Dr. McCullough’s Works of Art

Art:
something that is created with imagination and skill and that is beautiful or that expresses important ideas or feelings

https://www.britannica.com/dictionary/art

I was recently made aware of an article by Dr. Peter McCullough which supposedly contained “difficult to deny” evidence for the existence of “SARS-COV-2.” This evidence was supposed to shut up those of us who state that no “virus” has ever been properly purified and isolated directly from the fluids of a sick patient and proven pathogenic in a natural way. Thus, I was a bit curious to see what Dr. McCullough had in store for us. Was he finally going to show the evidence we have been asking for? Did he have an actual scientific study adhering to the scientific method which could meet the burden of proof required to claim the existence of these fictional entities?

Let’s jump right in and find out just what kind of “difficult to deny” evidence that Dr. McCullough has to share with the class. I have provided McCullough’s full article detailing a study where the researchers claimed to work with various strains of “SARS-COV-2” while using cryo-EM to image and study the particles. Please note that the title of McCullough’s article claims to be seeing “Covid” up close rather than “SARS-COV-2.” This is an interesting error by the Dr. right off the bat as “Covid” is the disease while “SARS-COV-2” is the “virus.” Not off to a good start:

Seeing COVID up Close Makes It Difficult to Deny Its Existence

The endless frustrations of the SARS-CoV-2 crisis and pandemic response has led some to push back denying existence of the virus altogether.  

Laboratory methods in virology are well accepted and utilize a series of experiments to demonstrate cellular invasion, replication, transfer and repeated infection.  Whole genomic sequencing has aided in identification of variants and subvariants and helped greatly in forecasting what is coming next.  The CDC Nowcast system is an excellent application of targeted sequencing of viral samples.[i]

Nonetheless, some have said if SARS-CoV-2 cannot be cultured like a bacteria and “isolated” then it does not exist.  I have always responded that the principles of laboratory virology, sequencing, and the mass production of viruses such as that done by the Max Planck Institute for Dynamics of Complex Technical Systems are concrete processes that rely on the presence of the virus.[ii]

My understanding from the body of medical literature and firsthand clinical experience are consistent with the conclusion that COVID-19 is indeed a unique illness distinguishable from influenza and other viral infections.   I have always been impressed with the absence of bacterial superinfection and micro- and macro-thrombosis being features that separate COVID-19 from influenza and other viral syndromes.

Calder, et al, at the Francis Crick Institute has gone a step farther with advanced forms of electron microscopy to see the virus up close and personal.[iii]

A picture speaks a thousand words and should help even the most skeptical “viral denier” come onto the rational team that is trying to treat high risk patients, end ridiculous contagion control measures, and bring our world back to normal.

So, the next time someone at a cocktail party says “COVID-19 is a hoax, the virus has never been isolated, “show them some of these works of art!”

https://www.theepochtimes.com/health/seeing-covid-up-close-makes-it-difficult-to-deny-its-existence_4870522.html?welcomeuser=1

A picture is worth a thousand words, right Dr. McCullough?

According to Dr. McCullough, the “difficult to deny” evidence required to definitively prove the naysayers wrong about the existence of “SARS-COV-2” is not purified and isolated “viral” particles proven pathogenic in a natural way. Dr. McCullough’s air tight evidence comes in the form of the provided cryo-EM images, or “works of art” as he calls them, an interesting choice of words to use when attempting to claim the accompanying cryo-EM images are the required proof of existence. A picture is worth a thousand words, right Dr. McCullough? We can therefore conclude that a picture of Santa Claus is direct proof for the existence of the magical man in red. Bigfoot has been photographed numerous times, so I guess it is settled that he is off the endangered mythological creatures list. The Loch Ness monster? Yep, that fits the bill as well as ol’ Nessy is famous for striking a pose for curious onlookers. Thus it seems that using pictures as direct proof of existence is a rational thought process. As I can see no faults in Dr. McCullough’s line of thinking here, I guess I’m on McCullough’s Team Rational now!

However, if one were to be nitpicky, how the images were created and obtained may be the perfect place to start in regards to finding some holes in McCullough’s “logic.” Obviously, that security camera image of Santa is most likely of some kid’s parent and was taken from the very “trustworthy” Youtube video of the top ten times Santa was caught in the act. That can hardly be considered evidence of the “difficult to deny” variety. We know that the controversial Bigfoot image most likely came from a man dressed in a monkey costume. The fanous Loch Ness monster photograph was of a toy submarine with a plastic head attached. Thus, perhaps the source of the image as well as how it was created and obtained is more important than the actual image itself. After a careful bit of contemplation, my commitment to Team Rational may be wavering a bit here. Let’s see what we can uncover about the creation of McCullough’s works of art.

Looking to some of the highlights taken from the paper that Dr. McCullough was so mesmerized by, we find that the researchers attempted to study the structure of four different strains (Wuhan, Aplha, Beta, and Delta) of “SARS-COV-2” through the use of cryotomography. This is a form of electron microscopy that, according to Nature, is a technique where an electron microscope is used to record a series of two-dimensional images as a biological sample held at cryogenic temperatures is tilted. The “virus strains” used for the imaging were cultured and “grown” in Vero cells offsite at the World Influenza Centre, Francis Crick Institute, London, UK. The “viruses” were then maintained in Dulbecco’s Modified Eagle Medium (DMEM) Gibco™, with 100 U/ml penicillin, 100 μg/ml streptomycin (Pen-Strep) and 10% (v/v) heat-inactivated fetal calf serum (FCS). Thus, we are already off to a bad start as the researchers are not working with assumed “viral” particles purified and isolated directly from the fluids of any sick human but from unpurified cell culture supernatant assumed to contain the “viral” particles.

Digging into the results from the study, the researchers claim that the particles observed were cylindrical in shape with spikes protruding from the surface. This is in direct contrast to earlier research which they admit showed particles that were spherical or ellipsoidal in morphology and shape. Thus, the slam-dunk evidence that McCullough is presenting oddly gives us a completely different shape for “SARS-COV-2.”

These two don’t quite match up, now do they? 🤔

The rest of the highlights detail some of the methods and programs used to reconstruct the 3-D images of the cultured particles such as fiducial alignment, motion correction, dose-weighting, phase flipping, backplotting, postprocessing, reference mapping, etc. In order to create the 3-D model, it is stated that the particles were symmetrised using the EMAN program in order to generate a crude model from the best views of various particles. A brief explanation of what this process involves:

“As single particle cryo-EM images are 2-D projections of the to-be- determined 3-D structure at random views, the inverse problem is to determine the 3-D structure from these 2-D images using computational image processing methods. Current image processing methods rely on iterative processes in which the 3-D reconstruction is iteratively improved. It is critical that the initial 3-D model is correctly constructed before proceeding to full refinement.”

Symmetry view method. This EMAN method intentionally searches for particle images with best five-, three-, and twofold symmetry characteristics and uses these particles to construct the first crude 3-D model that will be further refined. This method is available in the EMAN program starticos.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020923/

I will leave it up to the reader to decide whether or not the numerous processes and computer programs used throughtout this herculean reconstruction effort results in the creation of an actual image or an artists interpretation conjured up and designed by computer software:

Electron cryotomography of SARS-CoV-2 virions reveals cylinder-shaped particles with a double layer RNP assembly

“SARS-CoV-2 is a lipid-enveloped Betacoronavirus and cause of the Covid-19 pandemic. To study the three-dimensional architecture of the virus, we perform electron cryotomography (cryo-ET) on SARS-Cov-2 virions and three variants revealing particles of regular cylindrical morphology. The ribonucleoprotein particles packaging the genome in the virion interior form a dense, double layer assembly with a cylindrical shape related to the overall particle morphology. This organisation suggests structural interactions important to virus assembly.

Introduction

SARS-Cov-2, the causative agent of Covid-19, has been the object of intense investigation, including structural studies by cryo-EM of individual component proteins at high-resolution, as well as cryotomography of viruses1,2,3 and infected cells4,5. Cryotomography of frozen-hydrated virus particles enables the visualisation of particle exterior and interior in three dimensions and thus is an important method for understanding the three-dimensional architecture of pleomorphic lipid-enveloped viruses. These previous studies have described SARS-CoV-2 virions as spherical or ellipsoidal and in addition have mainly visualised the spike protein (S) in pre-fusion and post-fusion states on the virus surface and ribonucleoprotein assemblies in the virus interior. Other virus structural components such as the membrane protein (M), the envelope protein (E) and additional non-structural proteins may play important roles in virus structure and assembly.

Because a number of fundamental questions remain about the virus architecture, such as how virion components interact in three-dimensions to assemble particles and how the large positive strand genome is packaged, we apply cryotomography to study the architecture of virions of the original Wuhan strain of SARS-CoV-2 and three variants that arose during the first year of the pandemic. The tomograms show the particles are of a uniform cylindrical shape with spike proteins distributed over the whole envelope. The interior of the particle reveals an organised RNP assembly with implications for virus assembly.

Results and Discussion

We recorded tilt-series and reconstructed cryotomograms of frozen-hydrated SARS-CoV-2 virions from the original Wuhan strain and Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617) variants. The tomograms show that the virions are predominantly of a single and uniform morphology which is an extremely flat cylindrical shape.”

Methods

SARS CoV-2 virus seed stock production

“Four different viral strains were obtained as follows:

Wuhan (hCoV-19/England/02/2020), (GISAID EpiCov™ accession EPI_ISL_407073) from The Respiratory Virus Unit, Public Health England (PHE), UK.

Alpha variant B.1.1.7 (hCoV-19/England/204690005/2020) from PHE through Professor Wendy Barclay, Imperial College, London, UK.

Beta variant B.1.351 (501Y.V2.HV001) from the African Health Institute, Durban, South Africa.

Delta variant B.1.617.2 (GISAID accession EPI_ISL1731019) from Professor Wendy Barclay, through Genotype-to-Phenotype National Virology Consortium.

All viruses were grown at the World Influenza Centre, Francis Crick Institute, London, UK under Biosafety level 3 conditions in Vero V1 cells, provided by Professor Steve Goodbourne, St George’s Hospital, University of London, UK and maintained in Dulbecco’s Modified Eagle Medium (DMEM) Gibco™ 41965039, with 100 U/ml penicillin, 100 μg/ml streptomycin (Pen-Strep) and 10% (v/v) heat-inactivated fetal calf serum (FCS).”

Tomogram generation

Talos-acquired tilt series were fiducial aligned with the IMOD package18 and reconstructed with SIRT, 5 iterations. Resulting tomograms were binned 4x and filtered with 10 iterations of Nonlinear Anisotropic Diffusion.

Movie frames of Krios acquired tilt series were motion corrected, dose-weighted and fiducial-aligned using the IMOD package. The contrast transfer function was estimated with CTFFIND419, tomograms were CTF corrected by phase flipping and reconstructed with novaCTF20, producing a weighted back-projection tomogram and a SIRT-like filtered tomogram. For visual analysis the SIRT-like filtered tomograms were binned 4x and subjected to 20 iterations of Nonlinear Anisotropic Diffusion filtering using the Bsoft21 program bnad with default parameters (λ = 0.1).

Subtomogram averaging

268 whole virions from 20 tomograms were picked manually from the 4-fold binned SIRT/NAD-filtered tomograms using EMAN 2.9122. Particles were then extracted from the unbinned, CTF-corrected WBP tomograms with 4-fold downsampling (box size 1598 Å, 8.88 Å/pixel sampling) using Relion 3.123. Reference-free alignment of all subtomograms produced an initial map which was used as a reference for further 3D classification and alignment. A loose, soft mask around the outer surface of the virion envelope was used during further alignment, classification and postprocessing. The average map of 2-layered virions was estimated to have a resolution of 44 Å at the FSC = 0.143 cutoff by Relion postprocessing. After the alignment of all subtomograms, 3D classification into 5 classes without alignment was then carried out to examine the varying morphologies of the virions.

For PCA analysis, the virion subtomograms were re-imported into EMAN 2.9122, aligned against the Relion map and analysed using PCA-based classification, starting from 3 initial basis vectors and requesting 3 output classes.

Spike particles were picked manually from the 4-fold binned SIRT/NAD-filtered tomograms using IMOD. 4418 particles from 251 virions in 18 tomograms were then extracted from the full-size WBP tomograms with 2-fold downsampling (box size 444 Å, 4.44 Å/pixel sampling) for subtomogram averaging in Relion 3.1. Reference-free initial model generation in C1 produced a map with clear 3-fold features, which was symmetrised and used as a reference for further refinement with C3 symmetry applied and with a loose mask around the ectodomain. Upon convergence of refinement, the particles were reclassified with relaxation of C3 symmetry, revealing a class with the 1-RBD-up conformation. The classes were separated and the closed conformation particles were finally refined with C3 symmetry, while the 1-RBD-up particles were refined without symmetry. The final resolutions were estimated as 30 Å (closed conformation) and 28 Å (1-RBD-up conformation) at the FSC = 0.143 cutoff by Relion postprocessing.

The subtomogram averaging maps were backplotted into the frame of reference of the original tomogram using in-house scripts (available from the authors). Spike particles were visually inspected and removed if they were misaligned (i.e. had a relative tilt of over 90° with respect to the normal of the viral envelope) or duplicate particles which converged to the same position during alignment.”

https://www.nature.com/articles/s42003-022-04183-1

An electron microscopic image of a thin section of SARS-CoV within the cytoplasm of an infected cell, showing the spherical particles and cross-sections through the viral nucleocapsid. https://www.cdc.gov/sars/lab/images.html

Why were the images of the “SARS-COV-2 virions” in this study of a different shape and morphology than those seen in the many studies that came before? If we look into the various processes and limitations associated with electron cryotomography, we can get an idea as to why this may have been the case. It must be understood that the cryo-EM images are 3-D reconstructions which require different computer programs and software to combine and create the images seen. In this process, the sample is frozen and then put through a series of recordings in which the sample is tilted on a different axis and at various angles to obtain multiple images which are then merged together to create a 3-D reconstruction. However, in order to generate these “works of art,” there are some problems which are regularly encountered which can distort the final result.

For frozen biological samples, radiation damage is a major concern. The longer the sample is exposed to the electron beam through various tilts, the quicker the sample heats up and becomes unusable. As more electrons are used, the originally sharp edges of macromolecular structures degrade and eventually “bubbles” which causes distortions and creates difficulties with interpretation. Another issue is missing wedges of information which remain unmeasured due to limitations in acquiring images at a certain angle. This creates worse resolution of the 3-D reconstruction in the direction parallel to the electron beam than the resolution perpendicular. This in turn can cause spherical objects to appear ellipsoidal. Various computer software and algorithms were created to try and correct these issues yet they still remain and can make identifying structures challenging. This is all detailed in highlights from the below source:

Electron Cryotomography

INTRODUCTION—THE STORY OF FtsZ

“ECT can produce three-dimensional (3-D) reconstructions of intact cells in near-native states to “molecular” resolution (∼4 nm), and has thus begun providing unprecedented views into the ultrastructure of bacterial cells.”

Tilt-Series Acquisition and Fundamental Limitations

“The word “tomography” means imaging by sections or sectioning. The most familiar use of the word is the medical “CT,” or “computed tomography” scan, wherein X-ray projection images through a subject are recorded from a number of directions and then merged to produce a 3-D anatomical model. Similarly, in electron tomography, a “tilt-series” of projection images are recorded of a single object like a bacterial cell as it is incrementally tilted around one, and sometimes two axes, and these images are then merged to produce a 3-D “reconstruction” or “tomogram” (Fig. 2). The basic workflow is that a grid is inserted into the EM, a target is chosen and centered under the electron beam, a projection image is recorded, the sample is rotated (tilted) a degree or two, another projection image is recorded, and the cycle of rotation and imaging is repeated as far as useful images can be obtained (until the sample becomes prohibitively thick or the grid or grid holder begins to block the beam, usually ∼65°). Images of the inverse tilt angles (i.e., 0° to −65°) are recorded similarly, or alternatively, the tilt-series can begin at one extreme tilt angle (like 65°) and proceed through the untilted position to the opposite extreme (i.e., −65°). Unfortunately, for frozen-hydrated biological materials, radiation damage prohibits this. As the imaging electrons pass through the sample, they can remain unscattered, scatter elastically, scatter inelastically, or suffer multiple scattering events. Although image contrast (the information content) is produced by the interference of the unscattered and the elastically scattered electrons, the inelastically scattered electrons gradually destroy the sample. Inelastic scattering events break covalent bonds, deposit heat, and more rarely even knock atomic nuclei out of place. Because for every useful elastic scattering event there are approximately 3 damaging inelastic scattering events (Henderson 1995), as more and more electrons are used to build up an image, sample damage accumulates. The originally sharp edges of macromolecular structures degrade and eventually “bubbles” of (presumably) radiolytic fragments appear and catastrophically disrupt the structure (Comolli and Downing 2005; Iancu et al. 2006b; Wright et al. 2006). Thus the most fundamentally limiting factor in ECT is the total number of electrons that can be used to record images before the sample is destroyed.”

“Because with even the thinnest samples, useful images at tilt angles higher than ∼65–70° cannot usually be collected, there is a “wedge” of information (the tilt angles surrounding 90°) that remains unmeasured. As a result, the resolution of the 3-D reconstruction in the direction parallel to the electron beam is significantly worse than the resolution perpendicular. In simple visual terms, this causes spherical objects to appear somewhat ellipsoidal (smeared in the direction of the beam), and continuous objects such as filaments and membranes are more visible in some orientations than in others. This is why in “xz” or “yz” tomographic slices (such as Fig. 1D), the membranes do not appear to connect around the “top” and “bottom” of the cell. Although the missing wedge may be reduced to a missing “pyramid” by rotating the grid 90° and collecting a second, orthogonal tilt-series (a so-called “dual-axis” data set), this procedure is more than twice as time consuming, the dose that can be used per image is halved, and alignment errors between the tilt-series can erode the benefit (Nickell et al. 2003; Iancu et al. 2005). Thus a third fundamental limitation in ECT is the anisotropic resolution caused by tilt limitations (the “missing wedge”).”

3-D Reconstruction and Interpretation

“As mentioned earlier, because no goniometer is perfect, specimens move laterally and vertically within the column throughout the tilt-series. The images must therefore be precisely aligned before a 3-D reconstruction can be calculated. As an additional challenge, because of the physics of electron optics, changes in height/focus within the column cause images to rotate and show subtly different magnifications. Further, although the tilt angle of each image is approximately known, the actual angles reached must be determined more accurately. Sophisticated software has therefore been written to refine estimates of the translations, rotation, magnification, tilt axis, and tilt angle of each image in the tilt-series (Mastronarde 2008). The colloidal gold beads typically added to the samples provide precise fiducial markers to facilitate this process.

Once the images are aligned, 3-D reconstructions can be calculated with a variety of algorithms. The most intuitive is “back-projection,” in which a reconstruction is built up by “smearing” the densities in each image back through space in the opposite direction they were projected (Fig. 2B) (Crowther et al. 1970b). To understand this reconstruction in Fourier space, the key principle is that the 2-D Fourier transform of a projection image is a central slice of the 3-D Fourier transform of the object (the “Projection theorem”) (Crowther et al. 1970a). Thus the 3-D Fourier transform of the sample can be “filled” with the transforms of the 2-D images, and then re-sampled onto a regular (for instance Cartesian) coordinate system and inverse transformed to produce a real-space reconstruction (Lee et al. 2008). Various software packages have been written to perform these calculations, including IMOD (Mastronarde 2008), the TOM toolbox (Nickell et al. 2005), and RAPTOR (Amat et al. 2008). Once reconstructed, tomograms can be “denoised” to improve image contrast and enhance interpretability (Frangakis and Hegerl 2001Narasimha et al. 2008) and/or “segmented” to allow specific features to be visualized in isolation or as surfaces (Pruggnaller et al. 2008).”

“In summary, ECT produces 3-D images of intact cells in near-native states to “molecular resolution,” but the sample must be thin (< 0.5 µm), the interpretability of the resulting tomograms is limited by radiation damage, the resolution is anisotropic because of tilt limitations, the procedure is complicated and requires expensive electron cryo-microscopes, and identifying structures of interest in the tomograms can be challenging.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869529/

A visual representation of the steps involved to create this beautiful work of HIV-1 art. https://figshare.com/articles/figure/Cryo_tomography_of_HIV_1/645351

My reasoning for including this last source is because it provides a great amount of detail covering what goes into obtaining cryotomography images from the data generated. I have highlighted information on both single-particle imagery as well as tomograms from cryo-EM. This is to showcase the numerous steps involved in creating the fine art McCullough enjoys as well as other “works of art” constructed using this process. There is a great deal of interesting information within the article which I had to leave out for space considerations so I do recommend giving the full article a read if there is a desire for greater clarity on the subject. Once again, I will leave it up to the reader to decide what, if anything, can be gleaned from these computer-generated recreations.

In this source, it is stated that data preprocessing involves three separate steps for reconstruction of the images such as screening, boxing, and CTF determination. 3-D reconstruction of a “virus” from multiple single particle images is the last step in the computationally intensive process of particle alignment. The majority of 3-D maps assume an icosahedral symmetry and even though non-icosahedral reconstructions represent the ideal target, this remains complicated due to the requirement of far more data needed for reconstruction. There are numerous programs to choose from for 3-D reconstruction in regards to particle alignment and refinement and it is ultimately up to personal preference as to which one is used. These programs use alignment algorithms which require an accurate initial model, although there are some programs where it is stated that this is unecessary. In order to generate a model, it must be determined how many particles are to be used for reconstruction and which ones are high quality as lower quality images can contain aberrations which may do more harm than good in enhancing the resolution of a reconstruction. It is said that in order to determine the number of particles required to achieve a specific resolution reconstruction, assumptions must be made about the data, both without and with noise. After these various steps are performed, it is possible to reconstruct the 3-D model of the “virus” by merging all of the 2-D particle images, after CTF correction, into a single 3-D map using computer programs. For tomography, image alignment is crucial and is performed using a graphical user interface (GUI) computer program. The final step in the structural determination of a “virus” is interpretation of the 3-D density map in regards to resolution determination, segmentation, and model fitting and care must be taken not to overinterpret the map beyond its best approximated resolution. The rest of the process involves fitting to a model for visualisation:

1.16 Cryo-Electron Microscopy and Tomography of Virus Particles

“Virus reconstruction typically relies on one of two primary imaging modalities – single particle and tomography – the choice of which depends on the structural heterogeneity of the virus either in vitro or in the presence of a host cell. The major difference between the two techniques is the manner in which the data are recorded. In single particle data collection, individual virus particles on the grid are imaged only once, and it is the collection of thousands of these images that is used to reconstruct the virus (Figure 2 ). In cryo-ET, a single area of a grid containing many viral particles is imaged multiple times, at a variety of tilt angles, and it is the combination of these images that is used to generate a tomogram and subsequently an averaged density map (Figure 3 ). Although single particle data collection is often used for subnanometer resolution structural studies, in the case in which either structural or conformational non-uniformity exists (e.g., viral infection of a cell), tomography proves a more fruitful endeavor. Regardless of the technique used, implicit in each of these techniques is the need to process the raw data, and although the means of doing so are quite unique, the basic principle of aligning and averaging the data remains the same.

This chapter outlines common techniques used in the field of cryo-EM and cryo-ET for virus reconstruction, from data collection to processing and interpretation. Although many of these techniques have been well reviewed in the past,[18][21][19][20] because cryo-EM is a rapidly evolving field, advancements in both data collection procedures and data processing algorithms are a frequent occurrence. The methodologies discussed in this chapter are commonly used at the National Center for Macromolecular Imaging (NCMI) for the reconstruction of viruses by cryo-EM and cryo-ET.”

1.16.2. Single Particle Reconstruction

“As a result of the conformational uniformity with which virus particles are produced, and the ease with which they can be purified, single particle reconstruction has long been used to solve the structure of icosahedral viruses.[1][2] Single particle reconstruction relies on imaging hundreds to hundreds of thousands of viral particles, computationally isolating each particle, and then combining the individual particles into a single 3-dimensional (3-D) density map (Figure 2). Because cryo-EM provides a projection of the virus onto the recording medium, the lack of an even distribution in the 3-D orientation of the particles can introduce bias when reconstructing the data. Accordingly, a single particle imaging session should result in the collection of a series of 2-D images that taken together offer a well-sampled view of the many different orientations of the particles. The process of single particle reconstruction involves isolating each viral particle from a set of images, determining its orientation and center, and then using this information to stitch the data back together into a single 3-D representation of the virus.

Although the first single particle cryo-EM reconstructions were published approximately 40 years ago,[1][2][22] advancements are still being made in the field that have enabled the resolution of these reconstructions to push well beyond the subnanometer threshold.[4][6][8][10][5][9] The driving force behind these advancements is the ability to identify not only the secondary structural elements but also the Cα backbone and side chains of the individual subunits of a virus.

1.16.3. Tomography

Although purified samples with high structural uniformity are ideal for single particle reconstruction, it is not always possible to work within a system that lends itself to the single particle approach. In the presence of structurally diverse samples, a technique that can capitalize on the information content of just a few particles of similar or identical conformation is needed. The theory behind tomography is that by collecting a series of images from a sample, at a variety of tilt angles, it is possible to reconstruct a 3-D volume density from the 2-D projections (Figure 3(a)).[23][24] This approach allows for a great deal of low-resolution 3-D information to be extracted from the handful of particles in the tomogram. Under standard tomographic imaging conditions, a series of 71 images are taken from −70° to 70° at a step size of 2° (60° and 80° tilt holders are available as well). These images can then be aligned to each other using fiducial markers present in the images (Figure 3(b)), from which a volume (a tomogram) can be extracted (Figure 3(c)).25 From this volume, it is possible to extract individual subtomograms corresponding to each of the particles. These subtomograms can then be classified, aligned, and averaged to obtain a single 3-D model.[26][28][27]

One consideration for tomography is the high dose that the sample receives during the tilt-series imaging. In single particle data collection, the sample is often subjected to between 20 and 25 electrons per Å2 per micrograph.[20][29] In tomography, however, because a single area of the grid is imaged approximately 70 times, the sample is subjected to far more radiation, making damage a consideration. In general, during tomography, the dose per image is set to a point at which the total dose per tomogram (71 images) is between 80 and 100 electrons per Å2.27 Even at this dose level, radiation damage begins to become an issue and will limit the maximum achievable resolution through this technique.[30][31] Therefore, in order to limit the total dose delivered in a single tomogram, on average, the dose per image must be reduced to nearly 1/20th of what is commonly used for a single particle micrograph. Lowering the total dose per image reduces the signal-to-noise ratio (SNR) in the data and effectively lowers the maximum attainable resolution from the data, the gain from ‘dose fractionation’ notwithstanding.32 Due to the manner in which a single tomographic series is recorded, the maximum resolution achieved so far with these techniques only approaches the nanometer threshold,33 which is far below the current standard set by the single particle approach.[4][11][5][6][7][8][9][10]

1.16.6. Data Preprocessing

“The next step in the reconstruction workflow is preprocessing the electron micrographs collected from the microscope. For single particle reconstructions, preprocessing typically follows the same three steps: screening, boxing, and CTF determination. Each of these steps is essential to the reconstruction process, and although they can be as time-consuming as the data collection process, efforts have been made to automate them.[51][52] Alternatively, preprocessing tomogram data entails aligning the series of tilt images to each other. Due to the nature in which tomogram data are collected, the data are screened during data collection to ensure that if there is an aberrant image in the tilt series, another can be collected at the same tilt angle to prevent a loss of data for that angle.

1.16.7. Single Particle Reconstruction

The 3-D reconstruction of a virus from multiple single particle images is the last step in the computationally intensive process of particle alignment.1 The general principle behind the theory is that given a series of single particle images, once you have determined their icosahedral (or asymmetric) orientations, you can combine the data to form one cohesive 3-D model. Although this process can be applied to a small number of particles, unless a large number of particles are used, many features – such as preferred orientation, limited defocus range, and noise – keep a reconstruction from reaching high resolution. To circumvent these issues, it has been common practice to use an ever-increasing number of particles.”

1.16.7.1. Particle Alignment and Refinement

“Three-dimensional reconstruction has been the source of a great deal of research in the field and has led to the development of many programs, including EMAN,51 EMAN2,52 MPSA,58 Spider,61 XMIPP,62 IMAGIC,63 FREALIGN,64 AUTO3DEM,65 SPARX,66 and IMIRS.67 Although some of these programs are more efficient than others, the choice of which to use is usually a matter of personal preference.”

1.16.7.1.2. Asymmetric alignment 

“The majority of virus reconstructions published in the literature are of specimens for which there is some assumed symmetry to the map. For many viruses, the assumed symmetry is icosahedral; however, due to the nature in which these viruses are built and mature, there are important molecular components in their overall structure that are not symmetric. Accordingly, there has been an increased interest in the determination of the structure of these viruses without imposed symmetry because it will help shed light on the process of viral assembly and maturation.[6][12][16][70][13][14][15] The assumption of symmetry is often made because it simplifies the process of orientation determination. Furthermore, by icosahedrally averaging a map, the information content of a data set is enhanced, making it possible to achieve high-resolution reconstructions with nearly 60 times less data than for a reconstruction in which no symmetry is assumed.68 The process of orientation determination for an asymmetric reconstruction differs from the traditional symmetric orientation search in that one must be able to identify the non-icosahedral components in the virus particle.6 In the case of a phage such as P-SSP7, which has a large tail and portal structure, it is conceptually easy to understand how one would determine the asymmetric orientation of the particles because the non-icosahedrally symmetric feature is visible in the raw micrographs (Figure 15 (a)). However, in the case of a virus such as HSV-1 in which the structure does not have such a protruding feature that is readily identifiable in raw micrographs, it is more difficult to find the true asymmetric orientation of the particle (Figure 15(b)). Once the asymmetric orientation of every particle has been determined, the process of reconstructing the virus asymmetrically is identical to any other reconstruction in which symmetry is assumed.

1.16.7.2. Single Particle Data Subset Selection

Traditionally, the resolution of a map is improved as increasingly more particles are added to the reconstruction. However, this must be weighed against the observation that some data are of better quality than others, and inclusion of data that contain aberrations may in fact do more harm than good in enhancing the resolution of a reconstruction. The quality of a single particle can be thought of as a metric of conformational heterogeneity, icosahedral symmetry, and imaging conditions.56 The better all three of these factors are, the more likely the particle will contribute to producing a high-resolution reconstruction.

The number of particles required to achieve a specific resolution reconstruction depends on assumptions made about the data, both without and with noise.[71],”

“Often, the ‘best’ particles are those that have been determined, through the process of iterative alignment and refinement, to consistently score higher than other particles.58 Although there is no concrete method to determine a priori which particles are the best, there are many steps that can be taken to ensure that the quality of data you put into your reconstruction is of the highest quality possible. First, in the event that an accurate initial model for your sample exists, there is no need to collect data at a defocus higher than 2 μm. In addition, by carefully screening your data on the front end, it is possible to eliminate entire micrographs of poor-quality data. A second level of screening can be performed at the level of boxing, where it is possible to identify and remove particles that may be experiencing the effects of local charging. Although there is no way to directly control for conformational heterogeneity within your sample, if there are visible differences in the raw particle data, it is possible to ameliorate this problem by selecting subsets of the data (Figure 9). Nevertheless, even if ‘bad’ particles make it through these steps of screening, in some cases, the process of data refinement will eliminate particles that are not consistent with the existing pool of data.

1.16.7.3. Three-Dimensional Reconstruction

Once the orientations of all of the particles in a data set have been determined, it is possible to reconstruct the 3-D model of the virus. This process works by merging all of the 2-D particle images, after CTF correction, into a single 3-D map. 3-D reconstruction programs typically have a method to generate these 3-D density maps, and to achieve this task EMAN and EMAN2 use the programs make3d and e2make3d, respectively. These two programs are based on a direct Fourier inversion method to generate the 3-D volumetric data, and they have a variety of command line parameters that allow the user to specify symmetries or data preprocessing steps.”

1.16.8.2. Alignment and the ‘Missing Wedge’

“Just as in single particle cryo-EM, there are a variety of schemes for determining the orientation and alignment of the particles in the extracted subtomograms.[25][26][28][51][76] Accordingly, a well-resolved tomographic average requires the individual subtomograms containing the extracted particles to be aligned with respect to each other – a process that is rarely as straightforward as in single particle cryo-EM. Because most cryo-holders have a maximum tilt angle of ±70° (although some 80° holders are available), the total coverage for a tomogram is limited to 140° at best (Figure 17 (a)). Ideally, a tomogram would contain data collected across a full 180°; however, because this is not possible with the currently available cryo-holders, this lack of information from 70° to 90° is manifested as a missing wedge of data in Fourier space (Figure 17(b) and (c)), and if it is not corrected for, it can distort the final reconstruction. An additional complicating factor is that because the tilt angle for a group of particles in a single tomogram is identical, all the particles have the same missing wedge. However, because the particles are randomly oriented in the sample, each particle has different missing wedge data with respect to its orientation (Figure 17(b) and (c)). Because the missing wedge is manifest as a value of zero in the FFT of the data, calculation of the cross-correlation between two particles can result in the elimination of a great deal of information in Fourier space because cross-correlation involves multiplication by these zeros (Figure 17(c)). To address the fact that the presence of missing data in the Fourier space can hinder proper alignment of the subvolumes,[27][28] procedures have been developed to circumvent this problem by normalizing the cross-correlations calculated for two particles at different orientations (Figure 17(f)).28 Although this approach has been met with success, there is no guarantee that the missing wedge problem has been solved, especially for high-resolution tomographic reconstructions.

Alignment of tomographic data requires the three Euler angles (α, β, γ) for every computationally extracted particle to be determined through a series of orientation searches. This process is typically initialized through comparison with an approximate model of the virus. However, in the event that no adequate reference model is available, it is necessary to generate an initial model from the available data. The raw data can be used to generate an adequate initial model by comparing subtomograms to each other in a process known as ‘all-vs-all’ comparison.76 This initial model can be further improved through a series of iterative refinements in which the individual subtomograms are classified, aligned, and averaged to a single or multiple 3-D models. Furthermore, as in single particle cryo-EM, particles can be aligned with respect to their asymmetric features, making it possible to generate single particle cryo-ET maps without imposing symmetry in the map. Nevertheless, again as in single particle cryo-EM, the assumption of symmetry during reconstruction dramatically enhances map resolution.16 To generate a 3-D model from cryo-ET data, once the orientation search is complete and the particles have been aligned, the data can be reconstructed into a 3-D model by averaging the individual subtomograms together while accounting for the missing wedge information from each particle.28

1.16.9. Data Interpretation

The final step in the structural determination of a virus is interpretation of the resulting 3-D density map, specifically resolution determination, segmentation, and model fitting. However, care must be taken not to overinterpret the map beyond its best approximated resolution: 20 Å for gross structure, 12 Å for individual protein domains, 9 Å for long and smooth α helices and large β sheets, 4.7 Å for bumpy α helices and possibly β strands, less than 4.5 Å to possibly determine a Cα backbone trace and bulky side chain features, and less than 3.6 Å to resolve ambiguity in β strand and loop connectivity.[77][79][78]

1.16.9.2. Segmentation

One of the key components in data interpretation is segmentation of a map. Because most complex macromolecules can be broken down into smaller self-assembled protein complexes (a segment), differentiating these pieces from the whole is part of understanding the viral architecture and assembly process. For viruses, when symmetry is assumed in the reconstruction, it is possible to extract just the asymmetric unit and segment it accordingly;[29][48] however, as more viruses are reconstructed without imposed symmetry, it will become necessary to consider the map as a whole.[6][13][15][14] Although a map can be segmented manually by visual inspection of the density map, simultaneous visualization of the map with fit crystallographic homologs eases the process. Traditionally, segmentation is performed manually,[75][84][85] but tools have been developed to automate certain aspects of the segmentation process. These programs use a variety of approaches, such as watershed86 and principal component analysis87 or multiscale segmentation,88 to identify individual protein components in the virus. Although these algorithms save a great deal of time during segmentation, their accuracy depends on the resolution and the overlapping density between adjacent molecules.

Regardless of what software is used, accurate segmentation remains a challenging task. For example, a 9-Å resolution map of the bacteriophage did not reveal the presence of two separate coat proteins in the capsid. However, when a 4.5-Å map of ε15 was obtained, a second coat protein was discovered after the Cα backbone trace of that protein revealed that it was in fact two distinct proteins.4

1.16.11. Future Prospects

“As described in this chapter, the structural features of most viruses are lost in the noisy images recorded from the electron microscope. Fortunately, after extensive data processing and reconstruction, it becomes possible to resolve these features – in some instances at near-atomic resolutions. Although most of this work has been augmented by image processing techniques that computationally enhance image contrast, advances in fabrication techniques have enabled the microscopist to do so directly by altering the optics of the electron microscope.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7151817/#!po=21.0983

“If they don’t understand viruses, show them this work of art!” – something probably said by Peter McCullough at some point in time 🤷‍♂️

In Summary:

  • According to McCullough, some have said if “SARS-CoV-2” cannot be cultured like a bacteria and “isolated” then it does not exist
  • Wrong. We state that the existence of these particles must be proven within humans first through purification and isolation directly from the sample taken from a sick patient. Cell culturing shouldn’t even be a part of the conversation until this has been done.
  • McCullough states that his understanding from the body of medical literature and firsthand clinical experience is consistent with the conclusion that “COVID-19” is indeed a unique illness distinguishable from influenza and other “viral” infections
  • He believes that a picture speaks a thousand words and should help even the most skeptical “viral denier” come onto the rational team
  • McCullough advises the readers that the next time someone at a cocktail party says “COVID-19″ is a hoax or that the “virus” has never been isolated, they should “show them some of these works of art!”
  • The researchers performed electron cryotomography (cryo-ET) on “SARS-Cov-2 virions” and three variants which revealed particles of regular cylindrical morphology
  • This is rather odd as, according to the CDC, “SARS-COV-2” is spherical in shape
  • The researchers state that previous studies have described “SARS-CoV-2 virions” as spherical or ellipsoidal
  • They once again state that their tomograms show the particles are of a uniform cylindrical shape with spike proteins distributed over the whole envelope
  • They recorded tilt-series and reconstructed cryotomograms of frozen-hydrated “SARS-CoV-2 virions”
  • The tomograms showed that the “virions” are predominantly (i.e. not all of them) of a single and uniform morphology which is an extremely flat cylindrical shape
  • The researchers used 4 strains (Wuhan, alpha, beta, delta) which were “grown” at the World Influenza Centre, Francis Crick Institute, London, UK under Biosafety level 3 conditions in Vero V1 cells (monkey kidney cells)
  • The cell cultured creations were maintained in Dulbecco’s Modified Eagle Medium (DMEM) Gibco™, with 100 U/ml penicillin, 100 μg/ml streptomycin (Pen-Strep) and 10% (v/v) heat-inactivated fetal calf serum (FCS)
  • For tomogram generation, Talos-acquired tilt series were fiducial aligned with the IMOD package and reconstructed with SIRT, 5 iterations
  • Movie frames of Krios acquired tilt series were motion corrected, dose-weighted and fiducial-aligned using the IMOD package
  • The contrast transfer function was estimated with CTFFIND4, tomograms were CTF corrected by phase flipping and reconstructed with novaCTF, producing a weighted back-projection tomogram and a SIRT-like filtered tomogram
  • 268 whole “virions” from 20 tomograms were picked manually from the 4-fold binned SIRT/NAD-filtered tomograms using EMAN 2.9122 (i.e. they picked the best particles that they had reconstructed from multiple images)
  • Reference-free alignment of all subtomograms produced an initial map which was used as a reference for further 3D classification and alignment
  • A loose, soft mask around the outer surface of the “virion” envelope was used during further alignment, classification and postprocessing
  • After the alignment of all subtomograms, 3D classification into 5 classes without alignment was then carried out to examine the varying morphologies of the “virions”
  • For PCA analysis, the “virion” subtomograms were re-imported into EMAN 2.91, aligned against the Relion map and analysed using PCA-based classification
  • Spike particles were picked manually from the 4-fold binned SIRT/NAD-filtered tomograms using IMOD
  • 4418 particles from 251 “virions” in 18 tomograms were then extracted from the full-size WBP tomograms with 2-fold downsampling for subtomogram averaging in Relion 3.1
  • Reference-free initial model generation in C1 produced a map with clear 3-fold features, which was symmetrised and used as a reference for further refinement with C3 symmetry applied and with a loose mask around the ectodomain
  • The subtomogram averaging maps were backplotted into the frame of reference of the original tomogram using in-house scripts
  • Spike particles were visually inspected and removed if they were misaligned (i.e. had a relative tilt of over 90° with respect to the normal of the “viral” envelope) or duplicate particles which converged to the same position during alignment
  • ECT is said to produce three-dimensional (3-D) reconstructions of intact cells
  • In electron tomography, a “tilt-series” of projection images are recorded of a single object like a bacterial cell as it is incrementally tilted around one, and sometimes two axes, and these images are then merged to produce a 3-D “reconstruction” or “tomogram”
  • The basic workflow is that a grid is inserted into the EM, a target is chosen and centered under the electron beam, a projection image is recorded, the sample is rotated (tilted) a degree or two, another projection image is recorded, and the cycle of rotation and imaging is repeated as far as useful images can be obtained (until the sample becomes prohibitively thick or the grid or grid holder begins to block the beam, usually ∼65°).
  • Unfortunately, for frozen-hydrated biological materials, radiation damage is an issue
  • As the imaging electrons pass through the sample, they can remain unscattered, scatter elastically, scatter inelastically, or suffer multiple scattering events
  • Inelastic scattering events break covalent bonds, deposit heat, and more rarely even knock atomic nuclei out of place
  • Because for every useful elastic scattering event there are approximately 3 damaging inelastic scattering events as more and more electrons are used to build up an image, sample damage accumulates
  • The originally sharp edges of macromolecular structures degrade and eventually “bubbles” of (presumably) radiolytic fragments appear and catastrophically disrupt the structure
  • The most fundamentally limiting factor in ECT is the total number of electrons that can be used to record images before the sample is destroyed
    • With even the thinnest samples, useful images at tilt angles higher than ∼65–70° cannot usually be collected and there is a “wedge” of information (the tilt angles surrounding 90°) that remains unmeasured
    • As a result, the resolution of the 3-D reconstruction in the direction parallel to the electron beam is significantly worse than the resolution perpendicular and this causes spherical objects to appear somewhat ellipsoidal (smeared in the direction of the beam)
  • Although the missing wedge may be reduced to a missing “pyramid” by rotating the grid 90° and collecting a second, orthogonal tilt-series (a so-called “dual-axis” data set), this procedure is more than twice as time consuming, the dose that can be used per image is halved, and alignment errors between the tilt-series can erode the benefit
  • Thus a third fundamental limitation in ECT is the anisotropic resolution caused by tilt limitations (the “missing wedge”)
  • Because no goniometer is perfect, specimens move laterally and vertically within the column throughout the tilt-series and fhe images must therefore be precisely aligned before a 3-D reconstruction can be calculated
  • Sophisticated software has therefore been written to refine estimates of the translations, rotation, magnification, tilt axis, and tilt angle of each image in the tilt-series
  • Once the images are aligned, 3-D reconstructions can be calculated with a variety of algorithms
  • The most intuitive is “back-projection,” in which a reconstruction is built up by “smearing” the densities in each image back through space in the opposite direction they were projected
  • Various software packages have been written to perform these calculations, including IMOD (Mastronarde 2008), the TOM toolbox, and RAPTOR and once reconstructed, tomograms can be “denoised” to improve image contrast and enhance interpretability
  • Limitations of ECT include:
    • The sample must be thin (< 0.5 µm)
    • The interpretability of the resulting tomograms is limited by radiation damage
    • The resolution is anisotropic because of tilt limitations
    • The procedure is complicated and requires expensive electron cryo-microscopes
    • Identifying structures of interest in the tomograms can be challenging
  • In cryo-ET, a single area of a grid containing many “viral” particles is imaged multiple times, at a variety of tilt angles, and it is the combination of these images that is used to generate a tomogram and subsequently an averaged density map
  • There are common techniques used in the field of cryo-EM and cryo-ET for “virus” reconstruction, from data collection to processing and interpretation
  • Single particle reconstruction relies on imaging hundreds to hundreds of thousands of “viral” particles, computationally isolating each particle, and then combining the individual particles into a single 3-dimensional (3-D) density map
  • Because cryo-EM provides a projection of the “virus” onto the recording medium, the lack of an even distribution in the 3-D orientation of the particles can introduce bias when reconstructing the data
  • The process of single particle reconstruction involves isolating each “viral” particle from a set of images, determining its orientation and center, and then using this information to stitch the data back together into a single 3-D representation of the “virus”
  • In the presence of structurally diverse samples, a technique that can capitalize on the information content of just a few particles of similar or identical conformation is needed
  • The theory behind tomography is that by collecting a series of images from a sample, at a variety of tilt angles, it is possible to reconstruct a 3-D volume density from the 2-D projections
  • Under standard tomographic imaging conditions, a series of 71 images are taken from −70° to 70° at a step size of 2° (60° and 80° tilt holders are available as well)
  • For single particle reconstructions, preprocessing typically follows the same three steps: screening, boxing, and CTF determination
  • Alternatively, preprocessing tomogram data entails aligning the series of tilt images to each other
  • The 3-D reconstruction of a “virus” from multiple single particle images is the last step in the computationally intensive process of particle alignment
  • The general principle behind the theory is that given a series of single particle images, once you have determined their icosahedral (or asymmetric) orientations, you can combine the data to form one cohesive 3-D model
  • The majority of “virus” reconstructions published in the literature are of specimens for which there is some assumed symmetry (the quality of being made up of exactly similar parts facing each other or around an axis) to the map
  • The assumption of symmetry is often made because it simplifies the process of orientation determination
  • In the case of a “virus” such as HSV-1 in which the structure does not have such a protruding feature that is readily identifiable in raw micrographs, it is more difficult to find the true asymmetric orientation of the particle
  • Once the asymmetric orientation of every particle has been determined, the process of reconstructing the “virus” asymmetrically is identical to any other reconstruction in which symmetry is assumed
  • The resolution of a map is improved as increasingly more particles are added to the reconstruction but this must be weighed against the observation that some data are of better quality than others, and inclusion of data that contain aberrations may in fact do more harm than good in enhancing the resolution of a reconstruction
  • The number of particles required to achieve a specific resolution reconstruction depends on assumptions made about the data, both without and with noise
  • The ‘best’ particles are those that have been determined, through the process of iterative alignment and refinement, to consistently score higher than other particles but there is no concrete method to determine a priori which particles are the best,
  • Although there is no way to directly control for conformational heterogeneity (the quality or state of being diverse in character or content) within the sample, if there are visible differences in the raw particle data, it is possible to ameliorate this problem by selecting subsets of the data
  • Even if ‘bad’ particles make it through these steps of screening, in some cases, the process of data refinement will eliminate particles that are not consistent with the existing pool of data
  • The process of creating a 3-D model works by merging all of the 2-D particle images, after CTF correction, into a single 3-D map using 3-D reconstruction programs which typically have a method to generate these 3-D density maps
  • A well-resolved tomographic average requires the individual subtomograms containing the extracted particles to be aligned with respect to each other – a process that is rarely as straightforward as in single particle cryo-EM
  • Ideally, a tomogram would contain data collected across a full 180°; however, because this is not possible with the currently available cryo-holders, this lack of information from 70° to 90° is manifested as a missing wedge of data in Fourier space, and if it is not corrected for, it can distort the final reconstruction
  • An additional complicating factor is that because the tilt angle for a group of particles in a single tomogram is identical, all the particles have the same missing wedge
  • To address the fact that the presence of missing data in the Fourier space can hinder proper alignment of the subvolumes, procedures have been developed to circumvent this problem by normalizing the cross-correlations calculated for two particles at different orientations
  • Although this approach has been met with success, there is no guarantee that the missing wedge problem has been solved, especially for high-resolution tomographic reconstructions
  • Alignment of tomographic data requires the three Euler angles (α, β, γ) for every computationally extracted particle to be determined through a series of orientation searches
  • This process is typically initialized through comparison with an approximate model of the “virus” yet, in the event that no adequate reference model is available, it is necessary to generate an initial model from the available data
  • This initial model can be further improved through a series of iterative refinements in which the individual subtomograms are classified, aligned, and averaged to a single or multiple 3-D models
  • To generate a 3-D model from cryo-ET data, once the orientation search is complete and the particles have been aligned, the data can be reconstructed into a 3-D model by averaging the individual subtomograms together while accounting for the missing wedge information from each particle
  • The final step in the structural determination of a “virus” is interpretation of the resulting 3-D density map, specifically resolution determination, segmentation, and model fitting
  • However, care must be taken not to overinterpret the map beyond its best approximated resolution
  • One of the key components in data interpretation is segmentation of a map
  • For “viruses,” when symmetry is assumed in the reconstruction, it is possible to extract just the asymmetric unit and segment it accordingly; however, as more “viruses” are reconstructed without imposed symmetry, it will become necessary to consider the map as a whole
  • There are many programs which use a variety of approaches, such as watershed and principal component analysis or multiscale segmentation, to identify individual protein components in the “virus”
  • Although these algorithms save a great deal of time during segmentation, their accuracy depends on the resolution and the overlapping density between adjacent molecules
  • Regardless of what software is used, accurate segmentation remains a challenging task
  • These images can then be aligned to each other using fiducial markers present in the images from which a volume (a tomogram) can be extracted
  • From this volume, it is possible to extract individual subtomograms corresponding to each of the particles and these subtomograms can then be classified, aligned, and averaged to obtain a single 3-D model
  • In tomography, however, because a single area of the grid is imaged approximately 70 times, the sample is subjected to far more radiation, making damage a consideration
  • The structural features of most “viruses” are lost in the noisy images recorded from the electron microscope
  • After extensive data processing and reconstruction, it is said that it has become possible to resolve these features – in some instances at near-atomic resolutions
  • However, most of this work has been augmented by image processing techniques that computationally enhance image contrast
  • Advances in fabrication techniques have enabled the microscopist to do so directly by altering the optics of the electron microscope

Dr. Peter McCullough saw some pretty pictures claiming to be “SARS-COV-2” based on reconstructed 3-D images taken from cryo-EM. In his exuberance, he rushed to write an article about this ground-breaking evidence that he was sure would convince the naysayers to join him on “Team Rational.” Dr. McCullough was certain that the computer-generated recreations would be the visual evidence needed to sway these naysayers even though they had already meticulously poured over numerous TEM images claiming to be “viruses” and remained unconvinced after doing so. “A picture is worth a thousand words,” Dr McCullough gleefully sang! “Show them these works of art!” However, what Dr. McCullough somehow forgot to take into account during his reckless abandon is that the thousand words behind the picture matter. And in the case of his “works of art,” the words behind the creation of these images speak much louder than the images themselves. 

When we break down the methods behind the creation of Dr. McCullough’s fine art, we find that the particles are once again the direct result of unpurified cell culture supernatant. The “viruses” in question were “grown” off site in Vero cells and were then maintained in Dulbecco’s Modified Eagle Medium (DMEM) Gibco™, with 100 U/ml penicillin, 100 μg/ml streptomycin (Pen-Strep) and 10% (v/v) heat-inactivated fetal calf serum (FCS). Thus, we do not have any evidence that the particles imaged were ever in the fluids of a sick host nor is there any evidence that the particles are pathogenic in any way. What we have is evidence that when numerous toxic substances and foreign materials are mixed together in a petri dish with African green monkey kidney cells and incubated for days, they break apart and die leaving various particles left in the wake. The images claiming to be “viral” particles are either cellular debris or potentially artifacts created during the imaging process. There is absolutely zero evidence that the particles reconstructed and recreated are replication-competent “viruses.”

However, if that is not enough to make one doubt the “difficult to deny” power of these images, factor in the various processes that must be undertaken just to reconstruct these works of art. After the many alterations done during the cell culture process, the sample is flash frozen, thinned to the appropriate size, and then battered with intense heat from the electron beam in order to generate images. The sample is tilted along an axis while numerous recordings are obtained. Meanwhile, the longer the sample is subjected to this extreme heat, the further it is damaged and degraded, distorting the images. After the recording, the various 2-D images are aligned and merged using computer programs and different algorithms to fill in the missing data. The computer software maps the result onto a 3-D representation of what it has determined that the particles look like. Numerous assumptions, estimates, and interpretations are made throughout this process in order to generate the desired recreation. 

Thus, when one really thinks about it, McCullough’s description of these images as “works of art” may be the most accurate description for them. The images stem from human skill and imagination. They are used to express certain ideas, emotions, and feelings. The reconstructions are derived from a creative process and displayed for the subjective interpretation of the viewer. For all intents and purposes, the cryo-EM images are “works of art.” However, one thing they are not are slam-dunk proof for the existence of “SARS-COV-2” nor any other “virus.” They are images of random particles that only have meaning to the eye of the beholder. So while McCullough’s description may be perfect for these images, I’m not certain that it conveys the message that he thinks it does. If Dr. McCullough wants these cryo-EM images to be taken seriously as proof for the existence of “SARS-COV-2,” I think we can all agree on one thing he may not have thought about in his joyous proclamation.

105 comments

  1. If you liked this fairy tale, look into Stephanie Seneff and her nonsense about how misfolded proteins cause prion disease.

    How do these people forget basic biology? Misfolded proteins, or damaged proteins are caused by cell damage, they are the symptom, not the cause of the disease!
    The cause is most likely pesticides which cows get put around their necks. In the year of mad cow, they changed the dosage… But Nah look over there at bullshit prions.

    Same with Alzheimer’s, the beta amyloid has been exposed as a lie.
    But even before then, what causes plaque buildup? No, the morons think the plaque caused the damage.

    Never mind that Alzheimer’s brains have high levels of aluminum, let’s focus on the result of damage.

    I honestly think they are so brainwashed in academia that all they see is the lingo. Common sense isn’t common and Occam’s razor gets ignored.

    Liked by 1 person

  2. As far as I know, the sacrosanct, vaunted and preeminent CDC does not have any SARS CoV-2 samples in stock. If this is still true after nearly 3 years, why? How about having SARS CoV-1 in the fridge or elsewhere?

    You would expect the CDC, which is supposed to be at the forefront of ALL disease and virus invented pandemics, to have samples to study and test for furthering control of the coming Covid-3 outbreaks or whatever else the sordid demons of the WEF/elite have cooked up in 2023.

    Certainly they could find some covid cells during an autopsy in the deceased that were purported to be killed by the virus. Heck, it seems they are finding everything else in these poor people.

    Liked by 1 person

      1. The specific instruction to not get curious and do any autopsy on Covid deaths should have woken up more but didn’t. Its like the ukraine military spending where they didn’t want any audit of how the money is spent!

        Liked by 1 person

  3. No one has ever isolated, purified and visualized (not even on a microscope slide and two-dimensionally) any of the so-called molecules that are considered and declared to be hormones by so-called bio-medical scientists. In reality, so-called hormones only exist at a HYPOTHETICAL/THEORETICAL level… just like all other so-called molecules (ie, structures/particles in the sub-microscopic realm), such as, for example, proteins, enzymes, antibodies, etc. … The so-called INDIRECT proofs of the existence of molecular structures, which are invoked by so-called bio-medical scientists, are devoid of any real scientific relevance, because they are obtained following procedures carried out blindly, without any foreknowledge, only based on assumptions… and, they are INTERPRETED only based on criteria either completely random, or born of scientific prejudices, or dictated by the interests of the sponsors (governments, pharma-chemical concerns, modern western medicine).

    Liked by 2 people

    1. I haven’t heard about hormones being theoretical, too.
      Don’t get me wrong, I’m inclined to believe it, because of all the virology/genomics nonsense. Can you point me towards some studies where it is apparent that hormones are theoretical?

      Like

  4. For example, the claim that the sample is frozen and then put through a series of recordings in which the sample is tilted on a different axis and at various angles to obtain multiple images which are then merged together to create a 3-D reconstruction, is a sophisticated statement, beyond the realm of ordinary people’s knowledge, which almost no untrained person thinks fit to question. For almost all people, this kind of statements (about which they have no idea) that so-called science is full of, are considered truths that no longer require any kind of confirmation. This is one of the ways that people are manipulated into blindly believing all the aberrant and stupid scams of the so-called scientists.

    Liked by 1 person

  5. People without training in molecular biology, virology, immunology, genetics, do not know what it is about, so – impressed by the appearance of sophistication of extremely deep science – blindly approve all the conclusions of so-called scientists … in while almost all those who actually have training in the field (not just degrees) refuse any questioning of the official narratives, because they know it would endanger their positions, fame and material well-being.

    Liked by 1 person

    1. Thanks Jeffrey! I just finished listening to Dr. Cowan’s talk as I didn’t want to be influenced by it. I found out through his video that the Bailey’s also did a video. I’m excited to watch that one as well. Do you know if anyone else broke McCullough’s delusions down?

      Like

      1. Mike, not to my knowledge. It’s too bad, he actually makes for an easy target, gets it so wrong. Christine Massey does touch on it a bit in her new interview with Jimuphy Masters, briefly.

        Liked by 1 person

      2. ❗️Peter McCullough interprets cell structures as “viruses”❗️

        NEXT LEVEL analysis refutes Peter McCullough’s proof of virus existence

        ➖No virus isolation
        ➖No biochemical characterization and genome sequencing
        ➖Not a single documented control attempt
        ➖Cell structures are misinterpreted as viruses

        Like

  6. Was some type of cryo protective agent used in the process? The purpose of such agent is to prevent the formation of ice crystals. If ice crystals are found then tissue dehydration has occurred. Were the samples monitored for dehydration? The sample was frozen after the cytopathic effect – to what temperature? From what I understand, all unprotected tissue freezes below – 50 C. This would produce ice crystals and indicate dehydration. What does “frozen-hydrated SARS-COV-2 virions” mean?

    Liked by 1 person

  7. I remain on TeamNoVirus. Point and declare. CGI. I kept thinking it’s like a photo of a fire bombed city, devastated down to rubble. You could reconstruct it, imagine it, to be Dresen or Hiroshima or any other city you had previous images of. It’s nonsense. McCullough screwed the pooch on this one.

    Liked by 1 person

    1. Definitely Lynn. I got the impression that McCullough was super excited to share these images and did not give the paper any sort of critical nor logical thought. It’s obvious he also did not apply those skills to his writing. 😉

      Liked by 1 person

  8. Mike, thanks for the indepth analysis.

    The following is my commentary on Doctor McCullough’s claims, which I found to be particularly annoying.

    Laboratory methods in virology are well accepted

    (Accepted? By whom and on the basis of what? It is still called GERM THEORY isn’t it? If it’s theory then it remains unproven regardless of the methods used to establish it as a fact.)

    and utilize a series of experiments to demonstrate cellular invasion, replication, transfer and repeated infection.

    (Provide one experiment that does any of these things that hasn’t already been debunked. You can’t take us past cell theory. You have no facts to support any of these conditions.)

    Whole genomic sequencing has aided in identification of variants and subvariants

    (Of what? You can’t have a variant of something not proven to exist unless it is a variant of an in silico model of a fictional entity. In which case the variant is also fictional. What are you sequencing and to what limit? From where did your fragments originate? Did you multiply them before you aligned them? What was in the sample from a person? What did you mix your sample with? Did you obtain different results without adding the sample from a person to your mixture of ingredients or was it the same? I’m sure you forgot to do this because it would disprove your theory. How would you know if your specific sequence, which you arbitrarily selected from a multitude of possible sequences, ever existed outside of your computer?)

    and helped greatly in forecasting what is coming next.

    (The ancient Assyrians and Babylonians were ahead of you.)

    The CDC Nowcast system is an excellent application of targeted sequencing of viral samples.

    (Samples of what? It is presumptuous of you to assume there is a virus in any sample.)

    Nonetheless, some have said if SARS-CoV-2 cannot be cultured like a bacteria and “isolated” then it does not exist.

    (Virus particles are said to be much smaller than bacteria, which can be seen under a light microscope. This is not a valid comparison. What is pointed to on electron micrographs and called virus particles are indistinguishable from exosomes. There is no scientific basis for claiming a viral particle is anything other than an exosome.)

    I have always responded that the principles of laboratory virology, sequencing, and the mass production of viruses

    (The principles of laboratory virology and sequencing are a faith-based process. They proceed from an assumption, which has never been proven, namely, that viruses exist. Something that has never been proven to exist cannot be produced in any quantity.)

    such as that done by the Max Planck Institute for Dynamics of Complex Technical Systems

    (It does not matter what dynamic procedure is employed or how complex it is if it fails to produce even one viral particle. And neither would it matter who the institute was named after.)

    are concrete processes that rely on the presence of the virus.[ii]

    (A process is an action undertaken by a subject. If an action is caused by a virus then there should be no problem showing the virus. Similarly, we could say that the process of writing requires the presence of a pen. But we cannot say that writing is dependent upon a pen without showing the pen. You cannot show an action without showing what causes it. Now if you believe in ghosts then that is another story – you have left the material world and entered into the spiritual, where science is of no use.)

    My understanding from the body of medical literature

    (All peer reviewed, of course, by the same members of the club.)

    and firsthand clinical experience

    (And this is based on the myth of contagion?)

    are consistent with the conclusion that COVID-19 is indeed a unique illness

    (You can’t have a specific disease without first proving what causes it.)

    distinguishable from influenza and other viral infections.

    (Comparing one fallacy to other fallacies does not make it true.)

    I have always been impressed with the absence of bacterial superinfection and micro- and macro-thrombosis being features that separate COVID-19 from influenza and other viral syndromes.

    (A statement without any basis in scientific fact. Just because a person has symptoms or conditions does not prove their cause. And arbitrarily assigning symptoms and conditions to unproven causes is unscientific.)

    Calder, et al, at the Francis Crick Institute has gone a step farther with advanced forms of electron microscopy to see the virus up close and personal.[iii]

    (A step farther than what? You haven’t established any facts. You are still in the realm of theory – germ theory. Electron microscopy only produces micrographs. You cannot look into an electron microscope like an optical microscope and see something. There is no valid comparison between images on electron micrographs and what is in a living human being. Electron microscopes deal with dead matter. You cannot prove that the particles you see on the electron microscope images are anything other than artifacts or simply unidentifiable dead cell debris. You cannot prove that any particle you see is the cause of a disease. You cannot prove that it replicates. You cannot prove that it does anything.)

    Now I can sleep!

    Liked by 2 people

    1. Its not Germ Theory, its germ HYPOTHESIS.

      Theory, used colloquially, means unproven but in the scientific method (the ACTUAL definition), a theory is the thoroughly tested and the results recreated multiple times to show that they are unbiased and that you get the same results EVERY time.

      The reason this is germ HYPOTHESIS, is because they havent actually proven anything as they have never adhered to the scientific method, EVER, so technically they’ve only come up with the idea and never tested it with the scientific method, meaning that they havent even gotten to step 2 of it.

      I wish people would stop using the word theory to describe this pseudo-science garbage. Its the incorrect scientific term to describe it.

      Liked by 1 person

      1. The UNABRIDGED dictionary states
        “Theory, hypothesis”
        In scientific usage, a HYPOTHESIS is a provisional conjecture regarding the causes or relations of certain phenomena; a THEORY is a hypothesis which has undergone verification,and which is applicable to a large number of related phenomena. In ordinary usage, hypothesis may denote any assumption without proof; theory is opposed to practice, sometimes to fact.

        Like

  9. It is ridiculous to see so-called scientists that although they cannot provide any direct, uninterpretable evidence of the existence of viruses, this does not prevent them from talking about the existence of the hypothetical genetic material as if they have already brought direct, uninterpretable evidence of its existence. And they do this thing with regard to all supposed particles in the submicroscopic realm. In reality, so-called molecular biologists have never produced any direct, non-interpretable evidence for the existence of anything in the submicroscopic realm.

    Liked by 1 person

  10. In general the process of measuring something starts from the idea that the measurement will advance knowledge of what is being measured through its association with other elements of reality which have likewise been categorised and integrated within a particular framework of reality. It would be senseless therefore to devise a new scale or type of measurement for every new imagined reality or process of combining things in a new way, because you would have no way of relating the new knowledge to existing theory.
    In this way the calibration, and even the invention of a device to measure scales beyond known dimensions nevertheless proceeds from certain basic assumptions about reality. One has to have a hypothetical frame of reference in order to aim the focus of the measurement in the right ballpark. As such the invention of the electron microscope was a machine designed to prove the existence of the virus, which itself already formed the backbone of the macrobiotic scale of medical diagnosis and cure. No matter that the theory was on shaky ground.
    From the beginning the EM was an instrument built and calibrated to harmonise its results with a known worldview. Whatever results it produced were always shoehorned into the accepted worldview. At least by those who had a stake in that worldview and who probably funded the research.
    But if one is inclined to question theoretical constructs and even cast about for alternative theories certain somewhat gaping holes tend to appear. For example, if germ theory is correct in suggesting that viral infection occurs through aerosolised exchange of breath, then wouldn’t it be simple to obtain a sample of air and filter out the virus? Also, how does one know that a sick person has the new virus when their symptoms are generic and indistinguishable from a number of prevalent diseases? Surely the disease itself must be characterised by its symptoms before one can start taking samples and looking for a cause?
    So my view has always been one of healthy scepticism, that if, as in this case, it is impossible to filter such a small particle, then this begs the question as to how we can be so sure that our calculations at such a tiny scale are successfully integrated with the wider body of bio medical systems. Like with the fantastic claims for the effect of co2 on the atmosphere it is surely a case of putting the cart before the horse. The theory we want to exist because it somehow conforms to our other designs on life may not always turn out to be so innocuous!

    Liked by 1 person

    1. Is he this guy for real or part of a Monty Python script?
      A form of cross- infection via vaccination?

      Is he part of the plot fear creation to confuse the population?

      In an issue of w-plus 05, 2013, it was well observed that we must replace beliefs with understanding, otherwise in a confused populace other “successful” camps could offer dangerous explanations and gain the upper hand.

      What is amazing that clinicians fail to see the inconsistency of many of the purported theories that some of the so-called “scientists”/doctors are postulating as possible. Some of the claims lean toward science fiction.

      Liked by 1 person

    2. I love that there have been ZERO studies on “shedding” and yet everyones going crazy over it. This is a massive bone of contention I have with the fake “awakened” Japanese people as when I ask them, “Where are the studies adhering to the scientific method proving your claims of shedding?” (i’m an asshole I know (^_~) )

      First, they dont even understand the scientific method so we can throw that out the window, but then they say, “Well people on twitter say they are feeling sick being around jabbed people.” I then facepalm and tell them, “Without scientific proof of this shedding, you cannot claim it to be real. On top of that, why the hell are you believing the crap you read on twitter?” The response is usually, “But Doctor so and so of BLAH clinic (aka allopathic virus pusher against the clot shots) is saying it.”

      You can see where the conversation goes… I challenge them to prove the existence of Convaids… Nope.

      Liked by 1 person

      1. Sadly, they are easily fooled. I’ve seen very smart people aware of the fraud of contagion who still believe in this shedding crap. It is hard for everyone to break free from the indoctrination. We just have to keep demanding proof which adheres to the scientific method. Once they realize that they can not provide it, maybe this will be a wake-up call or at least plant a seed for one in the future.

        Like

  11. „The medical world has built an infinite literature without any ideas of cause (except some, erroneous and vacillating). Medicine is rich in science, but now, as well as in all past time, it suffers from a dearth of practical ideas. The average doctor is often educated out of all the common-sense he was born with. This, however, is not his fault. It is the fault of the System. He is an educated automaton. He has facts—scientific facts galore—without ideas. Millions have facts, but no ideas.Thousands of doctors have all the scientific data needed, but they have not harnessed their science to common-sense and philosophy. Without a clear conception of cause, cure must remain the riddle that it is.
    To me it means that diagnosis is a meaningless term; for, as used, it means discovering what pathological effects—what changes—have been brought about by an undiscovered cause. Diagnosis means, in a few words, discovering effects which, when found, throw no light whatever on cause.”

    TOXEMIA EXPLAINED By J. H. TILDEN, M. D.

    Like

  12. Mike, thank you again for your detailed work. I always feel like I’ve learned something, with each of these articles. For example, I didn’t realize they did this much computer-based modeling for structures which are supposed(?) to be “visible” via the elecron microscope. I guess this directly shows the limitations of this technology (I’m aware of all the potential for artifacts through Hillman, too).
    I don’t know if I agree with the dismissive/ridiculing tone. I agree that what is posed as “evidence” is very much ridiculous, and it’s gotten more and more ridiculous over the years. I just don’t know if shaming someone will get the desired result of the person seeing the error of their thinking, or if will just fuel ego-driven, aggressive responses. I guell we’ll see.

    The main point I wanted to state, though (towards McCullough as well):
    What good does (even indisputable) pictures of particles do… if the function of these particles has never been established?

    Liked by 1 person

    1. I definitely see your point regarding tone. McCullough has been a bit of a thorn in our side but I could have shown some restraint for sure. I highly doubt he would listen one way or the other. I believe there is a reason he gets as much exposure as he does.

      Like

  13. Thanks Mike . Great work .

    One thing I did not consider before , what have cell cultures got to do with reality.

    —-
    telegram translate:

    https://t.me/NextLevelOriginal/62

    NEXT LEVEL – Knowledge rethought:
    Answers to questions from the community

    🅾️ Today for VirusTHEORIE

    What are cell cultures?
    In order to work with so-called cell cultures in the laboratory, the tissues that have previously been removed from organs (e.g. a fetus) are separated by force. These “cells” must be constantly prevented from merging back into tissue (tissues in the lab can only be kept from dying and decomposing for a few days). This is achieved by adding fetal bovine serum, which slows the decomposition process. This is not possible with the serum of adult humans and animals.

    ❗️Forcibly dissected tissue = cell cultures❗️

    ➖➖➖➖➖➖➖

    The ghosts of HeLa: How cell line misidentification contaminates the scientific literature

    https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186281

    Introduction to Cell Culture

    https://www.thermofisher.com/uk/en/home/references/gibco-cell-culture-basics/introduction-to-cell-culture.html

    Like

    1. An addition on bovine serum

      On N L forum

      ——

      Question
      Hello, I’m new here.. the reason is first of all the analysis of the article by Peter McCullough. I have a question of understanding here. I read again and again that the culture cells are starved during “virus isolation”. Is this starvation process described somewhere (from when do the cells starve?) The analysis shown in the NL refers to Gibco DMEM 41965039, which is a “high glucose” variant. Is it because the medium is not changed during the specified 4-5 days and the glucose is then used up?

      Reply from Florian ( admin on site)

      https://t.me/NEXTLEVEL_OnlineForum/8886

      The calf serum is diluted and poisoned with antibiotics.

      https://t.me/NEXTLEVEL_OnlineForum/8887

      Lanka replicates the methods. As seen here 10% FCS vs. 1% FCS. And with vs. without Glutamax.

      ( there is a slide but does not work copy/ pasting

      ——

      Question:

      Is it really the case that the pregnant cow’s abdomen is cut open without anaesthetic and the calf is also “utilised” without anaesthetic so as not to damage the cells (tissue)?

      Answer:

      In order to meet the inexorably growing demand for foetal serum, 2,000,000 pregnant cows are opened alive every year without having been anaesthetised beforehand; their foetus, which is also unanaesthetised, is removed, cut open and its blood is taken from the beating heart.

      If one were to remove the foetus from the living mother animal in the conventional way (in the sense of a birth), the yield of serum would be much lower. If anaesthetics were administered to the mother animals and/or foetuses, the foetal serum would rapidly decompose because these drugs cannot be removed from the serum.

      The foetal serum is now produced from blood obtained in this way!

      LG
      NEXT LEVEL TEAM

      Comment:

      Why do you use calves? Because of the size and thus higher “yield”? What about aborted human embryos? Before the abortion, the women are anaesthetised…

      human fetuses are also used… Look what these cell lines like WI-38 or HEK-293 are… It’s so inhumane… By the way, the number stands for the experiment that finally worked…

      When you read that, it makes you sick to your stomach. And on the other hand, has this crime brought any benefit to people’s health?

      Unfortunately no, because it is used to produce and test medicines. No animal suffering in the world has any benefit.

      ————————-
      Telegram translate :

      https://t.me/Corona_Fakten/1318

      NEXT LEVEL – Knowledge rethought:
      Answers to questions from the community

      🅾️ Today for VirusTHEORIE

      Addition to the question “Why fetal bovine serum” (to the article):

      When organs are removed from an organism, they quickly die and decompose, even if they are refrigerated. If one removes individual organ tissues from organs in order to study “life” in the laboratory, or to “isolate”, multiply, “grow” alleged “viruses” in the laboratory, then these tissues die even faster and go to decompose even faster. Fetal serum slows down this tissue breakdown process.

      ➖➖➖➖➖➖➖

      Like

  14. Drop in COVID alertness could create deadly new variant, WHO says

    “Lapses in strategies to tackle COVID-19 this year continue to create the perfect conditions for a deadly new variant to emerge, as parts of China witness a rise in infections, the head of the World Health Organization said on Friday. . .”

    https://www.reuters.com/business/healthcare-pharmaceuticals/whos-tedros-says-new-covid-variant-concern-could-emerge-2022-12-02/

    From Johns Hopkins:

    “All RNA viruses mutate over time, some more than others. For example, flu viruses change often, which is why doctors recommend that you get a new flu vaccine every year.”

    “Coronavirus variants are classified in different categories by organizations such as the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC).

    A variant of interest . . .

    A variant of concern . . .

    A variant of high consequence . . .

    https://www.hopkinsmedicine.org/health/conditions-and-diseases/coronavirus/a-new-strain-of-coronavirus-what-you-should-know#:~:text=Variants%20of%20viruses%20occur%20when,distinct%20variants%2C%E2%80%9D%20he%20says.

    Hopkins also claims:

    “Variants of viruses occur when there is a change — or mutation — to the virus’s genes. Ray says it is the nature of RNA viruses such as the coronavirus to evolve and change gradually. “Geographic separation tends to result in genetically distinct variants,” he says.’

    Viruses are not technically living things—they invade living cells and hijack their machinery to get energy and replicate, and find ways to infect other living organisms and start the process over again.

    The mechanics of the process according to the theory:

    “How viruses mutate largely has to do with how they make copies of themselves and their genetic material, says Marta Gaglia, an associate professor of molecular biology and microbiology at the School of Medicine. Viruses can have genomes based on DNA or RNA—unlike human genomes, which are made up of DNA, which then can create RNA.”

    “Gaglia studies how viruses take control of infected cells and reprogram the cells’ machinery to reproduce themselves. “We’ve been working on a protein that the virus encodes that destroys the host RNA, blocking the cells from being able to express their own protein and blocking, among other things, antiviral response,” she says . . .”

    A little deeper into the mechanics and why coronaviruses are different from others viruses:

    “. . . Our DNA-synthesizing machinery tends to have an error correction mechanism. It will figure out if there’s a problem, usually because the structure is kind of weird; if it’s not the right pairing, it will excise and repair the mistake. That happens during replication. It’s as if, while you were copying down a text and made typos, you could proofread and fix them.

    The RNA-synthesizing machinery that most RNA viruses use to copy their genome doesn’t have this error correction mechanism. But coronaviruses have a special enzyme that allows them to do error correction, so they have a lower mutation rate than other RNA viruses. I don’t think it works quite as well as the DNA mechanism, though.

    There’s this idea that because most RNA viruses cannot error correct, they make lots and lots of mistakes. That’s not great for us, because it allows them to mutate rapidly and avoid the immune system. But if they make too many mistakes, it’s not good for the virus either, because the viruses will just break down. . .”

    There is more in the article and it leads to the vaccine after a discussion about the immune system.

    https://now.tufts.edu/2021/06/09/how-viruses-mutate-and-create-new-variants

    This is an interesting story, but the critical element of proof is missing. At this point I am questioning just why viruses are included in germ theory. By definition a theory is stronger than a hypothesis. If viruses or viral particles have not been shown to exist then there is no basis for a hypothesis about how they function and interact with cells. So if you don’t have a hypothesis you can’t have a theory.

    On the other side of the story one encounters the same problem with cell theory. Because there is no direct evidence of the existence of the necessary cellular components to support a hypothesis then neither can you have a theory.

    I realize all of what is presented here is found in the textbooks. Anyone obtaining a medical degree will be indoctrinated with this information. They will be tested and graded on how well they can recall this information. They will not be able to obtain a degree without answering the test questions correctly.

    After they obtain their degree they will be able to practice what is called medicine. But what happens if what they were taught isn’t how the real world works?

    Liked by 1 person

  15. Their insistence on ignoring the question of how they know the particle in the photo is the cause of a disease is very telling. This isn’t an ambiguous concept. A picture of a particle is not even remotely acceptable as evidence that the particle causes a disease. This is self evident. That someone as intelligent as McCullough fails to understand this does not seem credible. So he is exposing himself as a dishonest actor. At best, he is lying to himself. But even that is hard to believe

    Liked by 1 person

  16. Biochemical characterization and genome sequencing require a deep critical analysis, because they are two of the big lies of the so-called molecular biology.

    Like

  17. „Man has such a predilection for systems and abstract deductions that he is ready to distort the truth intentionally he is ready to deny the evidence of his senses only to justify his logic.”

    – Fyodor Dostoevsky

    Like

  18. „Today’s scientists have substituted mathematics for experiments, and they wander off through equation after equation, and eventually build a structure which has no relation to reality.”

    ― Nikola Tesla

    Like

  19. Hi Mike, your website is awesome. I was reading thishttps://www.nejm.org/doi/full/10.1056/nejmoa2001017
    They don’t use the typical toxins I hear mentioned. They didn’t do a control, which is highly suspicious as you and your colleagues have critically pointed out, but what might be driving the CPE they got if not the toxins? Could it be just lack of cell nourishment or maybe natural course of lung cancer cells?
    Thanks to all of you. Keep up the good work and let me.kmow if\how I can help.

    Liked by 1 person

    1. Thanks Albert! It is not very clear what was done during the culturing of the cells but we can see from the methods that “viral” transport media was used in conjunction with PBS. They also passaged the culture which in and of itself can induce CPE:

      “Bronchoalveolar-lavage fluid samples were collected in sterile cups to which VIRUS TRANSPORT MEDIUM WAS ADDED. Samples were then centrifuged to remove cellular debris. The supernatant was inoculated on human airway epithelial cells,13 which had been obtained from airway specimens resected from patients undergoing surgery for lung cancer and were confirmed to be special-pathogen-free by NGS.14

      Human airway epithelial cells were expanded on plastic substrate to generate passage-1 cells and were subsequently plated at a density of 2.5×105 cells per well on permeable Transwell-COL (12-mm diameter) supports. Human airway epithelial cell cultures were generated in an air–liquid interface for 4 to 6 weeks to form well-differentiated, polarized cultures resembling in vivo pseudostratified mucociliary epithelium.13

      Every 48 hours, 150 μl of PHOSPHATE-BUFFERED SALINE was applied to the apical surfaces of the human airway epithelial cells, and after 10 minutes of incubation at 37°C the samples were harvested. Pseudostratified mucociliary epithelium cells were maintained in this environment; apical samples WERE PASSAGED in a 1:3 diluted vial stock to new cells.”

      Like

      1. Thanks Mike. Sorry for so many questions.
        I’m a lazy idiot:
        https://en.m.wikipedia.org/wiki/Viral_transport_medium
        Some of the typical toxins seem to be included in VTM per the Ref2, which is CDC\FDA prep guide or whatever.
        Reading about PBS thought this doesn’t seem to be as much of a culprit. What’s your take on PBS?
        Ok then I searched “passage cell culture” and realized I’m even more lazy and shouldn’t have bothered you. It’s fascinating how much jargon this fields has and the depth. I have a lot of learning to do, which is a good thing IMO, cause it’s fun.
        I did search for virology text books. I found a few free PDFs, and ordered one on hard copy, but none of them had anything in-depth about isolation in the table of contents. It appears from searching the terms you pointed out from that paper was much more fruitful on terms of identifying materials for learning about isolation processes. Is it your experience that researching the jargon used in the isolation papers is a better pathway to learning compared to reading virology text books?
        I’ve already seen pretty much every Sam Bailey / Cowan/ your website / spacebusters videos, so I’m kind at the point where I’m stuck regurgitating statements in learned from those or really researching myself, and I’m more comfortable with the latter when it comes to discussing the topic with germ theory believers. Your website has been wonderful. Thanks.

        Liked by 1 person

      2. Yes, the same toxins are in the VTM which the sample is immediately subjected to after being taken from the sick patient. The PBS is considered non-toxic to cells but it is still not heavily studied for its effects on them. You can find some literature such as these studies which show a detrimental effect with cell burst and protein loss to cellular responses and increase in cytokine release:

        “Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss.”

        https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796241/

        “As heightened extracellular calcium concentrations can initiate an inflammatory cascade, we hypothesized whether the addition of −/− or +/+ PBS might have an influence on human peripheral blood mononuclear cells (PBMCs) under different culture conditions. These alterations of extracellular conditions might influence several functions including secretion of cytokines, proliferative responses, and cell death.11”

        “In this present study, we provide evidence of a significant effect of PBS containing calcium and magnesium on cell cultures of human PBMCs. Because PBS either in −/− or +/+ formulations is used ubiquitously in laboratories, cellular responses induced after contact or co-incubation with these solutions are evident. In support of this hypothesis, addition of EDTA-plasma significantly abrogates cytokine secretion, in particular of IL-1ra. As EDTA complexes free Ca++ ions and therefore inhibit calcium-dependent cellular functions,17 we believe that the effects seen by +/+ PBS are Ca++ triggered and cause interactions which can initiate increases in cytokine release.”

        https://academic.oup.com/labmed/article/40/5/290/2657559

        Like

  20. Virologists claim our bodies contain trillions of collective viruses (“virome”). I wonder how many unique viruses are supposed to be part of this soup? Assuming this is true, then how can they possibly isolate a particular virus that causes a novel pathogen?

    A lot of the health freedom movement is probably controlled opposition. Many of these doctors claim they treated thousands of COVID patients with HCQ+ Ivermectin, etc. What if that’s all fake and meant to promote the SARS-COV-2 narrative? AFLDS is under increasing scrutiny for example.

    https://reinettesenumsfoghornexpress.substack.com/p/americas-frontline-doctors-founder

    Liked by 1 person

      1. Yes, Even if we assume the supernatant has only viruses, they still need to be sure there is only one type of viruses there before sequencing or culture. Eleni caught on to this pretty early with the HIV p24 proteins. She has such a calm and clear way of explaining. RIP.

        Liked by 1 person

    1. The virus theory and the theory of evolution are actually bedfellows. They are identical insofar as both contain the idea of something evolving from one form into another. The virus theory, like its bedfellow, the evolution theory, has never been proven. Although, the evolution theory encompasses a far greater spectrum than the virus theory, one could reason that viruses are simply a subset of all that evolution is said to encompass.

      Both dispense with the idea of a supreme creator and lawgiver and defer to random chance, but no such thing as “chance” is ever explained, except within the concept of ignorance. Yet these are the same people who maintain that there are absolute laws as they strive for grand unification theories.

      The idea of intelligent design is repugnant to them for no reason whatsoever. If one were to ask them to consider that the Creator created all things by a initial act and then interposed at various intervals, they scoff. If one were to ask them to consider that the very forces observed within creation are actually tools in the Divine hand, they mock.

      Their science teaches them to look for a cause “for” all things, yet they refuse to look for the cause “of” all things. Let us pose a simple question to them, “which is greater, the thing made or the maker of the thing?” They will answer, “the maker of a thing is greater than the thing made.” And then they do an about-face and say, “we don’t know what caused the universe.” When one continues to ask, “if all things are governed by the operation of laws, from whence cometh these laws?” And again they will say, “we don’t know.” A pitiful lot they are indeed. One should not be surprised when confronted with their absurd ideas, and neither that their ideas are held in the highest esteem.

      The following is proof of my assertions concerning them.

      “Viruses undergo evolution and natural selection, just like cell-based life, and most of them evolve rapidly.

      When two viruses infect a cell at the same time, they may swap genetic material to make new, “mixed” viruses with unique properties. For example, flu strains can arise this way.

      RNA viruses have high mutation rates that allow especially fast evolution. An example is the evolution of drug resistance in HIV.”

      https://www.khanacademy.org/science/ap-biology/natural-selection/common-ancestry-and-continuing-evolution/a/evolution-of-viruses

      Liked by 1 person

  21. Another disappointment while they recently approved jabs for infants and babies.
    The existence of viruses is crucial.

    —-

    Squabbling + fighting about whether viruses exist or not has zero effect on stopping the control grid.

    ‘At this time in our history, if we don’t stop the control grid, we’re going to be slaves.’ #FinancialRebellion #CHDTV

    https://media2-production.mightynetworks.com/asset/49667654/trim.83E58642-64BB-4016-95E4-CCD81BBAFE9D.MOV?_gl=1*p1fqxj*_ga*MTIzMTkzOTQ4OC4xNjI1NzM1OTcw*_ga_T49FMYQ9FZ*MTY3MDM3MDMwMS4xNjIxLjEuMTY3MDM3MDgxNS4wLjAuMA..

    Like

  22. The so-called technique of X-ray diffraction (crystallography) of hypothetical (submicroscopic) DNA crystals… is a scam.

    The so-called technique of sequencing and characterizing hypothetical proteins and hypothetical nucleotides (which are supposed to exist as sub-microscopic structures) using a so-called laboratory technique called gel electrophoresis… is still a scam.

    In reality, all the laboratory techniques used in the so-called Molecular Biology are pure scams, because it is claimed that by means of them the so-called submicroscopic particles can be analyzed… that is, particles that are so small that no one has ever been able to bring , DIRECT, UNINTERPRETABLE and REPEATABLE evidence to prove their existence.

    This is also the reason why in the scam called Molecular Biology no real, correct and true control experiments can be done to confirm the validity of the results obtained by indirect methods, by comparison with results that would have been obtained by direct methods, which provides evidence that is not interpretable.

    Like

  23. All this scientifical mumbo jumbo reminds me of the cloaked priests in black reading a bible written in Latin to the ignorant masses, threatening them with death and hellfire if they did not obey everything they said to the letter.

    Liked by 1 person

  24. Whole-genome trove ties new genes, variants to autism

    Autism has nothing to do with vaccines – yea, right. You need to be qualified (in the club) for access.

    “The largest-yet analysis of whole-genome sequences from autistic people links new genes and variants to the condition. The findings and sequences — from 5,100 autistic and 6,212 non-autistic people — are on a cloud-based platform and are available to qualified researchers.”

    https://www.spectrumnews.org/news/whole-genome-trove-ties-new-genes-variants-to-autism/

    Like

  25. Did you know that no control experiments can be created to confirm that what is seen to be there and happening on the microscope slide corresponds to what is there and happening in the living organism for the simple fact that there is no technological possibility to to see, directly and live, what exactly exists and what exactly happens in the living organism? Then, if there can be no control experiments to confirm any of the microscopists’ claims, why do the so-called scientists who know this stuff hide this fact from public opinion?

    Like

  26. The fact that there is no visual confirmation by optical microscopy of the existence of so-called DNA molecules does not prevent so-called molecular biologists from claiming to have the technology to isolate, purify, sequence, and physically and bio-chemically describe the hypothetical molecules of DNA, whose existence has never been proven by direct, reproducible and, above all, non-interpretable methods.

    Liked by 1 person

  27. There is no Science of the sub-microscopic realm of existence for the simple fact that there is no real knowledge of that realm of existence.

    So-called scientists’ purported understanding of what exists and what happens at the sub-microscopic level is nothing more than conjecture.

    The so-called circumstantial evidence claimed by so-called scientists as the basis for describing what they claim exists and occurs at the sub-microscopic level is devoid of any real value as long as it is interpretable and cannot be confirmed by means of direct and uninterpretable evidence.

    Certainly there is something beyond the realm of “optical visibility” (ie, beyond the realm of optical microscopy).

    The problem is that man CANNOT find out (has not been gifted with the ability to find out) how existence is structured and functions in the sub-microscopic realm.

    Scams called “atom-molecular science” are fools for the naive.

    Atoms and molecules are nonsense supplied to the masses as science.

    The reality is that even in the field of light microscopy so-called scientists understand EXTREMELY little of what they see.

    Almost all people (regardless of educational background) believe that so-called scientists possess a deep knowledge of reality at the microscopic and sub-microscopic levels.

    An extremely small number of people have understood that the inventions of the so-called scientists are based exclusively on experiments carried out endlessly, by which they note what works and what does not work, that is, a real lottery, a wheel of fortune.

    Well, if so-called scientists really knew, then they wouldn’t need to do endless experiments.

    —————————–

    Below I post a material written by Harold Hillman, a former British scientist

    Distinction between science and empiricism

    This distinction is very important, but often elided over by lecturers and theoreticians, probably because they don’t like admitting that some of their beliefs may be unsoundly based. Some examples-

    • Anaesthetics. I was, personally, astonished when I was told that nobody knows how anaesthetics work. When chemistry improved to the point that pure chemicals which barely exist in nature could be isolated. some of their properties were unpredicted. Ether and chloroform, and nitrous oxide, were found to have anaesthetic effects, which—importantly—were reversible. Today, the mechanical side is far improved: the purity, the quantity delivered, the time measurement, the gas cylinders, the ambulances, are all far more efficient. But the way they operate on the body isn’t known. There are theories, and descriptions of their actions; for example, hypotheses including the cell ‘membrane fluid mosaic’. (These, probably mythical constructions, are what Margaret Thatcher worked on as a chemist). It’s not surprising that occasional anomalies occur.

    • Drug testing. If it does what they want, they say it works according to the theory. (‘Beta blockers’.) However, huge numbers of similar molecules are tested; the acid test is always empirical. Any unwanted effect is labelled a ‘side effect’ as though it might go away. The same sort of thing applies with insecticides, which have to be tried out on (for example) caterpillars or species of fly.

    • Blood transfusions. The blood types were all found empirically: the important thing was to avoid clotting, rather than work out what made them clot.

    • Antibiotics are another example: penicillin was found by accident when a mould, penicillium, was noticed to kill bacterial growths in a petri dish. Lithium, as an anti-manic depressive drug, was found by chance to make lab animals sleepy. Titanium was found by chance to work well in bone replacements: the new musculature adhered well to it. Stainless steel is another example: I doubt anybody really knows how it works. The trick is to alloy metals and test the result. An interesting point here is that many people will claim to know (e.g.) why stainless steel doesn’t rust: that there’s an oxide layer, for example. But if they can’t predict which alloys will work, this arguably is just a form of words restating what they’ve found by trial.

    Like

  28. You know what’s funny and sad?

    It is ridiculous and sad to see how people, regardless of their scientific training, believe any purported scientific nonsense as long as it is issued by those so-called scientists who are accredited by bankers, politicians, military men, etc.

    Mankind’s real problem is not the liars.

    Humanity’s real problem is those who blindly believe the lies of the liars.

    Unfortunately, regardless of their scientific training, too many people are too delighted with the demigod posturing of quacks they think are scientists.

    And, also unfortunately, many people are too comfortable to think it worth while to critically analyze the supposedly scientific claims of those they idolize as scientists.

    By this comment I make a general reference to human nature, not to any particular person.

    Like

  29. So-called scientists make an infinite number of supposedly scientific claims that they feel no moral obligation to ever prove to anyone (because they can’t).

    Therefore, whoever does not believe, unconditionally, in the statements of scientists who are accredited by the System, will be ridiculed, will be slandered, will be discredited, will be harassed and will be persecuted.

    As for the so-called System Sciences, remember the satire expressed in Hans Christian Andersen’s fairy tale about the emperor’s invisible clothes.

    Like

  30. Hi Mike,

    Have you seen Hammond’s latest hit-piece: “Mike Stone Proves My Point about the Dogma of Virus Denialism.”

    I have made a comment there. (The second one I posted. I messed up the first one.)

    Let’s see how he replies to my comment.

    Liked by 1 person

      1. Thanks. He is a difficult guy to talk to, isn’t he? I don’t think he will be able to wriggle out of this one, though. At least not without looking like a hypocrite. LOL.

        Liked by 1 person

    1. Hi Tom! I saw the hit-piece by Hammond and I feel truly blessed that he was gracious enough to highlight my work. I’ve already received subscribers from his site…which I don’t think was his intention. 😁 I also saw your comments. Excellent job as usual! I am banned so I can not join in but I have been having my own back and forth with Jeremy on Twitter if you’d like to check it out. 😉

      https://mobile.twitter.com/jeremyrhammond/status/1603495122414493696

      Like

      1. Jeremy Hammond makes money selling anti-vaccine books. If he had to admit there are no viruses, then there would be no need for vaccines and no need for books warning people against them. Then he wouldn’t make any money selling books. He is simply a hustler with an anti-vaccine book hustle. That is how he makes his money and gets donations. But people still live in fear of viruses when they follow after him.

        Liked by 1 person

  31. Mike, its a year old though but have you considered the points made by this molecular biologist Dr. Wilson who talks about 3 independent lines of evidence in a video. He says cryo EM for the Rona that’s why I was reminded of this.

    I’m sure Dr. Kaufman and Cowan addressed it earlier but can’t locate a video or article that rebuts this or in your site. Apologies if this is repetitive, but i always try and learn from critics and that expands our own understanding. Thanks.

    Liked by 1 person

      1. Thanks. Will look fwd to it. There is so much to learn and discern. Unfortunately there is no one book or expert to follow here. We have to meticulously construct the knowledge base, listen to all points of view, expand our own views and be willing to shed our own untested notions.

        Liked by 1 person

  32. In regard to all attempts at explaining virus isolation, one principle, entirely overlooked, is

    If you can’t explain something in simple terms, you don’t understand it

    In the early 1960s, Richard Feynman gave a series of undergraduate lectures that were collected into a book called the Feynman Lectures on Physics. Absent from the book was a lecture Feynman gave on planetary motion, but a later finding of the notes enabled David Goodstein, a colleague of Feynman’s, to write a book about it: Feynman’s Lost Lecture. From an excerpt of the book published in a 1996 issue of Caltech’s Engineering & Science magazine:

    Feynman was a truly great teacher. He prided himself on being able to devise ways to explain even the most profound ideas to beginning students. Once, I said to him, “Dick, explain to me, so that I can understand it, why spin one-half particles obey Fermi-Dirac statistics.” Sizing up his audience perfectly, Feynman said, “I’ll prepare a freshman lecture on it.” But he came back a few days later to say, “I couldn’t do it. I couldn’t reduce it to the freshman level. That means we don’t really understand it.”

    https://kottke.org/17/06/if-you-cant-explain-something-in-simple-terms-you-dont-understand-it

    Liked by 1 person

  33. He who will seek to understand the true Art of Healing will at some point succeed in understanding that it has nothing to do with the lies and errors of molecular biology, virology, immunology, genetics, histology, microbiology, human anatomy and physiology, etc. .

    In fact, we don’t even need to delve into the structural and functional mysteries of the tissues of our being to understand what is harming our health and how we can help the self-healing processes.

    The problem is that we cannot even penetrate the structural and functional mysteries of the tissues of our being because that is a domain reserved only for the Creator, as the physicist Richard Feynman well noted when he declared that what he cannot create he cannot understand.

    In reality, all the supposed sub-microscopic sciences are just big lies.

    When it comes to microbiology, 99.9% of it is guesswork.

    The structures observed on the slide of the optical microscope belong to tissues that are in the process of death and decomposition because, in addition to being disconnected from the vital energy flows of the living organism, they were also drastically damaged by the preparatory procedures and by the microscopy itself.

    In this context, to consider that what is seen on the glass slide of the optical microscope corresponds to what exists in the living organism and to the processes that take place in the tissues of the living organism, is a matter devoid of any elementary logic and, above all, of any scientific honesty.

    Most probably the so-called cells, the so-called cellular organelles, the so-called microorganisms of the pleomorphic cycle and all other structures observed on the glass slide are only fragments of organic tissue in various stages of decomposition to reach the level of microzymes.

    Most probably these residues resulting from the decomposition of tissues down to the level of microzymes play no active role in life processes, just as the rubble resulting from renovation processes plays no active role in the reconstruction of a house, but from it the bricks are recovered to be reused in the same way as the body recovers the microzymes it needs to regenerate its tissues.

    The point is that it has been proven for millennia that in terms of maintaining health and promoting self-healing processes there is no need to try to penetrate the mysteries of our being, especially since we cannot even do this.

    As Max Planck said:
    “Science cannot solve the ultimate mystery of nature. And that is because, in the last analysis, we ourselves are a part of the mystery that we are trying to solve.
    Experiment is the only means of knowledge at our disposal. Everything else is poetry, imagination.”

    Those who want to understand the true Art of Healing can begin by studying the writings of Herbert M. Shelton (critical study, of course… 1Thessalonians 5:21 but test them all; hold on to what is good,).

    “The course of every intellectual, if he pursues his journey long and unflinchingly enough, ends in the obvious, from which the non-intellectuals have never stirred.”
    – Aldous Huxley

    Like

  34. Awesome site. I highly commend you on your work. As funny as this may sound here, you’re wrong about ‘Bigfoot’. I understand the effect and the point you were making with the series of images near the beginning of the article, but that subject is one you should take a second look at.

    Liked by 1 person

Leave a comment

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: