Addressing Virus Skeptics

The Confusing misuse of the words isolate and cause

This is the first in a series of articles on the limitations of biomedical science research, how those limitations sometimes get glossed over when scientists disseminate information to the public and how that leads to widespread misconceptions about what theories have and have not been proven. I will begin with the example of viruses as the sole cause of pandemic illness.

            I was discussing the insanity of the COVID years with a friend and was surprised when he asked me (paraphrased from my memory), “Was there ever a virus isolated that causes the disease?” Despite being staunchly against the COVID-19 mandates, I firmly believed that the virus causing the disease had been isolated. I launched into an explanation of how SARSCoV2 had been purified and could be propagated in cells, that it had been sequenced, and that it bore striking similarity to its predecessor (SARSCoV1). I added that my collaborators and I had done experiments with it, infecting human epithelial cells with isolated virus. He pushed back asking, “But has anyone really isolated the virus from a patient?” At first, I was frustrated with this question because I had just told him we had a vial of virus and used it to infect cells so obviously it was isolated, but, at some point, I realized that I was missing his point entirely.  In fact, most scientists, as well as those that worship at the altar of the credentialed elite, miss the point he was making. The key to understanding his point is in the language used by the scientific community. The definition of a virus is “an agent, too small to be seen by light microscopy, that is able to multiply only within the living cells of a host and is capable of causing an infection in the host.” Isolate in chemistry means that something is purified away from all its contaminants. If you are trying to prove that you isolated a disease-causing agent from an infected person, it would be expected that you isolate it directly from the patient and show you can then elicit symptoms by exposing an animal to that isolated pathogen. Rephrasing his question in light of these definitions we get: “has anyone purified one of these submicroscopic agents from an infected person, removed all other contaminants and demonstrated that it causes COVID-19?”. I have to admit that, by that definition, the answer is no-but I didn’t admit it right away.

He asked if I could refer him to some studies that showed it had been isolated and I sent him a link to some of the first studies that reported isolation and characterization of SARSCoV2, published in Nature and The New England Journal of Medicine. I reread them with his skepticism in mind, and began to understand the criticism I’ve heard from some that virology is all based on circumstantial evidence and circular reasoning. The first thing I noted was that, in both studies, the authors began with the thought that this outbreak of “pneumonia of unknown cause” (meaning pneumonia they couldn’t attribute it to any of the usual pathogens) was similar to the SARS (severe acute respiratory syndrome) outbreak of 2003. The initial experiments they performed were looking for a beta-coronavirus similar to the SARSCoV1 virus, that was said to have caused that outbreak, in samples (oral/nasal swabs and bronchiolar lavage fluid) from those patients. No samples from healthy individuals were included. In the Nature paper, total RNA was isolated from 7 patients and, using primers directed at conserved regions of known beta-coronaviruses, the researchers were able to amplify DNA fragments by RT-PCR in 5 of the 7 samples.  What caused the pneumonia in the other 2? That’s anyone’s guess. Using a similar approach, in the NEJM paper they used samples from 10 patients, 3 with “pneumonia of unknown cause” and 7 who had pneumonia that they attributed to a different pathogen. They were able to amplify beta-coronavirus sequences from the 3 samples taken from the unknown cause patients, but not from other 7 samples. They also tested the samples for 22 other pathogens (including various influenzas, RSVs, rhinoviruses, a-coronaviruses associated with common colds and 2 bacterial pathogens), none of which were found in those 3 samples.

They go on to sequence the DNA they amplified and show that it has similarity to SARSCoV1 and several bat b-coronaviruses. The sequencing step has been another source of criticism because the sequence has to be pieced together from fragments. There are no sequencing methods that can accurately read more than 600-700 nucleotides at a time- another limitation scientists encounter. In a nutshell, most sequencing protocols rely on copying DNA of interest, using nucleotides (GATC) that are each labeled with a different fluorescent tag along with the enzyme (DNA Polymerase) that catalyzes DNA replication. As the synthesis of the DNA strand progresses each base will emit at a specific wavelength corresponding to the fluorescent tag and this is recorded until the synthesis is completed. It’s quite a clever invention. In an intact organism, the DNA polymerase can catalyze the replication of entire strand of genomic DNA; however, it loses steam after about 700 bases in a test tube. The average coronavirus genome is about 30,000 nucleotides, and of course plant and animal genomes are much larger. This is why the DNA first has to be cut into pieces that are small enough for the polymerase and then the sequence is pieced back together by using overlapping sequences to line it up. In summary, the fact that they found evidence of a beta-coronavirus, similar to one associated with a previous outbreak, in people who had pneumonia they couldn’t attribute to something else is a good starting point to hypothesize that this is a culprit in the disease, emphasis on starting point.

They then used protocols that are the gold standards in the virology field to attempt to isolate the virus and determine its “infectivity”. Those protocols begin with centrifuging a patient sample to remove cellular debris (the virus should remain in the supernatant) and then inoculating cultured cells with that supernatant to propagate the virus for subsequent purification and characterization. This propagation step is one that is frequently criticized by virology skeptics who claim: “if there is an insufficient quantity of virus to be able see it in a patient sample, it can’t be real and can’t be causing illness”. This is not really a valid argument because a very small amount of a pathogen (or an allergen, toxin or hormone for that matter) can trigger cellular signals that are amplified within the cell, ultimately leading to a physiological response. You often need more of a chemical product for visualization than you need for a cellular response. When performing biochemical isolation of anything from a complex tissue, there is a significant loss of product at each step and, without a large amount of starting material, it can be impossible to purify enough to visualize. That’s the reason for the propagation step.

Another thing to mention about this propagation step is that what the virology skeptics see as circumstantial evidence, the academic scientist views as more rigorous proof of concept, which leads to a large communication gap. One of the major hallmarks of viruses, as they are defined, is that they use the host machinery to express the proteins encoded by their genetic material, e.g., those necessary to replicate the genome and package it into new viral particles that can then be taken into surrounding cells. The propagation step demonstrates that samples from a patient can be added to cultured cells and you can then recover viral particles from the medium that are: 1) present in exponentially greater quantities than what was first added to the cell and 2) comprised of protein and nucleic acid containing the same sequences as those found in the patient sample. This supports the claim that a virus is present in those patients. As for whether it was actually isolated (another criticism I have heard from the viral skeptic camp), in the NEJM study (but not the Nature paper), the authors use sucrose gradient ultracentrifugation to purify the virus away from other material in the cell culture medium. It is a bit frustrating that the term “isolate” is used in various studies, regardless of whether the virus was actually purified away from other contaminants, and one must take that into consideration when interpreting the results.  In both the studies discussed here, the authors show electron micrographs of the “isolated” virus and confirm by RT-PCR that the same sequences that were seen in the patient sample are present in the “isolated” virus. While they include a saline solution negative control when they add the virus to cells, and the images showing the changes in overall cell morphology by light microscopy have this negative control, the EM images are performed only on the cells that were inoculated with the patient sample. This has led to the claim that these EM images of viruses are actually of cellular structures such as exosomes, which have similar features. The evidence against this would lie in the fact that a virus, as it is defined, will replicate in cells and you will get more out than you put in. Looking at a time course of this such as was done in this study on SARSCoV1, it is apparent that these particles are not observed until at least 15 hours after addition of patient sample, consistent with the need for the virus to replicate before there are sufficient quantities to visualize.  One could still argue that what we call viruses are actually cellular material that becomes amplified during certain physiological situations, including those associated with disease, but that is also unproven and there is still all the other evidence that would have to be dismissed. The nucleic acid sequences observed in the virus-positive sample are not universally observed in the human genome. Not all people test positive for SARSCoV2 (or any other virus) at any given time, nor do they produce antibodies to them. One could argue that individuals may have an array of viruses that exist symbiotically in their bodies, in the same way that individuals have distinct bacterial flora and that certainly calls into question the enthusiasm some in the public health field have for widespread vaccination programs and the goal of eradicating all viruses.

After this controversial purification step from cultured cells, the authors measure “viral load” by examining cytotoxicity in the cells exposed to increasing concentrations of the “isolated” virus (once again saline negative controls are included). Another criticism I have heard is that these experiments are meaningless because “scientists are throwing cow blood and drugs and other toxic agents onto cells and then attributing the resulting cell death to the virus.” This claim stems from a poor explanation by the scientific community of how cells are grown in the laboratory, so let me briefly explain cell culture protocol-something I have done thousands of times. All the dishes of cells used in those viral load studies are grown in the same culture medium which typically consists of buffered saline solution at physiological pH, supplemented with glucose, amino acids, calf serum and other nutrients needed for healthy cell growth. The calf serum is included (at 5-10%) as a source of growth factors and other signaling molecules to which cells would be exposed. The goal is to best mimic the physiological setting the cells would experience in vivo. Finally, an antibiotic cocktail (penicillin and streptomycin) is often included to prevent contamination of the cells in culture. Any effects that are observed only in the presence of the viral sample cannot be attributed to calf serum, antibiotic or other culture medium components as those are present in the negative controls as well. For the record, this is the same experimental set-up used to demonstrate cytotoxicity of other drugs and environmental toxins, and to elucidate thousands of cellular pathways.

There is really no way to get around the threshold of material needed for purification of a virus, but there are other ways to visualize it in vivo, such as the use of immunofluorescence to identify proteins, or in situ hybridization to identify nucleic acids specific to the virus. There have been some studies published, that use these methods to demonstrate the presence of spike protein, nucleocapsid protein or their respective coding RNAs in patient samples. With immunofluorescence an antibody that recognizes a specific protein can be conjugated to a fluorophore and added to tissue sections; the antibody will bind to its target protein if it is present and unbound antibody can be washed away. The sample can then be viewed under a fluorescence microscope to visualize where the protein was in the tissue. Similarly, a fluorescent probe that is complementary to a specific RNA sequence can be added to tissue and, if that sequence is present, the probe will hybridize to it. Unhybridized probes are then washed off and the samples observed under a microscope. In one study, antibody to nucleocapsid protein was examined in the lung tissue of  patients who had died of or with COVID-19 and fluorescence was observed in the epithelial cells of the bronchi. They also observed nucleocapsid RNA in the bronchial epithelium by in situ hybridization. Unfortunately, they did not include any negative controls (i.e. patients that died without a positive COVID  test). In another study, saliva from COVID positive-subjects who displayed only mild symptoms or who had severe symptoms were analyzed for the presence of SARCoV2 RNA, along with samples from healthy controls. They observed the presence of both sense and antisense SARSCoV2 nucleotide strands in the saliva of the subjects with severe symptoms, the latter being an indicator of active viral replication. (SARSCoV2 is a positive/sense-RNA virus and the negative/antisense strand will only be present when it is copying its genome. By using probes to both, they can identify the presence of the virus and evidence of replication.) In the healthy controls, no SARSCoV2 RNA was detected and, in the patients with mild symptoms, significantly less viral RNA was observed than in the patients with severe symptoms.  This is the closest I have seen to direct visual evidence for the presence of the virus in actual human samples.

I’ve addressed the question of whether SARSCoV2 (or any virus) were actually isolated, and you may be wondering whether my insightful and skeptical friend was satisfied. He immediately raised the question of whether this virus identified in these papers was shown to cause disease. After all, that IS an implicit part of viral pathology. This question raises a debate that has been going on for over a century-between “germ theory” and “terrain theory” believers. In my personal opinion, and I think the data on COVID-19 support this, most diseases attributed to outside pathogens are a combination of the two. In a significant percentage of people with multiple conditions associated with early death, exposure to SARSCoV2 appeared to trigger severe disease and often death. This, however, is not an example of a virus making a healthy person ill or being the sole cause of disease. It is obviously impractical to directly investigate viral transmission in humans because no one is going to approve an experiment in which you expose healthy individuals to a potential pathogen. This is yet another limitation to biomedical science research and this is where epidemiological observations, paired with animal model experiments, can provide insight. The viral transmission experiment has been performed in mice expressing human ACE2, the protein to which spike protein binds, in golden hamsters, and in rhesus macaques.  These studies all demonstrate that, after animals were exposed to isolated (by sucrose gradient ultracentrifugation) SARSCoV2, there was evidence of viral replication in specific tissues (determined by RT-PCR), along with some measurable symptoms including “ground glass opacities” in the lungs, such as were observed in human patients. In all cases, the symptoms reversed and there was no convincing evidence of severe disease. In one of the hamster studies, a healthy animal housed with an infected animal later tested positive, and histopathological evaluation of the lung showed infiltration of inflammatory cells and transient changes consistent with airway inflammation. Note that negative controls in which animals were administered the saline solution were included but the data for those negative controls were not consistently shown for every analysis. Importantly though, none of those studies show that the virus caused disease, only that it led to transient symptoms that are loosely related to those observed in humans. This is consistent with the hypothesis that viruses can contribute to disease and can trigger physiological responses in healthy animals but not that they make a healthy animal severely ill.

In summary, I could not really answer my friend’s questions satisfactorily. While there are data to suggest that SARSCoV2 (or agent being called a SARSCoV2 for those who remain skeptical) has a sequence can be passed from animal to animal and appears to cause alterations in the airways that could contribute severe disease, there is no way to prove it is the sole cause of the disease we call COVID-19. Epidemiological observations show a correlation between the presence of the viral sequences and increased reports of respiratory illness. Some people who test positive become very sick and die. My belief is still that elements of both germ theory and terrain theory can explain what we refer to as infectious disease and that, for the majority of people with healthy “terrains”, most viruses are not cause for alarm. If the goal is to treat sick individuals and prevent long-term damage and death, and a virus could be the major contributing factor in their illness, a positive test could be good information to have. It should NEVER have been used to demand society shut down or that people receive unnecessary medical interventions. I don’t think there would be as many skeptics if we treated these data as guides when treating patients as opposed to justification for authoritarian mandates.