In the last article in this series, we saw how the Chief Epidemiologist at China’s CDC Wu Zunyou admitted that the virus was never actually isolated, even though the Deputy Dean of Wuhan Jinyintan Hospital Chaolin Huang and his colleagues claimed to have done precisely that (Huang et al 498).
But the Chinese are not the only researchers who claim to have isolated SARS-CoV-2. At the end of that article we cited an Australian paper whose authors describe in detail how they isolated the virus:
- Anupriya Aggarwal et al, Platform for Isolation and Characterization of SARS-CoV-2 Variants Enables Rapid Characterization of Omicron in Australia, Nature Microbiology, Volume 7, Issue 6, Pages 896-908, Nature Portfolio, London (2022)
In this article we will be taking a close look at this paper—hereinafter referred to as Aggarwal—to see if the authors’ claims stand up to scrutiny.
No less than forty-two authors are listed in the article’s byline, though only the first three are singled out for special mention: Anupriya Aggarwal, Alberto Ospina Stella, and Gregory Walker. All but three of the authors are employed by medical colleges or laboratories in New South Wales, Australia. The three exceptions work at the following institutions: the Department of Bioanalytical Sciences in Melbourne, Australia : the Department of Bioanalytical Sciences in Bern, Switzerland : and the University of Erlangen-Nürnberg in Erlangen, Germany.
In this paper, the authors claim to have:
developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. (Aggarwal 896)
In the Methods section of the paper, the authors describe in meticulous detail how the virus was isolated from respiratory samples. Specimens were collected from participants in the study and PCR tests were carried out for SARS-CoV-2. Those that tested positive were saved for virus isolation:
Viral isolation from primary specimens. Respiratory specimens were collected and stored at 4 °C for same-day diagnostic RT–qPCR ... Specimens positive for SARS-CoV-2 were then frozen at −80 °C within 24 h of collection, and later transported to a certified BSL-3 [Biosafety Level 3] facility for primary isolate propagation. (Aggarwal 906)
These samples were then filtered:
Thawed viral eluate was sterile-filtered through 0.22 μm column filters (Merck, UFC30GVOS) at 10,000 × g for 5 min and then serially diluted (3-fold series) in quadruplicate. (Aggarwal 906)
- Elution is the process of extracting one material from another by washing with a solvent. This step is carried out to concentrate the alleged virus by removing extraneous substances:
The technique most commonly used to concentrate viruses from water samples is the virus adsorption-elution microporous filter method, or VIRADEL. ([Xagoraraki et al 6](file:///home/brendan/Downloads/Xagorarakietal.2014ASCEinvited.pdf))
Aggarwal and her colleagues do not describe the elution process.
Sterile Filtration is a procedure to remove biological agents—fungal spores, bacteria, unicellular organisms, prions—from a specimen by passing the specimen through very fine filters. A pore size of 0.22 μm (220 nm) is generally considered suitable to remove microorganisms while allowing the smaller viral particles to pass through.
Serial dilution is a series of sequential dilutions that are performed to convert a dense solution into a more usable concentration. In serial dilution, the density of particles is reduced in each step. In this particular case, each dilution reduced the concentration to one third of its initial value (hence 3-fold). The resulting solution was then divided into four separate specimens (in quadruplicate). These are the viral dilutions referred to in the next passage:
Viral dilutions were added to HAT-24 cells seeded in 96-well plates at 104 cells per well (final volume, 100 μl). Plates were incubated at 37 °C and monitored by brightfield microscopy every 24 h using high-content microscopy. (Aggarwal 906)
This is the crucial step in the process of virus isolation: the culturing of the virus in HAT-24 cells. So what are HAT-24 cells and why are they needed?
HAT-24 Cells
HAT stands for hypoxanthine-aminopterin-thymidine, a selection medium used for culturing mammalian cells in the laboratory. The purpose of such a medium is to allow only certain types of cells in the culture to divide and multiply—a form of artificial selection. For example, only those cells that contain a specific target gene will be able to reproduce in such a medium. HAT-24 is described by Aggarwal et al thus:
In this setting, we introduced a hyper-permissive HEK293T-based cell line that co-expresses ACE2 and TMPRSS2 (HAT-24) (Aggarwal 897 - I have corrected the typo: HEK239T)
ACE2 is an enzyme that can be found on the membrane of certain cells. It is believed to provide one of the pathways by which SARS-CoV-2 can enter a cell. The spike protein on the virus’s outer envelope allegedly damages the membrane of endothelial cells (such as those lining blood vessels) via downregulation of the ACE2 receptors.
TMPRSS2 is a similar enzyme that is believed to play an important role in the process whereby a virion infects a cell. It is found in the membranes of epithelial cells of the lungs and several other organs:
Viruses found to use this protein for cell entry include influenza virus and the human coronaviruses HCoV-229E, MERS-CoV, SARS-CoV and SARS-CoV-2 (COVID-19 virus). CoV are extracellular particles in an envelope with protruding spike proteins. In order for them to infect a cell, the viral spike protein needs to bind to the host cell receptor. Following binding, there needs to be proteolytic cleavage of the bound spike protein for fusion between viral and host cell membranes to occur. Exposure of the fusion peptide allows viral entry (S mediated cell–cell fusion). This process involves the conformational change of the spike protein until the viral mRNA genome is deposited into the host cell cytoplasm. These conformational changes and fusion require cellular proteases, the availability of which is a rate-limiting step in CoV entry. There are several proteases crucial to coronavirus viral entry and they are found at different subcellular locations. (Thunders & Delahunt)
TMPRSS2 is one such protease, an enzyme that catalyzes proteolysis, or the breaking down of proteins into smaller polypeptides, peptides, and amino acids.
So only cells that have these receptors (ie have the genes that encode them) will reproduce in the HAT-24 cell culture. The aim is to create a cell culture in which there is an abundance of cells that the virus can infect in order to reproduce itself, which in turn leads to an abundance of the virus. The cells chosen for the HAT-24 culture were a variant of HEK 293 Cells:
Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific immortalised cell line derived from a spontaneously miscarried or aborted fetus or human embryonic kidney cells grown in tissue culture taken from a female fetus in 1973. (Wikipedia)
HEK 293T is one of several variants of the original HEK 293 line. Note that on page 897 of Aggarwal, HEK293T is misspelled once as HEK239T.
HEK 293T cells are used because they are particularly vulnerable to viral infection—allegedly. This is what the term hyper-permissive means (permissivity):
Cell Culture
In order to “grow” the alleged virus, these infected HEK 293T cells are cultured in a nutrient medium:
Cell culture. HEK293T cells (Thermo Fisher, R70007), HEK293T derivatives and VeroE6-TMPRSS2 (CellBank Australia, JCRB1819) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11995073) with 10% fetal bovine serum (FBS) (Sigma, F423-500). VeroE6 cells (ATCC CRL-1586) were cultured in Minimum Essential Medium (MEM; Sigma, M4655) with 10% FBS and 1% penicillin-streptomycin (Gibco, 15140122). pBEC cultures were grown and differentiated until confluent in complete Bronchial Epithelial Cell Growth Basal Medium (Lonza, CC-3171) before use for air–liquid interface experiments. All cells were cultured and incubated at 37 °C, 5% CO2 and >90% relative humidity, unless otherwise indicated. (Aggarwal 905)
In other words, in addition to the infected human embryonic kidney cells and the alleged virus, the cell cultures also contained:
- Dulbecco’s Modified Eagle Medium
- Fetal Bovine Serum
- VeroE6 Kidney Cells
- Penicillin-Streptomycin
Dulbecco’s Modified Eagle Medium (DMEM) is a commonly used variation of Eagle’s Minimum Essential Medium (MEM):
Minimal Essential Medium (MEM) is a synthetic cell culture medium developed by Harry Eagle first published in 1959 in Science that can be used to maintain cells in tissue culture. It is based on 6 salts and glucose described in Earle’s salts in 1934 (calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium phosphate and sodium bicarbonate), supplemented with 13 essential amino acids, and 8 vitamins: thiamine (vitamin B1), riboflavin (vitamin B2), nicotinamide (vitamin B3), pantothenic acid (vitamin B5), pyrodoxine (vitamin B6), folic acid (vitamin B9), choline, and myo-inositol (originally known as vitamin B8). (Eagle’s Minimal Essential Medium)
DMEM was developed in 1959 by the Italian virologist Renato Dulbecco. It contains a slightly different mix of nutrients than MEM.
Many variations of this medium have been developed, mostly adding additional vitamins, amino acids, and/or other nutrients.
Fetal bovine serum is another mix of nutrients that promotes cell growth and division:
Fetal bovine serum (FBS) is the most widely-used growth supplement for cell culture media because of its high content of embryonic growth-promoting factors. When used at appropriate concentrations, it supplies many defined and undefined components that have been shown to satisfy specific metabolic requirements for the culture of cells. FBS is a complex mixture of biomolecules that includes growth factors, proteins, trace elements, vitamins, and hormones. These are important for the growth and maintenance of cells in vivo and in culture. (Merck)
Note that FBS contains many undefined components—its contents are not completely known by the researchers using it, which prompts the question: How can the researchers control for substances that are present in the cell culture but whose identity is unknown?
Sigma refers to the company Sigma-Aldrich, which supplies the FBS. It is part of the German chemical conglomerate Merck Group. F423-500 means 500 ml of a particular type of FBS with the product number F423. Several varieties of FBS are advertised on Merck’s website, but I could not find this particular product.
VeroE6 kidney cells refers to another popular cell line used for culturing viruses. They are not relevant to our study as they were not used in the initial isolation of the virus:
Derived from the kidney of an African green monkey (Cercopithecus aethiops [Chlorocebus aethiops]) in the 1960s, Vero cells are one of the most common mammalian continuous cell lines used in research. This anchorage-dependent cell line has been used extensively in virology studies ... In addition, Vero cells have been licensed in the United States for production of both live (rotavirus, smallpox) and inactivated (poliovirus) viral vaccines, and throughout the world Vero cells have been used for the production of a number of other viruses, including Rabies virus, Reovirus and Japanese encephalitis virus ... There are several lines of Vero cells commercially available (i.e., Vero, Vero 76, Vero E6), but they were all ultimately derived from the same source ... (Ammerman et al 1)
Penicillin-streptomycin, or Pen Strep, is a mixture of two common antibiotics, penicillin G and streptomycin, which is widely used in mammalian cell culture media to prevent bacterial contamination.
The presence of so many extraneous materials and chemicals—many undefined—introduces numerous confounding variables into the process of virus isolation. It is essential to eliminate each and every one of these as a contributing factor to any effects observed.
Cytopathic Effect
Let us return to Aggarwal and her colleagues in the laboratory. What happened next to the cell cultures in the 96-well plates?
Plates were incubated at 37 °C and monitored by brightfield microscopy every 24 h using high-content microscopy. Once extensive CPE became evident in at least 2 dilutions (Fig. 1e); the cells and supernatant from these cultures were collected and cleared from debris by centrifugation at 2,000 × g for 5 min, aliquoted and stored at −80 °C (passage 1). (Aggarwal 906)
This is the critical step which is believed to prove that the HAT-24 cells have indeed been infected by a pathogenic virus. The cell cultures were incubated at body temperature and kept under observation to see if any manifested CPE, which stands for Cytopathic Effect. This term refers to biochemical and morphological changes that take place in a cell when it has been infected by a pathogenic virus. The commonest cytopathic effect is lysis, the breaking down of the cell membrane. An infected cell may also die without experiencing lysis due to its inability to reproduce by cell division.
During the time that synthesis of viral components is occurring in the infected cell, the cell undergoes characteristic biochemical and morphological changes. Progression of these changes is most readily observed in cell culture, where infection of cells is more easily synchronized and where the cells can be observed and sampled frequently during the course of infection. Morphological changes in cells caused by viral infection are called cytopathic effects (CPE); the responsible virus is said to be cytopathogenic. The degree of visible damage to cells caused by viral infection varies with type of virus, type of host cells, multiplicity of infection (MOI) and other factors. Some viruses cause very little or no CPE in cells of their natural host. [In such cases,] Their presence can be detected visually only by hemadsorption or interference, in which infected cell cultures showing no CPE inhibit the replication of another virus subsequently introduced into the cultures, or in situ by viral antigen or nucleic acid detection. On the other hand, some viruses cause a complete and rapid destruction of the cell monolayer after infection. The microscopic appearance of the CPE caused by some of these cytocidal viruses may be sufficiently characteristic to allow provisional identification of an unknown virus. (American Society for Microbiology)
Once the CPE was observed in at least two of the wells in the 96-well plates, the cultures underwent centrifugation in order to remove cellular debris. The supernatant is the less dense fraction of a solution that floats to the top during or before centrifugation. The centrifuged solution was then divided into separate portions (aliquoted) and stored at −80 °C. This completed Passage 1. Passage 2, which does not concern us, involved a similar process using a different lineage of cells for the culture, namely vero cells.
The following descriptions are given of the visible and extensive CPE that occurred during Passage 1:
Fig. 1 ... b‒d, Rapid appearance of viral CPE (see large spherical viral syncytia) ... (b is mock uninfected control) ... (Aggarwal 897)
Fig. 4 ... Note: CPE is scored through the rapid appearance of large spherical syncytia. d, e, 96 h of culture: uninfected confluent well (d) and low-level infection with CPE revealed as a combination of spherical syncytia and the formation of plaques (e). f, g, 96 h of culture: low-level infection with CPE revealed by extensive fusion across the viral cell sheet and the formation of plaques (f); extensive infection where CPE has resolved early as spherical syncytia and has become granular in appearance over time (g). (Aggarwal 901-902)
Three effects are mentioned:
- The appearance of large spherical syncytia
- The formation of plaques
- Extensive fusion across the viral cell sheet
A syncytium is a mass of cytoplasm containing many nuclei, or a multinucleate cell resulting from the fusion of several mononuclear cells.
A viral plaque is a patch of abnormal tissue distinguishable from surrounding tissue, which is formed when a cell culture grown on some nutrient medium is infected with a sample of a virus. The virus replicates and spreads, generating regions of cell destruction known as plaques.
The third effect is essentially the same as the first, cell fusion being the process that creates syncytia.
Controls
In order to eliminate the possibility that the results of an experiment are due to confounding variables rather than the independent variable under study, scientific controls are used. Controls are a necessary part of the scientific method. Aggarwal et al only mention controls a few times in their paper:
(b is mock uninfected control) ... (Aggarwal 897)
Cell nuclei were enumerated with high-content microscopy and cell numbers normalized to mock-infected controls where 100% represents cell numbers for mock-infected controls and 0% represents cell numbers for the highest viral concentration ... (Aggarwal 898)
Brightfield images were visually inspected by two independent experienced operators and compared against negative and positive infection controls to score wells binarily for CPE (+ or −) to calculate TCID50 values according to the Spearman-Karber method. Fluorescence images were processed with IN Carta analysis software (Cytiva) to obtain total nuclei counts per well. For calculation of LD50 values, cell counts were normalized so that 100% represents the average cell number for mock-infected controls and 0% represents the average cell number for the highest viral concentration tested. (Aggarwal 906)
Cellular nuclei counts were obtained with IN Carta automated image analysis software (Cytiva), and the percentage of virus neutralization was calculated with the formula: %N = (D−(1−Q)) × 100/D, where ‘Q’ is a well’s nuclei count divided by the average count for uninfected controls (defined as having 100% neutralization) and D = 1−Q for the average count of positive infection controls (defined as having 0% neutralization). (Aggarwal 906)
The term mock-infected is defined online thus:
A control used in infection experiments. Two specimens are used one that is infected with the virus/vector of interest and the other is treated the same way except without the virus. Sometimes a non-virulent strain is used in the mock-infected specimen. (Holmgren Lab)
This sounds like a proper negative control—everything the same but without the presence of the alleged virus—but Aggarwal et al never describe, even in outline, how they prepared their mock-infected samples or how they treated them. Nor do they explain why they use the expression mock uninfected control on page 897 but mock-infected controls elsewhere. Are these the same? In a peer-reviewed paper it is essential that the authors describe every step of the procedure they followed in sufficient detail to allow other researchers to replicate their findings. This is impossible in this case without further details being provided. They also mention positive controls. These must be cell cultures which have been infected with viruses in order to produce a CPE, which can then be compared to the CPE of the cell cultures allegedly infected with SARS-CoV-2. But once again no details are given, so it is impossible for other researchers to replicate this part of the experiment.
Criticisms
Several criticisms have been levelled at this culturing process by those who say that the whole process of isolating viruses by culturing them in cells and observing the cytopathic effect is a flawed one.
The Necessity for the Culturing Process Why is it necessary to culture the virus in the laboratory? If a patient is infected with a pathogenic virus and is exhibiting the symptoms of the disease associated with this pathogen, then surely they are already culturing the virus in their own cells? In the case of a patient suffering from COVID-related pneumonia, specimens of alveolar fluid from the infected lungs ought to be teeming with the virus. Why not skip the complicated culturing process and simply image this fluid in an electron microscope? Scientific curiosity alone cries out for such a step to be carried out. And if COVID-19 is airborne and highly contagious, then having a COVID-positive patient cough into a test-tube ought to provide the researcher with all the virus they require. Why is this never done?
Inadequate Controls Independent researcher Mike Stone has the following to say on the subject of mock-infected controls on his website ViroLIEgy:
Before the culture experiments have even begun, there are numerous variables that can potentially be the cause of any observed effect created through experimentation. Thus, it is very important for virologists to perform proper controls to ensure that the results are valid and are in fact caused by their independent variable rather than a creation from their own experimental methods.
Unfortunately, this is not the case in virology. What is most often found in these papers, if found at all, is the mock-infected culture, which again is where they will take whatever cell line is chosen for the experiment and apply the same(?) compounds to it without the unpurified human sample. Sadly, it is often not clear exactly how the mock-infected culture was treated or if one was ever performed. However, even if done as described, is this a proper control which factors in all potential variables? No, absolutely not. The only way it would possibly be a proper control is if the virologists started with only the purified/isolated particles assumed to be the “virus” as the independent variable used for experimentation. As they do not do so, the unpurified human sample already has many host and foreign materials present along with many others added to it which need to be factored into the equation as potential causes of any observed effect. Thus, using only a cell without a human sample is not a proper control when an unpurified sample without isolated particles is utilized to try and prove cause and effect. In order to have a proper control, the virologists would need to use samples from both healthy humans and those who are sick with similar symptoms but said not to have the assumed “virus.”
In the case of Aggarwal et al, we simply don’t know whether their mock-infected or mock-uninfected samples were created by taking respiratory specimens from healthy individuals testing negative for COVID-19 or by simply preparing the same HEK293T cell culture but without including any respiratory specimens. Why are we not told? In the subsection Viral isolation from primary specimens of the Methods section, they only mention Respiratory specimens ... positive for SARS-CoV-2 (Aggarwal 906).
Stone then quotes from the paper The Misconception called Virus by virologist—or, as he now calls himself, ex-virologist—Stefan Lanka:
Virologists primarily believe in the existence of viruses, because they add allegedly “infected” blood, saliva or other body fluids to the tissue and cell culture, and this, it must be stressed, after having withdrawn the nutrients from the respective cell culture and after having started poisoning it with toxic antibiotics. They believe that the cell culture is then killed by viruses. The key insight, however, is that the death of the tissue and cells takes place in the exact same manner when no “infected” genetic material is added at all. The virologists have apparently not noticed this fact! According to the most basic scientific logic and the rules of scientific conduct, control experiments should have been carried out. In order to confirm the newly discovered method of so-called “virus propagation”, in order to see whether it was not the method itself causing or falsifying the result, the scientists would have had to perform additional experiments, called negative control experiments, in which they would add sterile substances or substances from healthy people and animals to the cell culture. This, of course, to check whether it is not the method itself that yields or falsifies the results.
These control experiments have never been carried out by the official “science” to this day. During the measles virus trial, I commissioned an independent laboratory to perform these control experiments and the result was that the tissues and cells die, due to the laboratory conditions, in the exact same way as when they come into contact with allegedly “infected” material.
The entire purpose of control experiments is to exclude the possibility that it is the applied method or technique which may cause the result. Control experiments, then, are the highest duty in science and also the exclusive basis of claiming that one’s conclusion is scientific. During the measles virus trial it was the legally appointed expert—Dr. Podbielski, see further in this article—who stated that the papers which are crucial for the entire science of virology contain no control experiments. From this we can conclude that the respective scientists have been working extremely unscientifically, and this without even noticing it. (Lanka 3)
Stefan Lanka has actually carried out the proper negative control experiments to see whether the cytopathic effect really is caused by viral infection. We will be looking at those experiments in a later article.
- The Cytopathic Effect Why does the occurrence of the so-called cytopathic effect indicate that a virus has been isolated? Without adequate controls, how do we know that the CPE was brought about by viral infection and not by any of the other ingredients of the cell culture—some of which are even undefined? Until proper control experiments are carried out, the argument that the CPE indicates the presence of a pathogenic virus is circular. Critics of this method of virus isolation do not deny that these cytopathic effects are observed : they simply hypothesize that they are not caused by viral infection.
Conclusions
Did Aggarwal and her colleagues isolate SARS-CoV-2? Certainly not to my satisfaction. Having said that, the complete lack of transparency concerning their mock-infected controls leaves me in the dark. I cannot say with complete confidence that the cytopathic effects they observed in the infected specimens were not also observed in the control specimens because the latter did not contain any virus. Without a proper detailed description of the procedures followed, I cannot draw any conclusions. And no other researcher can possibly replicate Aggarwal et al’s findings.
In the next article we will take a look at the subject of virus isolation in general, tracing its history from earliest times to the present day in an attempt to explain how such a bizarre and complicated procedure came to be regarded as the gold standard in virus isolation.
And that’s a good place to stop.
References
- Anupriya Aggarwal et al, Platform for Isolation and Characterization of SARS-CoV-2 Variants Enables Rapid Characterization of Omicron in Australia, Nature Microbiology, Volume 7, Issue 6, Pages 896-908, Nature Portfolio, London (2022)
- Nicole C Ammerman, Magda Beier-Sexton, Abdu F Azad, Growth and Maintenance of Vero Cell Lines, Current Protocols in Microbiology, November 2008, Appendix 4E, Wiley Online Library (2008)
- Chaolin Huang et al, Clinical Features of Patients Infected with 2019 Novel Coronavirus in Wuhan, China, The Lancet, Volume 395, Issue 10223, Pages 497-506, Elsevier, London (2020)
- Stefan Lanka, The Virus Misconception: Part 1: Measles as an Example, WissenschafftPlus magazin, Issue 01/2020, LK-Verlags UG, Werder (Havel) (2020)
- Michelle Thunders & Brett Delahunt, Gene of the Month: TMPRSS2 (Transmembrane Serine Protease 2), Journal of Clinical Pathology, Volume 73, Issue 12, Pages 773-776, BMJ Group, London (2020)
Image Credits
- COVID-19 Poster: © 2021 Dublin Region Homeless Executive, Fair Use
- Anupriya Aggarwal of the Kirkby Institute: © Anupriya Aggarwal (photographer), Fair Use
- A 96-Well Plate: © Azenta US, Inc, Fair Use
- HEK 293 Cells Imaged by Immunofluorescence: © Iznewton (microscopist), Creative Commons License
- The Grivet: A Species of African Green Monkey: © Rod Waddington (photographer), Creative Commons License
- Pen-Strep Chemical Formulas: Vaccinationist (designer), NEUROtiker (designer), Public Domain
- Cytopathic Effect (Aggarwal: Figure 1): © Anupriya Aggarwal et al, Fair Use
- Cytopathic Effect (Aggarwal: Figure 4): © Anupriya Aggarwal et al, Fair Use
- Mike Stone: © Dr Tom Cowan, Fair Use
- Stefan Lanka: © Coronadatencheck, Fair Use
- Isolation: Virology’s Black Sheep?: Copyright Unknown, Fair Use
Video Credits
- NBC Nightly News 23 January 2021: NBC Nightly News, © NBC Universal, Fair Use
Online Resources
