Enders Polio Paper (1949)

John Franklin Enders, the man responsible for the “isolation” of the Measles “virus” in 1954 using the cell culture process he supposedly perfected, was also heavily involved in Polio research. He was responsible for the cultivation of the Lansing Polio “virus” on non-nervous tissue culture experiments in 1949. Up to that time, Polio was only thought to be able to infect nervous system tissues as it was claimed to be a nervous system disease. Polio was only shown to “grow” on brain and nerve cells and various previous attempts by other researchers to grow the “virus” on liver, lung, spleen, and kidney cells all failed miserably.

Fortunately for virology, Enders miraculously “proved” that Polio was not one-dimensional as he was able to grow the “virus” with his disgusting tissue culture methods using the severed body parts of deceased human embryos and a premature infant. It was through these experiments that Enders observed what he called cytopathogenic effects in the cultures. This is what virologists to date claim as evidence of “viral” particles hijacking cells and causing morphological changes when in reality it is the breakdown of the cell due to starvation, poisoning, and environmental stress (among other factors). You may notice many similarities between the techniques used in this paper to the cell culture techniques used today.

While reading Enders detailed breakdown of his morbid hobby, keep in mind that when he claims successful replication of the Lansing strain of the Polio “virus,” what he is referring to is calculations estimating the supposed “viral” growth:

“Next, the team needed to figure out how much virus had been produced, using a technique called endpoint dilution. On the 16th day of the 67-day period, some of the infected fluids were taken from the muscle, skin, and connective tissue cultures and infected into mice and monkeys. Another set of mice and monkeys were infected with the original virus solution for comparison. 

The amount of virus was estimated based on something called an LD50, which means “the dose that kills 50% of the infected animals after a specified time.” The volume of virus solution needed to kill 50% of the test animals was 10¹⁵ (10 trillion) times lower after 16 days compared to day 1, meaning it was more concentrated after 16 days. So, the virus had been successfully grown to very high levels. In the intestinal cells, that factor was 10¹⁴ and in the nerve cell controls it was 10¹² .”

Laboratory methods behind the polio vaccine discovery

In other words, Enders guessed how much “virus” was present based on how much culture fluid was injected into the test animals and how fast it killed them compared to results from the original sample. This assumes a “virus” was present in the tissue culture supernatant as he was unable to physically see any “viruses” replicating. There were no attempts at purification/isolation of any “virus” particles directly from a sick human nor were any attempts made to do so with the tissue culture supernatant. No EM images of Polio “virus” particles were presented in his paper. Everything Enders claimed about the cultivation/replication of a Polio “virus” is based on guesstimates and assumptions:

Cultivation of the Lansing Strain of Poliomyelitis Virus in Cultures of Various Human Embryonic Tissues

“An extraneural site for the multiplication of the virus of poliomyelitis has been considered by a number of investigators (2, 6). The evidence that this may occur is almost entirely indirect, although recent data indicate that Theiler’s mouse encephalomyelitis virus as well as various mouse pathogcnic poliomyelitis-like viruses of uncertain origin may multiply in nonnervous tissue (1,3). Direct attempts by Sabin and Olitsky (4) to demonstrate in vitro multiplication of a monkey-adapted strain of poliomyelitis virus (MV strain) in cultures composed of certain nonnervous tissues failed. They obtained, however, an increase in the agent in fragments of human embryonic brain.

Thc general recognition that the virus may be present in the intestinal tract of patients with poliomyelitis and of persons in contact with them emphasizes the desirability of further investigation of the possibility of extraneural multiplication. Accordingly, experiments with tissue cultures were undertaken to determine whether the Lansing strain of poliomyelitis virus could be propagated in three types of human embryonic tissues. The results are summarized here in a preliminary manner.

The technique was essentially the same as that recently described for the cultivation of mumps virus (6). The cultures consisted of tissue fragments suspended in 3 cc of a mixture of balanced salt solution (3 parts) and ox serum ultrafiltrate (1 part). Tissues from embryos of 273 to 44 months as well as from a premature infant of 7 months’ gestation were used. These were: the tissues of the arms and legs (without the large bones), the intestine, and the brain. Each set of cultures included 4 or more inoculated with virus, and usually a similar number of uninoculated controls. The primary inoculum consisted of 0.1 cc of a suspension of mouse brain infected with the Lansing strain of poliomyelitis virus.4 The identity of the virus was verified by (a) the character of the disease it produced in white mice following intracerebral inoculation; and (b) its neutralization by specific antiserum. Subcultures were inoculated with 0.1 cc of pooled centrifuged supernatant fluids removed from the previous set of cultures.

The procedure of cultivation differed from that usually followed by other workers in that the nutrient fluid was removed as completely as possible and replaced at periods ranging from 4 to 7 days. Subcultures to fresh tissue were prepared at relatively infrequent intervals, ranging from 8 to 20 days.

Two experiments have been carried out employing cultures composed chiefly of skin, muscle and connective tissue from the arms and legs. The findings in each have been essentially the same. In the first, a series of cultures has now been maintained for 67 days. During this interval, in addition to the original set, three successive subcultures have been made to fresh tissue and the fluids have been removed and replaced 10 times (Table 1). Assuming that at each change of fluid a dilution of approximately 1:15 was effected and that at the initiation of each set of cultures the inoculum was diluted 30 times, it has been calculated that the 10% suspension of infected mouse brain used as the primary inoculum had been diluted approximately 10^17 times in the fluids removed from the third subculture on the 16th day of cultivation. These fluids, however, on inoculation into mice and monkeys, produced typical paralysis. Accordingly, since the mouse LD of the original inoculum was it would appear that the increase in virus during the course of the experiment was at least of the order of 10^15 imes. During the 67-day period of cultivation a progressive decrease in mouse infectivity was recorded (Table 1). On the other hand, in the second experiment, mentioned above, the calculated increase in virus during a 52-day period is now of the order of 10^16 times and no decrease in mouse infectivity has so far been observed.

The agent propagated in the first experiment continued to exhibit the principal characteristics of the Lansing strain during the period of cultivation, as indicated by the following observations: (a) fluids from each set of cultures produced paralysis and death in mice after intracerebral inoculation; (b) the agent present in the fluids of the second set of subcultures was neutralized by antiserum specific for the Lansing strain; (c) following intracerebral inoculation, the fluids from the third set of subcultures produced flaccid paralysis within 7 and 10 days, respectively, in two rhesus monkeys. Microscopic examination of the spinal cords of these animals revealed lesions characteristic of poliomyelitis.

Cultures of intestinal tissue were prepared with fragments from the entire intestine of human embryos, except in one experiment in which jejunum of a premature infant was used. In the latter, the bacteria were eliminated in the majority of cultures by thorough washing of the tissue and by the inclusion in the original nutrient fluid of 100 units/cc of penicillin and of streptomycin.

In one experiment with embryonic intestine, which included two subcultures and 7 changes of nutrient fluid, the calculated dilution of the original inoculum was of the order of 10^13.7 times. On the basis of the mouse LD, of the original inoculum and that of the last supernatant fluid, it was calculated that the virus had increased about 10^19.7 times. The identity of the agent thus cultivated in intestinal tissue has not yet been confirmed by neutralization tests or monkey inoculation, but it elicits a response in the mouse typical of the Lansing virus.

The cultures prepared with intestine of the premature infant have, so far, been maintained 17 days with 3 changes of nutrient fluid. Virus has been demonstrated, by mouse inoculation, in the fluids removed during the course of the experiment, including that of the 17th day. The calculated multiplication of the virus was approximately 10′ times. This finding suggests that multiplication occurred in this tissue which, from the embryologic point of view, is more mature.

To compare the increase of virus in nervous tissue with that in tissue of the intestine and the extremities, cultures of embryonic brain were prepared. The multiplication of the virus in this medium has been comparable to that in the other types. Thus in one experiment carried out contemporaneously with the series of embryonic intestinal cultures mentioned above, the calculated multiplication of virus was of the order of 10’^12 times.

No evidence was obtained which indicated that an agent other than the Lansing strain of virus was propagated in any of the three types of tissue. Mouse infectivity tests for the presence of virus in the supernatant fluids of uninoculated control cultures were negative. Aerobic and anaerobic cultures of supernatant fluids yielded no growth of bacteria.

On microscopic examination of fragments of the three types of tissue, removed after about 30 days of cnltivation, differences have been observed in cell morphology
between those derived from inoculated and uninoculated cultures. Many of the fragments from uninoculated cnltures contained cells which appeared to be viable at the time of fixation, as indicated by the normal staining properties of the nuclei and cytoplasm. In contrast, the nuclei of the majority of the cells in fragments from inoculated cultures showed marked loss of staining properties. Since the amount of material which has been studied is as yet relatively small, one cannot conclude that the changes observed in the inoculated cultures wcre caused by the virus.

It would seem, from the experiments described above, that the multiplication of the Lansing strain of poliomyelitis virus in the tissues derived from arm or leg, since these do not contain intact neurons, has occurred either in peripheral nerve processes or in cells not of nervous origin.

DOI: 10.1126/science.109.2822.85

In Summary:

• An extraneural site for the multiplication of the “virus” of poliomyelitis has been considered by a number of investigators
• Enders admits that the evidence that this may occur is almost entirely indirect
• Direct attempts by Sabin and Olitsky to demonstrate in vitro multiplication of a monkey-adapted strain of poliomyelitis “virus” (MV strain) in cultures composed of certain nonnervous tissues failed
• Thc general recognition that the “virus” may be present in the intestinal tract of patients with poliomyelitis emphasized the need of further investigation of the possibility of extraneural multiplication (keep in mind, this is 40 years after Polio was “isolated” by Landsteiner and Popper and they still did not know where to find the “virus”)
• The cultures consisted of:
  • Tissue fragments suspended in 3 cc of a mixture of balanced salt solution (3 parts) and ox serum ultrafiltrate (1 part)
  • Tissues from embryos of 273 to 44 months as well as from a premature infant of 7 months’ gestation were used
  • These were: the tissues of the arms and legs (without the large bones), the intestine, and the brain
  • Each set of cultures included 4 or more inoculated with “virus”
  • The primary inoculum consisted of 0.1 cc of a suspension of mouse brain infected with the Lansing strain of poliomyelitis “virus”
• The identity of the “virus” was verified by:
  1. The character of the disease it produced in white mice following intracerebral inoculation
  2. Its neutralization by “specific” antiserum
• Subcultures were inoculated with 0.1 cc of pooled centrifuged supernatant fluids removed from the previous set of cultures (pooled means they were all mixed together)
• The procedure of cultivation differed from that usually followed by other workers in that the nutrient fluid was removed as completely as possible and replaced at periods ranging from 4 to 7 days
• Subcultures to fresh tissue were prepared at relatively infrequent intervals, ranging from 8 to 20 days
• A series of cultures had been maintained for 67 days
• During this interval, in addition to the original set, three successive subcultures were made to fresh tissue and the fluids removed and replaced 10 times
• During the 67-day period of cultivation a progressive decrease in mouse infectivity was recorded
• Enders claims the “agent” propagated in the first experiment had the same characteristics of the Lansing strain based the following observations:
  • Fluids from each set of cultures produced paralysis and death in mice after intracerebral inoculation
  • The “agent” present in the fluids of the second set of subcultures was neutralized by antiserum specific for the Lansing strain
  • Following intracerebral inoculation, the fluids from the third set of subcultures produced flaccid paralysis within 7 and 10 days, respectively, in two rhesus monkeys
• Cultures of intestinal tissue were prepared with fragments from the entire intestine of human embryos, except in one experiment in which jejunum of a premature infant was used
• In the latter, the bacteria were eliminated in the majority of cultures (i.e. not all of them) by thorough washing of the tissue and by the inclusion in the original nutrient fluid of 100 units/cc of penicillin and of streptomycin
• One experiment with embryonic intestine included two subcultures and 7 changes of nutrient fluid and Enders calculated (i.e. guessed) that the “virus” had multiplied
• However, he admits that the identity of the “agent” thus cultivated in intestinal tissue was not confirmed by neutralization tests or monkey inoculation
• The cultures prepared with intestine of the premature infant had been maintained 17 days with 3 changes of nutrient fluid
• On microscopic examination of fragments of the three types of tissue, removed after about 30 days of cnltivation, differences were observed in cell morphology between those derived from inoculated and uninoculated cultures (Enders eventually coined the term Cytopathogenic Effect (CPE) to describe this)
• Enders admits that since the amount of material which was studied was relatively small, one cannot conclude that the changes observed in the inoculated cultures wcre caused by the “virus” (thus invalidating “viruses” as the direct cause of CPE which we know can be caused by numerous factors other than imaginary “viruses”)

There was no “virus” ever purified/isolated from these tissue culture experiments. The “virus” was only assumed to be present in the sample provided based on how sick the animals would get after intracerebral inoculation as well as results from non-specific neutralization tests using theoretical antibodies. The replication of the “virus” was estimated based on how fast the animals died when injected with toxic tissue culture soup after 16 days of incubation compared to the original sample. Enders added the practice of changing nutrient fluids and subculturing at regular intervals, both of which could have made the tissue culture supernatant more toxic to the animals thus killing them faster. There is absolutely no reason to conclude that a “virus” multiplied and killed the animals more quickly when the changes to the tissue culture can explain this outcome. Even Enders finding of the cytopathogenic effect was cast in doubt by the man himself when he concluded that one cannot conclude that the changes observed in the inoculated cultures wcre caused by the “virus.” There is nothing monumental in this paper other than showcasing the changes in culture techniques of a madman reveling in the macabre depravity that is virology.