARS-CoV-2 viral particles had an average diameter of 64 nm (range, 56 to 75 nm) (Figure 2). Tissue preservation is also critical, and poor preservation, as is common for autopsy material, compromises 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 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, immunohistochemistry, in situ hybridization, 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 sought. Experience with electron microscopy for diagnosis of kidney diseases alone is not sufficient to accurately discern subcellular organelles from novel viruses, and appropriate experience should be gained or sought.”

https://ajp.amjpathol.org/article/S0002-9440(20)30503-4/fulltext

Does anyone really know what these particles are?

In Summary:

  • There has been growing concern over the erroneous identification of “coronavirus” directly in tissues by authors using electron microscopy
  • The authors of the first article claim that most papers describe particles that resemble, but do not have the appearance of, “coronaviruses”
  • Some of the structures described as “virus” are clathrin-coated vesicles (CCVs), normal subcellular organelles involved in intracellular transport
  • They claim that the paper discussed shows only normal cell structures that, to the non-electron microscopist virologist, may resemble “coronavirus”
  • Identification of “viruses” is not always straight forward
  • They conclude that care should be taken to prevent mistaking cell organelles for “viral” particles
  • The researchers in the 2nd article believe most of the published images depicting the suspected “virus” are very similar, if not identical, to multivesicular bodies (MVBs)
  • They suspect that the EM images of “SARS-CoV-2” published to date are in fact MVBs
  • Seeing an MVB by EM is just a snapshot and it is not known how and when they evolved or how long they remain
  • While “free floating” cytoplasmic microvesicles could represent “viral” particles, they are nonspecific
  • The presence of coating proteins may cause an electron-dense area around these vesicles giving the appearance of a “viral corona”
  • Transmission EM of tissue sections is not a specific or sensitive method for the detection of “viral” particles; there are numerous structures found by EM that resemble “viruses” (so-called “viral-like” particles)
  • They conclude that caution should be suggested when identifying a “virus” by EM in tissue sections
  • The researchers in the 3rd article believe the EM images in the Correspondence do not show “coronavirus” particles but instead show cross-sections of the rough endoplasmic reticulum (RER)
  • These structures are surrounded by dark dots which might have been interpreted as spikes on “coronavirus” particles
  • It’s mentioned that there were two recent reports in which structures that can normally be found in the cytoplasm of a cell have been misinterpreted as “viral” particles
  • They conclude that care must be taken when interpreting cytoplasmic structures to correctly identify “virus” particles
  • The 4th article discusses a paper which showed circular inclusions in the cytoplasm of several syncytiotrophoblasts that were concluded to be “SARS-CoV-2 virions”
  • However, the author of this article came to a different conclusion and determined that the structures identified as “SARS-CoV-2 virions” look exactly like clathrin-coated pits or vesicles
  • In transmission electron micrographs in which tissue-thin sections are stained with uranyl acetate and lead citrate, coated vesicles have an electron-dense studded surface that appears identical to the “corona” comprising the spike protein that decorates all “coronaviruses,” including “SARS-CoV-2 virions”
  • The authors of the 5th article also state that ultrastructural details in autopsy tissues have been misinterpreted as “coronavirus” particles in recent papers
  • The images in these publications show putative “virus” particles that lack sufficient ultrastructure for an unambiguous identification as “virus”
  • Some of these particles definitely represent other cellular structures, such as rough endoplasmic reticulum, microvesicular bodies, and clarinth-coated vesicles
  • Bradley and colleagues quoted publications as a reference for their “virus” particle identification, which, in these authors opinions, both identified “non-coronavirus” structures as “coronavirus” particles
  • They conclude that this dilemma of diagnostic EM should alarm us all, as misleading information on the presence of “SARS-CoV-2” in tissue has already made its way into the scientific literature and seems to be perpetuated
  • The 6th article also points out that several studies have used EM to propose direct evidence of infection of the kidney and other tissues by “SARS-CoV-2”
  • However many of these studies are fraught with confusion over differentiating “virus” from normal structures within cells, leading to an explosion of misinformation
  • Understanding the biology of “viruses” is essential to accurately identify “viral” particles by EM because certain cellular organelles can mimic the structure of “viral” particles
  • Accurate interpretation of electron micrographs requires integration of morphology and biology
  • The putative “virions” appeared in a location that would not be expected for “coronavirus”
  • 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
  • Cells have many organelles comparable in size to the “coronavirus”
  • Cellular vesicles can be difficult to classify on the basis of morphology alone but can be deduced from their relationship with other membranes in the cell
  • The intraluminal vesicles found within larger vesicles have been confused with “SARS-CoV-2” particles
  • Microvilli captured in plasma membrane invaginations can also mimic multivesicular bodies and be confused for “viral” particles
  • To ensure the rigor and reproducibility for the identification of “viruses” in tissues by electron microscopy, the authors came up with a ser of criteria:
    1. Structure: morphologic features of the “viral” particles should conform to prior knowledge of the “virus”
      • However, 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
      • “Coronavirus” spikes are often difficult to visualize in thin sections using transmission EM
      • Tissue preservation is also critical, and poor preservation, as is common for autopsy material, compromises objective interpretation of electron micrographs and the ability to conclusively identify “viral” particles
    2. 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 to differentiate these particles from normal cellular structures
    3. Independent evidence to corroborate EM findings: additional validated tests, such as PCR, immunohistochemistry, in situ hybridization, and immunoelectron microscopy, should be performed independently to confirm “viral” infection and further support the interpretation of the EM findings
    4. Expertise: electron microscopy should be performed and interpreted by experienced individuals and aided by appropriate controls and bona fide images of the “virus” sought
      • Experience with electron microscopy for diagnosis of kidney diseases alone is not sufficient to accurately discern subcellular organelles from novel “viruses”

Virology is steeped in theories assumptions, and guesswork. This is easily demonstrated by the ridiculous lengths they go to claim certain particles as “viruses” while identical ones are said to be “non-viral.” It is admitted that in order to have an accurate interpretation of the EM image, prior knowledge of the morphology and morphogenesis of the “virus” in question needs to be known. If there is no a priori knowledge, then a model needs to be created through EM of an in vitro (outside of a living organism) cell culture. However, the cell culture process is the exact opposite of purification/isolation and seeing as “SARS-COV-2” is a “novel virus,” the sample would need to be properly purified/isolated first in order to not mistake any of the similar and/or identical particles guaranteed to be in the sample as the culprit implicated in disease. On top of that, in order to be certain the particles claimed as “SARS-COV-2” actually exist in a sick person and are not a creation stemming from the cell culture process itself, the sample (especially for a “novel virus”) would need to purified/isolated directly from the sick patient first, not cultured in cells.

“SARS-COV-2” was based on the prior knowledge of earlier “Coronaviruses.” Somewhere in this chain of “Coronaviruses” to which virologists base their knowledge upon, one of them would have had to come from a properly purified/isolated sample taken directly from a sick human. This purified/isolated sample would need to have been proven pathogenic in a natural way in human and/or animal studies. However, I have gone through every original “Coronavirus” paper and this process was never carried out. Not once. Knowing that there are numerous particles claimed to be similar and/or identical to “SARS-COV-2” (and the other “Coronaviruses”) that are guaranteed to be in the unpurified sample, how can virologists claim that the particles they image are actually a “virus” rather than any of the normal subcellular structures/particles? According to the above sources, it is very apparent that there can be no certainty.

In the end, we are left with static images of dead particles which are interpreted based on assumed morphology and the location the “virus” is expected to be in. If they don’t meet these subjective requirements, the same particles are interpreted as normal subcellular structures/particles. It is clear to see, the image of “SARS-COV-2” truly is in the eye of the beholder…or at least the one interpreting the TEM images.

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