Was Polio Ever Properly Purified and Isolated?: Part Two

In 1959, Carlton Schwerdt wrote an in-depth review of the evidence which had been gathered up to that time in relation to the purification and isolation of a Polio “virus.” The paper itself is about 41 pages long and covers everything from the various attempts to achieve purification, the difficulties of “viral” idenfification/characterization by means of electron microscopy, the methods used for separation and their limitations, the findings on the supposed chemical properties of the “virus,” the different means of inactivation, and the inconclusive results from serological studies. I’ve highlighted the relevant portions of the review covering the “purification” of the Polio “virus” and edited out most of the indirect chemical/serological sections due to length considerations. I do recommend taking a moment to read the full 41 pages sometime as it really showcases the overwhelming amount of guesswork and assumptions that go into virology. Contradicting and incompatible results abound while dubious connections between unrelated studies using different methods are regularly made. Keep in mind that it was later admitted that Schwerdt himself only achieved purity of 10% of the “virus” and that at no point were samples taken directly from humans ever purified/isolated and studied. All of these results relate to tissue cultures from the emulsified spine/brain of monkeys, rats, and mice:

PURIFICATION AND PROPERTIES OF POLIOVIRUS

I. INTRODUCTION

“Studies of the physical and chemical properties of the human polioviruses by direct means require, as is the case with any virus, preparations of the greatest possible degree of purity. This poses the problem of establishing criteria of purity that can be met experimentally. Perhaps the ideal operational concept of a pure virus preparation is one in which all particles are identical with respect to measurable physical properties and each possesses the property of infectivity. This concept may not necessarily be adequate for all viruses, since virus particles of detectable chemical and physical heterogeneity may nevertheless possess the same infectivity. An approach based on this concept may be applied, however, to the problem of purifying some of the small viruses, including the polioviruses, which seem to be relatively uniform in size and shape.

Until recently the usual source of human poliovirus for purification studies was limited to portions of the central nervous system (CNS) of infected primates (Loring and Schwerdt, 1942; Stulberg et al., 1948) or the entire CNS of rodents infected with Type 2 polioviruses (Gard, 1943; Loring and Schwerdt, 1946; Bachrach and Schwerdt, 1952, 1954). Attempts to purify virus from such sources were severely handicapped by the low physical concentration of virus in the CNS tissue, as well as by the presence of large amounts of so-called normal, macromolecular tissue particles of physical and chemical characteristics similar to those of the virus. With the advent of poliovirus propagation in tissue culture (Enders et al.,1949), however, purification studies progressed more rapidly. Tissue culture not only provided an abundant source of virus for purification, relatively free of contaminating, nonviral components (Schwerdt and Schaffer, 1955,1956), but also a means of accurate assay (Dulbecco and Vogt, 1954), which has served as an aid in following purification steps and in identifying the physical particle with which infectivity is associated (Schwerdt and Fogh, 1957).

The problems related to the purification of polioviruses have been considered briefly by Beard (1948) in his general review on purified animal viruses. The early, limited knowledge of physical and chemical properties of these viruses has been presented by Melnick (1953) and Gard (1955). A brief survey will be made here of the attempts to purify, identify, and characterize poliovirus particles with respect to their physical, chemical, and serological properties. Polioviruses of human origin will be considered which are classified immunologically as Types 1, 2, and 3 (Committee on Typing of The National Foundation for Infantile Paralysis, 1951, 1953). Occasional reference will be made to the FA strain of mouse encephalomyelitis virus, which is not classified as a poliovirus, although it shows some resemblance to murine poliovirus, Type TO (von Magnus et al., 1955).

II. PURIFICATION

Most published attempts to purify the polioviruses have been essentially singIe step procedures designed to increase specific virus activity, i.e., virus infectivity per unit mass of protein, by selective removal of nonviral materials. Evaluation of early trials is difficult because of the inaccuracies of virus concentration estimates made by assay in monkeys. With the discovery that the Lansing (Armstrong, 1939) and, subsequently, other strains of Type 2 poliovirus were pathogenic for Swiss mice and cotton rats, infectivity assays became more accurate and the results of virus concentration and purification studies more meaningful.”

“Other organic solvents as well as ether, notably N-butanol (Bachrach and Schwerdt, 1952), chloroform (Polson and Selzer, 1954), and fluorocarbon (Manson et al., 1957), have been used to extract or denature nonviral materials. The usual experience has been that emulsification of crude aqueous suspensions of poliovirus with these immiscible organic solvents results in the removal of as much as 90% or more of the nonviral contaminants, including lipids, without concomitant loss in total infectivity from the aqueous phase. Following the successful concentration of influenza virus (Cox et al., 1947) by precipitation with methanol, and the elution of infectivity from the precipitate with alkaline buffers, several attempts have been made to concentrate and partially purify polioviruses by the same technique (Stulberg el al., 1948; Pollard and Finegold, 1948; Ibenez and Jimenez-Castellanos, 1948). Methanol concentrations of 20 to 30% were used to precipitate poliovirus at approximately – 5” to 5OC. Specific infectivity increases of 10- to 80-fold were achieved in this way.

Among other methods of concentrating and partially purifying poliovirus has been the so-called “isoelectric” precipitation at pH 4 to 4.5 (Bachrach and Schwerdt, 1954; Polson and Selzer, 1954; Albano, 1956). It has not been established by these experiments, of course, that this pH range actually encompasses the isoelectric point of the polioviruses. It may be that the virus is only adsorbed to or occluded by the great mass of nonviral protein precipitated under these conditions. Nevertheless, the virus can be eluted preferentially from such precipitates with appropriate alkaline buffers.”

“In contrast to virus precipitation methods, nonviral contaminants have been precipitated preferentially from suspensions of poliovirus-infected mouse CNS by means of protamine sulfate, thus leaving the virus in the supernatant fluid (Warren el aZ., 1949). Such partially purified virus could then be concentrated by ultracentrifugation.

One of the earliest approaches to the partial purification of poliovirus involved the adsorption to alumina gel C and elution of infectivity with alkaline buffer (Sabin, 1932; Schaeffer and Brebner, 1933). More recently, LoGrippo and Berger (1952) used ion exchange resins, both cationic and anionic, for the concentration of the virus as well as removal of salts and impurities. Suspensions of mouse CNS infected with the Lansing strain served as source of virus. Mouse assays permitted fairly quantitative estimates of recovery and degree of purification, which were approximately 40%, and 45-fold, respectively. Bentonite has also been used as an adsorbent, removing, in this case, lipids and lipoproteins and leaving partially purified poliovirus jn suspension (Oker-Blom and Nikkila, 1955).”

“The physical methods applied to poliovirus concentration and partial purification have included ultrafiltration (Clark et al., 1933), pervaporation (Polson and Hampton, 1957), electrophoresis (Polson, 1953a) , and ultracentrifugation (Schultz and Raffel, 1937; Loring and Schwerdt, 1942; Gard, 1943). Ultrafiltration served only to concentrate the virus without significant purification. Similarly, pervaporation achieved a 1000-fold reduction in volume, permitting subsequent purification by dialysis and high-speed centrifugation. Preparative electrophoresis of the zone or convection type has found only limited application to poliovirus purification as yet, although it may be a potentially powerful tool for isolating the virus from suspensions in which it is present in fairly high concentrations.

Preparative vacuum ultracentrifugation has had perhaps the greatest appeal as a physical method of purification and concentration, as evidenced by its incorporation in many rnultistep purification procedures, some of which are described in Section 11,B. It permits one to take advantage of the relatively large size of the virus particle for separating it from many of the cellular contaminants. Its limitations are, of course, the inability to remove selectively those so-called “normal macromolecular” constituents with sedimentation characteristics similar to the virus component (Sharp, 1953).”

B. Multistep Procedures

In order to purify polioviruses sufficiently for characterization studies, more elaborate procedures have been developed, incorporating several of the individual steps already noted, as well as others, such as enzymatic digestion and sedimentation in a density gradient. Among the earlier concerted efforts to this end are those of Loring and Schwerdt (1942, 1946) and Gard (1943). The former investigators attempted to purify the MV and Lansing strains of poliovirus from monkey spinal cords and cotton rat CNS, respectively. Their procedure involved extraction of virus from CNS tissues with saline, freezing and thawing of these extracts, ether extraction, and several cycles of high- and low-speed centrifugation. Preparations obtained in this way were characterized by specific infectivity assays, sedimentation velocity analysis, and electron microscopy.

Gard (1943) reported in detail studies on both mouse encephalomyelitis virus and human poliovirus purification. His source of human poliovirus was either feces or brain and spinal cord from infected man. Precipitation with ammonium sulfate was included in his procedure, in addition to most of the steps employed by the former investigators. Infectivity was found to sediment within the range of 150 to 195 S (Svedberg units), in remarkably good agreement with those found by later workers (Melnick et al., 1951; Polson and Selzer, 1952; Schwerdt and Schaffer, 1955). Analytical ultracentrifugal analyses of these preparations revealed several components. Electron microscopy revealed filamentous particles 15 mp in diameter and of greatly varying lengths which, however, were also isolable from normal CNS and feces. These filaments are now thought to be fragments of bacterial flagella (Gard, 1955). It is clear in retrospect that these early attempts, although promising, did not yield preparations consisting solely of homogeneous poliovirus particles.

During the past few years highly effective purification procedures have been developed and applied to poliovirus propagated in cotton rat CNS tissue (Bachrach and Schwerdt, 1954) and in monkey kidney tissue culture (Schwerdt and Schaffer, 1956). They have resulted in virus preparations of high specific infectivity and sufficient purity to permit identification of the virus particle. The steps involved in the purification of virus from CNS tissue suspensions were: precipitation of the virus at pH 4.5, elution of infectivity with an alkaline buffer of high ionic strength, emulsification of the eluate with N-butanol, separation of the aqueous phase from the emulsion, and subjection of this phase to two cycles of high- and low-speed ultracentrifugation. Treatment of the virus preparation with crystalline pepsin, ribonuclease, and deoxyribonuclease intervened between the cycles of ultracentrifugation.

The method for the purification of polioviruses from tissue culture fluid
evolved from the above procedure but differs from it in two major respects. The initial precipitation of virus from the tissue culture fluid was aided by the addition of 15% methanol; after the final cycle of ultracentrifugation, the virus concentrate was fractionated by sedimentation in the ultracentrifuge through BL sucrose density gradient. This last step yielded several components, as illustrated in Fig. 1. Virus concentrates from the fluids of Maitland type cultures were fractionated into four visible components, labeled A, B, C, and D, in order of increasing sedimentation rates; those from monolayer tissue culture fluids revealed two visible components, namely, C and D. Fractions A, C, and D appeared as light-scattering bands; fraction B as a pigmented component. The infectivity was associated with fraction D, which contained a homogeneous population of spherical particles. Simplified modifications of this basic procedure, using monolayer tissue culture fluid, have been developed recently for serological studies on purified poliovirus (Mayer et al., 1957).

The specific infectivities of fraction D were of high order and are listed in Table I with those achieved by several other procedures, including that reported recently by Albano (1957), who purified tissue culture poliovirus by a combination of enzymatic treatment and subsequent dialysis at pH 4. Specific infectivity measurements indicate only the relative purity of a virus preparation and are limited by the efliciency of the assay method employed. Furthermore, they do not give information on the fraction of virus particles, which may have been inactivated during storage and purification, but which nevertheless survived the procedure to constitute
part of the final purified virus concentrate.

Rough estimates of the absolute degree of purity have been made from physicochemical (Schwerdt and Schaffer, 1956) as well as from serological studies (Mayer et al., 1957) and have been found to be 50% or greater. Such estimates are made on the basis of mass of characteristic physical particles without reference to the infectivity of these particles and do not, therefore, strictly fulfill our criteria for virus purity.

IDENTIFICATION OF POLIOVIRUS PARTICLES

The identification of a virus particle requires the establishment of a relationship between infectivity and an adequately described physical particle. With polioviruses, this relationship has been demonstrated for a spherical particle of 27-30 mp diameter (the major infective component). There is some suggestion, however, that poliovirus infectivity can also be associated with a smaller particle.

A. Major Infective Component

Frequent attempts have been made to observe poliovirus particles by electron microscopy, but, as Rhian et al. pointed out in 1949, no characteristic physical particle had been unequivocally identified as the infective virus particle up to that time. Not until Bachrach and Schwerdt (1954) correlated infectivity with physical particle count by analytical electron microscopy has any direct evidence been presented for the identity of the virus particle. In this instance, Lansing virus was purified from infected cotton rat CNS tissue. Characteristic spherical particles, approximately 28 mp in diameter, were observed and counted in these preparations by electron microscopy, using the spray droplet technique (Backus and Williams, 1950). These particles were not found in concentrates prepared from uninfected CNS tissue suspensions. The quantitative data from virus concentrates yielded a constant ratio of particle count to 50% infective doses (ID60) in cotton rats of approximately 21,000 from preparation to preparation. Furthermore, it was not possible to dissociate infectivity from these particles by separation cell experiments using the ultracentrifuge.”

B. Small Infective Component

Extensive investigations of MEF-1 poliovirus propagated in mice have been made in South Africa by Polson and co-workers. In 1954, a separation of infective particles of two sizes was reported (Selzer and Polson,1954). This was accomplished by sedimentation of the virus from suckling mouse brains in the preparative ultracentrifuge in the presence of sucrose or of hemocyanine from Caminella sincta. The hemocyanine served as an indicator of sedimentation velocity as well as providing a concentration gradient in the tube to prevent convection. Samples taken at different depths along the centrifuge tube were assayed in mice; results indicated approximately 1% of the infectivity was associated with particles with a sedimentation coefficient equal to that of the hemocyanine, namely, S = 100. The bulk of the infectivity of the preparations was associated with a sedimentation coefficient of about 170 S, corresponding to the usual value for polioviruses. Diameters of 24 and 30 mp were calculated for the small and major infective components, respectively, assuming spherical particles with density 1.3. These observations were made upon crude and upon partially purified preparations, but no indication of the specific infectivity of the fractions was given. Considering the relatively crude methods of separation and assay employed, the validity of such findings must be questioned. However, when the small component was isolated and again subjected to sedimentation, the infectivity pattern indicated a true separation.”

“The suggestion was made that small particles were a real variant, although passage in tissue culture or prolonged passage in mice had not been studied. It has also been reported (Polson el al., 1957) that the small infective component had the same electrophoretic mobility as the larger component. The published experimental details do not indicate that the small component was studied independently, however.”

“In addition to poliovirus, several other viruses propagated in mouse brain were reported to consist of infective components of more than one size (Polson, 1956; Kipps et al., 1957). The size (volume or mass) ratio reported for the two infectious components of poliovirus was approximately 2: 1, and it was suggested that the ordinary 30 mu particle might be composed of two minimal infectious units. If the earlier hypothesis of smaller complement-fixing subunits holds, the large (30 mu) and small (24 mu) infectious particles would correspond to 16 and 8 complement-fixing subunits, respectively.”

“The arguments for the existence and significance of the small infectious component of poliovirus have not been accepted without question, as evidenced by the discussion following the presentation of the investigations of the South African group at the 1956 Ciba Conference (Kipps et al., 1957). It must be emphasized that all the published work was with the virus from mouse brain. Whether the phenomenon is general for all tissues or for neural tissue, or is simply limited to mice or rodents, is not known. In a careful search of the literature on the small infectious component of poliovirus and other viruses, no mention could be found of critical experiments to establish the antigenic relationship of the small and large infectious particles. Problems associated with mixtures of viruses in single specimens in identification and diagnosis of human enteric viruses have been discussed by Melnick (1957). It is not inconceivable that poliovirus and some other (smaller) virus pathogenic to mice could be maintained in quasi-equilibrium
through many passages, and that separation could be achieved by selective passage or by physical techniques.”

“The concept of infectious units smaller than the usually accepted virus particle is of fundamental importance, especially in view of the current concepts of virus structure and infectivity of RNA (Williams, 1957; Fraenkel-Conrat et al., 1957). It is hoped that further experimental evidence will be offered to resolve the questions of existence and nature of the small infectious component. In this respect it would be preferable to use the less complex tissue culture systems, if such are applicable, and to achieve purification to such a degree that physical and chemical properties may be determined directly and the results expressed in terms of specific infectivities. Serological techniques should be used to establish the viral identity of units of different size separated by physical means.

IV. PHYSICAL PROPERTIES

Identification and purification of poliovirus particles has made it possible to observe their physical properties directly, and thereby supplement our early, limited knowledge of their properties from indirect measurements.

A. Electron Microscopy

The general problems of associating virus infectivity with particles observed by electron microscopy have been discussed by Williams (1954) and Rang (1955). Prior to the positive identification of infectivity of the Lansing strain with 28 mp particles (Bachrach and Schwerdt, 1954), electron microscopy of poliovirus preparations was open to question.”

“The measured diameters of particles of all strains of poliovirus in these studies have been in the range of 26 to 31 mp. An exception is a diameter of 41 mp, reported by Sabin et al. (1954) for the Leon strain of Type 3. These authors state, however, “the seemingly large size of the Type 3 particles may be an artifact.”

2. Internal Structure

“By the application of a collodion-over-agar filtration technique, Taylor and McCormick (1956) have presented electron micrographs of unshadowed particles showing electron dense cores of about 20 mp diameter. With prolonged drying in the same system, shadowed preparations showed flattened particles with “collapsed membranes” and protruding centers. When the virus was pretreated with glycerol, particles of varying sizes from 20 mp (the size of the %oI’~”) to 30 mp were observed. They suggested the virus particle was “cell-like,” and implied that the electron dense core represented the nucleus which contained the nucleic acid. It is not unreasonable to conceive of the poliovirus particle having a dense, nucleic acid-containing core and a less dense outer region. It seems premature, however, to attempt an explanation of the particle’s internal structure from these findings. Although the exact techniques of Taylor and McCarmick have not been followed in the authors’ laboratory, electron micrographs of poliovirus prepared under various conditions have not shown comparable forms. The electron dense core may represent an accumulation of salt upon drying. The effect of a low concentration of glycerol upon the particles is even more puzzling, since this substance is commonly used in virus storage, and we have recently used it routinely (rather than sucrose) in
density gradient sedimentation. Removal of high concentrations of glycerol by dialysis, or dilution to low concentration for direct spraying and electron microscopy have yielded no evidence for disintegration of particles or of variability in their size. Again the effects of salt and drying for electron microscopy may explain Taylor and McCormick’s observations. We do confirm their finding of no effect of glycerol upon the infectivity of the virus. Cramer et al. (1957) recently published an electron micrograph of strain FA mouse encephalomyelitis virus showing dense centers similar to those found in the unshadowed preparations of Taylor and McCormick. Details of technique were not given, and a proper comparison cannot be made.

Hampton (1955) observed particles of about 15 mw diameter, frequently as aggregates, after the rather drastic treatment of air drying, vacuum shadowing, wetting, redrying, and reshadowing preparations of poliovirus. It was suggested these small particles were subunits of the fundamental
virus particle. (This is discussed in Section VI1,B under Complement-Fixing Antigens.) In contrast to Taylor and McCormick (1956), Hampton stated there was no evidence for a containing membrane on the particle.

Electron microscopy should play an important part in the eventual elucidation of the substructure of the poliovirus particle. However, the techniques applied so far have not allowed proper correlation with other physical or chemical measurements or with the biological properties of the virus. Experiments should be performed in such a manner that the morphological observations by electron microscopy would be directly or quantitatively related to alteration of other properties, such as sedimentation, nucleic acid content, infectivity, or antigenicity.

3. Virus-Cell Interaction

Intracellular visualization of poliovirus particles by electron microscopy has not been clearly demonstrated. The identification of such small particles in the complex cellular background is almost impossible, unless one can find definite aggregates such as those observed in cells infected with adenovirus (Morgan et al., 1956). Ruska et al. (1956) published micrographs showing “virus-like” bodies in monkey kidney epithelial cell cultures infected with Type 1 poliovirus. The bodies were within the nucleus, frequently concentrated near the nucleolus, in the few cells remaining after the second day of infection. Neither control micrographs of uninfected cells nor micrographs of cells at or prior to the time of reIease of virus from the majority of the cells were shown. The identity of the bodies observed is doubtful in view of a recent study by Kallman et al. (1958) on the cytology of cells known to be infected, and in which the time of release of virus was correlated with cytopathic changes. No poliovirus-like particIes were distinguished from cellular material before or during virus release, but cytopathic changes continued over an extended period of time.

Claims have been made by Reagan and co-workers (1953, 1955, 1956) that virus-like particles were observed in or on erythrocytes of cotton rats inbculated with Lansing and of chickens inoculated with Lansing and Mahoney viruses. Their experimental controls are questionable and the published micrographs are of such poor quality as to permit no meaningful interpretation of the results. Mule (1955) presented electron micrographs showing objects of various sizes and shapes on erythrocytes of infected humans or animals. He inferred from this that the red cells serve as a mechanism for virus transport and even possibly as a site for virus replication. In neither of these studies has in vitro adsorption of poliovirus to red cells been demonstrated by electron microscopy.

These observations by Ruska et al., Reagan et al., and Mule contribute nothing to our knowledge of the morphology and structure of the poliovirus particle, and little if anything to knowledge of virus-cell interactions, but serve to illustrate the pitfalls one may encounter in uncritical use of the electron microscope in attempts to identify virus particles.”

B. Other Components

“In calculations based upon nitrogen content (e.g. , specific infectivity, RNA content, etc.), 16% nitrogen has been assumed for protein and nucleic acid in the authors’ laboratory. In the absence of dry weight analysis or of evidence for other components of the virus particle, 16% nitrogen has also been assumed for the whole particle.”

“Although complete chemical characterization of polioviruses may never be made, there remains much that can be accomplished in the elucidation of chemical components, subunits, or groups related to the immunological properties of the virus and to the fundamental virus-cell interactions.”

“In connection with rodent-adapted Type 2 poliovirus, it should be noted that in purified preparations of Lansing virus from infected cotton rat CNS, Bachrach and Schwerdt (1954) observed particles of about 12 mp in diameter, in addition to the characteristic virus particles. The CF test was not applied to the material examined by electron microscopy and no significance was attached to the small particles, since similar particles were observed in normal CNS controls.”

https://doi.org/10.1016/S0065-3527(08)60491-1

In Summary:

• Studies of the physical and chemical properties of the human polioviruses by direct means require, as is the case with any “virus,” preparations of the greatest possible degree of purity
• This poses the problem of establishing criteria of purity that can be met experimentally
• Schwerdt proposes a criteria for a pure “virus” preparation as one in which all particles are identical with respect to measurable physical properties and each possesses the property of infectivity
• This concept may not necessarily be adequate for all “viruses,” since “virus” particles of detectable chemical and physical heterogeneity (the quality or state of being diverse in character or content) may possess the same infectivity
• In other words, there was no standardized criteria for the definition of a pure “virus” preparation in 1959 even though purification/isolation had been claimed to be achieved over a decade before
• The usual source of human poliovirus for purification studies was limited to portions of the central nervous system (CNS) of infected primates or the entire CNS of rodents infected with Type 2 polioviruses
• Attempts to purify “virus” from these sources were severely handicapped by:
  1. The low physical concentration of “virus” in the CNS tissue
  2. The presence of large amounts of normal, macromolecular tissue particles of physical and chemical characteristics similar to those of the “virus”
• Tissue culture provided an abundant source of “virus” for purification, relatively free (i.e. to a certain degree) of contaminating, nonviral components
• The problems related to the purification of polioviruses were considered briefly by Beard (1948) in his general review on purified animal “viruses”
• Evaluation of early purification trials was considered difficult because of the inaccuracies of “virus” concentration estimates made by assay in monkeys
• The usual experience with organic solvents was that emulsification of crude aqueous suspensions of poliovirus tesults in the removal of as much as 90% or more of the “nonviral” contaminants (not 100% of the “nonviral” contaminants)
• Several attempts were made to concentrate and partially purify (oxymoron) polioviruses by precipitation with methanol and the elution of infectivity from the precipitate with alkaline buffers
• Among other methods of concentrating and partially purifying poliovirus has been the “isoelectric” precipitation at pH 4 to 4.5
• However, it was not established by these experiments that this pH range actually encompasses the isoelectric point of the polioviruses
• In contrast to “virus” precipitation methods, “nonviral” contaminants were precipitated preferentially from suspensions of poliovirus-infected mouse CNS by means of protamine sulfate, thus leaving the “virus” in the supernatant fluid so that the partially purified “virus” could then be concentrated by ultracentrifugation
• One of the earliest approaches to the partial purification of poliovirus involved the adsorption to alumina gel C and elution of infectivity with alkaline buffer
• The physical methods applied to poliovirus concentration and partial purification have included:
  1. Ultrafiltration
  2. Pervaporation
  3. Electrophoresis
  4. Ultracentrifugation
• Ultrafiltration served only to concentrate the “virus” without significant purification
• The limitations of preparative vacuum ultracentrifugation is the inability to remove selectively “normal macromolecular” constituents with sedimentation characteristics similar to the “virus” component (i.e. it can’t separate everything)
• Schwerdt says in order to purify polioviruses sufficiently for characterization studies (which would require 100% purification – this has never been achieved), more elaborate procedures were developed
• Schwerdt says he and Loring attempted to purify (i.e did not purify) the MV and Lansing strains of poliovirus in 1946 from monkey spinal cords and cotton rat CNS
• Analytical ultracentrifugal analyses of Gard’s preparations revealed several components
• Electron microscopy revealed filamentous particles 15 mp in diameter and of greatly varying lengths which were also isolable from normal CNS and feces
• These filaments were thought to be fragments of bacterial flagella (i.e. not a purified isolated sample)
• Schwerdt admits it was clear in retrospect that his, Loring, and Gard’s early attempts, although promising, did not yield preparations consisting solely of homogeneous poliovirus particles
• Schwerdt states they had developed better purification procedures that resulted in “virus” preparations of high specific infectivity and sufficient purity (what does that even mean?) to permit identification of the “virus” particle
• “Virus” concentrates from the fluids of Maitland type cultures were fractionated into four visible components, labeled A, B, C, and D
• They associated infectivity with fraction D, which contained a homogeneous population of spherical particles
• However, he admits that specific infectivity measurements indicate only the relative purity of a “virus” preparation and are limited by the efliciency of the assay method employed
• Rough estimates of the absolute degree of purity have been made from physicochemical as well as from serological studies and have been found to be 50% or greater
• Such estimates were made on the basis of mass of characteristic physical particles without reference to the infectivity of the particles and do not strictly fulfill his criteria for “virus” purity
• Schwerdt states that there is some suggestion that poliovirus infectivity can also be associated with a smaller particle
• Frequent attempts were made to observe poliovirus particles by electron microscopy but Schwerdt states that no characteristic physical particle had been unequivocally identified as the infective “virus” particle until his paper in 1954 (which was later admitted to be purification of at most 10%)
• In 1954, a separation of infective particles of two sizes was reported by Selzer and Polson
• This was accomplished by sedimentation of the “virus” from suckling mouse brains in the preparative ultracentrifuge in the presence of sucrose or of hemocyanine from Caminella sincta
• Diameters of 24 and 30 mp were calculated for the small and major infective components assuming spherical particles with density 1.3.
• These observations were made upon crude and upon partially purified preparations, but no indication of the specific infectivity of the fractions was given
• Schwerdt stated that, considering the relatively crude methods of separation and assay employed, the validity of such findings must be questioned
• The suggestion was made that small particles were a real variant, although passage in tissue culture or prolonged passage in mice had not been studied
• The published experimental details did not indicate that the small component was studied independently
• In addition to poliovirus, several other “viruses” propagated in mouse brain were reported to consist of infective components of more than one size
• The size (volume or mass) ratio reported for the two infectious components of poliovirus was approximately 2: 1, and it was suggested that the ordinary 30 mu particle might be composed of two minimal infectious units
• The arguments for the existence and significance of the small infectious component of poliovirus have not been accepted without question
• Schwerdt emphasized that all the published work was with the “virus” from mouse brain
• In a careful search of the literature on the small infectious component of poliovirus and other “viruses,” no mention could be found of critical experiments to establish the antigenic relationship of the small and large infectious particles
• Problems associated with mixtures of “viruses” in single specimens in identification and diagnosis of human enteric “viruses” have been discussed by Melnick (1957)
• It is not inconceivable that poliovirus and some other (smaller) “virus” pathogenic to mice could be maintained in quasi-equilibrium through many passages (i.e. two or more “viruses” in a sample = not purified/isolated)
• The concept of infectious units smaller than the usually accepted “virus” particle is of fundamental importance, especially in view of the current concepts of “virus” structure and infectivity of RNA
• Schwerdt hoped that further experimental evidence will be offered to resolve the questions of existence and nature of the small infectious component
• Schwerdt stated it would be preferable to use the less complex tissue culture systems and to achieve purification to such a degree that physical and chemical properties may be determined directly and the results expressed in terms of specific infectivities
• Prior to Schwerdt’s own 10% “purification” of Polio in 1954, early, limited knowledge of the “virus” properties came from indirect measurements (and still do to date)
• Schwerdt again highlights the results of his own “positive identification of infectivity” of the Lansing strain with 28 mu particles in 1954 and states that electron microscopy of poliovirus preparations prior to his work were open to question
• The measured diameters of particles of all strains of poliovirus in various studies have been in the range of 26 to 31 mp
• An exception is a diameter of 41 mp, reported by Sabin for the Leon strain of Type 3
• These authors stated, however, “the seemingly large size of the Type 3 particles may be an artifact”
• In 1956, Taylor and McCormick observed that when the “virus” was pretreated with glycerol, particles of varying sizes from 20 mp (the size of the “core”) to 30 mp were observed
• They suggested the “virus” particle was “cell-like,” and implied that the electron dense core represented the nucleus which contained the nucleic acid
• Schwerdt concluded that it was premature to attempt an explanation of the particle’s internal structure from their findings for a few reasons:
  1. Electron micrographs of poliovirus prepared under various conditions have not shown comparable forms
  2. The electron dense core may represent an accumulation of salt upon drying
  3. The effect of a low concentration of glycerol upon the particles was puzzling since it was commonly used in “virus” storage
• Schwerdt again states that the effects of salt and drying for electron microscopy may explain Taylor and McCormick’s observations
• In 1955, Hampton observed particles of about 15 mu by diameter, frequently as aggregates, after the rather drastic treatment of air drying, vacuum shadowing, wetting, redrying, and reshadowing preparations of poliovirus
• It was suggested these small particles were subunits of the fundamental “virus” particle
• While Schwerdt believes electron microscopy should play an important part in the eventual elucidation of the substructure of the poliovirus particle, the techniques applied so far have not allowed proper correlation with other physical or chemical measurements or with the biological properties of the “virus“
• Intracellular visualization of poliovirus particles by electron microscopy has not been clearly demonstrated
• The identification of such small particles in the complex cellular background is almost impossible
• In 1956, Ruska published micrographs showing “virus-like” bodies in monkey kidney epithelial cell cultures infected with Type 1 poliovirus
• The bodies were within the nucleus, frequently concentrated near the nucleolus, in the few cells remaining after the second day of infection
• Neither control micrographs of uninfected cells nor micrographs of cells at or prior to the time of reIease of “virus” from the majority of the cells were shown
• In images from Kallman in 1958, no poliovirus-like particIes were distinguished from cellular material before or during “virus” release, but cytopathic changes continued over an extended period of time
• From 1953 to 1956, Reagan et al presented images of “virus-like” particles in 1955 yet Schwerdt contends their experimental controls were questionable and the published micrographs were of such poor quality that no meaningful interpretation of the results could be made
• In 1955, Mule presented electron micrographs showing objects of various sizes and shapes on erythrocytes of infected humans or animals
• He inferred from this that the red cells serve as a mechanism for “virus” transport and even possibly as a site for “virus” replication
• In neither of these studies was in vitro adsorption of poliovirus to red cells been demonstrated by electron microscopy
• Schwerdt claims that these observations by Ruska et al., Reagan et al., and Mule contribute nothing to the knowledge of the morphology and structure of the poliovirus particle, and little if anything to knowledge of “virus-cell” interactions
• He says they serve as examples of the pitfalls one may encounter in uncritical use of the electron microscope in attempts to identify “virus” particles
• In Schwerdt’s lab, calculations based upon nitrogen content (e.g. , specific infectivity, RNA content, etc.), 16% nitrogen had been assumed for protein and nucleic acid
• In the absence of dry weight analysis or of evidence for other components of the “virus” particle, 16% nitrogen had also been assumed for the whole particle
• Schwerdt admits complete chemical characterization of polioviruses may never be made
• Schwerdt noted that in “purified” preparations of Lansing “virus” from infected cotton rat CNS, he observed particles of about 12 mp in diameter in addition to the characteristic “virus” particles but ignored them as similar particles were present in his controlls

It is very telling that even though claims of purification/isolation were made, according to Carlton Schwerdt, up to 1959 there was no accepted standardized criteria for the definition of a purified “virus.” While to the layman, a purified “virus” would be one that was free of any foreign elements, contaminants, pollutants, or substances that adulterate it, Schwerdt’s own criteria was that the particles are of the same size and that they are infectious. Even then, he made the caveat that his criteria may not fit all “viruses” as there are some that are heterogeneous (the quality or state of being diverse in character or content; consisting of dissimilar or diverse ingredients or constituents) and still considered infectious. In other words, his criteria were not really criteria at all. Schwerdt admitted that there was a problem with the establishment of a purification criteria that could be met experimentally. Perhaps this is why there were claims of purification of the Polio “virus” that were said to be 80% (Loring and Schwerdt 1946) yet were later admitted to be only 1%. Or Schwerdt’s later assertions that he had achieved greater purification in 1954, which while accurate to an extent, only consisted of 10% purity. Maybe this is why there was the accepted classifications of “partially purified,” “sufficiently purified,” “essentially purified,” etc.? Schwerdt knew, along with the rest of virology, that complete purification and isolation of particles assumed to be “viruses” is an impossible task.

Even with the “advancements” in technology today, it is said that the methods and technology for the complete purification/isolation of these particles does not exist. This is made abundantly clear in the area of exosome research and has been stated regularly by these researchers even as recently as May 2020:

“Nowadays, it is an almost impossible mission to separate EVs and viruses by means of canonical vesicle isolation methods, such as differential ultracentrifugation, because they are frequently co-pelleted due to their similar dimension. To overcome this problem, different studies have proposed the separation of EVs from virus particles by exploiting their different migration velocity in a density gradient or using the presence of specific markers that distinguish viruses from EVs. However, to date, a reliable method that can actually guarantee a complete separation does not exist.”

Click to access viruses-12-00571.pdf

If the technology for the purification and isolation of particles assumed to be “viruses” does not exist today and it was admitted that the claims of purification/isolation of the Polio “virus” in the 1940’s and 1950’s were nowhere near 100% with no standardized criteria to define purity, it is safe to say that the Polio “virus” was never properly purified nor isolated. It is also safe to say that this applies to every alleged “virus” to date and that the papers claiming so are nothing but the work of pure science fiction.