“Isolation and purification of a virus is necessary before a vaccine can be made.”
The above statement came from the New York Times obituary of Wendell Meredith Stanley, considered by some to be the “Father of Virology.” While it is technically a correct statement, it really should read:
Isolation and purification of the particles believed to be a “virus” is necessary before one can claim the discovery of a “virus.”
Purification and isolation of the particles assumed to be a “virus” are the very first steps needed to be fulfilled in order to try and claim the discovery of a new “virus.” They are essential and can not be skipped over. Unfortunately, while it is admitted that these steps are important, virologists have bastardized the meanings of these words and never provide evidence that these steps are properly satisfied.
So what exactly is meant by purification and isolation? Let’s look at the common definitions for these fairly simple concepts that virology is seemingly incapable of being able to grasp:
- to make pure; free from anything that debases, pollutes, adulterates, or contaminates:
- to free from foreign, extraneous, or objectionable elements:
- the act of separating something from other things : the act of isolating something
For a more in-depth breakdown of the processes used to attempt purification, please see this post:
Is Complete Purification/Isolation of a “Virus” Even Possible?
In order to claim that a “virus” has been discovered, it must first be shown that the particles have been purified and isolated directly from a sick patient. These particles must be free of foreign elements as well as anything that can contaminate, adulterate, or pollute them (i.e. purified) and they must be separated from everything else that is found within the sample as well (i.e. isolated). This is the only way a researcher could show that the assumed “virus” particles exist if they were to take EM images of just these particles with nothing else present. They could then use only these particles to try and prove pathogenicity in animal studies. The particles would then be able to be biochemically characterized and described. Only then would there be a legitimate claim as to the discovery of a “virus” as there would be direct physical evidence of particles that can be shown to cause the disease they are associated with. Sadly, this process is never done.
In the case of the influenza “virus” (both A and B), it was obvious there was no purification nor isolation of any kind, even though the researchers claimed (at least regarding isolation) that this had been done. Unpurified throat washings from sick humans assumed to contain a “virus” were mixed with additives and injected into ferrets until they became sick. Once this occurred, the ferret’s tissues (normally lungs and nasal turbinates) were ground up, mixed with additives, and injected into other ferrets. This process was eventually transferred from ferrets to mice and a “virus” was assumed to be passed from animal to animal. This process never accounted for the billions of foreign particles (human/animal DNA, chemicals, other cellular components, etc.) which would be contained within the injected mixtures. Thus it was clear that despite the claims of early influenza virologists, no purified/isolated influenza “virus” was ever presented.
While the lack of purification/isolation of the influenza “virus” was apparent, I was very interested in finding the first images of the particles assumed to be the “virus” as not a single “discovery” paper ever provided any. During my search into the first transmission electron micrograph images, I stumbled upon a 1945 study by Wendell Meredith Stanley which discussed the attempts to purify influenza “virus” for use in vaccines. What this led to was further evidence for the complete lack of any purification/isolation of the “virus” assumed to be influenza.
Before looking at his research, here are a few brief passages about Stanley which help to put his work into perspective:
“After his fellowship, Stanley returned to the United States and became an assistant at the Rockefeller Institute for Medical Research in New York. At the invitation of the director of the institute, Simon Flexner (AAI ’20), and plant pathologist Louis O. Kunkel, Stanley transferred to the Department of Plant and Animal Pathology to work on TMV in 1932, marking his introduction to the field of virology. With the success of his early research on TMV, he quickly rose through the ranks of the Rockefeller Institute, becoming an associate member (1937) and a member (1940). When it was announced that the Rockefeller Institute would close its Princeton campus in 1948, Stanley moved to UC Berkeley to launch and direct the Virology Laboratory. He remained at UC Berkeley for two decades, founding and chairing both the Department of Biochemistry (1948–53) and the Department of Virology (1958–64) before retiring from his position as the director of the Virology Laboratory in 1969.”
“Wendell M. Stanley was awarded the 1946 Nobel Prize in Chemistry with John Howard Northrop and James Batcheller Sumner. One-half of the prize was awarded to Sumner for “his discovery that enzymes can be crystallized,” and the other half was awarded jointly to Northrop and Stanley for “their preparation of enzymes and virus proteins in a pure form.” By isolating and crystallizing tobacco mosaic virus (TMV), Stanley demonstrated that viruses blurred the lines between the living organisms studied by biologists and the nonliving molecules studied by chemists.”
“Five years later, Stanley crystallized TMV and suggested that it, too, was a pure protein. By 1937, however, he recognized that TMV crystals were actually nucleoproteins. Furthermore, Stanley demonstrated that these nucleoproteins, apparently lifeless in crystallized form, sprang back to life and multiplied when dissolved and reintroduced into tobacco. This was a revolutionary discovery, for it challenged the widely held belief that diseases were transmitted only by living organisms.
Stanley continued to study virology and served as a spokesman for the field for the remainder of his career. During the Second World War, he experimented with various techniques for purifying influenza virus and devised a method for preparing the influenza vaccine by centrifugation.”
“He was awarded the Nobel Prize in Chemistry for 1946. His other notable awards included the Rosenburger Medal, Alder Prize, Scott Award, and the AMA Scientific Achievement Award. He was also awarded honorary degrees by many universities both American and foreign, including Harvard, Yale, Princeton and the University of Paris. Most of the conclusions Stanley had presented in his Nobel-winning research were soon shown to be incorrect (in particular, that the crystals of mosaic virus he had isolated were pure protein, and assembled by autocatalysis).“
The first thing that stood out to me was that Wendell Meredith Stanley was a researcher for the Rockefeller Institute who was invited there by Simon Flexner. If you are uncertain why this is a red flag, I recommend digging into how the Rockefeller’s overtook and reshaped our healthcare system with their oil money in the early 1900’s. Their tentacles are everywhere. As for Simon Flexnor, you can read a bit about the man, his research into Polio, his Rockefeller connections, and his infamous brother Abraham here:
The second thing I noticed was that Stanley was awarded a Nobel Prize (among numerous other awards) for the preparation of enzymes and “virus” proteins in a “pure” form even though most of his conclusions turned out to be incorrect, including the findings that his tobacco mosaic “virus” crystallized particles were pure protein.
Regardless of his blunders, Stanley’s 1945 paper is a very interesting yet long 29-page read. While I won’t go into the whole paper, a few things caught my eye. One was the “virus isolates” he used for “purification” provided by one Thomas Francis Jr., and the other is the admittance that extraneous proteins were present in the “virus” materials. Here are a few brief excerpts:
THE PREPARATION AND PROPERTIES OF INFLUENZA VIRUS VACCINES CONCENTRATED AND PURIFIED BY
“It is possible that the great variation that has been reported in the effectiveness of vaccination with inactive virus was due largely to three interrelated factors, namely, differences in the ratio of virus to extraneous protein in the vaccine, variation in the amount of virus administered, and differences in the degree of destruction of antigenicity of virus during inactivation. No exhaustive study of these factors has been reported although there are many suggestions in the literature as to the importance of each. For example, Andrewes and Smith (45) in a study of the effect of foreign tissue extracts on the efficacy of influenza virus vaccines concluded that “the relative poorness of vaccines made from heterologous species is probably accounted for by the fact that foreign tissue extracts interfere with the immunizing response,” and “that the antibody mechanism is so swamped by the large amount of foreign protein presented to it that it cannot deal so efficiently with the minute amount of virus it receives at the same time.” There is a wide variation in the amount of virus present in tissue extracts and in infectious extra-embryonic fluids, and hence, in vaccines prepared from such materials.”
As a whole, the results just described, as well as those of other investigators
(9, 20, 21, 38), provide ample evidence of the desirability of preparing concentrated and purified vaccines, of determining the minimal amounts of treatment necessary for inactivation, and of establishing the optimum dosage for optimum antibody response and persistence of that response. From a practical standpoint, such vaccines should be purified sufficiently so that the virus represents the major antigenic component, concentrated sufficiently so that the virus content is several times that present in infectious allantoic fluid, and inactivated in such a manner that appreciable loss of immunizing potency does not occur. The present report records the preparation and properties of influenza virus vaccines concentrated and purified by means of differential centrifugation and inactivated by different procedures.
Materials and Methods
Virus Strains.–The PR8, Lee, and Weiss strains of influenza virus were all obtained from Dr. T. Francis, Jr., of the University of Michigan. The recent passage history of the PR8 strain has been described (48). When received in this laboratory the Lee strain had been isolated from ferret B75 following 8 passages in ferrets and then passed 137 times in mice and 21 times in chick embryos. The Weiss strain had been passed 3 times in ferrets, 23 times in mice, and 4 times in chick embryos when received. Following 2 or 3 passages in chick embryos in this laboratory large pools of the three strains were prepared as described earlier (48).”
From the above excerpts, it is clear that the material used to create a vaccine were anything but purified. There was extraneous proteins within the samples that Stanley believed was affecting the ability to instill immunity. Looking at the serial passaging process used for the creation of each strain gives a good idea as to why there was an abundance of proteins in the “virus” samples:
The Lee strain (Influenza B) was “isolated” from a ferret. However, this was proceeded by 8 passages into other ferrets. It was then passaged 137 times into mice and 21 times in chick embryos.
The Weiss strain (Influenza A) was passed 3 times in ferrets, 23 times in mice, and 4 times in chick embryos.
These two strains (along with PS8…but more on that one a bit later) were passaged 2-3 times again in chick embryos and then all three were POOLED (i.e mixed) TOGETHER.
When talking about serial passaging, what they are referring to is a process created by Louis Pasteur where tissues from “infected” animals are ground up and “passed” through invasive inoculations into healthy animals in an attempt to make them sick. They continue this process (often through multiple animals) until they get the desired result:
“Alternatively, an in vivo experiment can be performed where an animal is infected with a pathogen, and this pathogen allowed time to grow in that host before a sample of it is removed from the host and passed to another host. This process is repeated for a certain number of hosts; the individual experiment determines this number.”
“To solve this problem, Pasteur worked with the rabies virus in vivo. In particular, he took brain tissue from an infected dog and transplanted it into another dog, repeating this process multiple times, and thus performing serial passage in dogs. These attempts increased the virulence of the virus. Then, he realized that he could put dog tissue into a monkey to infect it and then perform serial passage in monkeys. After completing this process and infecting a dog with the resulting virus, Pasteur realized that the virus was less virulent. Mostly, Pasteur worked with the rabies virus in rabbits. Ultimately, to create his vaccine for rabies, Pasteur used a simple method that involved drying out tissue. As is described in his notebook:
In a series of flasks in which air is maintained in a dry state…each day one suspends a thickness of fresh rabbit spinal tissue taken from a rabbit dead of rabies. Each day as well, one inoculates under the skin of a dog 1 mL of sterilized bouillion, in which has dispersed a small fragment of one of these desiccated spinal pieces, beginning with a piece most distant in time from when it was worked upon, in order to be sure that it is not at all virulent.
Pasteur mostly used other techniques besides serial passage to create his vaccines. However, the idea of attenuating a virus through serial passage still holds.“
As I stated earlier, in the case of influenza, researchers used unpurified throat washings from a human (usually mixed with saline and other chemicals), injected this into the nose of a ferret, killed the ferret and then ground up its lungs and nasal turbinates (also mixed with other compounds/chemicals), injected this up the nose of a healthy ferret, and repeated this process until they saw the symptoms they wanted to see.
Upon seeing those symptoms, they killed the ferret, ground up its lungs/nasal turbinates, and injected this goo up the nose of a mouse. This mouse would be killed, its lungs/nasal turbinates ground up, and its tissues shot up the nose of another mouse. Once again, this process was repeated until the researchers saw the symptoms they wanted to see.
Then the ground up mouse goo was injected into chick embryos.
The whole time, a “virus” was ASSUMED to be in the toxic tissue goo passed from human to ferret to mouse to chick embryo, ignoring the countless times the animals did not get sick.
I don’t know about you, but an inter-species jamboree of ground up tissue goo with added compounds/chemicals doesn’t sound like a PURIFIED substance to me, let alone an isolate of any “virus.”
In any case, now that we cleared that up, on to the passage history of strain PS8 that was referenced under  in the above study. This is the first strain of influenza A “isolated” by Thomas Francis Jr. in 1934. The reference took me to another interesting Wendell Meredith Stanley paper from 1944 about the LACK OF PURIFICATION of the influenza “virus.” Keep in mind, this is a good 11 years after the influenza “virus” was said to be isolated:
AN EVALUATION OF METHODS FOR THE CONCENTRATION AND PURIFICATION OF INFLUENZA VIRUS
“The concentration and purification of influenza virus are of special importance in connection with studies on the fundamental chemical and physical properties of the virus itself. The lack of knowledge of such properties is emphasized at the present time by the disagreement concerning so simple and yet so fundamental a property as the true size of the infectious unit of influenza
virus. For some years it was generally accepted that the virus was represented
by a sphere about 80 to 120 mu in diameter (1, 2). Recently, however, Chambers and coworkers (3) and Bourdillon (4) secured data, some of which reasonably could be interpreted as indicating that at least a portion of the virus possesses a size of the order of 10 mu. Friedewald and Pickels (5) and Taylor and associates (6), on the other hand, consider, on the basis of recent experiments, that the size of the virus cannot be 10 mu and is more nearly that
accepted earlier. This general situation, which is illustrative of the lack of
definite knowledge of the fundamental physicochemical properties of influenza virus, emphasizes the necessity for studies on the concentration and purification of the virus, for with the preparation of purified virus it should become possible to conduct experiments in which a given physical entity is correlated with virus activity. The direct positive correlation of virus activity with a given physical entity on fractionation of that entity by different procedures would provide good evidence that the preparation is essentially pure and hence suitable for studies on its fundamental physicochemical properties.
The concentration and purification of influenza virus also appear to be of
importance in connection with the prevention or control of influenza. Numerous investigators (7-17) have shown that the administration of active or inactive virus results in an increase in circulating antibodies, and Francis (18, 19) has demonstrated that this increase is accompanied by an enhancement of the virus-inactivating capacity of the nasal secretions. Irrespective of the mechanism by means of which a solid immunity to influenza is achieved, the importance of securing concentrated virus preparations is indicated by the findings of Hirst and coworkers (20) that the average antibody response of human beings isdirectly related, though not strictly proportional, to the amount of virus administered. The widespread use of the chick embryo for the cultivation of viruses (21) and the current increase in the use of infectious materials from this source for human vaccination make it prudent to attempt to purify influenza virus obtained from chick embryos in order to avoid or reduce difficulties due to sensitization of human beings to chick embryo proteins. If not used directly, such concentrates could be used for the production of antisera which could be employed after the manner described by Smorodintseff and associates (22).”
Materials and Methods
Source of Virus.–The PR8 strain of influenza virus, generously supplied by Dr. T. Francis, Jr., of the University of Michigan, was used in the present work. When received in the form of frozen and dried ailantoic fluid, the strain had been isolated from ferret 198, passed 70 times in mice, 717 times in tissue culture, and 23 times in chick embryos. It was passed twice in chick embryos in this laboratory, and a pool of ailantoie fluid from the second passage was distributed in a large number of sealed ampules which were stored in a COs ice box. At weekly or bimonthly intervals, single ampules of this stock inoculum were thawed, allowed to stand at room temperature for about 30 minutes with occasional stirring, and a 10 -5 dilution was prepared with sterile 0.1 m phosphate buffer at pH 7. The residual stock inoculum was stored at 4°C. for subsequent use and 0.1 or 0.2 co. portions of the diluted inoculum were injected through a small opening in the shell above the air sac into the allantoic sac of White Leghorn chick embryos brought to 10 days of age at 39°C. The infected embryos were incubated at 36°C. for 36 to 48 hours (28), then chilled for 2 or more hours at 4°C., and the allantoic fluid was harvested and used as starting material.
Chicken Red Cells.–Red cells obtained from adult chickens by severing the cervical vessels were prepared as described by Hirst and Pickels (29). The ceils were stored at 4°C. in a 15 to 20 percent suspension in saline and were used within 1 week of preparation.
Red Cell Agglutination Titrations.–The agglutination titrations were carried out
by Hirst’s method (29) as modified for use in this laboratory (30). Unfortunately, during the major portion of the present work a virus standard was not available for purposes of comparison, hence the values reported in this paper are not standardized.
Chick Embryo Titrations.–The titrations in chick embryos were carried out as described by Hirst (31), except that sterile 0.1 M phosphate buffer at pH 7 was used as a diluent in the place of saline (32).
Nitrogen Determinations.–Since essentially all of the nitrogen in solutions of materials sedimented two or more times at high speed was found to consist of protein nitrogen, total nitrogen determinations by the Nessler method (28) were made on such solutions and the values obtained were regarded as protein nitrogen values. In all other cases the samples were treated with an equal volume of hot l0 per cent trichloroacetic acid, then quickly cooled, and the precipitate collected by centrifugation. The precipitate was dissolved in a small volume of 0.2 N sodium hydroxide and precipitated with 5 per cent trichloroacetic acid. Analyses by the Nessler method were made on the washed precipitates. Because of the possibility that different preparations might contain different percentages of nitrogen, the concentrations of protein are usually expressed in terms of protein nitrogen. However, preliminary results indicate that approximate protein concentrations may be obtained from the protein nitrogen values by multiplying by the factor 10.
High-Speed Centrifugation.–Two vacuum centrifuges of the air turbine type described by Bauer and Pickels were used (33). The centrifuge heads, which were 8 inches in diameter, each accommodated 14 Lusteroid tubes 3 inches in length and 0.75 inch in diameter at an angle of 33 ° from vertical. About 10 to 15 minutes were required for acceleration or deceleration, and in the present experiments centrifugation was continued for 2 hours at 24,000 R.P.M. However, it should be noted that in subsequent experiments reported elsewhere (34) centrifugation for 20 to 30 minutes was found sufficient to sediment influenza virus. During the latter portion of the work, a commercially available Sharples laboratory centrifuge was used. The centrifuge was equipped with a celluloid liner and a cooling coil, and the experiments were conducted in a manner similar to that used in work with tobacco mosaic virus (35).
Sedimentation Constants.–Determinations of sedimentation constants of the purified virus preparations were kindly made by Dr. M. A. Lauffer and Mr. H. K. Schachman by means of a Bauer-Pickels type ultracentrifuge (36, 37) equipped with a Svensson-Philpot optical system (38, 39). The solutions used usually contained about 2 to 3 rag. of protein per cc. in 0.1 M phosphate buffer at pH 7. The constants corrected to water at 20°C. are given in Svedberg units. A Svedberg unit, symbolized by S, is a sedimentation rate of 10 “13 era. per second per unit centrifugal field.”
“High-speed centrifugafion of infectious allantoic fluid appears to result in the almost quantitative removal of CCA activity from the supernatant fluid, whereas treatment with adult or embryonic chicken red cells or the freezing and thawing method effects only a 50 to 90 per cent removal of CCA activity. A second elution of red cells or of the precipitate obtained on freezing and thawingor a second treatment of the supernatant allantoic fluid with fresh red ceils or by the freezing and thawing method yields only a small amount of activity; hence, such additional processing of allantoic fluid appears to be of no practical importance. Since the eluates of red ceils and the extract of the precipitate obtained on freezing and thawing of allantoic fluid contained over 80 per cent of non-specific protein, it is obvious that such eluates or extracts should not be used directly for vaccination purposes or for studies on the physicochemical properties of influenza virus. Differential centrifugation provides a direct means of obtaining a concentrated and purified preparation, presumably free of large amounts of inactive protein, and therefore appears to represent the method of choice for the large scale preparation of purified virus for purposes of vaccination or for the study of physicochemical properties.”
“The concentration and purification of influenza virus by means of differential
centrifugation in a vacuum type centrifuge, by adsorption on and elution from adult chicken red cells, by elution of the precipitate formed on freezing and thawing of allantoic fluid, by adsorption on and elution from embryonic chick red cells, and by combinations of the first method with each of the three succeeding methods, have been studied. Over-all yields of virus of about 50 to 70 percent were obtained by these methods and combinations of methods except for somewhat lower yields when adsorption on and elution from adult chicken red cells was employed. However, the purified products obtained by methods involving only the use of red cells or the freezing and thawing technique were found to contain about 80 per cent of non-virus protein. The purified products obtained when differential centrifugation was used either alone or in combination with any one of the other methods were found to be indistinguishable and to consist of a fairly homogeneous component having a sedimentation constant of about 600 S. Such preparations possessed about 22,000 chicken red cell agglutinating units per mg. of protein nitrogen and solutions containing only about 10 -14 gm. of the materials gave 50 per cent infectivity end points in chick embryos. The Sharples centrifuge was found to be almost as efficient as the vacuum type centrifuge for the concentration and purification of influenza virus and, because of its larger capacity, is recommended for the preparation of purified virus on a large scale.”
- The great variation that has been reported in the effectiveness of vaccination with inactive “virus” was due largely to three interrelated factors, including differences in the ratio of “virus” to extraneous protein in the vaccine
- Andrewes and Smith (of Influenza A fame) in a study of the effect of foreign tissue extracts on the efficacy of influenza “virus” vaccines concluded that “the relative poorness of vaccines made from heterologous species is probably accounted for by the fact that foreign tissue extracts interfere with the immunizing response”
- They also stated “that the antibody mechanism is so swamped by the large amount of foreign protein presented to it that it cannot deal so efficiently with the minute amount of virus it receives at the same time.”
- There was a wide variation in the amount of “virus” present in tissue extracts and in infectious extra-embryonic fluids, and hence, in vaccines prepared from such materials
- Stanley states that as a whole, the results described, as well as those of other investigators, provided ample evidence of the desirability of preparing concentrated and purified vaccines
- He states that from a practical standpoint, such vaccines should be purified sufficiently (not completely apparently) so that the “virus” represents the major antigenic component
- The PR8, Lee, and Weiss strains of influenza “virus” were all obtained from Dr. T. Francis, Jr., of the University of Michigan
- When received, the Lee strain had been “isolated” from ferret B75 following 8 passages in ferrets and then passed 137 times in mice and 21 times in chick embryos
- The Weiss strain had been passed 3 times in ferrets, 23 times in mice, and 4 times in chick embryos when received
- Following 2 or 3 passages in chick embryos in his laboratory, large pools of the three strains were prepared
- In other words, Stanley took the unpurified serially passaged strains provided to him by Thomas Francis Jr., passaged them 2-3 more times in chick embryos, and then mixed all together…does that sound purified/isolated?
- The concentration and purification of influenza “virus” are of special importance in connection with studies on the fundamental chemical and physical properties of the “virus” itself
- The lack of knowledge of such properties was emphasized by the disagreement concerning so simple and yet so fundamental a property as the true size of the infectious unit of influenza “virus”
- Size estimates ranged from 10 mu to 80-120 mu
- This disagreement in the size of the “virus” is illustrative of the lack of definite knowledge of the fundamental physicochemical properties of influenza “virus,” and according to Stanley, it emphasized the necessity for studies on the concentration and purification of the “virus”
- He stated that with the preparation of purified “virus,” it should become possible to conduct experiments in which a given physical entity is correlated with “virus” activity
- The direct positive correlation of “virus” activity with a given physical entity on fractionation of that entity by different procedures would provide good evidence that the preparation is essentially pure and hence suitable for studies on its fundamental physicochemical properties
- The concentration and purification of influenza “virus” also appeared to be of importance in connection with the prevention or control of influenza
- The widespread use of the chick embryo for the cultivation of “viruses” and the current increase in the use of infectious materials from this source for human vaccination made it prudent to attempt to purify influenza “virus” obtained from chick embryos in order to avoid or reduce difficulties due to sensitization of human beings to chick embryo proteins
- Stanley received PS8 from Francis Jr. in the form of frozen and dried ailantoic fluid, which had been “isolated” from ferret 198, passed 70 times in mice, 717 times in tissue culture, and 23 times in chick embryos
- It was passed twice in chick embryos in Stanley’s laboratory, and a pool of ailantoic fluid from the second passage was distributed in a large number of sealed ampules which were stored in a CO2 ice box
- Red cells were obtained from adult chickens by severing the cervical vessels and the ceils were stored at 4°C. in a 15 to 20 percent suspension in saline and were used within 1 week of preparation
- During the major portion of Stanley’s work, a “virus” standard for red cell agglutination titration was not available for purposes of comparison, hence the values reported were not standardized
- For nitrogen determination, he assumed all values obtained were protein nitrogen values since essentially all of the nitrogen in solutions of materials sedimented two or more times at high speed was found to consist of protein nitrogen
- Because of the possibility that different preparations might contain different percentages of nitrogen, the concentrations of protein were usually expressed in terms of protein nitrogen
- The experiments were conducted in a manner similar to that used in work with tobacco mosaic “virus” (which were later determined to be inaccurate)
- High-speed centrifugafion of infectious allantoic fluid appeared to result in the almost quantitative removal (in other words, not a complete removal) of CCA activity from the supernatant fluid, whereas treatment with adult or embryonic chicken red cells or the freezing and thawing method effects only a 50 to 90 percent removal of CCA activity
- Since the eluates of red ceils and the extract of the precipitate obtained on freezing and thawing of allantoic fluid contained over 80 percent of non-specific protein, it is obvious that such eluates or extracts should not be used directly for vaccination purposes or for studies on the physicochemical properties of influenza “virus“
- Stanley concluded that differential centrifugation provided a direct means of obtaining a concentrated and “purified” preparation, presumably free of large amounts of inactive protein, and therefore appeared to represent the method of choice for the large scale preparation of purified “virus” for purposes of vaccination or for the study of physicochemical properties
- Methods of “purification” studied included:
- Differential centrifugation in a vacuum type centrifuge
- Adsorption on and elution from adult chicken red cells
- Elution of the precipitate formed on freezing and thawing of allantoic fluid
- Adsorption on and elution from embryonic chick red cells
- Combinations of the first method with each of the three succeeding methods
- Overall yields of “virus” of about 50 to 70 percent were obtained by the methods and combinations of methods studied except for somewhat lower yields when adsorption on and elution from adult chicken red cells was employed
- The “purified” products obtained by methods involving only the use of red cells or the freezing and thawing technique were found to contain about 80 percent of “non-virus” protein
- The “purified” products obtained when differential centrifugation was used either alone or in combination with any one of the other methods were found to be indistinguishable and to consist of a fairly homogeneous component
- Such preparations possessed about 22,000 chicken red cell agglutinating units per mg. of protein nitrogen and solutions containing only about 10 -14 gm. of the materials gave 50 percent infectivity end points in chick embryos
According to Stanley, the purification of influenza is of special importance in studying the fundamental chemical and physical properties of a “virus.” However, this was never accomplished which is why there was disagreement over the size of the influenza “virus.” The researchers could not actually see any “virus” so they attempted to guess a size based on indirect measurements. This showcased the lack of knowledge up to that point in time about the assumed “virus.” In fact, without the complete purification/isolation of the particles assumed to be a “virus,” there could be no direct positive correlation between a physical entity and the disease it was said to produce. Even with this admittance by Stanley that no purified/isolated “virus” existed (11 years after the supposed isolation of an influenza “virus” took place in 1933), it is obvious looking at the creation of PS8 by Francis Jr. in 1934 that the “virus” was an unpurified mess:
“The PS8 strain supplied by Thomas Francis Jr. was received in the form of frozen and dried allantoic fluid, and had been isolated from ferret 198, passed 70 times in mice, 717 times in tissue culture, and 23 times in chick embryos”
Keep in mind that the purification and isolation of any “virus” must be shown in the original paper(s) claiming the discovery of any “virus.” This is not a step that can be left up to “future studies” by different researchers using different materials and methods. This process must be carried out first before any claims can ever be made. Sadly, virology has a very bad habit of always announcing purification and isolation of “viruses” without ever directly proving it.
It should be clear that serial passaging an assumed “virus” in animals is not purification and there was NO PHYSICAL ENTITY that the researchers could use for direct positive correlation to “virus” activity hence no isolation 11 years after a “virus” was said to be isolated. While it was nice to have Stanley’s confirmation as to the lack of a purified/isolated influenza “virus,” it is obvious that the steps required were not carried out when you read the original papers:
Richard Shope Swine Flu Paper (1931)
Did Thomas Francis Jr. Isolate Influenza A in 1934?
Did Thomas Francis Jr. Isolate Influenza A in 1937?
Did Thomas Francis Jr. Isolate Influenza B in 1940?
This lack of purification/isolation is why the CDC admitted in March 2021 that it could not find any records of any kind on the purification/isolation of the influenza “virus.”
In other words: