ViroLIEgy

A Conversation With the Terrain Theory Podcast About ViroLIEgy

Last month, I had the great pleasure of speaking with both Ben Hardy and Mike Merenda, creators and hosts of the Terrain Theory Podcast. If anyone is unfamiliar, this is an excellent podcast that has had many wonderful guests on, such as Dr. Tom Cowan, Dr. Andrew Kaufman, Dr. Amandha Vollmer, Dr's. Sam and Mark Bailey, Dawn Lester and David Parker, Alec Zeck, Steve Falconer, Jacob Diaz, Michael Wallach, and Eric Coppolino. I feel extremely honored and privileged to be a part of the Terrain Theory Podcast's list of guests. I had an amazing time doing the show, and I hope that you enjoy this conversation.

Terrain Theory is hosted by Ben Hardy and Mike Merenda, two childhood friends on a journey to tear down the old fear-based germ theory paradigm and usher in a better, brighter approach to health and wellness. Discover interviews with guests from the alternative medicine space and find inspiration in real-life stories of Terrain Transformations. Reclaim agency and discover methods both old and new to improve and optimize your health and your terrain. You are your primary healthcare provider.

Episode 60: Mike Stone on Viroliegy.com, reading the method section of a virus isolation paper, and the rabies myth

Mike Stone is the founder of Viroliegy.com, a veritable database of articles and research exposing the lies of virology and Germ Theory – using their own sources. In this conversation with Mike we discuss:

• How exploring the HIV/AIDS fraud led to viroliegy.com
• "But what about rabies?"
• Reading the method section of a virus isolation paper
• The myth of "gain of function"
• Lack of evidence for nanotech in the jabs
• Patents don't equal proof
• "So what causes COVID?"

https://www.terraintheory.net/blogs/podcast/episode-60-mike-stone-on-viroliegy-com-reading-the-method-section-of-a-virus-isolation-paper-and-the-rabies-myth

https://bit.ly/3JzJ8vI

https://bit.ly/407aNLG

Questioning the Culling With Tom Quinn

Recently, I had a conversation with Tom Quinn about the current travesty occurring with our chickens due to the "avian flu." This is the latest scam used by those in power to scare the populace over the invisible boogeyman. It is being used to inhumanely and grotesquely murder chickens, damaging our food supply and the economy. I recently wrote about this massacre on my Substack, which you can read here. We also discussed the differences between germ and terrain theory as well as the dangers of vaccination. Our conversation starts at about the 17-minute mark. I hope you enjoy it!

You can find out more about Tom Quinn and his excellent radio show from the following links:

https://ksco.com/shows/60112-tom-quinn

https://www.zbsradio.com/show_detail/id/125

Diving Into ViroLIEgy With Patrick Timpone

I recently sat down for another chat with Patrick Timpone, and we went back down the rabbit hole related to germ thoery and virology. We covered quite a few topics in our latest conversation, such as:

• The Rosenau Spanish flu experiments
• Pasteurs fraudulent rabies experiments
• The lack of an immune system
• Symptoms of disease are a sign of healing and detoxification
• Koch's Postulates disprove bacteria as pathogens
• Fear and the Nocebo effect
• There is no new disease
• The 100% PCR false-positive Whooping Cough "epidemic"
• The HIV = AIDS Hoax
• Using effects to claim a cause
• Allowing the body to heal itself

And much more!

I hope that you enjoy our latest dive into the fraud of germ theory and virology and are able to take something from it.

https://m.soundcloud.com/oneradionetwork/022123-stone-mike

Please check out Patrick's recent conversation with Steve Falconer, the creator of many excellent documentaries exposing germ theory, as well:

And be on the lookout for an upcoming conversation between Patrick with both Drs. Sam and Mark Bailey.

Questioning the “Viral” Paradigm With Richard Cox

A few weeks ago, I was privileged to speak with Richard Cox on his Deep State Consciousness podcast, where we examined the "viral" paradigm. It was a conversation that was a long time coming as we had meant to speak much sooner, but scheduling conflicts kept getting in the way. When the time finally arrived, we unfortunately had some technical difficulties, but luckily, we were able to cover the areas that we wanted to discuss. I am excited that our conversation is now available for everyone to listen to. We discussed quite a few topics, including:

• What made me begin to question the "viral" paradigm?
• What was my mindset like in 2017 when I started challenging my own long-held beliefs?
• Are there any diseases where an alternate cause outside of a "virus" is difficult to explain?
• What is my approach to examining the different "viruses?"
• What pushback have I received from those defending the "viral" paradigm?
• What are the particles virologists see under the electron microscope if not "viruses?"
• What factors outside of a "virus" could have led to disease during the "Covid pandemic?"
• What is being done to bridge the gap between the "No Virus" and the "Bioweapon" camps?
• And much more!

It was a fun and engaging conversation that I hope you will enjoy!

Mike Stone is the author of the Viroliegy blog, where he investigates the scientific evidence for the existence of viruses. In this interview I ask Mike about journey into that research, what it's like to communicate a position most people find so radical and – if the 'no virus' paradigm is correct – what on earth that would mean for our belief in a scientific worldview.

Please check out Richard's site and podcast for more great content and conversations!

https://www.deepstateconsciousness.com/

https://deepstateconsciousness.podbean.com/

https://podcasts.apple.com/us/podcast/the-deep-state-consciousness-podcast/id1338867921

https://www.audible.com/pd/Podcast/B08JJN72YX

Respiratory Syncytial “Virus” (RSV)

Due to the media hype of the "Tripledemic" (previously known as the "Twindemic"), most are probably well aware and fairly familiar with the name "respiratory syncytial virus," otherwise known as "RSV." We've been warned over the last few months that there was an alarming increase in cases of this "virus," primarily amongst children, and that it was adding to an escalating burden on our healthcare system along with "Covid" and "influenza." Obviously, as "RSV" is said to attack primarily infants, it has set concerned parents off into a panicked hysteria over how best to protect their children from yet another "virus" floating in the increasingly "viral-filled" air. However, is it necessary to be alarmed at the mere mention of the threat of "RSV" or is this yet another in a long line of media-hyped figments of the imagination? As I've covered "SARS-COV-2" and "influenza" quite a bit, it's time to set the sights on "RSV" and see what kind of scientific evidence they have in store for us in order to justify this supposed "threat."

To begin with, what exactly is RSV? What symptoms are we potentially dealing with here that set this "virus" apart from the two that are said to be currently causing mayhem around the world? Let's take a look at a CDC list of the common symptoms between "RSV," "Covid," and "influenza" and see what distinguishes the "viruses" from one another:

It looks like the symptoms match up to a T beyond the regularity of occurrence. From the article accompanying the image, we get some more information regarding whether these "viruses" can be distinguished based upon symptoms alone:

Suffering from flu, RSV or COVID-19? How you can tell the difference

The three viruses have many symptoms that are similar.

"All three viruses have symptoms that are similar, which can make them difficult to tell apart. But knowing which virus a person has can help them receive proper treatment or, if need be, let them know if they need to isolate."

"COVID-19, flu and RSV are more similar to each other than they are different in terms of symptoms."

"However, public health experts told ABC News the absence of one of the symptoms does not mean a patient doesn't have a particular virus and that the only way to be sure is to get tested."

"In most cases, if anybody has generic symptoms, such as fever, cough, runny nose, there's going to be no real way to distinguish which one is which without a test," Dr. Scott Roberts, an assistant professor and the associate medical director of infection prevention at Yale School of Medicine, told ABC News."

https://www.google.com/amp/s/abcnews.go.com/amp/Health/suffering-flu-rsv-covid-19-difference/story%3fid=94211146

According to the list provided by the CDC, it looks as if every single symptom is shared between all three "viruses." The only difference is claimed to be in regards to the regularity of a particular symptom occurring. However, it is stated that even the absence of a particular symptom can not rule in or out any of the "viruses" and that testing is necessary in order to obtain a definitive diagnosis. Thus, it is clear that there is no way to clinically diagnose any of these "viruses" by way of symptoms alone. I dealt with the problem of differential diagnosis in an article at my Substack.

The CDC confirms that the symptoms associated with RSV are nonspecific and overlap with many "viral" and bacterial diseases. As clinical diagnosis based on symptoms alone is impossible, the testing that is used to identify "RSV" is the usual culprits with PCR and antigen tests. "Viral" culture and antibody tests are said to be less commonly used with antibody testing limited to research and surveillance:

Clinical Laboratory Testing

"Clinical symptoms of RSV are nonspecific and can overlap with other viral respiratory infections, as well as some bacterial infections. Several types of laboratory tests are available for confirming RSV infection. These tests may be performed on upper and lower respiratory specimens.

The most commonly used types of RSV clinical laboratory tests are

• Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR), which is more sensitive than culture and antigen testing
• Antigen testing, which is highly sensitive in children but not sensitive in adults

Less commonly used tests include:

• Viral culture
• Serology, which is usually only used for research and surveillance studies

Some tests can differentiate between RSV subtypes (A and B), but the clinical significance of these subtypes is unclear. Consult your laboratorian for information on what type of respiratory specimen is most appropriate to use."

https://www.cdc.gov/rsv/clinical/index.html

However, there is a major problem with relying on these tests to diagnose cases of a particular disease. As "RSV" can not be accurately diagnosed clinically due to nonspecific and overlapping symptoms, there is no way to identify cases in order to determine the prevalence of the disease within a given population. In order for the results of the PCR test to be considered accurate, disease prevalence must be known first. As defined by the CDC, prevalence is "the proportion of persons in a population who have a particular disease or attribute at a specified point in time or over a specified period of time." This is how the CDC calculates disease prevalence:

Disease prevalence of "RSV" can only be determined through cases identified by clinical diagnosis, which the CDC admits is an impossibility due to the nonspecific and overlapping symptoms. There is no way to distinguish "RSV" from "Covid" nor the "flu." As the PCR test results are unable to be interpreted accurately without establishing disease prevalence first, the PCR test is useless as a diagnostic tool. Thus, the health institutions are creating cases of "RSV," "Covid," and the "flu" through PCR testing in order to claim the legitimacy of the PCR results used to generate the cases. This is obviously fraudulent and illogical circular reasoning as the test itself can not be used to create the cases needed in order to determine a disease prevalence rate used to legitimize the results of the test.

It is clear to see for anyone looking at this situation both critically and logically that the same symptoms of disease have been given numerous names associated with various "viruses" over the decades. The rise and reliance on molecular testing to distinguish between these "viruses" is a recent phenomenon that can only be valid if the "viruses" being tested for were purified and isolated directly from the fluids of a sick host and proven pathogenic in a natural way to begin with. However, this has never once been done once for any of these "viruses" and it will be seen that it is no different for "RSV." Let's take a quick peek at the story provided for the discovery of this "virus" before diving in-depth into the papers supplied as evidence:

History

"RSV was discovered in 1956 but was not initially associated with respiratory illness among infants. Indeed, when a group of 14 chimpanzees were noted to be suffering from colds and coryza, Morris and co-workers isolated a new virus originally named chimpanzee coryza agent (CCA). Subsequently, Chanock and co-workers confirmed that the agent caused respiratory illness in humans when they obtained isolates from two children, one with laryngotracheobronchitis and the other with bronchopneumonia, that were indistinguishable from CCA. When specific neutralizing antibody to CCA was found to be present in most school-aged children, "chimpanzee coryza agent" was more appropriately renamed respiratory syncytial virus to denote its clinical and laboratory manifestations."

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173590/

As can be seen from the above passage, "RSV" was originally discovered in 1956 by Morris et al. in chimpanzees, not in children. It wasn't until a year later that Chanock et al. "confirmed" that the chimpanzee coryza agent (CCA) was capable of "infecting" humans based off of "isolates" obtained from two children. However, is this story true? Did either team really isolate a new "virus" which was found in both chimpanzees and humans that was capable of producing respiratory disease? Was a "virus" actually properly purified and isolated directly from the fluids of a sick host and subsequently proven pathogenic in a natural way? Were proper controls carried out for any of the experiments performed and what, if anything, did they show? Let's examine these questions and more by looking at the three papers supplied as the evidence for the discovery of "RSV." I am presenting the entirety of the Morris et al. 1956 paper as well as the first paper in 1957 from Chanock et al. I edited out some of the antibody experimentation from a follow-up study by Chanock et al., also from 1957, for length consideration and because the information was redundant. All three papers are available for download or to look up via the DOI number. To break down the evidence for "RSV," I am trying a different format where I am providing commentary throughout the study rather than summarizing important points beforehand. Let me know in the comments if you prefer one way over the other.

Recovery of Cytopathogenic Agent from Chimpanzees with Coryza.

Right off the bat, we can see that Morris et al. did not seem wildly confident in their discovery as they described the "virus" as being of "apparent" (i.e. seeming to be real or true, but not necessarily so) etiologic significance. They claimed to have established an association between the agent and respiratory illness in a lab worker but then admit that the serological evidence suggested that it could either be the agent that they "discovered" or one that was closely related.

During October, 1955: a respiratory illness characterized by coughing, sneezing and mucopurulent nasal discharge occurred in a colony of 20 "normal" chimpanzees at the Walter Reed Army Institute of Research. The present paper describes the isolation of a virus of apparent etiologic significance in the epizootic, establishes an etiologic association between the chimpanzee coryza agent and respiratory illness in a laboratory worker and finally, presents serologic data suggesting that a number of human beings have experienced infection with the chimpanzee coryza virus or an agent closely related to it.

The materials used for the "isolation" of the "virus" were throat swab samples from 14 of 20 young monkeys said to be clinically ill with coryza, a catarrhal inflammation of the mucous membrane in the nose. A group of older monkeys, which had been previously experimented on for human hepatitis "virus," were used in transmissibility experiments. This invalidates the monkeys as a proper control. The "virus" was grown in tissue cultures which consisted of epithelial-like cells derived from human liver that were incubated in roller tubes with nutrient medium consisting of Eagle's basal medium, inactivated horse serum, and L-glutamine. Penicillin and streptomycin were added to control adventitious bacterial contaminants.

Matcrials and methods. Chimpanzees and collection of specimens. The chimpanzees in the epizootic were 15 to 20 months old and were obtained from a commercial breeder in Dania. Fla., 3 to 24 weeks prior to their illness. They were housed at the Forest Glen Annex of the Walter Reed Army Institute of Research (WRAIR). Blood specimens for serological study were obtained from individual chimpanzees at outset of the epizootic on Oct. 13. 1955, when 5 of the 20 animals were suffering from clinical coryza, and periodically thereafter until Apr. 18. 1956. Throat swabs
were obtained from all animals in the colony on Oct. 17. 1955 when 14 of the 20 animals were clinically ill with coryza; the swabs provided the material employed for viral isolation studies. Another group of somewhat older chimpanzees was used in studying experimental transmissibility of the coryza. The 6 animals in this group had been inoculated previously with material presumed to contain the virus of human infectious hepatitis: they were housed in a different location from the others and had had no direct contact with other chimpanzees for over a year.

Tissue cultures. Cultures of epithelial-like cells derived from human liver (Chang strain) were prepared by the method of Chang(1). The cultures were grown in roller tubes (1.5 x 13 cm) and stationary bottles (4 x 4 x 14 cm) in nutrient medium consisting of 8 parts Eagle's basal medium (2), 2 parts inactivated horse serum, and 0.2 part L-glutamine. Penicillin (100 uniits/ml) and streptomycin (20 ug/ml) were added to control adventitious bacterial contaminants. Tubes and bottles contained 1 ml and 15 ml of nutrient fluid, respectively. The cells were fed on the 3rd or 4th day by replacing the old nutrient. fluid with an equal amount of fresh nutrient. Cultures were incubated at 36°C and at the time of use were usually 4 to 6 days old.

The "virus" was said to be "isolated" from the throat swab specimens in 1 out of the 14 monkeys. The sample was immediately rinsed in nutrient fluid containing antibiotics and then centrifuged for a bit to remove larger particles. At no point was the sample ever checked by electron microscopy for evidence of the assumed "viral" particles within the sample before it was added to either the nutrient fluid or the tissue culture. In fact, there are zero EM images of the supposed "virus" in any of the three papers presented, even after the cell culture experiments were performed. After 4 days of incubation, the fluids were replaced with fresh nutrients/antibiotics, which resulted in the death of the cells and signs of the cytopathogenic effect 4 days later. None of the samples from the 13 remaining monkeys resulted in the same cytopathogenic effects.

Isolation of coryza agent. A fresh (witthin the hour of collection) throat swab from a chimpanzee (Sue) involved in the epizootic was washed in 2 ml of tissue culture nutrient fluid containing antibiotics. After centrifugation at 3000 rpm for 15 minutes to remove large particles, 0.2 ml of the supernatant was inoculated into a roller tube culture of 4-day-old Chang liver cells. After 4 days incubation the original cell nutrient was replaced with fresh nutrient. Four days later cellular degeneration characterized by rounding, granulation, and sloughing from the tube wall was noted. Serial transmission of the cytopathogenic agent to other tube- or bottle-cultures of Chang liver cells was readily
accomplished by passage of suspensions of degenerated cells in their infected fluids. Simlilar isolation attempts which were made with materials obtained on October 17th from 13 other ill chimpanzees gave negative results.

I am glossing over the antibody results, which are nothing but nonspecific chemical reactions stemming from, in this instance, the mixture of human and horse blood. In order for antibody results to have any meaning, antibodies and the antigens themselves must be properly purified and isolated along with the assumed "viral" particles. As this has never occurred once in either case, it is impossible to use results obtained from one invisible hypothetical entity to indirectly claim the presence of another invisible hypothetical entity.

Serologic procedures. Virus. Seed virus was obtained by inoculating bottles of liver cells with the chimpanzee coryza agent (CCA) and harvesting cells and fluids 8 davs later when the infected cells characteristically showed complete degeneration. After grinding in a TenBroeck grinder the mixture was clarified by centrifugation at 3000 rpm for 15 minutes. The resulting supernatant constituted the seed vilrus. Infectivity was preserved by storage at -70° tin sealed glass ampoules.

Neutralization tests. Serial 2-fold dilutions of serum which had been inactivated at 56° for 30 minutes (0.15 ml) were mixed with a constant amount of virus (100 to 1000 tissue culture LD50) contained in 0.15 ml of infected tissue culture material. The mixtures were incubated in a water bath at 37°C for 1 hour after which 0.1 ml of each mixture was added to each of 2 tubes containing normal liver cells. The cultures were examined microscopically for cellular degeneration after a 6- to 8-day incubation period. The neutralization titer was considered the highest dilution of serum completely inhibiting cellular degeneration. Appropriate cell and serum controls and a virus titration were included in each test.

Complement fixation tests. Satisfactory complement fixing antigen was prepared from infected liver cells grown in medium containing 20% inactivated horse serum. When the horse serum component of the medium was not heated at 56° for
1/2 hour the material was anticomplementary if used in the complement fixation (CF) procedure employing overnight fixation in the cold, in accordance with the standard technic of the Department of Virus Diseases, WRAIR (3) which was used in the current studies. For use in CF tests human and chimpanzee sera were inactivated for 30 minutes at 56°C and 60″C, respectively. The serum titer was expressed as the reciprocal of the highest dilution giving 75% or greater fixation of complement after overnight incubation at 4°C in the presence of 2 units of antigen and 2 full units of complement. Controls included in each test were antigen (prepared from uninfected liver cell culltures propagated in inactivated horse serum), positive serum (obtained from a man who experienced a labratory infection, patient B1 in Text Fig. 1, and saline.

The next two sections are rather revealing. In the inoculated culture taken from the sample of one monkey, the cytopathogenic effect was observed after 8 days, and inclusion-like bodies were noted in the liver cell cultures. Inclusion-like bodies are defined as "aggregates of virus particles or virus-induced proteins or special structures characteristic of infection by viruses either in the cytoplasm or the nucleus." Thus, these effects are supposed to be specific to "viruses." However, the exact same inclusion-like bodies were observed in the uninoculated control cultures showing that the presence of an imaginary "virus" was unnecessary for this nonspecific effect to occur. Sadly, this damning information did not faze Morris et al. as they continued to believe that they had a new "virus."

However, if this information was not damaging enough to their conclusions, the attempts to prove pathogenicity should have sunk their battleship entirely. The researchers attempted intracerebral and intraperitoneal inoculations into one-day-old mice, weanling hamsters and young adult rabbits and guinea pigs. They intranasally inoculated 8 to 10 gram mice, young adult rats, and 16 to 20 lb chimpanzees. Groups of chick embryos (7 to 11 days old) were inoculated on the choriolallantoic membrane and into the amnionic, allantoic and yolk sacs. None of the inoculated animals or embryonated eggs, other than chimpanzees, developed signs of disease during observation. The one exception was a single guinea pig that developed a persistent fever beginning on the 3rd day, but this was eventually declared to be bacterial in nature. On top of this lack of pathogenicity, the cytopathogenic effect observed differed based upon the cell line used during culturing. When monkey kidney cells were used, the CPE obtained was difficult to interpret because of the presence in the cultures of adventitious simian "viruses." These are the same invisible foamy agents said to produce CPE in uninoculated cultures as observed by Enders, Rustigian, Cohen, Von Magnus, and Hull. Thus, this is yet another study supplying evidence that cell cultures and the cytopathogenic effect are an invalid and unreliable method to determine the presence of any "virus."

Results. Behavior of chimpanzee coryza agent (CCA) in liver cell culture. Inoculation of CCA obtained from the culture of the throat swab of chimpanzee Sue into liver cell cultures produced little or no change during the first 5 or 6 days. On about the 7th day scattered islands of round and granular cells appeared and a few cells were disintegrated and dislodged from the glass wall of the container. Once begun, the process of degeneration spread rapidly and within 24 hours practically all cells were dead and some were floating in the nutrient fluid. Intranuclear and intracytoplasmic inclusions which are eosinophilic in Giemsa-stained cell preparations, were observed in culltures of liver cells infected with CCA. However, similar inclusion-like bodies were demonstrated in uninoculated cells, grown in inactivated horse serum. At the present time the significance of the inclusion-like structures found in infected and uninfected cells cannot be stated with certainty.

Pathogenicity of CCA for laboratory hosts. Tissue culture materials containing 100 to 10,000 TC LD50 of CCA were inoculated by the intracerebral and intraperitoneal routes into one-day-old mice, weanling hamsters and young adult rabbits and guinea pigs. Other 8 to 10 gram mice, young adult rats and 16 to 20 lb chimpanzees were inoculated intranasally. Groups of chick embryos (7 to 11 days old) were inoculated on the choriolallantoic membrane and into the amnionic, allantoic and yolk sacs. With the exception of a single guinea pig that developed persistent fever beginning on the 3rd day, none of the inoculated animals or embryonated eggs other than chimpanzees developed signs of disease during observation periods ranging up to 28 days. The etiology of fever in the guinea pig was ultimately traced to a bacterial infection. Further, the fluids obtained from chick embryos inoculated by various routes failed to agglutinate chicken and human "0" erythrocytes. Tube cultures of human cells derived from conjunctiva (Chang) , intestine (Henle) and human embryo fibroblasts (otbtained from Microbiological Associates) were found to be less susceptible to the cytopathogenic effect of CCA than liver cells; these cells showed only incomplete degeneration after 16 days incubation. Monkey kidney cells underwent complete degeneration 8 days after infection with CCA but the cytopathogenic effect obtained was sometimes difficult to interpret because of occasional presence in the cultures of adventitious simian viruses (4).

Once again, unreliable antibody results were utilized to claim a relation between animals said to be stricken with the same imaginary "virus." Even when healthy animals developed a rise in antibodies, they were assumed to have had the "virus" even though there was zero evidence that the animals were ever sick. In the healthy animals, which showed neither illness nor antibody rises, it was assumed that they had no contact with the "virus." There is always an escape clause available for unfavorable findings.

Relation of CCA to epizootic coryza in chimpanzees at Forest Glen. CCA was found to be related to the epizootic disease of chimpanzees at the Forest Glen Annex of the WRAIR by the use of serologic technics. Illustrative results of CF and neutralization tests performed on sera from 2 of the chimpanzees in the Forest Glen epizootic are shown in Table I. It is seen that in both animals CF antibody against CCA was undetectable in the early sera but titered 1:40 or 1:80 in the sera taken approximately 2 months later. During the same period there was no significant change in CF antibody titer against the RI-APC-ARD virus or in HA1 antibodies for any of 5 strains of influenza virus. Finally, neutralizing antibody against CCA developed in both chimpanzees during the period between bleedings. The etiologic relation between CCA and the epizootic disease in the chimpanzees is supported further by the data obtained when sera of all 20
animals involved in the Forest Glen epizootic were examined for specific complement fixing antibody. As shown in Table II all 14 chimpanzees that experienced clinical coryza during the 3rd week of Oct., 1955, subsequently developed specific antibody. Four other animals that did not suffer clinical coryza likewise produced antibody, hence, they presumably experienced unrecognized infection. The remaining 2 animals apparently escaped infection; they neither suffered clinical disease nor developed CCA CF antibody.

Morris et al. attempted to experimentally recreate inflammation of the mucous membrane by taking toxic unpurified cell culture supernatant said to contain the "virus" and inject it directly into the noses of three monkeys. Two of the three monkeys developed sneezing, coughing, and nasal discharge after the nose goo injections while the third monkey remained unfazed. In another damning blow to their "viral" theory, two of the three control animals also developed disease after having uninoculated toxic cell culture goo injected into their noses. Thus, no "virus" was necessary to recreate the disease, and it could be easily surmised that the injection of toxic soup with or without an imaginary "virus" resulted in nasal discharge.

Experimentally induced coryza in chimpanzees. Three chimpanzees, 20 to 24 months of age and weighing 16 to 20 lbs were inoculated intranasally on Feb. 2, 1956 with 1.0 ml of 11th passage tissue culture material containing 10,000 TC ID50 of CCA. At the same time 3 other chimpanzees housed in the same room were injected intranasally with an uninfected Chang liver cell preparation. Results of this experiment are presented graphically in Fig. 1. Three days (Feb. 5) after inoculation 2 of the 3 chimpanzees receiving CCA developed respiratory illnesses characterized by sneezing, coughing and subsequently mucopurulent nasal discharge. These signs increased somewhat in severity and persisted at this level for 4 to 5 days; however, the animals were not febrile. By the 14th day the affected chimpanzees were free of signs of respiratory disease. The third chimpanzee in this group (Babe in Fig. 1) remained well throughout the period of observation. Of particular interest is the finding that this anlimal possessed CF and neutralizing antibodies (titers 1:40 and 1:20, respectively) at the time of inoculation. Two of 3 control chimpanzees that were housed with animals inoculated with CCA also developed disease. Onset of illness in these control animals occurred on the 7th (Feb. 9) and 9th (Feb. 11) day after receiving the non-infectious cell cultures, or 4 and 6 days, respectively, after the test animals had first exhibited symptoms. None of these chimpanzees developed fever.

Here we see more evidence that even from the control animals subjected to "non-infectious" materials, the same CPE was produced in the cell culture as seen from that observed in samples from "infected" monkeys thus showing no "virus" was necessary to produce the effect.

From each of the 4 chimpanzees that developed obvious respiratory illness, i.e., 2 test and 2 control animals, an agent cytopathogenic for liver cells was recovered from throat swabs taken on Feb. 8. In addition, a cytopathogenic agent was recovered on Feb. 15 from throat materials from the third chimpanzee receiving non-infectious materials. This animal, (Blondy in Fig. 1) did not show recognizable respiratory disease; nevertheless she developed complement fixing and neutralizing antibodies in minimum amounts. Each of the 5 recovered agents was shown to be similar to or identical with CCA in neutralization tests with specific antisera prepared in rabbits against the Sue strain and in complement fixation tests with a human serum that was known to react with CCA antigen.

It may be mentioned here that the 6 chimpanzees in this experiment (Fig. 1) were challenged by the intranasal instillation of 1 ml of tissue culture material containing 1000 TC LD50 of CCA on March 28, 1956, 55 days after the original exposure when each animal possessed demonstrable CF (range 1:20 to 1:160) and neutralizing (range 1:10 to 1:20) antibodies. All 6 chimpanzees remained free of clinical evidence of disease during an observation period of more than a month; moreover, during the same 30 day period there was no appreciable change in serum antibody titers.

Morris et al. tried very hard to claim that a lab worker was infected with "CCA" by way of antibody results as they were unsuccessful in producing the CPE in the culture from the sample taken from this person. Thus, he was presumed to have been infected based upon nonspecific indirect antibody results after the "gold standard" cell culture failed.

Infection of laboratory worker with CCA. During the second week of February 1956, an illness diagnosed clinically as "upper respiratory infection" occurred in a laboratory
worker who was working with CCA and who had had intimate contact with the experimentally infected chimpanzees. His illness was characterized by several days of nasal snuffiness, rhinorrhea, cough, malaise. followed by several days of low grade fever and frontal headache. CF and neutralizing antibodies against CCA were undetectable in the sera of the patient taken on Feb. 8, 1956. but titered 1:80 on Feb. 22, 1956. The single attempt to recover a cytopathogenic agent from throat washings taken on the 6th day of this man's illness was not successful. The serologic findings which are shown graphically in Fig. 1 (Patient B1) are taken as presumptive evidence that the CCA was of etiologic significance in the patient's illness.

Serologic reaction of CCA with sera obtained from animals immunized against different viruses. Sera obtained from animals immunized against a variety of viruses were examined in complement fixation and neutralizing tests for their ability to react with CCA. The antisera included those prepared in monkeys against the Enders strain of measles virus* (5) , Chanock's croup virus* (6) and Sabin's chimpanzee rhinitis 19 54 virus* (7): in rabbits against several strains of Coxsackie virus, (Group A, type 9 and Group B. types 1, 2, 3 and 4): certain "orphan" viruses (8) [Walter Reed prototypes 7043 (untyped), 7045 (ECHO type 6) and 7054 (ECHO type 2)] and simian virus SV5 (4) and in chickens against simian virus SV59 (4) .) All these anti-sera failled to react with CCA in complement fixation or neutralization tests.

In even more "highly-reliable" antibody testing results, Morris et al. showed that people who were said to have other "viral" infections also possessed the same antibody said to be specific to "CCA."

Occurrence of antibody against CCA in human sera. Results of CF tests to determine the occurrence of CCA antibody in different age groups in the human population are given in Table III. The sera were obtained from patients at the Walter Reed Army Medical Center with a variety of illnesses. It is evident from the tabular data that a number of human beings possessed CF antibodies that react with CCA antigen. Furthermore, such
antibodies were uncommon in children but were present in about 20% of the persons in the small group of adolescents and young adults examined. It is of some interest that the 40 young adults listed in the table were barrack mates of patient B1. Paired sera from groups of patients with common cold, bronchitis, cold agglutinin positive primary atypical pneumonia, and RI-APC-ARD infection (3 pairs in each category) were tested for complement fixing antibody against CCA. Certain of these tests were performed by Dr. Sidney Katz in Dr. John Dingle's laboratory in Cleveland using their sera and antigen supplied by us. None of the patients displayed a significant increase in CCA antibody. Nevertheless, certain of the patients possessed throughout their illnesses constant amounts of CCA antibody with titers ranging up to 1:80.

The summary provides a nice overview of how Morris et al. worked hard to convince themselves that they had uncovered a new "virus" from one monkey even though it was not pathogenic to laboratory animals, could only be presumptively associated by way of indirect nonspecific antibody results with a sickened lab worker, and could only be considered a "virus" if all of the contradictory evidence obtained via controls was ignored. Note that they attempted to say that despite being unable to directly link the new "virus" to human disease, the indirect nonspecific antibody results seen in people with unrelated respiratory diseases suggested that they had contact with "CCA" or an unknown related "virus."

Summary. A virus was recovered from throat materials of a chimpanzee with coryza during an epizootic of (respiratory disease in a colony of these animals. The new agent produced degenerative changes in tissue culture, but was not pathogenic for common laboratory animals. The donor chimpanzee as well as other chimpanzees involved in the epizootic developed specific antibodies against the coryza agent during the months following the outbreak. Susceptible chimpanzees following intranasal instillation of tissue culture materials infected with the coryza agent developed clinical coryza and subsequently made specific antibody. A presumptive etiologic association was established between the new agent and respiratory illness in a laboratory worker, but has not been implicated in the illnesses of small groups of patients with several common types of respiratory disease. However, a number of human beings, particularly adolescents and young adults, have antibodies in their sera directed against the coryza agent suggesting that these individuals have experienced infection with the new agent or one closely related to it.

doi: 10.3181/00379727-92-22538.

morris1956Download

In all honesty, the above paper may very well be the worst evidence of a new "virus" that I have ever had the displeasure of reading. The lengths that Morris et al. went to in order to deny the contradictory evidence seen in the controls so that they could convince themselves that they had a new "virus" was impressive. In order to state with a straight face that they had discovered a "virus," Morris et al. had to:

• Disregard only being able to "isolate" the agent from 1 out of 14 chimpanzees with the same symptoms of disease
• Overlook the fact that the control monkeys used in pathogenicity experiments were older and had already been experimented on for hepatitis "virus" research
• Ignore that the same "viral" inclusion bodies were seen in cultures from both the "infected" and the "uninfected" control monkeys
• Turn a blind eye to the inability to make numerous animals sick through different modes of injection
• Dismiss finding the same "non-viral foamy agents" seen in previous studies using monkey kidney cells which created indistinguishable CPE making interpretation difficult
• Make excuses for why healthy animals showing no signs of illness experienced a rise in antibodies
• Disregard the fact that only 2 out of 3 "infected" animals became sick while 2 out of 3 "uninfected" control animals also became sick
• Dismiss the fact that cytopathogenic "agents" were found in 3 control animals and only in 2 "infected" animals
• Look past the fact that they were unable to isolate a "virus" from a lab worker said to be "infected" and could only presume "infection" based upon nonspecific indirect antibody results
• Rationalize finding the same antibody responses in cases of respiratory disease not said to be caused by their chimpanzee coryza agent

Essentially, Morris et al. completely ignored the contradictory evidence from their control experiments that should have led them to the conclusion that the experimental conditions created the effects observed rather than an imaginary "virus." Instead, Morris et al. found ways to try and rationalize and explain away the contradictions so that they could claim the successful "isolation" of an invisible "virus" that they never observed directly, nor could they prove indirectly through experimentation. We could end this investigation right here with "RSV," as every paper afterward was built upon the fraudulent foundation of the unscientific conclusions and evidence drawn from this single paper.

However, my curiosity always gets the better of me. As the 1957 paper by Chanock et al. was also referenced as being essential to the discovery of "RSV" in children, I had to take a look to see what kind of evidence these researchers uncovered a year later which could potentially salvage the train-wreck I had just read. Presented next is the full 1957 paper with commentary.

Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (CCA). I. Isolation, properties and characterization

To begin with, Chanock et al. claim that two agents recovered from children with respiratory disease were indistinguishable from Morris et al.'s CCA. As usual, no assumed "viral" particles were ever purified and isolated directly from the fluids of the sick children. Instead, throat swabs from infants with respiratory illness and those with nonrespiratory illness were immediately immersed in 5 ml of Hanks' solution containing 2,000 units of penicillin, 2,000 micrograms of streptomycin and 150 units of mycostatin per ml. before culturing. These samples were added to unpurified cell cultured creations consisting of either:

1. Human epidermoid carcinoma grown in either human or horse serum, washed in Hank's solution, and maintained in Eagles medium with chicken serum
2. Human liver epithelium grown in Eagles medium with 20% horse serum which was also maintained in Eagles medium with 4% horse serum
3. Human amnion cultures prepared in Medium 199 with horse serum
4. Monkey kidney cultures maintained in Medium 199 with calf serum added for growth

As can be seen, this process is the exact opposite of purifying and isolating any "virus" and consists of creating a toxic mixture of human and animal fluids with synthetic chemicals.

INTRODUCTION

The present study was undertaken in an attempt to recover new cytopathogenic agents from infants with severe lower respiratory illness (bronchopneumonia, bronchiolitis, and laryngotracheo-bronchitis) and to assess their etiologic significance in this disease complex. During the course of these investigations, two similar viruses were recovered -which were demonstrated to be indistinguishable from an agent associated with an outbreak of coryza in chimpanzees (CCA virus) studied by Morris, Blount and Savage (1). It is the purpose of this communication to describe the isolation, properties and characterization of these agents. In the second paper of this series (2) the epidemiologic aspects of human infection with these viruses will be discussed.

MATERIALS AND METHODS

Tissue culture. The KB strain of human epidermoid carcinoma was grown in the chemically defined medium of Eagle (3) containing either 10 percent normal inactivated (56 C) human or horse serum. Prior to use, the cultures were washed 3 times with Hanks' solution. Maintenance medium consisted of Eagle's medium with 2 percent inactivated chicken serum. The Chang strain of human liver epithelium (4) was grown in Eagle's medium with 20 percent inactivated horse serum and maintained in Eagle's medium with 4 percent inactivated horse serum. Maintenance medium for KB and liver cultures was changed every 3 to 4 days.

Human amnion cultures were prepared according to the method of Takemoto and Lerner (5). Maintenance medium for these cultures consisted of Medium 199 with 4 percent inactivated horse serum. Monkey kidney epithelial cultures were prepared and maintained in Medium 199 (6). For growth, 2 percent inactivated calf serum was added whereas the maintenance medium contained no serum.

Specimens for virus isolation. Throat swabs from infants with respiratory illness and infants with nonrespiratory illness were immersed in 5 ml of Hanks' solution containing 2,000 units of penicillin, 2,000 micrograms of streptomycin and 150 units of mycostatin per ml. Specimens were tested immediately or after storage at — 50 C. Two tenths ml of throat swab fluid was inoculated into each of 3 to 4 monkey kidney epithelium, KB and human amnion cultures.

Once again, I will be glossing over the indirect nonspecific antibody testing and results for the same reasons, as explained with regards to the previous paper.

Infectivity titrations and neutralization tests in tissue culture. These tests were performed as described previously (7). Twenty to 200 TCD50 of virus as measured by simultaneous titration were employed in the neutralization
tests, which were read on the third or fourth day. Chang liver cultures were used in all neutralization tests.

Complement fixation. The method of Osier, Strauss and Mayer (8) was employed. Briefly, 0.2 ml of serum dilution, 5 C'H|s0 (50 percent hemolytic dose) of complement contained in 0.5 ml, and 0.5 ml of antigen were incubated at 3 to 5 C for 18 hours followed by 20 minutes at 37 C, after which 0.3 ml of sensitized sheep erythrocytes was added and the test incubated at 37 C for 1 hour. Positive serum controls, anticomplementary controls and a complement titration were included in each test.

Complement-fixation (CF) antigens were prepared from infected KB cultures which were frozen and thawed twice and centrifuged at 1,500 rpm for 30 minutes. The supernatant fluid containing the antigen was stored at 50 C.

In this next section, we can see that the size of the "virus" was indirectly estimated as no actual "viral" particles were ever observed.

Measurement of particle size. Virus particle diameter was estimated by a modification of the method of Pardee and Schwerdt (9) which will be described in the text. The swinging bucket rotor (SW 39) of the Spinco Model L ultracentrifuge was used in the present experiments. The titrations for infectivity were performed with 3.2-fold dilutions and 8 culture tubes per dilution. The standard error of these titrations, as calculated by the method of Pizzi (10), was less than 10v0.2.

Chanock et al. used the density and particle diameter of "purified polio" to determine the centrifuge constant. However, there is a slight problem as polio was never purified nor isolated, as explained here, here, and here.

The centrifuge constant (K) was determined experimentally with purified type 1 poliovirus whose density and particle diameter are known. A suspension containing 1,000 PFU (plaque-forming units) per ml was employed. Infectivity titrations were carried out at 3-fold dilutions with four 100- millimeter monkey (Erythrocebus patus) kidney monolayer plates per dilution. The value of K obtained was 3.1 X 10^6.

Look at the ridiculous and inconsistent methods used to create antiserum. Why were the rabbits given more injections than the guinea pigs, and why were there additives such as 2 ml of a mixture of Mycobacterium, butyricum, paraffin oil and arlacel? Also note that the CCA agent was provided to Chanock et al. by Morris et. al as was the case for the antiserum of other "viruses," which were all provided by different researchers (including Enders) that used their own methods and recipes to create these substances.

Production of antiserum. Guinea pigs were immunized by 3 weekly intraperitoneal inoculations of 1 ml of infected tissue-culture fluid. Rabbits were given 3 weekly intravenous inoculations of 1 ml each followed by 2 weekly intramuscular inoculations of 1 ml of infected culture fluid combined with 2 ml of a mixture of Mycobacterium, butyricum, paraffin oil and arlacel.

Other viruses. The chimpanzee coryza agent (CCA) was kindly provided by Dr. J. A. Morris and Maj. E. Buescher.

Immune sera for other viruses. Specific antiserum for various viruses was kindly supplied by the following investigators: chimpanzee sera for CCA virus by Dr.'J. A. Morris and Maj. E. Buescher; monkey serum for measles by Dr. J. F. Enders, rabbit and chicken serum for mumps by Dr. F. B. Bang; human serum for psittacosis by Dr. A. G. Osier; and rabbit and human serum for primary atypical pneumonia (PAP) virus by Drs. Chien Liu and M. D. Eaton.

RESULTS

Chanock et al. based their "virus" off of 2 instances of CPE observed in 2 out of 59 patients (one with bronchopneumonia and the other with laryngotracheobronchitis) which could only be produced in the KB and liver cultures but not in the monkey kidney or human amnion cultures. The other 57 patients suffering through the exact same symptoms must not have had any "virus" based on these findings. It was noted that serial passaging the culture led to a quicker observation of the CPE. However, serial passaging is known to affect the cell culture as it is disturbed, and the toxic chemicals are refreshed, leading to quicker cell death.

Isolation of agents. An unusual cytopathogenic agent was recovered in KB culture from the throat swab of one of 41 patients with bronchopneumonia (patient Long) and from one of 18 individuals with laryngotracheobronchitis (patient Snyder). Henceforth these agents will be referred to as Long virus and Snyder virus. Attempts to recover these agents after inoculation of throat swab fluid into monkey kidney and human amnion cultures were unsuccessful, although these cultures were held for 3 to 4 weeks in good condition.

The behavior of these agents during their early tissue-culture passages is shown in table 1. The interval before cytopathogenic effects were observed after inoculation of Long virus decreased during the second and third KB passages. The behavior of the virus during the third KB passage was typical of the virus in subsequent passages.

Long virus was reisolated from the original throat swab fluid in cultures of human liver epithelium (Chang strain). It is of interest that the incubation period during the first passage in liver was shorter than that observed in KB culture.

Patient Long developed both CP (a titer of < 1 in 4 in the acute serum and 1 in 80 in the convalescent serum) and neutralizing antibodies (a titer of < 1 in 4 in the acute serum and 1 in 64 in the convalescent serum) for Long virus. Patient Snyder developed CF antibody for Long virus ( a titer of < 1 in 4 in the acute serum and 1 in 8 in the convalescent serum) but tests with the homologous virus could not be performed because of insufficient serum.

Here, we can see the lengths the researchers will go to in order to create the cytopathogenic effect that they want to observe. While Chanock et al. could not culture their "virus" in human amnion cells from the throat swab fluids of sick patients, they claimed that after culturing the "virus" in KB cells that they could get an effect in human amnion cells, albeit after a much longer culturing period and never a complete effect. In other words, they created an unpurified mixture in KB cells and added this to the human amnion culture, which was obviously more toxic to the cells than the original throat swab samples containing less contaminants, and claimed success even though the CPE took much longer to observe and was incomplete.

Cytopathogenic effect in tissue culture. Both Long and Snyder agents produced cytopathogenic effects in liver and KB cultures. With undiluted tissue-culture fluid these effects were observed within 2 to 3 days while minimal quantities of virus required 5 to 8 days. After isolation in KB cultures, Long virus produced an effect in human amnion culture. A longer interval was required in amnion cultures before cytopathogenic changes were observed and progression was very slow. Thus Long KB passage 1, when inoculated into KB cultures, produced an effect within 3 days and almost complete destruction of the cell sheet occurred by the fifth day, whereas amnion cultures inoculated simultaneously with the same material did not show an effect until 9 to 14 days, and the entire cell sheet was not involved by the twenty-second day.

The sensitivity of KB and liver cultures for Long virus was the same. A simultaneous titration of Long KB passage 5 in liver and KB cultures gave the same titer, i.e., 10^5 TCDv50 per ml.

The KB cells, which succumbed to the toxic recipe faster, were shown to have further damage than the liver and amnion cultures by the appearance of round cell degeneration.

The most striking effect of Long virus in tissue culture was the formation of syncytial areas. In KB cells a round cell degeneration also occurred but syncytium formation predominated. In liver and amnion cultures only syncytial areas were seen. The syncytial areas were characterized by a loss of cell boundaries and the accumulation of a varying number of intact nuclei in a region of homogeneous cytoplasm (figures 1 and 2). The chromatin pattern of the nucleus was altered (figure 3). Thick chromatin strands appeared to radiate peripherally from a thick central mass indistinguishable from an inclusion body. Involved areas of infected liver cultures were similar to those seen in KB cultures.

Cytopathogenic changes were first seen as small circumscribed syncytial areas which were randomly distributed and which enlarged over a 24-hour period, at which time numerous other syncytial areas made their appearance. Usually, within 1 to 4 days the entire cell sheet was involved.

As no "viral" particles were ever observed, Chanock et al. utilized indirect means to estimate the size of the invisible particles. They extrapolated (the action of estimating or concluding something by assuming that existing trends will continue or a current method will remain applicable) from centrifugation in sucrose solutions. The researchers admitted that the method they used was a crude estimate and needed confirmation by more refined measures. Perhaps having the actual "viral" particles visually on hand would help?

Physical properties. The diameter of Long virus was determined by centrifugation in sucrose solutions of varying concentrations by a modification of the method of Pardee and Schwerdt (9). Supernatant fluids were titrated for the residual infectivity. When the percentage of unsedimented virus was plotted against sucrose density the sucrose density at which all virus was sedimented was obtained by extrapolation. The sucrose density at which no virus was sedimented was also obtained by extrapolation. The viscosity corresponding to the extrapolated sucrose density values was calculated from the data given by Bates (11). From these values the particle diameter was derived by the formula given in table 2. In two independent experiments particle diameters of 105 and 107 millimicrons were obtained. The corresponding hydrated particle density values were 1.19 and 1.26. If these density values are taken as a range then the particle diameter may be estimated to be within a range of 90 to 130 millimicrons. It should be noted that the method employed for the determination of size merely provides a rough estimate that will require confirmation by more refined methods.

The antigen responsible for complement fixation appears to have a smaller sedimentation constant than the infectious particle. Thus, centrifugation at 30,000 r. p. m. for 2.5 hours in the number 30 rotor of the Model L Spinco ultracentrifuge (78,410 X G average) failed to diminish the CF antigen titer of the supernatant fluid. Following additional centrifugation at 40,000 r.p.m. for 3 hours in the number 40 rotor (105,000 X G average) the potency of CF antigen in the supernatant fluid remained unchanged.

It was possible to dissociate infectivity and CF antigen activity by centrifugation as shown in table 3. CF antigen was quantitatively recovered from the supernatant fluid while the pellets contained 90 percent of the recoverable infective virus without any detectable CF antigen.

In this section, Chanock et al. tried every which way to get their "virus" to infect chickens or multiply in embryonated eggs but were unsuccessful at every attempt. They could also not get their "virus" to sicken one-day-old mice by way of injection into the brain or into the space between the muscles and organs in the abdomen.

Other properties. Exposure of Long virus to 20 percent ether for 16 hours at 4 C resulted in complete loss of infectivity. Attempts to demonstrate a hemagglutinin for chicken or human "O" erthrocytes were unsuccessful. No pocks were seen 3 days after inoculation of 10 and 100 TCDv50. of Long virus onto the chorioallantoic membrane of the 11-day embryonated egg. Multiplication of Long virus could not be demonstrated when 9-day embryonated eggs were inoculated with 10,000 TCD50 into the amniotic sac and incubated for 6 days. Long virus was not pathogenic for 1-day-old mice by the intracerebral or intraperitoneal route. Long virus could be filtered through gradocol membranes of 590 and 960 millimicrons average pore diameter.

Chanock et al. attempted to connect the Long and Snyder "virus" to Morris et al.'s CCA by way of the pattern of CPE observed in liver and KB cultures as well as indirect nonspecific antibody results. Note that neither the CCA nor the Long/Snyder "viruses" had ever been observed in order to be characterized nor compared. The researchers also used these same indirect means, mostly relying on antibody results, to claim that their "virus" was unrelated to other "viruses."

Relationship to CCA virus. CCA virus was received in the laboratory after
Long virus had been isolated. CCA virus produced a cytopathogenic effect in liver and KB cultures which was identical to that seen with Long and Snyder viruses. By CF tests with paired human and chimpanzee sera (table 4) Long and Snyder viruses were indistinguishable from CCA virus. In neutralization tests with sera from immune guinea pigs and rabbits (table 5). Long, Snyder and CCA viruses reacted in a similar manner except for the tests with CCA immune guinea-pig serum. Although the postimmunization CCA guinea-pig serum neutralized Long virus, the homologous titer with CCA virus was 8-fold higher.

Relationship to other viruses. The cytopathogenic effects produced by Long virus was distinct from that characteristic of the adenovirus group. Long virus was not related antigenically to the adenoviruses as shown by the nonassociation of rises in CF antibody for Long and adenoviruses in infants with
or without respiratory illness (table 6). The history of these infants will be described more completely in the accompanying communication (2).

Long virus was not antigenically related to other human viruses which produce syncytial changes in tissue culture (table 7).

Long virus was distinct from the primary atypical pneumonia (PAP) virus of Liu and Eaton (12). A 1-in-4 dilution of a serum from a PAP immune rabbit (homologous titer of 1 in 256) failed to neutralize Long virus. In addition, CF antibody for Long virus was not detected nor was a change in Long neutralizing antibody observed with paired serum specimens from a PAP patient who developed neutralizing antibody for PAP virus during convalescence.

Long vims was not related to the psittacosis-LGV group of viruses since a human serum with a psittacosis CF titer of 1 in 160 failed to fix complement in a l-in-5 dilution with Long CF antigen.

DISCUSSION

In the discussion section, we get the statement that the CPE and antigenic properties between Long/Snyder and CCA were similar (not identical as previously stated) and yet they claim their agents were indistinguishable from each other. However, a pretty revealing statement was made where Chanock et al. admit that their findings did not prove that Long and Snyder "viruses" were responsible for the associated illnesses in infants. Enough said.

Morris, Blount and Savage recovered a virus (CCA) from an outbreak of mild respiratory illness in chimpanzees (1). This illness, was reproduced in susceptible chimpanzees by the intranasal instillation of tissue-culture CCA virus. In the present study two viruses (Long and Snyder), which were similar in tissue-culture cytopathogenic effect and antigenic properties with CCA virus, were recovered from infants with severe lower respiratory illness. The development of antibody to Long virus during the convalescence of these infants confirmed that Long and Snyder viruses were derived from human sources. It is clear, therefore, that agents indistinguishable from CCA virus are capable of causing human infection. However, this does not prove that Long and Snyder viruses were responsible for the associated illnesses in these infants. This problem and the role of this group of agents in respiratory illness of infancy will be considered in the accompanying communication (2).

The most striking property of Long and Snyder viruses was their ability to produce large syncytial areas in KB and liver cultures. Of the viruses which infect human beings only measles, mumps and CA viruses have been previously shown to produce syncytial changes in cultures of various types (7, 13, 14). Antigenically these viruses are distinct from the agents isolated in this study. In addition, mumps and CA viruses can be distinguished by their ability to agglutinate human and chicken erythrocytes. Measles virus produces syncytial changes in KB cultures but at least 5 weeks are required for complete cell destruction (15) whereas Long and Snyder viruses destroy the cell sheet within 3 to 6 days.

Similar to the adenoviruses, Long virus produces a CF antigen distinct from the infectious particle. However, the type of cytopathogenic effect produced in KB cultures, ether sensitivity and the lack of antigenic relationship in CF tests with human sera clearly distinguish Long virus from the adenovirus group.

SUMMARY

Two viruses, which were indistinguishable from an agent associated with coryza in chimpanzees (CCA virus), were recovered from infants with lower respiratory illness. The agents isolated in this study were characterized by the occurrence of a syncytial cytopathogenic effect in KB or human liver tissue culture and the production of a CF antigen which was separable from the infectious particle by centrifugation. The particle diameter was estimated to be 90 to 130 millimicrons. These agents were not related to the adenovirus group nor to other currently known viruses which cause respiratory illness.

https://doi.org/10.1093/oxfordjournals.aje.a119901

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What we got from Chanock et al.'s first paper from 1957 was the usual cell culture concoction that was apparently very difficult to create. It took many cell lines and various tricks by the researchers to try and get the preconceived cytopathogenic effect that they wanted to see. Even then, the researchers were largely unsuccessful with their attempts and had to rely on indirect nonspecific antibody results to try and claim the "virus" was present and that it was somehow related to the CCA "virus" that Morris et al. "discovered" in chimpanzees the year prior. It is therefore understandable, given the incredibly flimsy evidence obtained, why Chanock et al. stated their evidence did not prove that Long and Snyder "viruses" were responsible for the associated illnesses in infants. Thus, they left it to a second paper to provide this evidence for them. Ironically, it did no such thing.

With Chanock et al.'s second paper from 1957, you will see that the "proof" that was promised was just as poor, if not more so, than that presented within the first paper. I provided commentary and highlights for the pertinent information but edited out much of the invalid antibody results. If you desire to see the researchers' attempt to use one hypothetical entity to try and prove another, please feel free to download the linked paper.

Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (CCA). II. Epidemiologic aspects of infection in infants and young children

In the introduction, Chanock et al. attempt to make a case that Morris et al. had discovered a "virus" in captive chimps by "finding" evidence of a "virus" in 1 out of 14 chimpanzees. However, they were unable to "isolate" the same "virus" from a sickened lab worker. Fortunately, the researchers were able to use indirect nonspecific antibody results to claim that the invisible "virus" truly was present even though it could not be found nor observed.

INTRODUCTION

"Morris, Blount and Savage (1) studied an outbreak of coryza in a colony of chimpanzees held under observation for 3 to 24 weeks prior to the onset of illness. A virus (CCA) was recovered from one of the 14 affected chimpanzees and the remaining 13 animals developed antibody to this virus during convalescence. A person working with the infected chimpanzees subsequently experienced a respiratory infection and, although virus isolation attempts were unsuccessful, a rise in antibody for CCA virus was observed during convalescence. When susceptible chimpanzees were inoculated intranasally with tissue-culture CCA virus, coryza was observed after a 3-day incubation period. These findings suggested the possibility that CCA was a virus of human origin which produced an outbreak of mild respiratory illness when introduced into a susceptible population of chimpanzees.

Of their own findings, Chanock et al. stated that antibody results from children where "viruses" were recovered led them to believe that their invisible "viruses" were indistinguishable from Morris et al.'s CCA chimpanzee "virus." However, they admitted that the results could not be interpreted to mean that these "agents" were the cause of the illnesses in the infants.

During a study of infants with lower respiratory disease (2) two agents (Long and Snyder) were recovered which were indistinguishable from CCA virus. The infants from whom the viruses were recovered developed antibody during convalescence. From these findings it is clear that viruses which are indistinguishable from CCA virus are capable of infecting human beings. These results cannot be interpreted to mean that these agents were the cause of the illnesses in the infants, since the temporal association of disease and infection with an agent is only the first step in the chain of evidence required for etiologic significance (3).

Oddly enough, only infants considered to have severe lower respiratory disease were studied. Children with mild respiratory disease were not studied as these symptoms were said to be common and more difficult to diagnose accurately.

This communication will describe preliminary investigations which were designed to provide an understanding of the epidemiology and pathogenicity for infants of the CCA-Long-Snyder group of agents. Infants with severe lower respiratory illness and a control group of infants without such illness were studied. Mild respiratory illnesses were not studied because of the frequency with which these illnesses occur during the winter months in this age group (4), and since clinical diagnosis is less accurate than in infants with severe illness.

MATERIALS AND METHODS

In the methods section, it can be seen that the children studied were those with severe lower respiratory illness and that controls consisted of infants and children without respiratory illness. The children with respiratory illness were diagnosed with non-bacterial pneumonia as their illness was said not to resemble bacterial infection, even though both bacterial and "viral" pneumonia have the same clinical picture. The controls differed from the respiratory illness cases in that the proportion of hospitalized individuals was smaller and were, on average, older than the patients with respiratory illness.

Study population. The study population consisted of infants and children under 4 years of age with severe lower respiratory illness and a control group of infants and small children who did not have respiratory illness at the time specimens for virus isolation were obtained. The subjects were from a low socioeconomic environment.

Infants and small children up to the age of 4 years who were admitted to either the Harriet Lane Home of the Johns Hopkins Hospital, Baltimore, Md., or the Pediatric Division of the Baltimore City Hospitals with the diagnosis of bronchopneumonia or bronchiolitis constituted the hospital respiratory group. Patients up to 4 years of age with bronchopneumonia who were seen in the outpatient clinic of the same institutions constituted the outpatient respiratory group. Bronchopneumonia was diagnosed when tachypnea, fine moist rales and, in most instances, pulmonary infiltration occurred. The clinical course of the patients selected for this study did not resemble that of a bacterial pneumonia. Chest x-rays were taken on all ward patients with pneumonia and on the majority of the outpatients with pneumonia. Bronchiolitis was diagnosed when tachypnea, expiratory wheezing, prolongation of expiration, and evidence of emphysema were observed. In addition, it was not uncommon for moist rales and x-ray evidence of pulmonary infiltration to occur in infants with bronchiolitis.

The controls were children with non-respiratory illnesses from the outpatient clinics of the Harriet Lane Home and Baltimore City Hospitals and from the pediatric ward of the latter hospital. Patients with infectious disease were not admitted to this ward.

The study extended from October, 1956, to March, 1957. The respiratory patients and the controls were distributed over this 6-month intervaL

The same procedure was employed in studying the various groups. A throat swab and a blood specimen were obtained at the time of admission to the hospital or during the first clinic visit. An attempt was made to obtain a second serum sample 4 weeks later. In many instances a second sample could not be secured until after an interval of 6 to 10 weeks, and in several instances 12 weeks.

Table 1 shows the number of children in each group, their average age in months and the mean intervals between serum samples. It will be seen that the controls differed from the respiratory illness cases in that the proportion of hospitalized individuals was smaller. The controls were, on the average, older than the patients with respiratory illness.

Laboratory procedures. The techniques for virus isolation, complement-fixation (CF) and neutralization tests in tissue culture have been described (2)."

Amazingly, we find out that the Long "virus" was only able to be "isolated" from one infant with bronchopneumonia. Attempts to "isolate" the "virus" from 12 infants with bronchopneumonia, 28 infants with pneumonia, and 90 infants with bronchiolitis were all unsuccessful. They were also unable to "isolate" the "virus" from 151 controls. When attempting liver epithelium cultures for the respiratory patients and controls who showed an antibody response, all results were negative except for the one infant that the "virus" was recovered from. The incidence of "infection" with the "virus" as identified by CP and neutralization tests were not significantly different from the control group. This should show that the antibody results are entirely meaningless.

"Relation of Long virus to pneumonia and bronchiolitis of infancy. Long virus was isolated once in KB tissue culture from the throat swab fluid of a clinic infant with bronchopneumonia. Twelve other isolation attempts with throat swabs from clinic infants and children with bronchopneumonia were unsuccessful as were isolation attempts with specimens from 28 infants with pneumonia and 90 infants with bronchiolitis who were hospitalized. Long virus was not isolated from the throat swabs of 151 control individuals. The throat swabs of respiratory patients and controls who developed a rise in antibody to Long virus were tested in liver epithelium cultures with negative results except for the one patient from whom virus was isolated in KB culture.

In the group of infants hospitalized with pneumonia or bronchiolitis the incidence of infection with Long virus as determined by CP (table 6) or the neutralization technique was not significantly different from that of the control group. The control group was not completely satisfactory in that the average age was considerably older than the average for either respiratory group (table 1). However, the control infants whose age matched that of the respiratory patients exhibited the same incidence of CF antibody development (3 of 12 (25 percent)) as the older controls (4 of 17 (24 percent)). It is possible that some of the respiratory illnesses which required hospitalization were associated with Long virus but this would be difficult to establish because of the high rate of infection in the control group.

The incidence of Long virus infection among outpatients with bronchopneumonia and among outpatient controls is shown in table 7. The mean age and mean interval between serum samples were similar in both groups (table 1).

While Chanock et al. attempted to make a link between their "virus" and bronchopneumonia by way of indirect nonspecific antibody results, they admitted that the small sample size meant that the conclusion could only be tentative and that any association between the two awaited future studies.

It is important to note that the 13 individuals with bronchopneumonia were distributed over the 6-month period of the study and that cases of bronchopneumonia with an antibody rise for Long virus occurred during each of the months except December. The significantly higher rate of infection among the outpatients with bronchopneumonia suggests that an etiologic association exists between Long virus and a certain segment of the bronchopneumonia syndrome. However, since the number of outpatients with bronchopneumonia was small (13 individuals), any conclusions derived from this study must be regarded as tentative in nature. The firm establishment of an etiologic association between Long virus and bronchopneumonia must await future studies.

Detailed information on the 13 clinic patients with bronchopneumonia is
given in table 8. The isolation of pneumococcus or B hemolytic streptococcus from 3 of the 10 nose and throat cultures does not constitute an unusually high proportion of positive findings during the winter months as judged by the results presented by Babe (5). The clinical course of the 3 patients from whom bacterial pathogens were recovered was similar to that of the other patients with pneumonia and did not resemble a bacterial illness.

DISCUSSION

In the discussion, we can see how they try to utilize non-specific antibody reactions to indirectly claim the presence and relation of different "viruses." In this case, 41 infants with disease and 16 control infants without disease had some form of antibody response. Even though they could not "isolate" the "virus," it was claimed that these antibody results showed that "infections" were frequent. They compared their inability to "isolate" a "virus" with that of Morris et al. as if this somehow meant that both teams of researchers were successful in identifying the same "virus" even though neither team could "grow" the "virus" regularly. Interestingly, Chanock et al. admitted that CF antibody responses were assumed to indicate and measure infection and then tried to come up with various explanations for why their antibody results did not reflect their expectations. It was stated that it was not possible to determine which of the alternatives they dreamt up offered the correct explanation.

The isolation from infants with lower respiratory disease of two viruses which were indistinguishable from CCA virus indicated that agents of this group are capable of infecting human beings (2). Additional support for this conclusion was the finding that 41 infants with lower respiratory illness and 16 control infants without respiratory disease developed CF antibody, neutralizing antibody, or both, to Long virus during the course of this study.

The frequent occurrence of infection when compared with the infrequent isolation of virus suggests that Long virus is recoverable from the throat during a very short interval following infection. However, it is probable that many of the infections diagnosed by serologic methods in this study occurred after the throat swab was obtained and during the interval between the collection of serum samples. Morris, Blount and Savage (1) experienced similar difficulty in isolating CCA virus from chimpanzees with respiratory illness. In the epizootic of coryza studied by these investigators virus was recovered from only one of 14 affected chimpanzees 4 days after the first respiratory signs were noted.

Acquisition of neutralizing antibody to Long virus occurred very early in life and at a rapid rate. In the present study population, 77 percent of children 3 years of age and 80 percent of children 4 years of age possessed neutralizing activity in their serum. From these findings one would anticipate that infection of older individuals would occur infrequently. However, Morris, Blount and Savage (1) reported that 24 percent of individuals in the age group 10 to 18 years possessed CF antibody for CCA virus. We have observed a similar incidence of CF antibody for Long virus in a group of 67 prisoners (21 through 29 years old) from a Federal prison in Ohio.

Assuming that CF antibody is a measure of recent or persisting infection the findings just described could result from: (a) the occurrence of reinfection during the second and third decade at a time when neutralizing antibody levels have decreased below a certain critical level, (5) the occurrence of persisting infection in a certain proportion of individuals, (c) differences in the make-up of the various study populations, or (d) the occurrence of antigenically related but distinct agents which share a common CF antigen with Long virus. At the present time it is not possible to determine which of these alternatives offers the correct explanation."

In this next section, we get a pretty glaring admission from Chanock et al. as it is admitted that they could not demonstrate an association between their "virus" and lower respiratory disease in infants. Today, "RSV" is considered the leading cause of lower respiratory disease in infants, so it is quite telling that they could not demonstrate a relationship. The researchers theorized that they could not make a link due to the high rate of "infection" in the control group. It was also admitted that they could make no definitive statement on the pathogenic spectrum of the "virus" in man. All they could say was that from their study, in the majority of infants, "infection" resulted in either mild illness or no discernible disease. All the researchers could state was that they suspected an association with lower respiratory disease in infants.

"An association between Long virus and lower respiratory disease in hospitalized infants was not demonstrable. Possibly, however, such an association does exist but was overshadowed by the high rate of infection among the hospital control group. The lower rate of infection among the clinic controls was considerably less than that for outpatients with bronchopneumonia. Although this observed difference appears to be significant, the number of clinic bronchopneumonia patients was sufficiently small (13 individuals) that the conclusions derived from the data must be considered as tentative.

At the present time a definite statement cannot be made regarding the spectrum of pathogenicity of Long virus for man. It is possible to state that this virus was prevalent among young children in this community during the study period and that in the majority of infants infection resulted in either mild illness or no discernible disease.

Since (a) CCA virus has been shown to produce mild upper respiratory illness in chimpanzees, (b) Long and Snyder viruses were recovered from the throat of infants with lower respiratory illness, (c) Long and Snyder viruses are indistinguishable from CCA virus, (d) Long virus is suspected of an association with bronchopneumonia in infancy, and (e) the striking characteristic of these viruses is the production of syncytial areas in tissue culture, it is suggested that these agents be grouped together and named "respiratory syncytial" (RS) virus until their epidemiology and pathogenicity are better understood.

SUMMARY

In the summary, Chanock et al. wanted to remind us that their flimsy case rested on evidence that was based upon the "recovery" of a "virus" from one infant with bronchopneumonia and that an association of Long "virus" with severe lower respiratory illness in hospitalized patients could not be demonstrated. In other words, they had nothing.

A study was carried out to elucidate the epidemiology of an agent (Long virus) which was recovered from an infant with bronchopneumonia and shown to be indistinguishable from chimpanzee coryza agent (CCA). The neutralization test was found to be twice as sensitive as the complement-fixation technique in the serologic diagnosis of infection in infants and small children. In the present study population, 80 percent of children 4 years of age possessed neutralizing activity for Long virus in their serum. Seven percent of nonhospitalized control infants and children, without overt respiratory illness at the time of initial bleeding, developed antibody for Long virus during an average interval of 6 weeks in the winter months. The rate of infection among a similar control group who were hospitalized on a ward which did not admit patients with infectious disease was 6 times greater than among the outpatient controls from the same hospital.

An association of Long virus with severe lower respiratory illness in hospitalized patients could not be demonstrated, possibly because of the high rate of infection occurring in the ward control group. The data suggested an association between Long virus and bronchopneumonia occurring in outpatients. However, the number of out-patients with bronchopneumonia was small (13 individuals) and thus the conclusions derived from these data must be considered tentative.

It is proposed that the agents isolated in this study and the CCA virus be grouped together and named "respiratory syncytial" (RS) virus."

https://pubmed.ncbi.nlm.nih.gov/13478579/

chanock1957-1Download In Summary:

• "RSV," "Covid," and the "flu" have symptoms that are similar, which can make them difficult to tell apart
• The three "viruses" are said to be more similar to each other than they are different in terms of symptoms
• The absence of one of the symptoms does not mean a patient doesn't have a particular "virus" and the only way to be sure is to get tested
• If anybody has generic symptoms, such as fever, cough, runny nose, there's no real way to distinguish which one is which without a test
• The CDC confirmed that clinical symptoms of "RSV" are nonspecific and can overlap with other "viral" respiratory infections, as well as some bacterial infections
• "RSV" was discovered in 1956 but was not initially associated with respiratory illness among infants
• Morris and co-workers "isolated" a new "virus" from chimpanzees originally named chimpanzee coryza agent (CCA)
• Subsequently, Chanock and co-workers "confirmed" that the agent caused respiratory illness in humans when they obtained "isolates" from two children

• Morris et al. stated that the paper described the "isolation" of a "virus" of apparent etiologic significance (seeming to be real or true, but not necessarily so) in the epizootic
• They felt that they had established an etiologic association between the chimpanzee coryza agent and respiratory illness in a single laboratory worker
• Morris presented serologic data suggesting that a number of human beings experienced infection with the chimpanzee coryza "virus" or an agent closely related to it (there goes any claim of specificity)
• Throat swabs from 14 of 20 young monkeys with coryza (inflammation of mucous membrane) provided the material employed for "viral isolation" studies
• Another group of somewhat older chimpanzees was used in studying experimental transmissibility of the coryza and the 6 animals in this group had been inoculated previously with material presumed to contain the "virus" of human infectious hepatitis (thus invalidating them as a valid control)
• Tissue cultures of epithelial-like cells derived from human liver were grown in roller tubes in nutrient medium consisting of 8 parts Eagle's basal medium, 2 parts inactivated
  horse serum, and 0.2 part L-glutamine along with Penicillin (100 uniits/ml) and streptomycin (20 ug/ml) which were added to control adventitious bacterial contaminants
• A throat swab from one chimpanzee (Sue) involved in the epizootic was washed in 2 ml of tissue culture nutrient fluid
  containing antibiotics
• After 4 days incubation the original cell nutrient was replaced with fresh nutrient/antibiotics
• 4 days later, the cytopathogenic effect was observed as the cell died and a "virus" was claimed to be "isolated"
• Similar "isolation" attempts, which were made with
  materials obtained from 13 other ill chimpanzees, gave negative results (i.e. they could only "isolate" the "virus" from 1 of the 14 monkeys)
• CPE was observed from the one cultured sample after 8 days and inclusion-like bodies (i.e. aggregates of "virus" particles or "virus-induced" proteins or special structures characteristic of infection by "viruses" either in the cytoplasm or the nucleus) were seen in the inoculated culture
• However, similar inclusion-like bodies were demonstrated in uninoculated cells, grown in inactivated horse serum
• The researchers did not know the significance of the inclusion-like structures found in infected and uninfected cells and that nothing could be stated with certainty
• In other words, they found the exact same "viral" structures in the uninoculated control cultures as observed in the one inoculated culture thus proving this effect was not dependent on the presence of an imaginary "virus"
• With the exception of a single guinea pig that developed persistent fever beginning on the 3rd day, none of the inoculated animals or embryonated eggs other than chimpanzees developed signs of disease during observation periods ranging up to 28 days
• The etiology of fever in the guinea pig was ultimately traced to a bacterial infection
• Monkey kidney cells underwent complete degeneration 8 days after infection with CCA but the cytopathogenic effect obtained was difficult to interpret because of the presence in the cultures of adventitious simian "viruses"
• CCA was found to be related to the epizootic disease of chimpanzees at the Forest Glen Annex of the WRAIR by the use of serologic techniques
• Four other animals that did not suffer clinical coryza likewise produced antibody, hence, they presumably experienced unrecognized infection
• The remaining 2 animals apparently escaped infection; they neither suffered clinical disease nor developed CCA CF antibody
• Three monkeys were inoculated intranasally with the "virus" soup and three days later, 2 of the 3 chimpanzees receiving CCA developed respiratory illnesses characterized by sneezing, coughing and subsequently mucopurulent nasal discharge
• Two of 3 control chimpanzees that were housed with 
  animals inoculated with CCA also developed disease after receiving the non-infectious cell cultures
• In other words, no "virus" was necessary to experimentally recreate respiratory disease using either "infectious' or "non-infectious" toxic cell culture soup injected directly into the nasal cavities of monkeys
• From each of the 4 chimpanzees that developed obvious respiratory illness, i.e., 2 test and 2 control animals, an "agent" cytopathogenic for liver cells was recovered from throat swabs taken on Feb. 8
• In addition, a cytopathogenic agent was recovered on Feb. 15 from throat materials from the third chimpanzee receiving non-infectious materials
• Thus, the same CPE was observed in the cell cultures prepared from the 3 "non-infected" control animals as observed in the 2 "infected" animals which once again demonstrates that no "virus" is necessary to produce this effect
• The single attempt to recover a cytopathogenic "agent" from throat washings of a lab worker who experienced respiratory symptoms was not successful
• Thus, indirect nonspecific serologic findings were taken as presumptive evidence that the CCA was of etiologic significance in the patient's illness
• Paired sera from groups of patients with common cold, bronchitis, cold agglutinin positive primary atypical pneumonia, and RI-APC-ARD infection (3 pairs in each category) were tested for complement fixing antibody against CCA
• None of the patients displayed a significant increase in CCA antibody yet certain patients possessed throughout their illnesses constant amounts of CCA antibody with titers ranging up to 1:80
• In other words, the antibodies claimed to be specific to "CCA" were found in patients not said to be suffering from "CCA"

• During the course of the investigations by Chanock et al., two similar "viruses" were recovered which they claimed were demonstrated to be indistinguishable from an agent associated with an outbreak of coryza in chimpanzees (CCA)
• The KB strain of human epidermoid carcinoma was grown in the chemically defined medium of Eagle containing either 10 percent normal inactivated human or horse serum
• Prior to use, the cultures were washed 3 times with Hanks' solution and maintained in medium consisting of Eagle's medium with 2 percent inactivated chicken serum
• The Chang strain of human liver epithelium was grown in Eagle's medium with 20 percent inactivated horse serum and maintained in Eagle's medium with 4 percent inactivated horse serum
• Maintenance medium for KB and liver cultures was changed every 3 to 4 days
• Human amnion cultures were prepared according to the method of Takemoto and Lerner with maintenance medium for these cultures consisting of Medium 199 with 4 percent inactivated horse serum
• Monkey kidney epithelial
  cultures were prepared and maintained in Medium 199 while 2 percent inactivated calf serum was added for growth
• Throat swabs from infants with respiratory illness and infants with nonrespiratory illness were immersed in 5 ml of Hanks' solution containing 2,000 units of penicillin, 2,000 micrograms of streptomycin and 150 units of mycostatin per ml
• "Virus" particle diameter was estimated by a modification of the method of Pardee and Schwerdt (as they never observed any "viral" particles)
• The centrifuge constant (K) was determined experimentally with "purified" type 1 "poliovirus" whose density and particle diameter were said to be known (that's odd as "polio" was never purified nor isolated….)
• To create antiserum, guinea pigs
  were immunized by 3 weekly intraperitoneal inoculations of 1 ml of infected tissue-culture fluid while rabbits were given 3 weekly intravenous inoculations of 1 ml each followed by 2 weekly intramuscular inoculations of 1 ml of infected culture fluid combined with 2 ml of a mixture of Mycobacterium, butyricum, paraffin oil and arlacel
• An unusual cytopathogenic "agent" was recovered in KB culture from the throat swab of one of 41 patients with bronchopneumonia (patient Long) and from one of 18 individuals with laryngotracheobronchitis (patient Snyder)
• Attempts to recover these agents after inoculation of throat swab fluid into monkey kidney and human amnion cultures were unsuccessful
• In other words, they could only find the "virus" in 2 out of 59 patients suffering from the same symptoms of disease and they could only culture it in KB and liver cultures but not in monkey kidney nor human amnion cultures
• The interval before cytopathogenic effects were observed after inoculation of Long "virus" decreased during the second and third KB passages (not surprising as serial passaging speeds up cell death as the culture is disturbed and the toxic chemicals are refreshed)
• CPE could only be observed in human amnion cultures after the addition of the Long "virus" which was "isolated" in KB cultures first, albeit much more slowly and incomplete
• In KB cells a round
  cell degeneration occurred that was not observed in liver and amnion cultures
• Cytopathogenic changes were first seen as small circumscribed syncytial areas which were randomly distributed and which enlarged over a 24-hour period, at which time numerous other syncytial areas made their appearance (hence the name of the "virus")
• The size of the particles was extrapolated (i.e. the action of estimating or concluding something by assuming that existing trends will continue or a current method will remain applicable) using centrifugation and sucrose gradient solutions without any visual evidence of the particles they were supposed to be measuring
• Chanock et al. stated that it should be noted that the method employed for the determination of size merely provided a rough estimate that required confirmation by more refined methods
• Attempts to demonstrate a
  hemagglutinin for chicken or human "O" erthrocytes were unsuccessful
• Multiplication of Long "virus" could not be demonstrated when 9-day embryonated eggs were inoculated with 10,000 TCD50 into the amniotic sac and incubated for 6 days
• The Long "virus" was also not pathogenic for 1-day-old mice by the intracerebral or intraperitoneal route
• Chanock et al. attempted to make the case that the Long/Snyder "virus" was the same as Morris et al.'s CCA by way of indirect CPE patterns and antibody results and relied mostly on unreliable antibody results to claim that they were unrelated to other "viruses"
• In the discussion, Chanock et al. changed their description of the CPE and antigenic properties observed between their "virus" and CCA as similar rather than the previous descriptor of identical
• While they then claimed that agents were indistinguishable from CCA "virus" and were capable of causing human infection, it was admitted that this did not prove that Long and Snyder "viruses" were responsible for the associated illnesses in these infants
• Of the "viruses" which infect human beings only "measles," "mumps" and "CA viruses" were previously shown to produce syncytial changes in cultures of various types thus showing that this effect is not specific to "RSV" and the only difference between the "viruses" is unreliable serological results

• Chanock et al. claimed that Morris et al. recovered a "virus" (CCA) from one of the 14 affected chimpanzees
• They stated that the "virus" was linked to a sickened lab worker even though "virus" isolation attempts were unsuccessful
• It was stated that these findings suggested the possibility that CCA was a "virus" of human origin which produced an outbreak of mild respiratory illness when introduced into a susceptible population of chimpanzees
• Only infants with severe lower respiratory illness and a control group of infants without such illness were studied as mild respiratory illnesses were disregarded because of the frequency with which these illnesses occur during the winter months in this age group, and since clinical diagnosis is less accurate than in infants with severe illness
• Keep in mind that the researchers were studying lower respiratory illness with their newly discovered "virus" as this will be important later
• The controls differed from the respiratory illness cases in that the proportion of hospitalized individuals was smaller and were, on average, older than the patients with respiratory illness
• Long "virus" was "isolated" once in KB tissue culture from the throat swab fluid of a clinic infant with bronchopneumonia
• However, twelve other "isolation" attempts with throat swabs from clinic infants and children with bronchopneumonia were unsuccessful as were "isolation" attempts with specimens from 28 infants with pneumonia and 90 infants with bronchiolitis who were hospitalized
• The throat swabs of respiratory patients and controls who developed a rise in antibody to Long "virus" were tested in liver epithelium cultures with negative results except for the one patient from whom "virus" was "isolated" in KB culture
• Incidence of "infection" with Long "virus" as determined by CP or the neutralization technique was not significantly different from that of the control group
• The researchers felt that it was possible that some of the respiratory illnesses which required hospitalization were associated with Long "virus" but this was difficult to establish because of the high rate of infection in the control group
• They felt that significantly higher rate of infection among the outpatients with bronchopneumonia suggested that an etiologic association existed between Long "virus" and a certain segment of the bronchopneumonia syndrome
• However, it was admitted that since the number of outpatients with bronchopneumonia was small (13 individuals), any conclusions derived from this study must be regarded as tentative in nature
• The firm establishment of an etiologic association between Long "virus" and bronchopneumonia required future studies
• Pneumococcus or B hemolytic streptococcus was isolated from 3 of the 10 nose and throat cultures
• The clinical course of the 3 patients from whom bacterial pathogens were recovered was similar to that of the other patients with pneumonia and did not resemble a bacterial illness
• What exactly the difference was between a bacterial pneumonia and a "viral" pneumonia was not defined and probably for good reason as it is stated that distinguishing between "viral" and bacterial cases can be challenging as both share similar symptoms and biomarkers.
• The researchers attempted to claim antibody results showed the presence and relation of their "virus" with CCA when 41 infants with lower respiratory illness and 16 control infants without respiratory disease developed CF antibody, neutralizing antibody, or both
• The frequent occurrence of infection when compared with the infrequent "isolation" of "virus" suggested to them that Long "virus" was recoverable from the throat during a very short interval following infection
• They reiterated that Morris, Blount and Savage experienced similar difficulty in "isolating CCA virus" from chimpanzees with respiratory illness
• Chanock et al. admitted that it was an assumption that CF antibody was a measure of recent or persisting infection
• From their findings, they anticipated that infection of older individuals would occur infrequently yet this was not the case
• They offered up possible explanations for this contradiction but concluded that it was not possible to determine which of the alternatives offered the correct explanation
• Chanock et al. admitted that an association between Long "virus" and lower respiratory disease in hospitalized infants was not demonstrable
• Remember how I said to keep in mind the information about the study looking into an association with lower respiratory disease? Chanock et al. point blank admitted that they could not prove such a relationship existed with their "virus." Game over.
• They felt that such an association could exist but was overshadowed by the high rate of "infection" among the hospital control group
• A definite statement could not be made regarding the spectrum of pathogenicity of Long "virus" for man
• In the majority of infants, "infection" resulted in either mild illness or no discernible disease

In order to accept the discovery of "RSV," we must ask ourselves a few questions. Is the "isolation" of an invisible agent from one chimpanzee, while failing with 13 others, strong enough evidence to state the existence of a new "virus?" Does the inability to create the same disease in various animals logically lead to the conclusion that the new "virus" was pathogenic? Should indirect cytopathogenic effects in cell cultures and nonspecific antibody results be considered a valid substitute for direct proof of the existence of this "virus?" Is the "isolation" of a "virus" from a single infant by another team of researchers that also relied upon indirect cytopathogenic effects and nonspecific antibody results proof that the two invisible "viruses" are related? Should contradictory results from control experiments be ignored and/or explained away in order to keep the belief in the new "virus discovery" alive? If the original researchers admitted that they could not establish an etiological relationship between their chimpanzee "virus" and illness in humans, what does this tell you about the invisible entity's ability to cause disease in humans? What if the second group of researchers also admitted to not being able to demonstrate a relationship between their human version of this invisible entity and the lower respiratory disease that they were attempting to associate it with?

To anyone looking at the foundational evidence for "RSV" critically and logically, it is clear to see that no "virus" was ever proven to exist nor cause disease in humans. Instead, we were presented with the same unpurified cell cultured creations, small sample sizes, contradictory results from controls, indirect and nonspecific antibody measurements, and the inability to establish an etiological relationship between the invisible entity and the disease being studied. In other words, just another typical day in the pseudoscience known as virology.

Virology’s Lack of Control

There has been quite a bit of debate over the years in regards to whether or not virology adheres to the scientific method, with much of this debate focused on the lack of proper controls. The argument has centered on whether the controls that are sometimes used yet rarely described, known as mock infections, are even a valid control to begin with. For those who are unfamiliar, scientific controls are a check and balance system that are utilized during experimentation when researchers are attempting to determine the cause of an effect. Controls are designed to ensure that the presumed cause, known as the independent variable, is the only thing that could be causing the observed effect, known as the dependent variable:

"A study with control(s) is designed to ensure that the effects are due to the independent variables in the experiment. The use of controls allows to study one variable or factor at a time. It is, however, important that both the control and other (experimental) group(s) are exposed to the same conditions apart from the one variable under study. Doing so will help draw conclusions that are more accurate and reliable."

https://www.biologyonline.com/dictionary/control

One of the main reasons I have not really focused as much on the lack of valid controls in virology is that there are other far more pressing issues which need to be addressed first before even getting to the experimental stage in order to discuss proper controls. Take a look at the steps of the scientific method for a moment:

1. Observe a phenomenon
2. Alternate hypothesis
   • Independent variable (the presumed cause)
   • Dependent variable (the observed effect)
   • Control variables
3. Null hypothesis
4. Test/experiment
5. Analyze the observation/data
6. Validate/invalidate hypothesis

Determining the IV, DV, and controls come after observing a natural phenomenon and are established when creating a hypothesis. Virologists have a difficult time completing the first steps of the scientific method as, beyond finding people with similar symptoms, they are unable to observe how this natural phenomenon occurs. They are unable to see the assumed "virus" particles in nature. They can not witness these particles entering a human being and causing illness, nor can they see these particles being transferred from person-to-person and causing disease. Without the observation of how this natural phenomenon occurs, there is no ability to establish a valid hypothesis in order to design an experiment in order to test for the cause of an observed effect. This means that step number 2 of the scientific method is off the table. With the inability to successfully satisfy the first two steps of the scientific method, so too goes the ability to design and perform a scientific experiment. Thus, the argument in regards to whether or not the controls used during pseudoscientific cell culture experiments are valid or not is too far ahead of the game. The entire premise behind the cell culture experiments themselves is invalid, as was shown by the man who created the method.

The Establishment of the Cell Culture

Up to 1952, the virologists believed that a virus was a toxic protein or enzyme directly poisoning the body, and that it was somehow multiplied by the body itself and would spread 
in the body as well as between people and between animals. Medicine and science gave up on this idea in 1951, because the suspected virus had never been seen in an electron microscope and, above all, no control experiments had ever been carried out. It was acknowledged that even healthy animals, organs and tissue would release the same decay products during the decomposing process that had been previously misinterpreted as "viruses". Virology had refuted itself."

-Dr. Stefan Lanka https://viroliegyhome.files.wordpress.com/2022/08/wissenschafftplus-the-virus-misconception-part-1.pdf

In the early 1950's, virology was on its deathbed after decades of failing miserably in the attempts to purify and isolate the assumed "viral" particles in order to directly prove the existence and pathogenicity of these invisible entities. In all of the years of experimentation, virologists had nothing to show but indirect evidence of decay from human and animal tissue culture experiments claimed to be caused by the "virus" in question.

However, the experimental results were not specific to the presence of any assumed "virus" as the same state of decay was witnessed in tissues from healthy hosts. As Dr. Stefan Lanka pointed out in the above quote, virology had refuted its own pseudoscientific tissue culture experiments. On top of this, the findings of effects associated with a specific "virus" from one group of researchers could not be reproduced nor replicated by other groups of researchers. In fact, results were often contradictory towards what was considered established evidence. Researchers could not even agree on what exactly a "virus" was nor whether the experimental evidence offered was in fact valid or not. A great summary of this rift was presented in a 1999 essay by Karlheinz Lüdtke:

On the history of early virus research.

"With the "filterable" virus, something had been discovered which, according to the traditional concepts, which after all had mostly proved their worth in research into infectious diseases, could not be described in a way that all researchers could have shared. Very different interpretations of the nature of this phenomenon arose, which were put forward against each other. No experimental evidence for this or that concept, which all researchers should have accepted, could be presented by any side. In other words, the decision as to whether this or that explanation most accurately expresses the "true" nature of the virus could not be "objectified" empirically. Every version of the interpretation of the phenomenon remained open to attack, facts presented to the expert public could often be reinterpreted into fictions by opponents, who brought into play the dependence of the findings on the conditions of observation, the local situation of the experiments, the research-related nature of the attributions of characteristics, etc. as sources of error. For example, findings often reported by certain virus researchers at the time were not confirmed by other researchers as a result of their own experiments, or the observations could not be reproduced by all scientists working with the virus. Often, findings to the contrary were reported, or the findings that had been examined were considered artefacts. As with justification, reasons of various kinds could be invoked to reject the positions debated. Findings that were used to empirically confirm a suspected connection were often soon joined by negative findings reported by other researchers. However carefully and deliberately the techniques used in the experiments were employed, and despite the fact that each party could offer credible reasons for defending their respective positions and provide empirical evidence – which explains why "the various opponents 'constructed' widely diverging research objects which they identified as the 'virus'" (van Helvoort 1994a: 202) – at no time did they offer compelling reasons that would have led the other party to finally abandon artifact accusations."

Lütke THE HISTORY OF EARLY VIRUS RESEARCH _ENGLDownload

While things were obviously not going well for the field, virology got a booster in the form of a new method purporting to establish the existence of these invisible entities. This was the cell culture method which was introduced in 1954 by John Franklin Enders during his attempts to identify a measles "virus." As virologists could still not properly purify nor isolate the assumed "virus" particles directly from the fluids of a sick host, it was decided that the particles had to be grown in a culture instead as it was claimed that there were not enough particles present within the fluids. The "virus," which could not be found directly inside the fluids in order to be studied properly, was somehow determined to need a host cell in order to replicate itself so that it could then be found and studied. The cell culture method was said to produce supposedly superior indirect evidence to that which had come before and it ultimately ended up reviving the dying field. To achieve this superior indirect evidence, Enders, a virologist who was awarded a Nobel Prize (also in 1954) for the discovery of the ability of poliomyelitis "viruses" to grow in various types of tissue cultures, replaced animal and human tissue cultures with animal and human cell cultures. In other words, Enders was ironically recognized with the Nobel Prize for the evidence he had gathered using the old refuted tissue culture practices which were subsequently replaced by his new cell culture method within the very same year.

When Enders created his cell culture method, it must be noted that steps one and two of the scientific method still had yet to be achieved, thus he was jumping into experimentation without observing a natural phenomenon, identifying the dependent variable (i.e. the effect), nor isolating the independent variable (i.e. "virus") in order to establish a hypothesis to test against. Yet this did not stop his attempt to create pseudoscientific experiments designed to convince the world that these fictional entities exist. While I have covered the invalid cell culture practice in great detail here, I will provide a quick explanation for what Enders' method consisted of. In his seminal measles paper, Enders took throat washings from suspected measles patients (which were obtained in gargled fat-free milk) and added the samples to human and monkey kidney cells. He mixed into the culture bovine amniotic fluid, beef embryo extract, horse serum, antibiotics, soybean trypsin inhibitor, and phenol red as an indicator of cell metabolism. This mixture was then incubated for days and the fluids were passaged on the 4th and 16th days. Enders eventually observed what is called the cytopathogenic effect, which is a pattern of damage appearing in the culture as the cell breaks apart and dies. This effect was assumed by Enders to be the direct result of the invisible "virus" within the fat-free milk throat washings as it breaks into the cell through lysis of the cell wall membrane which resulted in the death of the cell and the replication of the "virus." In other words, he assumed that the cellular debris from a poisoned cell was not the broken pieces of a once intact cell but were instead the newly created "viral" copies. Despite the unscientific nature of the method, the cell culture was quickly established as the "gold standard" for "virus isolation" and is still used by virologists today:

Cell culture provides the optimum setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the "gold standard" for virus discovery.

-WHO https://www.google.com/url?sa=t&source=web&rct=j&url=https://apps.who.int/iris/rest/bitstreams/1088173/retrieve&ved=2ahUKEwiQstb8yOj7AhWOIzQIHeqcDKAQFnoECB4QAQ&usg=AOvVaw2RBaX3pO2PZ3JyW0s7U3Ol

What needs to be understood is that, along with not having a valid independent variable in purified and isolated "viral" particles, Enders created his own dependent variable in the cytopathogenic effect. This effect is not a naturally observed phenomenon. It is only observed through experimentation and manipulation in the lab. Enders already assumed that the "virus" existed and that he would observe an effect if he added a sample containing the assumed "virus" to a cell culture. Once he witnessed this effect, Enders claimed that this was a direct result of the presence of a "virus" even though he could not see the "virus" within the cell culture. By doing so, Enders engaged in what is known as an affirming the consequent logical fallacy, normally expressed as such:

This is also an example of begging the question and circular reasoning as beautifully illustrated in this graph by Alec Zeck and Dr. Andrew Kaufman:

Thus, we can see that not only is the method that Enders created unscientific as it does not adhere to the scientific method, his conclusions were steeped in logical fallacies as well. However, these were not the only issues with Enders' experimental method.

Enders Refuted Himself

Even though the cell culture was accepted as the "gold standard" proof for the "isolation" of a "virus," one thing that is regularly neglected to be mentioned is that Enders himself was uncertain whether his method was even valid to begin with. In his 1954 paper, Enders questioned whether the experimental results created in a lab (in vitro) could be said to reflect what happens inside of a body (in vivo):

"The pathologic changes induced by the agents in epithelial cells in tissue culture resemble, at least superficially, those found in certain tissues during the acute stage of measles. While there is no ground for concluding that the factors in vivo are the same as those which underlie the formation of giant cells and the nuclear disturbances in vitro, the appearance of these phenomena in cultured cells is consistent with the properties that a priori might be associated with the virus of measles."

Enders also admitted that his indirect evidence, which he used to associate with an invisible measles "virus," was incomplete:

"Although we have thus already obtained considerable indirect evidence supporting the etiologic role of this group of agents in measles, 2 experiments essential in the establishment of this relationship remain to be carried out."

However, the most damning revelation was the admission that during his experiments with assumed measles "virus" using the cell culture method that he invented, Enders observed the exact same cytopathogenic effects he had associated with the measles "virus" in normal control cultures without any "virus" present whatsoever:

"Monkey kidney cultures may, therefore, be applied to the study of these agents in the same manner as cultures of human kidney. In so doing, however, it must be borne in mind that cytopathic effects which superficially resemble those resulting from infection by the measles agents may possibly be induced by other viral agents present in the monkey kidney tissue (cf. last paragraph under G) or by unknown factors."

"A second agent was obtained from an uninoculated culture of monkey kidney cells. The cytopathic changes it induced in the unstained preparations could not be distinguished with confidence from the viruses isolated from measles. But, when the cells from infected cultures were fixed and stained, their effect could be easily distinguished since the internuclear changes typical of the measles agents were not observed. Moreover, as we have already indicated, fluids from cultures infected with the agent failed to fix complement in the presence of convalescent measles serum. Obviously the possibility of encountering such agents in studies with measles should be constantly kept in mind."

In other words, John Franklin Enders established through the use of uninoculated cultures that the cytopathogenic effect that he assumed was caused by the invisible measles "virus" occurred even when there was no assumed "virus" present within the culture. Thus, it was the cell culture process itself involving the starvation of the cell with minimal nutrients as well as the poisoning of the cell with toxic chemicals, additives, and foreign materials which eventually lead to the death of the cell resulting in the cytopathogenic effect. Therefore, it should have been concluded that the cytopathogenic effect was not the work of any fictional "virus." This revelation should have ended virology right then and there.

However, Enders control results were instead swept under the rug and ignored. Rather than calling the time of death on virology as should have been done by anyone with an ounce of intellectual honesty, those in charge doubled down on the invalid cell culture method as the standard that every virologist must adhere to in order to confirm the presence of a "virus" within a sample. This reliance on the cell culture evidence built upon a fraudulent cytopathogenic foundation cemented the entire field of virology even further into the mad world of pseudoscience as the scientific method continued to be forsaken.

Enders Refuted by Others

Interestingly, as more researchers began to attempt to recreate Enders results, the flaws in the methodology continued to pop up. In fact, different teams of researchers over the next five years found the exact same cytopathogenic results as Enders did when they performed uninoculated controls for themselves. Dr. Cowan did an excellent overview of three of these studies in this recent video:

Let's take a look at excerpts from the three papers Dr. Cowan discussed in order to see exactly what these researchers discovered. I've included highlights from each paper as well as the methods sections showing the ridiculous culturing steps each team utilized to try and "isolate" their "viruses."

In this first study from 1955 by Rustigian et al., it is stated that the researchers discovered an unidentified "agent" within their uninoculated controls while attempting to adapt dengue "virus" to roller tube cultures of rhesus monkey kidneys. This "agent" produced the exact same cytopathogenic effect that was observed by Enders with his uninoculated culture during his measles experiments. After preparing monkey kidney cultures for a poliomyelitis study, 3 more unidentified "agents" appeared in uninoculated cultures which created the very same cytopathogenic effect. The source of these kidney cultures were healthy monkeys exhibiting no symptoms of disease, thus there was no "virus" assumed to be present within the culture. After investigation into whether or not the observed CPE could have come from the media, the researchers concluded that there was something present within the healthy monkey kidney cells that brought about the cytopathogenic effect:

Infection of Monkey Kidney Tissue Cultures with Virus-Like Agents.

"The wide current interest in tissue cultures for the study of virus-host cell relationships and applied problems of virus diseases has emphasized the need of employing cells and tissues free of microbial agents. The hazard of introducing viruses into tissue cultures with contaminated media has been stressed recently (1). Of equal, if not greater importance, is the problem of the presence of viruses in tissue cultures as a result of unrecognized infection of cells and tissues employed as primary explants. Detection of such viral infections, in certain instances, may be complicated by the fact that no readily recognized changes are manifested. On the other hand, some viruses under the same conditions may subsequently interfere markedly with cellular growth as evidenced by inhibition of acid production or
cytological changes.

In our attempts, in 1953, to adapt mouse-adapted Hawaii dengue virus(2) to roller tube cultures of rhesus monkey kidney, an unidentified agent was encountered which induced cytopathogenic changes in cultures of monkey kidney and cancer HeLa epithelial cells. The agent was filterable and could not be cultured in various non-living media. Subsequently, 3 additional agents with identical cytopathogenic characteristics were passed from uninoculated monkey kidney cultures prepared for poliomyelitis studies, to HeLa cell cultures. Enders and Peebles(3) have recently reported recovery of an agent from an uninoculated monkey kidney culture which appears to have the same cytopathogenic characteristics in monkey renal cultures as our agents have. However, other than a description of its cytopathic effect in monkey kidney cultures, no other data was given concerning their agent. In view of the wide use of monkey kidney cultures in virus studies it seems of importance to report our observations with these agents. These include their cytopathic effect in rhesus monkey renal tissue, in HeLa cell and certain other tissue cultures, the manner of their recovery and passage in tissue culture and other characteristics. In addition, evidence is presented that kidneys of apparently healthy monkeys are the source of these agents in monkey kidney tissue cultures and not the medium constituents."

Materials and methods. Tissue culture technics. A. Roller tube cultures. Technics of Robbins, Weller and Enders (4) were essentially followed for preparation of roller tube cultures. In addition to rhesus monkey kidney, cultures were prepared frum monkey testes, human embryonic kidney, human embryonic skin and musclet and mouse infant kidney. In early studies the culture medium consisted of Hanks-Simms solution (70%) , beef embryo extract (10%) and horse serum (20%). Penicillin and streptomycin were added for final concentration of 100 units and 100 ug respectively. Later, bovine amniotic fluid was used in place of Hanks-Simms solution (5). Soybean trypsin inhibitor, in final concentration of .05 mg, was incorporated in medium for all cultures. One ml of medium was added to each culture tube containing 10 to 15 fragments of tissue. Culture fluids were changed every 3 to 4 days. At time of inoculation of agents, the medium was modified to contain 85% Hanks-Simms or bovine amniotic fluid, 10% beef embryo extract and 5% horse serum.

B. Stationary cultures of monkey kidney were prepared by trypsinization technic of Youngner(6) with bovine amniotic fluid containing 5% beef embryo extract and 10% horse serum. Following standardization of cell suspensions(7) 0.5 ml containing about 500,000 cells was added to culture tubes. When good cell growth occurred (7 to 9 days), the medium was changed to 95% bovine amniotic fluid and 5% horse serum.

C. HeLa cell strain of cancer epithelial cells, kindly supplied by Dr. J. T. Syverton of the University of Minnesota, was cultured as described by Scherer, Syverton, and Gey(8) and Syverton (9). Culture tubes were seeded with approximately 40,000 cells in 1 ml, and in 4 to 5 days  a layer of growth was obtained containing 90,000 to 160,000 cells. The nutrient fluid was then replaced with maintenance solution containing 10% chicken serum(8,lO). With prolonged incubation of cultures in this fluid, degenerative changes were noted (granulation, rounding of cells). When this occurred the maintenance medium was replaced with nutrient fluid which restored normal morphology in 1 to 2 days. Accordingly, in experiments involving long incubation periods, nutrient fluid was substituted for maintenance medium and left on the cells for brief intervals.

"Recovery of agents MK1, MK3, and MK4 from uninoculated monkey kidney cultures.  Shortly after encountering agent MK-D in attempts to adapt dengue virus to monkey kidney cultures, syncytial masses and vacuolatinn were again observed in an uninoculated roller tube culture 12 days after its preparation.

This culture and 16 others prepared from the same kidney were set aside for further study. By 15th day degeneration was marked in this culture and had appeared in 3 other cultures; and by 34th day in 7 of the 16 cultures. Fluids were harvested on 14th and 15th day from cultures showing degeneration, pooled and 0.2 ml inoculated into HeLa cell cultures. Lytic-like arm were present in 10 days. Fluids from these cultures were then harvested, pooled and 0.1 ml passed to fresh HeLa cultures. Similar degeneration with this second passage occurred in 6 days. Subsequent passages in HeLa cells were made with 0.1 to 0.2 ml of pooled fluids harvested at 1- to 2 day intervals for 4 to 6 days following definite signs of degeneration. The fluids were stored in a COz cabinet for varying periods between passages. This agent, designated MK1 has 
now been passaged serially 101 times in HeLa cell cultures. Agents MK3 and MK4 were recovered similarly from uninoculated cultures prepared from kidneys of different rhesus monkeys. Thus, MK3 was recovered from a series of uninoculated cultures in which 8 of 72 roller tubes revealed moderate to strong degeneration 12 days after preparation and MK4 from 11 of 41 stationary cultures with degeneration 20 days after preparation with trypsinized cell suspension. These 2 agents have now been serially passed 10 and 6 times respectively in HeLa cultures. MK3 following 10 HeLa cell passages caused vacuolation and formation of syncytial masses in monkey kidney cultures. Same results were obtained with MK4 after 6 HeLa cell passages. Information has been obtained which suggests that frequency of occurrence of these agents in uninoculated cultures may be relatively high. Thus in 6 of 9 series of cultures prepared from kidneys of different monkeys one or more of 7 to 41 cultures in each series held 20 to 55 days showed characteristic degeneration (Table I); and the recovery of an agent from cultures of 3 different series was successful. One attempt to recover an agent from uninoculated cultures of a kidney series which did not show degeneration was unsuccessful after 3 blind passages in HeLa cells and subsequent passage in monkey kidney cultures. We have consistently observed in practically all cultures prepared from different monkey kidneys occasional small vacuoles apparent from the onset of cell outgrowth. In some cultures they persisted, but in others they apparently disappeared with continued incubation."

"These findings provide evidence that the agents were derived from renal tissue of monkeys. Evidence that other tissue culture constituents are not the source of the agent is as follows: One agent was recovered at time Hanks-Simms solution was used instead of bovine amniotic fluid and the reverse was true for other 3 agents. Beef embryo extract has been routinely employed for cultivation of HeLa cells for over a year without resulting in progressive characteristic degeneration which occurred following inoculation of such cultures with the agents. Horse serum has been heated at 56°C for 30 minutes prior to its use as a medium constituent, and, as previously noted, 2 of the 4 agents so treated failed to produce cytopathic effects after 21 days. Finally 3 blind passages in HeLa cells and subsequent passage in monkey kidney cultures of fluids from one monkey kidney series failed to reveal an agent. But agents were recovered from cultures of 2 other monkey kidney series set up shortly prior to and after this series with the same lots of medium constituents."

"Summary. During attempts to adapt dengue virus to rhesus monkey kidney cultures, an unidentified agent which causes formation of syncytial masses and vacuolation in such cultures was encountered. Subsequently, 3 additional agents with similar cytopathogenic effects were passed and maintained in HeLa cell cultures from uninoculated monkey kidney cultures. Renal tissue and not the medium constituents is the source of the agent. Bacteriological studies with one agent were negative. The same agent passed through a Selas filter. Accordingly it is considered to be virus-like in nature. Similar experiments were not done with 3 other agents but because of certain common characteristics are believed to be of the same nature."

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In this second study, also from 1955, Cohen et al. found the same cytopathogenic effect in their uninoculated control cultures as was observed in their inoculated samples supposedly containing a measles "virus." Once again, this showed that the CPE was not a specific effect due to any "virus" and was therefore brought about by the experimental conditions. Cohen et al. realized that they could not use this effect to recognize the measles "virus" so they claimed that antibody results, which they admitted to presuming were due to a measles "virus," was sufficient criteria to distinguish between the cultured samples:

Fluorescent Antibody and Complement-Fixation Tests of Agents Isolated In Tissue Culture from Measles Patients.

"Enders and Peebies(1) observed that human or monkey kidney tissue cultures inoculated with specimens from measles patients undergo, after one or more passages, characteristic nuclear and other cytologic changes. Both infectious virus and specific complement-fixation antigen appeared concurrently in the cultures and the cytopathogenic effect  was neutralized by convalescent-phase measles serum. We have applied the fluorescent antibody technic of Coons and his colleagues (2,3) to similar cultures and find that the results parallel those of complement-fixation tests and thus provide immunochemical support for the data oi Enders and Peebles and in addition evidence that measles antigen(s) are present in the nuclei as well as in the cytoplasm of infected cells.

Methods. Patients were selected for study during a mild outbreak of typical rubeola. Clinical diagnosis was aided in certain cases by recognition of giant cells of the Warthin-Finkeldey type in stained films of nasal discharge obtained early in illness, as described by Tompkins and Mcaulay(4).

Collection of specimens and preparation of tissue cultures. The procedures outlined by Enders and Peebles (1) were followed. Blood, throat swabs or washings, or nasal discharge were obtained before or within a day after the appearance of the rash. When possible, specimens were inoculated into tissue cultures within a few hours after their collection. Tissue cultures consisted of trypsinized monkey kidney cells grown in sheets (5). Tubes were inoculated with 0.2 or 0.3 ml of the prepared specimens, fed with nutrient fluid to bring the volume to 1.0 ml, and incubated as stationary slants at 36.5°C. Nutrient fluid consisted of beef amniotic fluid containing 5% inactivated horse serum and 5% beef embryo extract. The first passage contained 100 units of penicillin, 100 ug of streptomycin, and 50 ug of nystatin (fungicidin) (6): others, 50 units and 50 and 10 ug, respectively. Transfers to fresh tubes were carried out at 14-day intervals with 0.2-ml aniounts of pooled fluids taken 5, 9, and 14 days after inoculation. Controls consisted of uninoculated cultures and of cultures passaged serially with fluids from uninoculated tubes.

Results. Transmissible agents, presumably viruses, were isolated from both blood and a throat swab of one patient, and from nasal discharge and a throat swab, respectively, of 2 others whose blood was not obtained. The throat swab specimens had been stored in a dry ice chest for 7 days. No agents were detected in specimens from 5 additional cases. Inocula from 3 of the latter had been stored at 4°-6°C for 2 days before culture; tests of inocula from the other 2 were discontinued after 3 apparently negative serial passages. These circumstances may have contributed to our failure to isolate agents.

Enders and Peebles(1) and Rustigian et al. (10) encountered latent virus-like agents that induce marked vacuolization and syncytial masses in monkey kidney tissue cultures. The cellular degeneration characteristic of these "monkey-kidney agents" frequently appeared in our cultures, both in those inoculated with specimens from measles patients and in controls; hence cytologic criteria for recognition of measles agents were difficult to apply. In some tissue culture series the "monkey-kidney agents" destroyed the cell sheets in 10 to 14 days. We therefore relied on tests for the presence of measles antigen to identify the 3 agents cultivated from measles inocula."

"Discussion. Demonstration that tissue cultures inoculated with specimens from measles patients produce antigens that react specifically with convalescent-phase measles sera substantiates the findings of Enders and Peebles( 1). We did not repeat their filtration experiments, but were unable to recover fungi, nodes from rabbits immunized with diphtheria or bacteria including pleuropneumonia-like organisms and leptospira from infected tissue cultures and presume, therefore, that the antigens that produced specific reactions in fluorescent antibody and complement-fixation tests were derived from measles virus. On the whole, antigen in infected tissue cultures was detected earlier by the fluorescent antibody method than by complement-fixation tests. Preliminary data suggest, however, that the latter are of value in differential diagnosis of exanthemata and encephalitides of unknown origin."

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This final study which Dr. Cowan highlighted is from Von Magnus et al. in 1959. In lockstep with the previous researchers, they also came across the same exact cytopathogenic effect in uninoculated cultures from healthy monkeys. The researchers claimed that the changes were due to what they referred to as "foamy agents" and stated that these "agents" are regularly found in healthy monkey kidney cells. The CPE generated was indistinguishable from that which was associated with the measles "virus." Thus, like Cohen et al. before them, Von Magnus et al. decided that this effect was of limited use in attempting to identify "viruses" and instead relied upon unreliable antibody tests to determine whether the measles "virus" was present in a sample. Also of interest is that these researchers failed in numerous attempts to "isolate" the measles "virus" as they were only "successful" in 5 of 13 cases. All attempts to isolate a "virus" failed 24 hours after onset of a rash. Regardless of their failures, Von Magnus et al. decided that their indirect evidence agreed with the assumption that the antibody results identified the measles "virus:"

Studies on Measles Virus in Monkey Kidney Tissue Cultures. 1. Isolation of Virus from 5 Patients with Measles.

"As described by Enders & Peebles (6), and later by Rustigian et al. (13) and by Cohen el al. (3) cytopathic changes similar to those caused by measles virus may be observed also in uninoculated cultures of monkey kidney tissue (Figs. 4-5). These changes are probably caused by virus-like agents, so called "foamy agents", which seem to be frequently present in kidney cells from apparently healthy monkeys. Specific measles antigen is, however, produced only in cultures infected with measles virus. Iu the present study the ability of tissue culture passage material to fix complement in the presence of convalescent phase measles serum was therefore used as a criterion for the presence of measles virus."

MATERIALS AND METHODS

Tissue Cultures: Cell of trypsinized kidneys from rhesus monkeys, cynomolgous monkeys or baboons were u sed routinely. The technique of Younger (15) for the preparation or these tissue cultures was used with some modifications as previously described (10). During the outgrowth of the cells, the growth medium consisted of lactalbumln hydrolysate 0.5 cent (11) in Hank's solution (5) with 2 per cent horse serum.

Before seeding with the virus the medium was changed. Each tube received 1.8 ml of either synthetic medium 199 (12) or bovine amniotic fluid (5) containing phenol red as an indicator (final dilution 0.02 per cent). All media contained penicillin (100 U/ml) and streptomycin (0.1 mg/ml).

Collection of specimens: isolation of the virus was attempted from throat washings and blood.

Throat washings: Measles patients were askcd to gargle either with 15 ml of a mixture of one part ox-heart infusion broth and 2 parts of buffered salt solution or, in later experiments, with 15 ml of distilled water containing l percent Bacto tryptose "Difco". Penicillin, 100 U. per ml and streptomycin 0.1 mg per m l were added to the fluid. From the throats of very young children specimens were obtained by cotton swabs which were subsequently immersed into 2 ml of one of the two fluids just described. In all instances, the specimens were immediately froten in C02-ice and then stored in an electrical deep-freeze (-60° C). Before inoculation into tissue culture the materials were thawed rapidly at 37°C in running tap water.

Blood: ln earlier experiments heparin was added to the blood (2 ml of an O.O5 per cent of heparin per 10 ml of blood). In later experiments the blood was allowed to coagulate. Red cells which had not become attached to the clot were resuspended In the serum, and this mixture was used as inoculum for tissue cultures. The blood samples were stored at +4° C until the time of inoculation.

Inoculation of tissue cultures: Tissue cultures were inoculated with 0.5 rnl of throat washing material or with 0.25 ml of blood. The final amount of fluid in the tubes was about 2 ml. The cultures were kept staUonary or placed in a rotating drum (1 rotation per minute).

Subcultures from the first passage were carried out between the 6th and the 16th day, usually on the 8th day after inoculation. The nutrient medium was as a rule not exchanged in the course of a passage. Subcultures from the later passages were made between the 6th and the 12th day of incubation. The passage material consisted of a suspension of cells and cell debris in culture medium. This mixture was obtained by loosening the cells still adhering to the glass through scraping with a pipette. The amount inoculated varied, but it was usually 0.2 mi, so that the final volume of fluid in the inoculated tubes was about 2 ml. Serial passages of material from control tubes were carried out in the same way.

DISCUSSION 

"The present work confirms the findings of Endel's & Peebles (G) and Cohen et al. that throat washings and blood frotn patients in the early stage of measles contain a virus which is able to produce characteristic cytopathic changes in cultures of human and simian kidney cells. In the inoculated cultures the same syncylical formations were observed as those described by Enders & Peebles (6). In addition it was found that in the tissue culture system employed in this other specific lesions developed on continued incubation. This second step of the degeneration resulted in the accumulation of cell debris with circular gritty-looking formations ,vhich had either a quite smooth or a wrinkled margin. These cytopathic changes appear to be as specific for measles virus as are the synctia. 

However, monkey kidney viruses or "foamy agents," may give rise to cellular degenerations which microscopically are indistinguishable from those caused by measles virus. For this reason the cytologic manifestations are of limited value in the study of measles and additional criteria ate required to establish the identity of the cultivated agents. This can be achieved by the demonstration of intranuclear inclusion bodies in the cell cultures (6) or by tests for the presence of measles anligen using either the fluorescent antibody technique (3) or the complement fixation reaction (3, 6). Because of its simplicity this last mentioned method has been employed in the present study. Observations on the development of measles antigen in the various cultures will be described in detail in a forthcoming publication (1).

In the study presented in this paper measles virus was isolated from five out of nine throat washings collected withln 24 hours after the outbreak of exanthem. The reason why the isolation failed in one patient (No. 12) may be that he had a vomiting immediately before gargling. In the remaining three cases (Nos. 2, 7 and 9) no obvious explanation for the negative results can be offered. Possibly it was because these isolations were all attempted with throat swabs in young children who were very excited and the sampling thus difficult to perform.

Virus was recovered from the blood in only one out of eight attempts made within 24 hours after the onset of the rash. This isolation rate is low compared with that obtained by Enders & Peebles (6) who recovered virus from the blood of four out of five patients. These authors used heparinized blood from which it may be easier to recover virus than from serum containing resuspended blood cells. In our laboratory the only isolation from the blood was made from one of the three samples to which heparin had been added. Also, Enders and Peebles used a larger inoculum (0.5 ml to 2 ml) than employed in this study (0.25 ml).

The possibility was considered that the failure to recover virus from 8 of the 13 patients examined might be due to an insusceptibility to measles virus of the particular cells employed. This, however, was apparently not the case, for when tubes prepared simultaneously with those employed in the unsuccessful isolation experiments were inoculated with measles infected fluid typical cytopathic changes developed together with the appearance of complement-fixing antigen in the nutrient media.

The observations presented in this paper agree well with the assumption that the isolated agents are the cause of measles."

SUMMARY

(1) Virus agents have been isolated in trypsinized monkey kidney tissue cultures from throat washings and blood from 5 out of 13 patients examined during the acute phase of measles. In all instances, virus was isolated from throat washings or throat swahs while one strain was recovered from the blood. All attempts to isolate virus later than 24 hours after onset of the rash failed.
2) The cytopathic manifestations observed in measles virus infected tissue cultures as well as in uninoculated tubes are described. Complement fixation tests for the presence of measles antigen have been used as criterion for the presence of this virus in the infected cultures.
(3) Intranasal and oral administration of material from late passages of one of the isolated agents to two Rhesus resulted in a pronounced measles-like rash in one of the animals, and both monkeys developed antibodies against the inoculated strain.
(4) In serological studies of acute and convalescent-phase sera from measles patients using measles virus infected cultures of monkey kidney tissue as antigen in complement fixation tests a clearcut rise in antibodies was observed in all cases.

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What we can take away from these three studies in combination with Enders' own uninoculated controls is that no "virus" was ever necessary in order for the cytopathogenic effect to occur. This effect was witnessed in cultures of kidney cells from healthy monkeys. It was stated that these findings were not rare as witnessing this cytopathogenic effect in uninoculated cultures from healthy monkeys was actually rather frequent. Thus, it is clear to see that it is the cell culture method itself, which consists of stressing and starving the cells which are outside of their natural environment and dousing them in kidney toxic antibiotics as well as mixing in foreign animal materials and other chemical additives that leads to the death of the cell. As this cytopathogenic effect was not specific to a "virus" and occurred in cultures without any "viruses" present, it can not be used to claim the presence of a "virus" as both Cohen et al. and Von Magnus et al. stated. Thus, there is no value to any results gained from this pseudoscientific experimentation.

While the above studies should be enough to wash away any doubt left about the fraudulent use of a cytopathogenic effect as confirmation that a "virus" is present in the sample, I am throwing a 1956 publication by Hull et al. into the mix as well which provides even more damning evidence supporting the discontinuation of this pseudoscientific practice. In this paper, the researchers stated that the use of the cell culture method led to the discovery of many unknown cytopathogenic agents that can only be detected through the use of this practice. The researchers used slight variations in the CPE observed during the culture for poliomyelitis vaccines to claim that there were at least 10 other invisible "virus-like agents" present at various times within the cultures. While Hull et al. believed that these agents most likely originated within the monkey kidney cells, they said there was a potential that these "agents" could represent contaminants from human sources, horse serum, nutrient medium or other solutions used in the preparation of the cultures. Regardless, they decided that these invisible agents, which were "recovered" from normal or uninoculated control cultures from healthy monkeys, were "simian viruses." The researchers did so despite the fact that they were unable to make any animals sick through various routes of injection beyond two exceptions where two of the "viruses" caused death upon injection in the brain. However, upon injection of these two "viruses" into the muscles, no illness occurred. In other words, Hull et al. observed cytopathogenic effects that they subjectively determined were not caused by the polio "virus" in both inoculated and uninoculated cultures and decided that there must be different "viruses" present within the culture based on the pattern of cell death observed. These mixtures assumed to contain invisible "viruses" were consistently demonstrated not to be pathogenic, hence the unobservable entities from uninoculated cultures with the same pattern of CPE from healthy monkeys did not meet the definition of a "virus:"

Viral Agents Recovered from Tissue Cultures of Monkey Kidney Cells

"Increased use of the technique of cell cultivation for isolation, maintenance and study of viruses has resulted in the discovery of many hitherto unknown cytopathogenic agents. In screening human stool samples or rectal swabs for poliomyelitis viruses, Melnick (1) isolated several "orphan viruses" which do not type with poliovirus antiserums, and from the same source Sabin (2) obtained 5 viruses referred to as He1; He2, etc. The new RI series of viruses was isolated in tissue culture by Hilleman and Werner (3) from throat washings of patients with respiratory disease. Rowe, Huebner et al. (4) isolated a similar group of viruses, the APC group, which first appeared in tissue cultures of human adenoid and tonsillar tissue and later were recovered from eye washings of patients with a conjunctivitis. Recently Rustigian et al. (5) reported recovery of two cytopathogenic agents from cultures of monkey kidney cells. Thus, a number of viral agents are present in tissues and excreta of man and lower animals which defy detection by methods other than tissue culture.

During production and testing of poliomyelitis vaccine, hundreds of thousands of monkey kidney cultures prepared from thousands of monkeys were observed in our laboratories and in others participating in this program. Numerous filterable, transferable cytopathogenic agents other than poliovirus were also encountered. Although these agents were probably introduced into the cultures with the monkey kidney cells, there remains the remote possibility that some represent contaminants from human sources, horse serum, nutrient medium or other solutions used in the preparation of cultures. This report describes 8 immunologically distinct agents isolated and studied in our laboratories, and makes reference to 2 from other sources."

"The agents isolated will be referred to as "Simian viruses" (S.V.) until such time as a definite association with some other host or identification can be established. These agents will be referred to as S.V.1, S.V.2, etc. Of the original group of agents which included S.V., through S.V.15, eight (S.V. 1, 2 4, 5. 6, 11, 12 and 15) will be described in this paper. The missing numbers were held by agents that were identified or reclassified after antiserum studies, or which have not yet been adequately studied for inclusion at this time. The latter category includes S.V.13 which is
the "lacy or foaming" agent frequently seen in cultures of monkey kidney cells.

RESULTS

The following is a brief account of the original isolation and description of the 8 agents referred to above. S.V.1 was recovered from roller-tube cultures of trypsinized monkey kidney cells planted on February 8, 1954. This series included 1,700 tubes prepared from a pool of kidneys from 4 apparently healthy cynomolgus monkeys. After 6 to 8 days of incubation the cultures appeared satisfactory and were used for titrations of poliovirus. After 6 more days of incubation a type of cell destruction was seen in some cultures beyond the end point of the poliovirus that was atypical of the damage produced by the latter. This atypical damage was observed in 17 percent of the cultures. This eytopathogenic effect (C.P.E.) was characterized by a reduction in size and distinct rounding of the cells."

"Monkeys were inoculated intracerebrally, intramuscularly, and in some instances by other routes, with all the viruses. Serum from each monkey inoculated was first assayed for antibodies against the specific virus to be injected. Only negative animals were used. With the exception of S.V.12 and S.V.15 no evidence of clinical illness or of gross or histopathological lesions was seen. Monkeys inoculated intracerebrally with high titer undiluted S.V.12 and S.V.15 viruses succumbed within 4 to 6 days after inoculation. The specific type of virus inoculated was readily recovered from brain and cord tissues of these animals. These same two viruses, however, when inoculated intramuscularly into monkeys failed to produce clinical disease or gross or histopathological lesions. The histopathological lesions observed in the intracerebrally inoculated monkeys revealed necrosis and complete destruction of the choroid plexus plus a generalized aseptic type of meningitis. No other brain pathology or lesions resembling those of poliomyelitis were seen. The various organs studied exhibited no recognized lesions and it appeared that all involvement was limited to the central nervous system in the region of the virus inoculation. The lesions produced by S.V.12 and S.V.I5 were indistinguishable.

Although no recognizable disease was produced in the monkeys inoculated with these viruses, with the above noted exceptions, some evidence was obtained in our laboratories, and in others, that S.V.2 and S.V6, may have some etiological significance in enteric infections."

"None of these monkey viruses infected embryonated eggs as determined by death of the embryo. However, one agent from the A.M.S.G.S. identified as S.V., had been recovered from embryonated eggs inoculated with monkey kidney tissue-culture fluids. Groups of adult and suckling mice were inoculated intracerebrally and intraperitoneally with each virus. However, no evidence of disease nor death of the mice was observed. Rats and rabbits which were inoculated with live virus for the production of antiserum also did not succumb to infection with any of these agents."

DISCUSSION

The isolations and characteristics of 8 apparently new viruses have been described. Although definite proof has not been presented, these agents probably have been simian in origin. What diseases, if any, they might be responsible for in monkeys also has not been established. S.V.2 and S.V. 6, may be etiological agents in intestinal infections of monkeys inasmuch as both have been isolated from stool samples of animals suffering from diarrhea. None have produced experimental infections when inoculated intramuscularly although both S.V.12 and S.V.15 caused a prostrating type of paralysis and death in monkeys following intracerebral inoculation. Attempts which are being made to infect monkeys by other routes of inoculation are not yet completed."

The greatest significance of these viruses has been their appearance in tissue cultures used to produce and to test poliomyelitis vaccine. This has been especially troublesome during tissue-culture safety testing of the vaccine as the occurrence of these viruses has in many instances invalidated tests for poliovirus, making retesting necessary and thereby delaying the release of vaccine. There has also been the problem of proving in some cases that the contaminating " simian virus" isolated during a vaccine safety test was not in the vaccine sample under test. In general, however, this has not been a problem since many of these agents have been recovered from normal or uninoculated control cultures."

SUMMARY

"Eight apparently new submicroscopic, filterable, cytopathogenic agents recovered in tissue cultures of rhesus and cynomolgus monkey kidney tissue have been described. No definite associations have been made between any of these agents and naturally occurring diseases of monkeys or other animals. The significance of these viruses in the poliomyelitis vaccine program and the problems created by them during vaccine safety testing in tissue culture have been discussed."

hull1956Download

These studies in and of themselves should be enough to show that, when controls are done, the cytopathogenic effect observed and attributed to the presence of an invisible "virus" is shown not to be specific to any "virus." In fact, no "viruses" are necessary to create this effect whatsoever. Luckily, we are fortunate enough that Dr. Stefan Lanka did not just rest on the work of others to show this and took it upon himself to verify these findings by doing his own set of control experiments, thus putting the proverbial final nail in the coffin of this pseudoscientific practice. As Dr. Cowan did an absolutely excellent job breaking down Dr. Lanka's control experiments, I am providing excerpts from his book Breaking the Spell:

"Here is the essence of Lanka's experiment, done by an independent professional laboratory that specializes in cell culturing. As seen in this series of photographs, each of the four vertical columns is a separate experiment. The top photo in each column was taken on day one, and the bottom photo was taken on day five. 

In vertical column one, normal cells were cultured with normal nutrient medium and only a small amount of antibiotics. As you can see, on neither day one nor day five was any CPE found; the cells continued their normal, healthy growth. 

In vertical column two, normal cells were again grown on normal nutrient medium and a small amount of antibiotics, but this time, 10% fetal calf serum was added to enrich the medium. Still, the cells in the culture grew normally, both on day one and day five.

The third vertical column shows what happened when Dr. Lanka's group used the same procedures that have been used in every modern isolation experiment of every pathogenic virus that I have seen. This included changing the nutrient medium to "minimal nutrient medium"—meaning lowering the percentage of fetal calf serum from the usual 10% to 1%, which lowers the nutrients available for the cells to grow, thereby stressing them—and tripling the antibiotic concentration. As you can see, on day five of the experiment, the characteristic CPE occurred, "proving" the existence and pathogenicity of the virus—except, at no point was a pathogenic virus added to the culture. This outcome can only mean that the CPE was a result of the way the culture experiment was done and not from any virus.

The fourth and final vertical column is the same as vertical column three, except that to this culture, a solution of pure RNA from yeast was added. This produced the same result as column three, again proving that it is the culture technique—and not a virus—that is causing the CPE."

Invalid Experiment = Invalid Controls

It should hopefully be clear by now why virology has a control problem. The cell culture method itself is not a valid experimental set-up as it was never designed through the adherence to the scientific method. The experiment creates the effect (CPE) and then assumes a cause ("virus") without ever verifying that the assumed cause exists in the first place. Even if we were to give them leeway with the use of the cell culture as a valid experiment to prove the presence of a "virus," the cytopathogenic effect is known to be caused by many other factors unrelated to any "virus" thus making the explanation of a fictitious "virus" as the culprit entirely unnecessary. The blaming of invisible "viruses" for observed CPE in place of other explanations was pointed out in a technical bulletin for cell culturing:

Understanding and Managing Cell Culture Contamination

"Since cytopathic viruses usually destroy the cultures they infect, they tend to be self-limiting. Thus, when cultures self-destruct for no apparent reason and no evidence of common biological contaminants can be found, cryptic viruses are often blamed. (See Figures 3a and 3b.) They are perfect culprits, unseen and undetectable; guilty without direct evidence. This is unfortunate, since the real cause of this culture destruction may be something else, possibly mycoplasma or a chemical contaminant, and as a result will go undetected to become a more serious problem."

https://www.google.com/url?sa=t&source=web&rct=j&url=https://safety.fsu.edu/safety_manual/supporting_docs/Understanding%2520and%2520Managing%2520Cell%2520Culture%2520Contamination.pdf&ved=2ahUKEwjPy_a7nPbuAhVPVs0KHbMbAoAQFjAAegQIARAC&usg=AOvVaw3M6StZvPbYyw30IqM9YhAY

It is known that the CPE can be caused by factors other than "viruses" such as:

1. Bacteria
2. Amoeba
3. Parasites
4. Antibiotics
5. Antifungals
6. Chemical contaminants
7. Age and cell deterioration
8. Environmental stress

And lest I forget, the also fictional "virus-like" entities (that are admitted to be impossible to separate from "viruses") known as exosomes are also said to create cytopathogenic effects.

To reiterate, the cytopathogenic effect is not a valid dependent variable as it is not a naturally observed phenomena, and it can be explained by various factors other than an invisible "virus." The unpurified fluids used during the culture is not a valid independent variable as the "virus" assumed to be within has never been shown to exist in a purified and isolated state before the experiment takes place. Thus, performing the cell culture as evidence for a "virus" is entirely unscientific as the scientific method can not be followed. However, for arguments sake, let's allow virologists to have the cell culture and the cytopathogenic effect as a valid experiment. This would mean that they would need to have valid controls performed alongside the cell cultures every time. One would think that this would be commonly seen in virology papers as we saw in those from the 1950s. Yet, more often than not, either no mention of the controls are ever found within the studies provided as evidence for the existence of "viruses" or what was done to the control culture is ill-defined. Perhaps this is due to the disastrous conclusions made by the authors of the 1950's studies, which irreparably damaged the claim of a "viral" cause of the CPE observed? In any case, if virologists do perform a control, they usually do what they refer to as mock infections:

mock-infected

"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."

https://www.genscript.com/biology-glossary/10558/mock-infected

What this means is that the virologists are supposed to use the same cell with the same additives (antibiotics, antifungals, minimal nutrient media, fetal bovine serum, etc.) but without the "virus" added in. Let's take a look at a few examples in order to see if this is what they do. In this first instance, the authors of a CDC study state that the mock-infected culture was treated with the medium only, so this appears to match up to the above definition. However, it is not stated whether the medium was exactly the same as that of the experimental culture. We must assume this to be the case, which is a problem as will be shown later.

SARS–associated Coronavirus Replication in Cell Lines

This second study is one of the pivotal early "SARS-COV-2" studies by Zhu et al. We are only told that the control is mock-infected and thus we must once again assume that the researchers used exactly the same medium/ingredients as no other details were provided.

A Novel Coronavirus from Patients with Pneumonia in China

This third example is from an Australian study in April 2020 which is infamous for adding trypsin, a protein digester, to the culture in order to create the "spike" corona in EM images. The researchers give us what they call uninfected control cells. Again, we must assume that the uninfected cell was treated exactly the same as the infected cell as no details were provided.

Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia

In this final study from Zhou et al., which is one of the pillars of the "SARS-COV-2" fraud, we are told that a mock "virus" was used. What is a mock "virus?" Zhou et al. do not say.

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Or they did not say so in public. However, in a private correspondence through e-mail, some further details were shed on this situation. From Dr. Mark Bailey's amazing essay "A Farewell to Virology," we find out that in the experimental culture, the antibiotics were doubled during the culture experiments to achieve a cytopathogenic effect in 1 out of 24 cultures. Not only is this a stunning failure rate to culture a "virus," the addition of more antibiotics to the experimental culture completely invalidates the results as the control was not treated the same.

As can be seen in these examples, what was done to the mock-infected controls is not well-defined and must be taken as an assumption that the two cultures were treated the same minus the assumed "virus." However, the Zhou et al. admission is exactly why we can not assume that the cultures are treated equally as this was obviously not the case. The addition of more antibiotics to the experimental culture was never mentioned anywhere within the paper. Zhou et al. committed scientific fraud. How many other "virus" studies would be shown to have done the same if the researchers were as honest as Xing-Lou Yang was in an e-mail exchange? The details of what was done to the mock infected controls must be provided with every paper yet this is rarely, if ever, the case.

However, there is an even bigger underlying problem for virology than a lack of ill-defined mock-infection controls even if all of the same additives are used. Remember, a control is supposed to eliminate only the one variable under study, i.e. the assumed "viral" particles. As the fluids that are utilized for the "inoculated" culture are admitted to not contain only purified and isolated "virus" particles but a whole gamut of potential substances such as host materials, bacteria, fungi, microvesicular bodies, etc., mock-infections where no human fluids are added to the culture are not proper controls. A proper control would be to use a sample from a healthy human which is treated in the exact same manner as the fluids with the assumed "virus." This includes adding the healthy human fluids to "viral" transport media which contains added chemicals, nutrients, fetal bovine serum, antibiotics/antifungals, etc. as this step is done immediately to the "viral" sample upon collection. Leaving fluids from healthy subjects out of the control invalidates the mock-infection as there are numerous confounding variables present within the experimental culture that are missing from the mock-infected culture. Thus, while a mock-infected control can be said to be a control, it is not the proper control for the pseudoscientific cell culture experiments.

In Summary:

• A study with control(s) is designed to ensure that the effects are due to the independent variables in the experiment
• The use of controls allows to study one variable or factor at a time
• It is important that both the control and other (experimental) group(s) are exposed to the same conditions apart from the one variable under study
• Relating to virology, that one variable would be the assumed "viral" particles and thus it is imperative that since virologists use an unpurified sample from sick humans, they would need to use an unpurified sample from a healthy human as a control as well
• Throughout the early 20th century, very different interpretations of the nature of "viruses" arose, which were put forward against each other
• No experimental evidence for this or that concept, which all researchers should have accepted, could be presented by any side
• Findings often reported by certain "virus" researchers at the time were not confirmed by other researchers as a result of their own experiments, or the observations could not be reproduced by all scientists working with the "virus"
• Often, findings to the contrary were reported, or the findings that had been examined were considered artefacts
• Every version of the interpretation of the "virus" phenomenon remained open to attack
• Facts presented to the expert public could often be reinterpreted into fictions by opponents, who brought into play the dependence of the findings on the conditions of observation, the local situation of the experiments, the research-related nature of the attributions of characteristics, etc. as sources of error
• Findings that were used to empirically confirm a suspected connection were often soon joined by negative findings reported by other researchers
• At no time did one party offer compelling reasons that would have led the other party to finally abandon artifact accusations
• In 1954, the tissue culture practice, which had been shown to produce the same results in healthy tissues, fell out of favor for John Franklin Enders new cell culture method
• The cell culture relied on the creation of the cytopathogenic effect (CPE), patterns of cell death, as being specific to "viruses" in order to identify that they were present within the culture
• Even though this method was quickly accepted as the new gold standard for "virus isolation," Enders himself questioned his results as he observed indistinguishable cell death in the healthy control cultures as seen in those with the measles "virus"
• According to Enders, a second agent was obtained from an uninoculated culture of monkey kidney cells
• The cytopathic changes it induced in the unstained preparations could not be distinguished with confidence from the "viruses" isolated from measles
• He felt it was obvious that the possibility of encountering such agents in studies with measles should be constantly kept in mind
• Other researchers over the next 5 years came to the same conclusions as they also observed the same cytopathogenic effect in their own healthy control cultures

• The hazard of introducing "viruses" into tissue cultures with contaminated media has been stressed recently
• Detection of such "viral" infections, in certain instances, may be complicated by the fact that no readily recognized changes are manifested (a.k.a. the CPE-less "viruses" escape clause)
• In 1953, the researchers attempted to adapt mouse-adapted Hawaii dengue "virus" to roller tube cultures of rhesus monkey kidney and an unidentified agent was encountered which induced cytopathogenic changes in cultures of monkey
  kidney and cancer HeLa epithelial cells
• Three additional agents with identical cytopathogenic characteristics were passed from uninoculated monkey kidney cultures prepared for poliomyelitis studies, to HeLa cell cultures
• Enders and Peebles reported recovery of an agent from an uninoculated monkey kidney culture which had the
  same cytopathogenic characteristics in monkey renal cultures as their agents
• Evidence was presented that kidneys of apparently healthy monkeys are the source of these agents in monkey kidney tissue cultures and not the medium constituents
• Shortly after encountering agent MK-D in attempts to adapt dengue "virus" to monkey kidney cultures, syncytial masses and vacuolatinn were again observed in an uninoculated roller tube culture 12 days after its preparation
• Agents MK3 and MK4 were recovered similarly from uninoculated cultures prepared from kidneys of different rhesus monkeys
• Information was obtained which suggested that the frequency of occurrence of these agents in uninoculated cultures may be relatively high
• The researchers consistently observed in practically all cultures prepared from different monkey kidneys occasional small vacuoles apparent from the onset of cell outgrowth
• The researchers stated that these findings provided evidence that the agents were derived from renal tissue of monkeys and that it was the renal tissue and not the medium constituents which was the source of the agents

• Enders and Peebies observed that human or monkey kidney tissue cultures inoculated with specimens from measles patients undergo, after one or more passages, characteristic nuclear and other cytologic changes
• In cell culture experiments for ruebella by Cohen et al., controls consisted of uninoculated cultures and of cultures passaged serially with fluids from uninoculated tubes
• Transmissible agents, presumably "viruses," were said to be isolated from both blood and a throat swab of one patient, and from nasal discharge and a throat swab, respectively, of 2 others whose blood was not obtained
• No agents were detected in specimens from 5 additional cases
• Inocula from 3 of the latter had been stored at 4°-6°C for 2 days before culture; tests of inocula from the other 2 were discontinued after 3 apparently negative serial passages and Cohen felt that these circumstances may have contributed to their failure to isolate agents
• Cohen reiterated that Enders and Peebles and Rustigian et al. encountered latent "virus-like" agents that induce marked vacuolization and syncytial masses in monkey kidney tissue cultures
• Keep in mind that they did not observe any "virus-like" agents, they just assumed that they must be present as the same CPE was observed in controls said to contain no "virus"
• The cellular degeneration characteristic of these "monkey-kidney agents" frequently appeared in our cultures, both in those inoculated with specimens from measles patients and in controls; hence cytologic criteria for recognition of measles agents were difficult to apply
• In other words, the CPE was identical as they could not tell any difference between the CPE observed in the uninoculated controls and the inoculated samples and could not use CPE as criteria to recognize the measles "virus"
• Cohen et al. did not repeat Enders filtration experiments, but were unable to recover fungi, nodes from rabbits immunized with diphtheria or bacteria including pleuropneumonia-like organisms and leptospira from infected tissue cultures so they presumed that the antigens that produced specific reactions in fluorescent antibody and complement-fixation tests were derived from measles "virus"
• In other words, as they could not use CPE to determine measles cases, they used non-specific antibody results which they presumed were specific to a measles "virus"

• As described by Enders & Peebles, and later by Rustigian et al. and by Cohen el al. cytopathic changes similar to those caused by measles "virus" may be observed also in uninoculated cultures of monkey kidney tissue
• These changes are probably caused by virus-like agents, so called "foamy agents", which seem to be frequently present in kidney cells from apparently healthy monkeys
• This second step of the degeneration resulted in the accumulation of cell debris with circular gritty-looking formations , which had either a quite smooth or a wrinkled margin (cell-debris with a circular formation sure sounds a lot like a "virus" )
• Monkey kidney "viruses" or "foamy agents," may give rise to cellular degenerations which microscopically are indistinguishable 
  from those caused by measles "virus"
• They decided for this reason the cytologic manifestations are of limited value in the study of measles and additional criteria are required to establish the identity of the cultivated agents
• In the study presented in this paper measles "virus" was isolated from five out of nine throat washings collected within 24 hours after the outbreak of exanthem
• The reason why the isolation failed in one patient was thought to be because he had vomited immediately before gargling
• In the remaining three cases, no obvious explanation for the negative results can be offered but they thought it may possibly  be because these isolations were all attempted with throat swabs in young children who were very excited and the sampling thus difficult to perform
• "Virus" was recovered from the blood in only one out of eight attempts made within 24 hours after the onset of the rash which was low compared with that obtained by Enders & Peebles who recovered "virus" from the blood of four out of five patients
• The possibility was considered that the failure to recover "virus" from 8 of the 13 patients examined might be due to an insusceptibility to measles "virus" of the particular cells employed but this theory was abandoned
• They stated that the observations presented in this paper agreed well with the assumption that the isolated agents were the cause of measles
• All attempts to isolate "virus" later than 24 hours after onset of the rash failed
• The cytopathic manifestations observed in measles "virus" infected tissue cultures as well as in uninoculated tubes were described
• Intranasal and oral administration of material from late passages of one of the isolated agents to two Rhesus resulted in a pronounced measles-like rash in one of the animals, and both monkeys developed antibodies against the inoculated strain

• Hull et al. stated that the increased use of the technique of cell cultivation for isolation, maintenance and study of "viruses" has resulted in the discovery of many hitherto unknown cytopathogenic agents
• Various researchers had found a number of "viral agents" which are present in tissues and excreta of man and lower animals which defy detection by methods other than tissue culture
• During production and testing of poliomyelitis vaccine, hundreds of thousands of monkey kidney cultures prepared from thousands of monkeys were observed in our laboratories and in others participating in this program and numerous filterable, transferable cytopathogenic agents other than "poliovirus" were also encountered
• Hull stated that although these agents were probably introduced into the cultures with the monkey kidney cells, there remained the remote possibility that some represent contaminants from human sources, horse serum, nutrient medium or other solutions used in the preparation of cultures
• The agents isolated were referred to as "Simian viruses" (S.V.) until
  such time that a definite association with some other host or identification could be established
• One of these included S.V.13 which is the "lacy or foaming" agent frequently seen in cultures of monkey kidney cells discussed in the studies above
• For S.V. 1, 1,700 tubes prepared from a pool of kidneys from 4 apparently healthy cynomolgus monkeys were used
• After 6 to 8 days of incubation the cultures appeared satisfactory and were used for titrations of "poliovirus" and after 6 more days of incubation a type of cell destruction was seen in some cultures beyond the end point of the "poliovirus" that was atypical of the damage produced by the latter
• In other words, a longer incubation period resulted in further cytopathogenic effects which were (subjectively) considered not due to polio so they determined it must have been caused by another "agent"
• This atypical damage was observed in 17 percent of the cultures
• Monkeys were inoculated intracerebrally, intramuscularly, and in some instances by other routes, with all the "simian viruses" and with the exception of S.V.12 and S.V.15, no evidence of clinical illness or of gross or histopathological lesions was seen
• Monkeys inoculated intracerebrally with high titer undiluted S.V.12 and S.V.15 "viruses" succumbed within 4 to 6 days after inoculation
• These same two "viruses," however, when inoculated intramuscularly into monkeys failed to produce clinical disease or gross or histopathological lesions
• It can be seen the assumed S.V.'s showed no signs of pathogenicity at any point, except for S.V. 12 & 15 which killed monkeys upon injections in the brain but did nothing upon injections in the muscles, thus they are not "viruses"
• No recognizable disease was
  produced in the monkeys inoculated with these "viruses"
• None of these monkey "viruses" infected embryonated eggs as determined by death of the embryo
• Groups of adult and suckling mice were inoculated intracerebrally and intraperitoneally with each "virus" and yet no evidence of disease nor death of the mice was observed
• Rats and rabbits which were inoculated with live "virus" for the production of antiserum also did not succumb to infection with any of these agents
• The "isolations" and characteristics of 8 apparently new "viruses" have been described yet no definite proof had been presented and these agents were thought to be probably simian in origin
• What diseases, if any, they might be responsible for in monkeys had not been established
• In other words, the researchers could not produce disease with these "agents" found in both inoculated and uninoculated cultures based upon identification by CPE and thus they were not "viruses"
• Many of these agents have been recovered from normal or uninoculated control cultures
• Hull et al. reiterate that eight apparently new submicroscopic, filterable, cytopathogenic agents recovered in tissue cultures of rhesus and cynomolgus monkey kidney tissue were described
• No definite associations were made between any of these agents and naturally occurring diseases of monkeys or other animals

• Dr. Stefan Lanka performed his own cell culture control experiments exposing the CPE fraud
• In control #1, normal cells were cultured with normal nutrient medium and only a small amount of antibiotics and on neither day one nor day five was any CPE found; the cells continued their normal, healthy growth
• In control #2, normal cells were again grown on normal nutrient medium and a small amount of antibiotics, but this time, 10% fetal calf serum was added to enrich the medium and still, the cells in the culture grew normally, both on day one and day five
• Control #3 included changing the nutrient medium to "minimal nutrient medium"—meaning lowering the percentage of fetal calf serum from the usual 10% to 1%, which lowers the nutrients available for the cells to grow, thereby stressing them—and tripling the antibiotic concentration
• On day five of the experiment, the characteristic CPE occurred, "proving" the existence and pathogenicity of the "virus"—except, at no point was a pathogenic "virus" added to the culture
• This outcome can only mean that the CPE was a result of the way the culture experiment was done and not from any "virus"
• Control #4 is the same control # 3 except that to this culture, a solution of pure RNA from yeast was added which produced the same result as column three, again proving that it is the culture technique—and not a "virus"—that is causing the CPE
• According to a cell culture technical bulletin, when cultures self-destruct for no apparent reason and no evidence of common biological contaminants can be found, cryptic "viruses" are often blamed
• It was stated that this is unfortunate, since the real cause of this culture destruction may be something else, possibly mycoplasma or a chemical contaminant, and as a result will go undetected to become a more serious problem
• The other factors which are said to be able to produce the CPE include:
  • Bacteria
  • Amoeba
  • Parasites
  • Exosomes
  • Antibiotics
  • Antifungals
  • Chemical contaminants
  • Age of the cell
  • Environmental stress
• Cell culture experiments are supposed to include detailed mock-infections yet rarely do
• Mock-infections are supposed to be treated the same way as the experimental culture except without the "virus"
• Sometimes a "non-virulent strain" is used in the mock-infected specimen
• In a pivotal "SARS-COV-2" study by Zhou et al., the antibiotics in the experimental culture were doubled during the culture experiments to achieve a cytopathogenic effect in only 1 out of 24 cultures and this information was left out of the published study
• Regardless, mock-infections are not proper controls as only the variable being studied (the "virus") should be eliminated
• As the unpurified fluids used also contain other host and foreign materials, contaminants, and pollutants, many confounding variables are eliminated as well if no human sample is used with the mock-infected culture
• Thus, a proper control for these pseudoscientific cell culture experiments would be utilizing a sample from a healthy host treated exactly the same way as the sample from the unhealthy host, "viral" transport media and all

If virology wants to be seen as a true science, it must adhere to the scientific method. This means that the experiments and the controls championed as the "gold standard" evidence for the field must be designed in adherence to this method. However, virology has it foundations rooted squarely in pseudoscience as there was never any naturally observed phenomena for which virology was built upon. The "virus" is an imaginary construct dreamt up in the minds of researchers who regularly failed to find a bacterial cause of disease who then assumed that there must be something else smaller and impossible to see contained within the fluids of sick humans. Like the very best Bigfoot hunters, virologists searched for these mythological entities for decades and failed to find direct evidence of them either directly within the fluids of a sick host or through various grotesque tissue cultures and inhumane animal experiments. The attempts to try and prove the existence of the fictional creations was on a downward trajectory, and by the 1950s, all was lost, and the field was ready to die its natural death.

However, things took a turn for the better for virology when John Franklin Enders introduced his fraudulent cell culture method in 1954 and ultimately revitalized the dying field. Virologists were now able to kill cells by combining various toxins in a Petri dish in order to create the picturesque works of art plastered all over the media as the representation of these mythological beings. The subsequent evidence and killing of the cell, referred to as the cytopathogenic effect, was redefined as a "virus" specific effect in order to establish an invalid dependent variable for the non-existent independent variable. Instead of direct evidence for the existence of these entities, a new form of indirect evidence was established in order to fool the masses with. Virology had successfully conjured up its own lab-created variables to play with rather than proving cause and effect through the study of any real-world phenomena.

While the field clung to the cell culture method in order to generate the pseudoscientific evidence needed to keep virology on life-support, there was a major problem that immediately popped up, as pointed out by Enders, the inventor of the technique, which he described witnessing during his initial experiments. The CPE blamed on the "virus" was observed during both the "infected" cultures as well as in his "uninfected" control cultures. This should have been the death knell for virology as it was clear that the toxic experimental design created the effect being blamed on the invisible "virus" as shown by the controls. If this wasn't damaging enough, various other researchers throughout the remainder of the 1950's reported the same findings as the identical CPE blamed on the "virus" was observed in healthy monkey control cultures said to contain no "viruses" whatsoever. Some researchers admitted that this effect could not be used to claim a "virus" was present and then relied on equally fraudulent antibody results to support their "virus" claims instead. Others attempted to state that the healthy control cultures contained "virus-like" agents yet failed miserably in attempts to prove pathogenicity using these fluids, thus showing instead that nothing meeting the definition of a "virus" was present. On top of this, many other factors became known to cause this same cytopathogenic effect other than the assumed "viruses" such as bacteria, amoeba, the antibiotics used, the age of the cell, etc. Virology had successfully refuted itself.

As the detailed descriptions of the control cultures proved the "virus" was not to blame for the observed CPE, many subsequent papers either stopped detailing what was done to the controls or simply avoided using controls all together. Controls known as mock-infections, if present in studies today, are ill-defined and have been shown in cases to be completely fraudulent, such as in the Zhou et al. paper, which treated the mock-infected control differently than the experimental culture, thus invalidating the results of a seminal "SARS-COV-2" paper. While these mock-infections are a form of control, they can hardly be considered proper controls as they do not eliminate only the "virus" particles from the equation. Also eliminated are the rest of the potential organisms, contaminants, chemicals, etc. within the fluids, which have been added to the "viral" sample that are subsequently left out of the mock-infected cultures, which contain no fluids from humans whatsoever. Thus, it can not be said that mock-infected cultures only change up the one variable being studied as there are many variables removed from the equation. Therefore, mock-infections without a comparable human sample minus the assumed "virus" is not a proper control.

If virology wants to be a true science, it must go back to the drawing board. Virologists must attempt to observe a natural phenoma where the independent variable (i.e. the cause; "virus") can be observed in nature. At the very least, this means that they must find the particles that they believe are "viruses" directly in the fluids of a sick host and determine a way to get these particles away from everything else within the fluids. Virologists must determine a valid dependent variable (i.e. the effect being studied; symptoms of disease) in order to establish a testable and falsifiable hypothesis. Only then would a valid experiment be able to be designed, including the establishment of proper controls. Nowhere was this process carried out in order for Enders to create the cell culture method. As the cell culture is not a valid scientific experiment adhering to this process, any and all controls are scientifically invalid as a result. Thus, it really is a waste of time arguing whether or not virology uses controls as their methods are built upon a pseudoscientific foundation. We can argue for virologists to implement what would be proper controls given the pseudoscientific parameters of their experiment, but it is clear that even despite their lack of use of proper controls, virology disproved itself long ago.

It is time to focus instead on the heart of the issue, the lack of adherence to the scientific method. The best control of all is to have virologists walk us through how their field adheres to the scientific method. What natural phenomena was observed? How did the virologists identify and establish a valid IV? What is the DV? What hypothesis led to the creation of the cell culture method, and how was this determined? How was this cell culture method validated without first having successfully purified and isolated the "virus" in order to learn how to grow one? How can virologists justify using a lab-created effect to claim a cause that can not be observed until after the experiment takes place when this is the antithesis of the scientific method? Asking virology to show how it adheres to the scientific method is a control that they will fail at every time. It is the only control needed.

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

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."

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

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 2001; Narasimha 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/

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

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.

Closing the Book on Gain of Fiction with Dr. Tom Cowan

Last Friday, I had the honor of joining Dr. Cowan for a conversation on the mess that is gain of function research. We attempted to make sense of the scarytales that we are currently being sold by those in the mainstream media as well as by some prominent voices in the health freedom movement. While it is interesting to delve into the pseudoscientific methods utilized in these studies, we must understand that these GOF studies are simply nothing but a distraction. To even be able to manipulate "viruses" in a lab, the "virus" in question must be proven to exist first. As we have shown over and over again, this has never once been the case in over a century. What we are left with is the same fraudulent unscientific experiments which have been used to generate frightening headlines in order to terrify the population into submission. Hopefully this conversation will help to shed some light as to what this research involves while serving as a reminder that there is no need to even entertain GOF studies until virology can share evidence adhering to the scientific method supporting the existence of any "virus" first.

Today, we have Mike Stone from Viroliegy. Mike writes brilliant pieces, I must say. They're extremely well researched and well presented. Recently, he wrote one called "Gain of Fiction." I thought it would be good to let Mike go through exactly they're doing in these gain of fiction experiments. I think it will help people understand exactly why it's fiction.

https://drtomcowan.com/blogs/podcasts/57-mike-stone

I also want to share Dr. Cowan's webinar also from last Friday that does an excellent job of shutting down this fictional narrative:

GAIN OF FICTION: DISCUSSING LAB-CREATED "VIRUSES" WEBINAR FROM 11/11/22

In this webinar, we discussed lab-created "viruses," including the new Boston variant.

I have included Dr.'s Sam and Mark Bailey's excellent "Virologie Nights" video here as well. As usual, they do a masterful job pointing out the absurdity of it all:

Virologie Nights

The "Gain of Function" narrative is reaching all new heights. Boston University claimed they engineered a "virus" with an 80% lethality rate. But what actually killed these poor mice?

Let's have a look at some of the "fear-porn" promoters of these stories and why they are leading people astray with pseudoscience.

Virologie Nights

For easy reference, here are the two articles I have done on this topic:

Gain of Fiction It's Gain of Fiction Story Time with RFK Jr. and Friends!

Hopefully, with all of this information, we can finally turn the page on this story and put this gain of fiction novel right where it belongs.

Talking ViroLIEgy with Tom Quinn

On Saturday, I had the privilege of being on the Tom Quinn radio show. Tom is a longtime teacher of ancient and modern religions, philosophy, and health systems. It was evident from the start of my conversations with him that Tom is incredibly knowledgeable about many areas including the fraud of Germ theory and virology. We had two chats before the radio show and my regret is that those were not recorded as they were fun and enlightening conversations on many topics outside of virology as well. I highly recommend tuning in to Tom's show as he covers many topics that may be of interest to many of you. His show is on KSCO 1080 AM on most Saturdays from noon to one PST.

Here is a link to my appearance on the Tom Quinn show which starts at about 21 minutes in:

Michael Wallach, director of the excellent docu-series The Viral Delusion, was also recently on the show on October 29th, 2022. It was because of Michael that I was introduced to Tom who had been wanting to have me appear on his show but did not know how to contact me. I am excited to add their fascinating conversation, which starts around the 12 minute mark, here as well:

The Chemical Structure of Antibodies?

People don't realize that molecules themselves are somewhat hypothetical, and that their interactions are more so, and that the biological reactions are even more so.

-Kary Mullis

Rodney Porter and Gerald Edelman share the credit for "discovering" the chemical structure of the unseen and entirely theoretical antibodies said to provide some form of immunity from infectious disease. They were jointly awarded the Nobel Peace Prize in 1972 for their "discoveries." The researchers both used techniques to break down immunoglobulins into smaller fragments by means of chemical and enzymatic treatments. They then claimed to analyze the physiochemical and biological characteristics of these fragments and proceeded to create hypotheses on how the fragments formed together. Keep in mind, neither researcher purified nor isolated any antibodies and they were still unable to see these entities. In fact, no one had ever seen an antibody as they were simply hypothetical molecules dreamt up in the late 1800's to explain chemical reactions created in the lab. Many theories were proposed over the decades attempting to explain how these invisible "protectors" looked, formed, and functioned but no one had ever laid eyes on them nor had any direct evidence that an antibody existed as believed. Yet somehow, through the magic of chemistry experimentation, Porter and Edelman were said to be able to chemically define the structure of these imperceptible molecules.

A brief historical background of their work:

"The 1972 Nobel Prize in Physiology or Medicine was awarded jointly to Gerald M Edelman and Rodney R Porter "for their discoveries concerning the chemical structure of antibodies" [1,2]. Porter's work used the protein-splitting enzyme papain to identify three fragments, two smaller very similar ones, both with capacity of combining with the antigen, and one larger piece lacking this capacity. Edelman's contribution was the demonstration that immunoglobulin molecules, like most biologically active proteins, were composed of chain structures that were held together by sulfur bonds and could be separated. None of the resultant fragments retained the specific reactivity of the intact immunoglobulin molecule. Since then, progress has been made in understanding the finer characteristics of the various domains of the individual chains of the immunoglobulin molecule.

https://www.uptodate.com/contents/structure-of-immunoglobulins/print

Rodney Porter 1959

According to the above source, Rodney Porter was able to use papain, a protein-splitting enzyme, to break down the antibody into its three distinct fragments. However, was this truly the case? Below are highlights from Rodney Porter's contribution to this antibody puzzle.

The Hydrolysis of Rabbit y-Globulin and Antibodies
with Crystalline Papain

"The molecular size of rabbit y-globulin is such that any direct attempt to relate structure to biological activity is not feasible at present. An alternative approach is to degrade antibody molecules in such a way that activity will persist in smaller fragments and so to reduce the structural problems involved.
In an earlier investigation of this type (Porter, 1950b) it was found that, if rabbit y-globulin containing antiovalbumin was digested with crude papain, a fragment with a molecular weight of about 40,000 could be produced which retained the ability to combine specifically with ovalbumin though it would no longer form a precipitate. It seemed probable that this fragment contained an antibody-combining site.

In that work, the amounts of crude enzyme used relative to y-globulin were large, making subsequent investigation of the products of digestion rather difficult. As crystalline papain can now be prepared easily (Kimmel & Smith, 1954) and fractionation techniques have improved, these earlier experiments have been repeated. It has been found that y-globulin is split by papain into three large pieces with very little release of amino acids or small peptides. If the y-globulin contains antibody against any of the several antigens investigated, then two of these pieces retain combining though not precipitating power. The third piece, which may be readily crystallized, has no antibody activity but it has most of the antigenic sites of the original molecule. The isolation and properties of these three fractions will be described.

A preliminary account of this work has been given (Porter, 1958a).

EXPERIMENTAL

Materials

Antisera. Rabbit antiovalbulmin, antibovine serum albumin and antihuman serum albumin were prepared by intravenous injection of the alum-precipitated protein (Porter, 1955).

Rabbit antipneumococci polysaccharide type 3 was prepared by injection of a suspension of the formalin-killed bacteria in 0-9 % NaCl solution.

Goat antirabbit y-globulin was prepared by intravenous injection of alum precipitate and subcutaneous injection of y-globulin with adjuvant (Freund & McDermott, 1942).

Rat antiserum was prepared according to the following schedule. The rats were given two intramuscular injections of protein and adjuvant into different thighs, with 1 week's interval between injection. Five weeks later they were given three injections, with 3-day intervals between injections, of alum-precipitated protein into the tail vein. They were bled by heart puncture 6 days after the last injection.

Rabbit y-globulin. This was prepared either by chromatography, with diethylaminoethylcellulose (Sober, Gutter, Wyckoff & Peterson, 1956), or by Na2SO4 precipitation according to the method of Kekwick (1940).

Crystalline papain. This was prepared from crude enzyme powder purchased from Hopkin and Williams Ltd., London. The enzyme was crystallized once as the free enzyme and twice as the inactive Hg dimer (Kimmel & Smith, 1954). It was freeze-dried and stored as the dimer.

Methods

Quantitative estimation of precipitating antibody. This was carried out according to Kabat & Mayer (1948).

Estimations of inhibitory power. These were made by quantitative antibody assay in the presence of the inhibitor or by measure of the delay in precipitation caused by the inhibitor with the antibody and antigen mixed in optimum proportions.

Chromatography. Diethylaminoethylcellulose and carboxymethylcellulose were prepared according to Peterson & Sober (1956). Column size was 2-4 cm. (diam.) by 30-35 cm. (ht.); volume of mixing chamber was 1200 ml. Buffers used on carboxymethylcellulose columns were O OlM-sodium acetate, pH 5 5, with gradient to 0.9M-sodium acetate, pH 5.5. In the refractionation of fraction I on diethylaminoethylcellulose, the following system was used: 001m-sodium phosphate, pH 64, with gradient to 0 2 M-sodium phosphate, pH 6-2. All buffers were saturated with toluene. The pH was measured with an EIL Direct Reading pH meter (Electronic Instruments Ltd.).

Enzyme digestion. y-Globulin (150 mg.) and Hg papain (1.5 mg.) were dissolved in 10 ml. of buffer (0 1M-sodium phosphate, pH 7 0, 0*01 m-cysteine, 2 mM-ethylenediamine-
tetra-acetate). This solution was incubated at 370 for 16 hr. in the presence of toluene. It was then dialysed against water with vigorous stirring and several changes of the outer liquid over 48 hr. This procedure, which removed the cysteine and ethylenediaminetetra-acetate, and facilitated oxidation, inactivated the enzyme. N-Ethyl maleimide was also used to inactivate the enzyme but as there appeared to be no advantage this was not continued. The non-diffusible digestion products were either freeze-dried or fractionated directly by chromatography after dialysis against acetate buffer, pH 5-5.

Protein determinations. Protein concentrations were determined by reading the absorption at 280 and 260 mu in a 1 cm. cell in a Unicam SP. 500 spectrophotometer.

Radioactivity measurements. Injection of hydrolysed algal [14C]protein and counting of the y-globulin fraction were carried out as described previously (Askonas, Humphrey & Porter, 1956).

Analytical methods. Amino acid analysis was by the method of Moore & Stein (1951) as modified by McDermott & Pace (1957).

N-Terminal amino acids were estimated by the fluoro-dinitrobenzene technique (Porter, 1957a). Hexose as 'glucose' was estimated by the anthrone method of Mokrasch (1954). Hexosamine as 'glucosamine' was
estimated, after separation from amino acids on a cation-exchange resin (Boas, 1953), by a modification of the method of Elson & Morgan (1933).

RESULTS

When a digestion mixture, prepared as described above, was dialysed against water at 2° a precipitate formed which appeared to be crystalline. If, however, the dialysis was against 0 04N-acetic acid there was no precipitation and the recovery of the non-diffusible digestion products could be estimated either by measuring the absorption at 280 mp or by freeze-drying and weighing the dry powder. This has been done in a number of experiments, and by either method the recovery of y-globulin protein taken has fallen in the range 85-95 %. In view of the probable handling losses in dialysis and freeze-drying, it is considered that the higher figure is the most accurate. When such a digest was examined in the ultracentrifuge in 0O1M-phosphate, pH 6.7, some crystals again formed on dialysis but the supernatant showed only one peak (S20., 3.5). As y-globulin has S20,w 6-5, it was clear that the protein had been split into large fragments of similar size with very little production of diffusible peptides. Attempts to fractionate this mixture by zone electrophoresis were not successful, but resolution could be achieved by chromatography on carboxymethyl-cellulose. Acetate buffer, pH 5 5, was chosen because under these conditions most of the
carboxyl groups of the ion-exchanger are dissociated; if the digest was brought nearer to neutrality crystals formed, indicating a low solubility of at least one component. To help to keep all the material in solution chromatography was carried out at room temperature (20-23o). With a
gradient of increasing salt concentration at this pH, three components could be resolved which have been named fractions I, II and III in order of elution from the column (Fig. 1).

If the gradient on the column was reduced, fractions II and III were more spread and III 'tailed' badly, but there was no suggestion of any further resolution. Fraction I appeared very close to the solvent front and this was re-run on a diethylaminoethylcellulose column at pH 6-4. No fractionation was obtained but again with a slow
gradient there was considerable tailing. By these limited criteria the three fractions appeared to be single components and to be the only significant products of the digestion of y-globulin by papain.

Results with shorter times of hydrolysis under the same conditions suggest that the splitting is complete in very much less than 16 hr., the period which has always been used. It follows that these three fractions are exceptionally resistant to further digestion by papain.

Yields of the three fractions were measured by summing the absorption at 280 mp in each peak. The ratios of yield varied somewhat from experiment to experiment but averaged (I: II: III) 1: 0-8: 0-9, and total recovery from the column was 85-90%. When re-run, fractions I and III were recovered in about 95 % yield and fraction II in about 85% yield. The absorptions of peaks I, II and III at 280 m, at a concentration of 1 mg./ml. in water or 0-02N-acetic acid were 1-4, 1-4 and 1-0 respectively. If the relative yields are corrected for the lower recovery of fraction II, and the lower specific absorption at 280 m,p of fraction III, then the corrected relative yields (I:II:III) are 1:0-9:1-25 by wt. In some experiments the fractions were concentrated by pressure-dialysis in the cold against water or 0-02N-acetic acid; in-soluble material was discarded and the solution freeze-dried and weighed. The yields of I and III were similar to those above but II was somewhat lower. Considerable error can occur because of denaturation in dilute solution, and variable losses on chromatography, but it is considered that the average yields calculated from chromatography approximate to the yields of the fractions produced in the digestion.

Fraction III is readily identifiable as the material with a low solubility near neutrality. It may be crystallized and recrystallized by dialysing a solution in 0-02N-acetic acid against sodium phosphate buffer, pH 6-0-7-0, at 20. The crystals are diamond-shaped plates, often of considerable size but thin and easily broken (Fig. 2).

Molecular weights. The three fractions were studied in the ultracentrifuge (see Addendum) and the results for normal y-globulin are simmarized in Table 1. The sum of the molecular weights of the three fragments is very close to the molecular weight of the original y-globulin. This is in agreement with the high recovery of non-diffusible digestion products. The relative sizes of the three fragments were (I:II:III) 1:1-05:1-6, which is in
approximate though not exact agreement with the calculated relative yields of the fragments."

"These results have been recalculated in terms of amino acid residues per mol. of fraction and per mol. of y-globulin. It has been assumed that the ash-free, moisture-free fractions have the same nitrogen content of 16 % as found for y-globulin by Smith, McFadden, Stockell & Buettner-Janesch (1955). In view of the difference in basic amino acid content the figure may be rather high for fractions I and II and low for fraction III. The results of this calculation are given in Table 3. The amino acid residues accounted for by the three fractions range from 83 to 116% of the figures for whole y-globulin. These discrepancies are probably explicable by errors in the different estimations, the assumption of 16 % of nitrogen in each fraction and by the peptide material lost in the dialysis. The overall recovery of amino acid residues in this calculation is 94 %.

"Carbohydrate estimations were made by Dr H. R. Perkins of this Institute, and the results with whole y-globulin and the fractions are given in Table 4. About two-thirds of the carbohydrate of the original molecule is found with fraction III, one third with fraction I and a small amount, which may not be significant, is with fraction II. It seems likely therefore that the carbohydrate moiety of y-globulin is in the two pieces, the larger with that part of the molecule which corresponds to fraction III and the smaller with the part corresponding to fraction I. If the total recovery of carbohydrate in the three fractions is compared with that present in the original, the yields are approximately 110 and 120 % for hexose and hexosamine respectively.

N-Terminal assay has been carried out on fractions I and II, and in both alanine was found to be the main terminal amino acid (about 0-9 mol./50 000), together with smaller amounts of aspartic acid (0.2 mol./50 000) and trace amounts of serine and threonine (together about 0- 1 mol./50 000).This is a very similar result to that obtained for whole rabbit y-globulin (Porter, 1950a; McFadden & Smith, 1955), except that the N-terminal amino acid content is more than three times as great as in the whole globulin. It is also in approximate agreement with the results of the digestion of rabbit antiovalbumin with papain powder, when an immunologically active fraction containing one N-terminal alanine per 40 000 was reported (Porter, 1950a). The significance of finding N-terminal alanine in both I and II is not certain, as there are about 110 alanine residues per molecule of y-globulin. An attempt to determine the N-terminal sequence was unsatisfactory owing to shortage of material, but it appeared that the sequence of I was alanylaspartyl and of II alanyl-leucyl. If this is correct, it suggests that II may derive from the N-terminal part of the molecule, as the sequence there is alanyl-leucylvalylaspartylglutamyl (Porter, 1950a; McFadden & Smith, 1955).

Immunological properties. The three fractions were prepared from digests of y-globulin which had been obtained from rabbit antisera against ovalbumin, bovine serum albumin, human serum albumin and antipneumococci polysaccharide type 3. None would precipitate with the corresponding antigen but fractions I and II prepared from y-globulin containing antiprotein antibodies inhibited the precipitation of the antigen by the homologous antiserum. This effect is specific; for example, I and II from antihuman serum albumin y-globulin had no effect when added to bovine serum albumin and its antiserum; fraction III, on the contrary, had no effect whatever its source or whatever the test system. Testing fraction III is difficult owing to its low solubility near neutrality, where antibody-antigen precipitation is carried out, but it is soluble to about 0 3 mg./ml. at 37* at pH 7-2 and it could be shown that it had less than one-thirtieth of the activity of the corresponding fractions I and II.

Quantitative estimates of the inhibitory power of I and II are shown in Fig. 3. In order to assess this on a molar basis it has been assumed that the fractions would have the same proportions of active molecules as the proportion of antibody in the y-globulin taken, and the weight of each fraction has been corrected accordingly when plotting the graph."

"Antigenic activity. The power of the fractions to precipitate with goat antirabbit y-globulin serum was now tested and I and II showed neither precipitation nor inhibitory activity. Fraction III precipitated 70% of the antibody precipitated by y-globulin.

Rat antiserum was prepared against rabbit y-globulin and fractions I, II and III. Groups of three rats were used for each antigen and, after the
immunization course described above, the serum of each group was pooled, and gave antibody contents of 4-2, 7-8, 8-1 and 4-0 mg. of antibody/ml. for y-globulin, I, II and III respectively. All the fractions were at least as effective antigens as the original molecule, and I and II were the most effective, but as only three animals were used per group the difference may not be significant. When rat anti-y-globulin was tested with the fractions, rather different results from those with goat antiserum were obtained. Fraction III precipitated 50% of the antibody, and I and II 15% each. The antigenic specificities of the fractions were compared by measuring the precipitates formed by 1, II and III with rat anti-II serum. The precipitation curves are shown in Fig. 5. It can be seen that the curves for I and II are almost identical, but III gives almost no precipitate. This again emphasizes the great similarity of I and II and their sharp distinction from III.

Synthesis of the fraction in vivo. In view of the distinctive characters of the three fractions the possibility was considered that they might be synthesized separately and joined into the whole molecule in a separate step. If this were to happen the fractions would probably not all be synthesized from the same pool of amino acids at the same time, and hence if radioactive amino acids were injected into a rabbit the incorporation of radioactivity after a short time interval would vary from fraction to fraction. Hydrolysed algal [14C]protein was used as the source of labelled amino acids; 240uc was injected intravenously, and the rabbit
was bled after 1 and 4 hr. The plasma y-globuilin is not labelled until 30 min. after injection (Askonas et al. 1956). The y-globulin was prepared and hydrolysed with papain, and the fractions were isolated, precipitated with trichloroacetic acid, and counted, at infinite thickness on a 1 cm.2 disk. The specific activities are given in Table 5 and it can be seen that there is no significant difference between the different fractions. There is therefore no evidence to suggest that these fractions or any large parts of them are synthesized independently.

DISCUSSION

The results show that papain-digestion of rabbit y-globulin causes limited and highly selective splitting to give three large fragments and very few small peptides. As papain shows rather a wide specificity in the digestion of the B chain of insulin (Sanger, Thompson & Kitai, 1955), it is apparent that the structure of the native molecule must be such that many potentially hydrolysable bonds are protected by steric and other factors. Ultracentrifuge studies of the splitting of y-globuilins from different species, by a variety of enzymes (Petermann & Pappenheimer, 1941; Petermann, 1942, 1946), have all shown that a small number of fragments are the main products in each case. It is possible that in y-globulins only small parts of the peptide chain are accessible to proteolytic enzymes, so that even though different enzymes break different bonds in these vulnerable sections the principal large digestion products are similar.

Our results with the papain-digestion of rabbit y-globulin suggest that the single polypeptide chain (Porter, 1950a; McFadden & Smith, 1955) have been split into three distinct sections, which together comprise some 90% of the original molecule. However, fractions I and II are so similar that the question arises whether they could be derived very largely from the same section of the chain. They are almost indistinguishable in N-terminal amino acid, amino acid analysis, molecular weight, antigenic specificity and, if derived from antibody, in their antigen-binding capacity. They differ in chromatographic behaviour and carbohydrate and cystine content.

There are at least three possible ways of splitting a single chain to give results such as this, and they are illustrated in Fig. 6: A shows the obvious split into three distinct sections, B the production of two fractions from the same section of chain and C the result if y-globuilin consists of two types of molecule, such as euglobulin and pseudoglobulin, one of which gives rise to I + III and the other to II+ III. If either B or C were correct then the yield of III should exceed the sum of the yield of I and II. In fact the yield of each of the three fractions is very similar, as would be expected in A. In B and C the overall recovery, in view of the molecular weights found, would be 130,000/188,000, i.e. 70 %, considerably lower than the experimental figure of about 90%, which again is that which would be expected if A were correct. Similarly, with the individual amino acid residues, the recoveries would be only 70%, with the possibility of big variations in individual cases as much more material would be lost. In fact the recovery of residues is 90-95%, and the variation 83-120% in
the recoveries of individual amino acids is close to the range expected from the errors in the different measurements and the assumptions made in the calculation.

Further, in B it would be expected that if I and II were digested further by papain, either interconversion would occur or both would be reduced to a slightly smaller common product. In fact both appear to be stable to a further 16 hr. digestion with papain at 370, being unchanged in chromatographic behaviour and other properties. The simplest explanation of our results therefore seems to be that y-globulin has been split into three distinct fractions, as shown in A. It is probable that the inhibitor described in the earlier work (Porter, 1950b) was a mixture of fractions I and II, and that fraction III was prevented from crystallizing by the impurities introduced with the crude enzyme preparation.

It follows that two large sections of y-globulin (each about 30 % of the whole) are extremely similar in chemical, physical and biological properties. The finding of such close agreement between the amino acid analysis of these two fragments and also an almost identical antigenic specificity suggests that there may be almost a repeat of the amino acid sequence and configuration. This is in contrast with the properties of fraction III, which differs in every respect, so that there appears to be a large repeating unit (I or II) joined to a larger section (III) of entirely different character. This unusual make-up of the y-globulin molecule is presumably related to its antibody activity. The similarity of the pieces with mol.wt., 50 000, where the antibody-combining sites may be very much smaller (Kabat, 1956), raises the question whether large sections are required to maintain the structural integrity of a small combining site or whether antibody-combiing sites may occur anywhere in these pieces.

The big quantitative difference between the inhibitory power of fractions I and II, when derived from antiprotein or antipolysaceharide antibodies, may reflect important differences between the two types of combining site, but perhaps more probably arises from differences in the speed and mechanism of precipitation between the two systems.

The significance of the part of the y-globulin molecule represented by fraction III in antibody-antigen reactions is not known. The ease of crystallization could be taken as evidence of greater identity of structure among individual molecules in ImI than in the whole molecule, which appears to be complex by all available physical and biological data (see Porter, 1958b). Preliminary electrophoretic examination, however, suggests that it is as complex as the original molecule by this criterion. Most of the antigenic sites of y-globulin appear to be on m, but as these sites can only be defined in relation to a given antiserum (Porter, 1957b), variable results might be expected, and there is in fact a difference in this respect between the goat and rat anti-y-globulin serum. Fraction III is remarkably stable. After precipitation by 5% trichloroacetic acid at room temperature, washing with ethanol and ether and drying for 1 hr. at 1100, it will redissolve in 0-05N-acetic acid and it retains its power specifically to precipitate goat antirabbit y-globulin.

The finding that y-globulin appears to be built of three sections, one of exceptional stability and the other two containing the antibody-combining
sites, is reminiscent of Pauling's (1940) theory of antibody formation, in which it is suggested that antibody molecules consist of a rigid centrepiece and two flexible ends capable of taking up configurations complementary to the antigen and hence forming antibody-combining sites on these flexible parts. Pauling further suggested that the flexibility might be due to a high content of proline residues. There is no evidence on the relative positions of fractions I, II and III in the whole molecule, except that II may be from the N-terminal end. Nor is there any evidence on the essential feature of Pauling's theory, that the amino acid sequence of all antibodies is identical and that the different antibody-combining sites are formed only by refolding of the same polypeptide chain. The proline content of I and II is less than that of III, whereas the cystine content, often an important feature in determining the stability of a protein molecule, is lower in III than in I and II.

The equal rate of incorporation of radioactive amino acids into the different fractions is in agreement with the view that the whole molecule is synthesized simultaneously from the same pool of amino acids.

SUMMARY

1. Rabbit y-globulin, when digested by crystalline papain, gives three fragments which together form 90% or more of the original molecule.
2. If the y-globulin contains antibodies, two fragments (I and II), of molecular weight 50,000-55,000, retain the power to combine with the antigen. The third fragment (III), molecular weight about 80,000, crystallizes easily and has much of the antigenic specificity of the original molecule.
3. I and II are extremely similar in chemical and biological properties, and III differs very widely in all respects. This has led to the suggestion that rabbit y-globulin is formed of two pieces with very similar structure joined to a third piece of quite different character.

doi: 10.1042/bj0730119.

Rodney Porter is given credit for helping to determine the chemical structure and the Y-shape of the unseen antibodies. He did so by what appears to be reverse engineering the antibodies by way of breaking the protein substances down into the smallest pieces possible, analyzing them chemically, and then creating a theoretical framework and model for how they supposedly joined together. At no point were the assumed antibodies seen as a whole unit before being digested by papain. Unless you can convince yourself the Superman-like diamond trap image in the study is an antibody fragment, there were no credible images of any of the three fragments matching what an antibody is said to look like, fragmented or whole.

Even if there were accompanying images, it is apparent from his own conclusions that Rodney Porter, after supposedly breaking down the y-globulin into the three fragments, was uncertain if they were antibodies at all as he was apparently not convinced yet if the y-globulin contained antibodies. However, that did not stop Porter from creating a hypothesis based on the results from the chemical reactions of his experiments. He then applied his hypothesis to previously established antibody theories, such as that presented by Linus Paulimg with the Direct Template theory in 1940, which itself was eventually rejected by most immunologists in favor of Frank MacFarlane Burnett's Clonal Selection Theory in 1957.

It is interesting to note that within the paper, the purification of the antibody fragments is never stated, even though different methods, such as ultracentrifugation and chromotograohy, were listed as having been used. Perhaps this is due to the fact that to achieve the most efficient purification levels required today, it is considered vital to have knowledge of the structure of the antibody first:

"Rodney Porter and Gerald M Edelman first elucidated the characteristic Y-shaped structure of antibodies. In 1972, they were awarded the Nobel Prize for Medicine and Physiology for their findings. These Y-shaped molecules were eventually identified as immunoglobulin G (IgG), the structure of which is composed of four polypeptide chains – two heavy (50 kDa) and two light chains (25 kDa) – linked by noncovalent bonds and disulfide bridges (Figure 1)."

"However, to perform the most efficient purification possible it is vital to have good knowledge of the structure of the antibody (or antibody derived structures) and its cognate antigen and ideally the affinity of their interaction."

https://www.dovepress.com/technology-advancements-in-antibody-purification-peer-reviewed-fulltext-article-ANTI#F1

This appears to leave us with a chicken and the egg scenario. How would Rodney Porter have achieved the required purification needed in order to elucidate the chemical structure and the Y-shaped composition of an antibody if he lacked the vital knowledge of the structure of an antibody to begin with? Logic would dictate that he would not be able to do so. Also, Porter was working with what would be considered polyclonal antibodies as monoclonal antibodies were not created until the 1970's. Polyclonal antibodies are said to be a complex mixture of antibodies contained within the serum of the host whereas monoclonal antibodies are supposedly a specific antibody cloned and cultured in the lab. Antigen-specific affinity purification is said to be required for polyclonal antibodies to prevent the inclusion of non-specific antibodies as more than one type is thought to be within the sample. This procedure was not utilized by Porter. In fact, depending on the chromatography equipment he would have utilized, these methods are said to be unable to purify antibodies from other proteins and macromolecules:

"By contrast, for polyclonal antibodies (serum samples), antigen-specific affinity purification is required to prevent co-purification of nonspecific immunoglobulins. For example, generally only 2–5% of total IgG in mouse serum is specific for the antigen used to immunize the animal."

"Dialysis, desalting, and diafiltration can be used to exchange antibodies into particular buffers and remove undesired low-molecular weight (MW) components. Dialysis membranes, size-exclusion resins, and diafiltration devices that feature high molecular weight cut-offs (MWCO) can be used to separate immunoglobulins (>140 kDa) from small proteins and peptides. However, except with specialized columns and equipment, these techniques alone cannot purify antibodies from other proteins and macromolecules that are present in typical antibody samples. More commonly, gel filtration and dialysis are used following other purification steps, such as ammonium sulfate precipitation [1]."

https://www.thermofisher.com/us/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-methods/antibody-purification-methods.html#:~:text=Antibody%20purification%20involves%20selective%20enrichment,cell%20line%20(monoclonal%20antibodies).

Thus, Porter appears to be left with a pretty important knowledge and equipment gap that is required in order to properly purify antibodies away from other antibodies, proteins, and macromolecules that would also be contained within the sample. It is important to ask how would he have known that the fractions he was working with were even from an antibody at all if they were unable to be properly purified and isolated away from other components/impurities. It seems as long as one takes whatever results are achieved through experimentation and then fits them into a hypothetical model for what these invisible particles could possibly look like, a Nobel Prize awaits.

GERALD EDELMAN 1961

Gerald Edelman is the other half of the dynamic duo awarded for figuring out the chemical composition and structure of an antibody. It was said that he succeeded in splitting the IgG sulphide bonds, thus showing how these unseen fragments were supposedly held together. As was the case with Porter, Edelman tried to reverse engineer the theoretical molecules no one had ever seen before and fit his results into a model:

"Dr. Edelman spent the next several years working backward to recreate a model of the principal antibody molecule, which he achieved in 1969. During that time, he also hypothesized — and was later proven correct — that the vast diversification exhibited by antibodies is an example of the body turning a developmental flaw into an advantage. When cells divide, miniscule errors in transcription often occur, leading to the development of proteins with differences that in the immune system amount to a system of "strength through diversity."

https://www.rockefeller.edu/our-scientists/gerald-m-edelman/2476-nobel-prize/

If it wasn't clear that both researchers had no idea what an antibody looked like as they were conducting their experiments, it is stated in an article in Immunology Letters that after his experiments, Edelman proposed the Y-shape of an antibody which was subsequently "confirmed" by electron microscopy and x-ray diffraction:

"In the same year, Edelman showed that reduction of the disulfide bonds of antibodies in the presence of denaturizing agents led to dissociation of the molecule into smaller pieces, now known to be the light (L) and heavy (H) chains [6]. Because the molecular weight of the original IgG molecule is 150 kDa, he concluded that the IgG molecule consisted of two heavy and two light chains linked by disulfide bonds and noncovalentinteractions. A Y-shaped configuration was proposed and then confirmed through electron microscopy and X-ray diffraction study. Thereafter, two antigenic types of light chains, denominated and chains were described (Fig. 6)."

https://doi.org/10.1016/j.imlet.2015.02.005

Interestingly, no source was cited for this "confirmation" nor were any real images of an antibody supplied as evidence. Instead, we get a drawing and a photo of Edelman next to his model at the Rockefeller University:

Many of the methods Edelman utilized in his paper were similar to Porter. As the paper is 24 pages long, I am presenting just a few highlights from the beginning and the ending to give an overview of his work. You will see that Edelman tentatively (subject to further confirmation; not definitely) concluded that the fragments were held by disulfide bonds and he then created a hypothesis to explain his findings. As with Porter's work, no images of purified and isolated antibodies nor antibody fragments are presented in the paper. Feel free to read the rest of Edelman's work with the provided link if you desire the full breakdown of his chemistry experiments:

Studies on Structural Units of the Y-globulins

"In studying the molecular structure of the y-globulins, one is confronted at the outset with the problem of whether these proteins consist of one or of several polypeptide chains. The solution of this problem has significance in determining the chemical basis of antibody specificity, and in formulating a detailed theory of antibody production. In addition, it bears upon the relation between normal y-globulins and those of disease.

A previous report (1) showed that normal human 7S y-globulin and a pathological macroglobulin dissociated to components of lower molecular weight when treated with reagents that cleave disulfide bonds. The present study is concerned with an extension of this approach to a variety of normal and abnormal y-globulins. Partial separation of the dissociation products of y-globulin has been achieved by means of column chromatography and starch gel electrophoresis. The results suggest that y-globulin molecules are composed of several discrete subunits or polypeptide chains linked by disulfide bonds."

"Porter (45) has recently demonstrated that rabbit y-globulin is cleaved by papain treatment into three fragments which together form over 90 percent of the original molecule. These fragments have molecular weights ranging from 50,000 to 80,000. Similar fragments have been found for papain-treated human y-globulins (46). The fragments obtained by treatment with papain do not seem to be identifiable with the subunits described above. It is likely, however, that the products of papain treatment are composed of portions of these subunits. This interpretation is strengthened by the finding that the two active antibody fragments obtained by treatment with proteolytic enzymes are linked by a single disulfide bond (47).

A unifying hypothesis may be formulated for the structure of proteins in the y-globulin family based on the findings presented above as well as on findings of other investigators. 7S y-globulin molecules appear to consist of several polypeptide chains linked by disulfide bonds. Bivalent antibodies may contain two chains that are similar or identical in structure. The 19S y-globulins would be composed of 5 or 6 multichain units of the size of 7S y-globulin. A provisional explanation for the wide molecular weight range of antigenically related globulins from Bence-Jones proteins to macroglobulins is suggested by this model. Heterogeneity and differences in isoantigenicity (48, 49) may arise from various combinations of different chains as well as from differences in the sequence of amino acids within each type of chain.

The finding that y-globulin contains dissociable subunits has a possible bearing upon the pathogenesis of diseases of y-globulin production. A primary defect in macroglobulinemia and multiple myeloma may be a failure of specificity and control in production and linkage of the various subunits to form larger molecules. Bence-Jones proteins may be polypepfide chains that have not been incorporated into the myeloma globulins because of a failure in the linkage process. Myeloma globulins may consist of combinations of subunits differing from those of the normal y-globulins, although both types of protein appear to contain subunits that are alike. This may explain in part the antigenic and chemical differences and similarities that have been found between the y-globulins of disease and normal y-globulin (36, 37). The hypothesis outlined above is capable of experimental test, since the products of various chemical treatments may now be separated and compared.

SUMMARY

When human and rabbit 7S y-globulins were reduced in strong urea solutions by a number of procedures, their molecular weights fell to approximately of the original values. Partial separation of the reduction products was achieved using chromatography and starch gel electrophoresis in urea solutions. One of the components of reduced human 7S y-globulin was isolated by chromatography, identified by starch gel electrophoresis, and subjected to amino acid analyses. The amino acid composition of this component differed from that of the starting material and also from that of the remaining components.

A reduced pathological macroglobulin dissociated to components with an average molecular weight of 41,000. Several reduced human myeloma proteins, when subjected to starch gel electrophoresis, yielded individual patterns that nevertheless had features in common with those of reduced normal v-globulins. Reduction of normal and abnormal y-globulins was accompanied by the appearance of titratable sulfhydryl groups. Chemical treatments other than reduction were used to determine the type of bond holding the subunits together. It was tentatively concluded that they were linked by disulfide bonds. An hypothesis is presented to relate the structural features of the various y-globulins in terms of the multiplicity of polypeptide chains in these molecules."

doi: 10.1084/jem.113.5.861.

In Summary:

• Porter started off by admitting that the size of Rabbit y-globulin is such that any direct attempt to relate structure to biological activity was not feasible
• It seemed probable that rabbit y-globulin digested by crude papain contained fragments of antibody molecules
• Porter claimed that y-globulin split by papain formed three fractions
• His experiments were to see if y-globulin had antibody properties in two of the fractions and stated that there was none in the third fraction
• The amount of precipitating antibodies and the inhibiting power were estimated through various methods and calculations
• N-Terminal amino acids, glucose, and glucosamine content were also estimated
• As y-globulin has S20,w 6-5, Porter stated by this indirect measurement that it was clear that the protein had been split into large fragments of similar size with very little production of diffusible peptides
• Attempts to fractionate this mixture by zone electrophoresis were not successful
• Porter admitted that by his limited criteria, the 3 fractions seemed to be of a single molecule and were the only significant substances in the papain digestion
• Results with shorter times of hydrolysis under the same conditions suggested that the splitting is complete in very much less than 16 hr., the period which had always been used, and thus it was assumed that these three fractions were exceptionally resistant to further digestion by papain (i.e. he decided it was not necessary to attempt splitting the fragment for any longer than 16 hours as it could only be broken into three fragments and he assumed no further degradation would occur if a longer time was attempted)
• Porter admitted the yields are affected by considerable errors due to denaturation by dilute solution and variable losses on chromatography
• Fraction 3 formed crystals that were diamond-shaped plates, often of considerable size but thin and easily broken (that doesn't sound like any image of an antibody fragment I've ever seen…)
• The sizes of the 3 fractions were in approximate but not exact agreement with calculated yield
• It was assumed that the ash-free, moisture-free substance of y-globulin was the same 16% nitrogen concentration as found by previous researchers
• Porter admitted that there were discrepancies which were probably explicable errors in the different estimations, the assumption of 16% nitrogen in each fraction, and the peptide material lost in dialysis
• He believed it was likely that the carbohydrates were in fractions 1 and 2
• He admitted that the significance of finding the N-Terminal in fractions 1 and 2 was unknown
• Attempts to determine the N-Terminal sequence were unsatisfactory
• The three fractions were prepared from digests of y-globulin which had been obtained from:
  • Rabbit antisera against ovalbumin (rabbit blood infused with chicken egg protein)
  • Bovine serum albumin (cow blood)
  • Human serum albumin (human blood)
  • Antipneumococci polysaccharide type 3 (bacterial origin)
• There was no immunologic effect on fraction 3 no matter what source they used/tested
• It was assumed that the fractions would have the same proportion of active molecules as the proportion of antibody in the y-globulin taken
• The power of the fractions to precipitate with goat antirabbit y-globulin serum was tested and I and II showed neither precipitation nor inhibitory activity
• When rat anti-y-globulin was tested with the fractions, rather different results from those with goat antiserum were obtained as fraction III precipitated 50% of the antibody, and I and II 15% each
• As the precipitation curves for I and II were almost identical, but III gives almost no precipitate,  this emphasized the great similarity of I and II and their sharp distinction from III
• Porter stated that in view of the distinctive characters of the three fractions the possibility was considered that they might be synthesized separately and joined into the whole molecule in a separate step
• He decided after injecting a rabbit, bleeding it, and then radioactively labelling the precipitates, that there was no evidence to suggest that these fractions or any large parts of them are synthesized independently
• Ultracentrifuge studies of the splitting of y-globuilins from different species, by a variety of enzymes, all showed that a small number of fragments are the main products in each case
• He claimed that it was possible that in y-globulins, only small parts of the peptide chain are accessible to proteolytic enzymes, so that even though different enzymes break different bonds in these vulnerable sections the principal large digestion products are similar
• Porter stated that his experiments suggested that the 3 fractions were from one molecule split into separate sections yet only made up 90%
• Fractions I and II were so similar that the question arose whether they could be derived very largely from the same section of the chain
• Porter stated that fractions I and II were almost indistinguishable in N-terminal amino acid, amino acid analysis, molecular weight, antigenic specificity and, if derived from antibody, in their antigen-binding capacity (thus, he was unsure if these fragments even belonged to an antibody)
• Porter stated that the range in recovery of residues and recovery of individual amino acids was close to the range of expected errors due to the different measurements and the assumptions made in the calculations
• He stated that the simplest explanation for his results was that y-globulin was split into 3 different fractions
• Porter stated that the discrepancies with his earlier work was probably due to impurities introduced in the crude enzyme preparation
• He also stated that the unusual make-up of the y-globulin is due to his presumption of it being an antibody
• The significance of the part of the molecule that fraction III represented (as they were trying to fit it into a predetermined model) was unknown
• Porter admitted that his findings of the 3 fractions of antibody molecule was similar to Pauling's antibody formation theory in 1940
• However, he was not certain what position the 3 fractions were in the make-up of the molecule
• In the summary, Porter posed the question "If Y-globulin contains antibodies" thus showing the uncertainty in his own findings
• If they are antibodies, Porter surmised that the y-globulin is made of up two similar, nearly identical fractions and one very different one

• According to Edelman, in studying the molecular structure of the y-globulins, one is confronted at the outset with the problem of whether these proteins consist of one or of several polypeptide chains
• The solution of this problem has significance in determining the chemical basis of antibody specificity, and in formulating a detailed theory of antibody production
• The results suggested that y-globulin molecules are composed of several discrete subunits or polypeptide chains linked by disulfide bonds
• Edelman reiterated that Porter demonstrated that rabbit y-globulin is cleaved by
  papain treatment into three fragments which together form over 90 percent of the original molecule (what happened to the remaining 10 percent?)
• Edelman stated that the fragments obtained by Porter were not the same as those which he discovered
• He did, however, make the interpretation that Porter's fragments may be made up of the smaller subunits he himself obtained
• Edelman felt his interpretation was strengthened by the finding that the two active antibody fragments obtained by treatment with proteolytic enzymes were linked by a single disulfide bond
• Edelman believed that his findings along with those of other researchers could be combined into a unifying hypothesis
• He stated that antibodies may contain two chains that are similar or identical in structure
• His model suggested an explanation for the wide weight ranges between the various molecules
• The finding that y-globulin has dissociable subunits may have a bearing on pathogenesis of disease (and then again, it may not…)
• A primary defect in macroglobulinemia and multiple myeloma may be due to a failure of specificity and control in the linkage of smaller units into larger ones
• Myeloma globulins may consist of a combination of subunits differing from normal y-globulin
• This may explain the antigenic and chemical differences and similarities between y-globulins of disease and normal y-globulin (in other words, he stated that antibodies are found in both diseased and healthy)
• Edelman claimed that his hypothesis was able to be tested experimentally (i.e. it was a guess that had not been proven scientifically)
• He admitted that only partial separation of the reduction processes was achieved
• Edelman tentatively concluded through his experiments that the subunits were linked by disulfide bonds
• He then created a hypothesis to relate the many structural features of the various y-globulins in terms of the multiplicity of the polypeptide chains in these molecules

In order to break down and identify the chemical structure of an antibody, logic would dictate that an antibody, like "viruses," would need to be properly purified and isolated from everything else and shown to actually exist first. However, from 1890 when antibodies were initially dreamt up in the mind of Emil Von Behring on up to this point in 1961 when it was decided that these entities were chemically defined structurally by two different researchers, antibodies had never been seen. They were (and still are) nothing more than hypothetical constructs based off of the results from grotesque animal and chemistry experiments which are used to explain the theoretical concept of immunity. As antibodies had never been observed in nature nor in the fluids taken from a host, they have only ever existed as a concept rather than as a fully functioning particle. This is why decades of different researchers have come up with their own guesses as to how these entities looked, formed, and functioned. This is why both Porter and Edelman had to try and reverse engineer an antibody and then fit their results into a conceptual model. Antibodies have never been scientifically proven to exist. Thus, any claims that Porter and Edelman were able to deduce the chemical structure and form of particles never shown to exist which they themselves never witnessed is ludicrous. All these two researchers did was add their own paragraphs to the immunology chapter in the long-form fiction known as germ theory.