D.A. Tyrrell’s 2nd “Coronavirus” Discovery Paper (1966)

This is the second paper by D.A. Tyrrell that is considered as preliminary evidence for the existence of human “coronaviruses.” Notice, as in his first paper from 1965, that once again there are numerous assumptions which are made, the least of which is that there is a “virus” present in the samples to begin with. The nasal washings from sick patients are mixed with additives such as saline and nutrient broth. The utterly horrific and grotesque fetal tissue and organ cultures are the main methods used to “grow” unknown and invisible “viruses” and the culture supernatant from both are pooled and mixed together. This disgusting unpurified material was used in attempts to make people sick. There are no accompanying EM images of any kind showing “crown-like” particles nor any “viruses” whatsoever. The only fortunate thing about this paper is that it is a short piece of pseudoscientific fiction:

CULTIVATION OF VIRUSES FROM A HIGH PROPORTION OF PATIENTS WITH COLDS

WITH present methods of tissue culture and testing, it is usually possible to cultivate a virus from about a quarter to a third of adult patients with common colds. Organ cultures of human foetal tracheal or nasal epithelium have been shown to support the growth of representative strains of all known respiratory viruses. These cultures have also been used to cultivate some apparently “new”viruses-namely (a) 2 rhinoviruses which will produce a cytopathic effect in human diploid-cell strains only after passage in organ culture 3 (b) a rhinovirus which will not grow at all in such strains 4; and (c) an ether-labile virus apparently unrelated to any of the known ether-labile viruses of the human respiratory tract.

Using organ cultures we have recently attempted to cultivate viruses from a series of patients with colds and similar acute respiratory illnesses.

METHOD

The specimens consisted of nasal washings collected at irregular intervals between 1961 and 1964, and more regularly since then from 21 adults and 2 children shortly after the onset of illness. The patients were laboratory staff and their families and members of the general public in whom spontaneous colds developed while they were staying at the Unit. Specimens were not collected when it seemed likely on epidemiological grounds that the infection had been caused by a virus that had already been collected. We also tested four pools of washings taken from volunteers infected with strains of virus which we had repeatedly failed to cultivate. The washings were made with isotonic phosphate-buffered saline, pH 7-1, and were mixed with an equal volume of nutrient broth and stored at -70°C until tested.

All specimens were tested in “standard” tissue cultures of monkey kidneys, HeLa cells, human diploid cell strains, and human foetal kidney. The specimens were also inoculated into organ cultures; medium was collected daily from these between the second and tenth day and stored with broth as for nasal washings. The fluids were then combined, and the pool was retested in standard cultures; if negative, it was inoculated intranasally into 6 or more volunteers. If 1 or more volunteers got colds the specimen was taken to contain a virus. In most cases two passages were made in organ cultures.

RESULTS

The results obtained so far are outlined in the accompanying table. The rate of virus isolation in standard cultures was about that expected, and the viruses were of
various types with rhinoviruses predominating. The rhinoviruses were identified by their biological properties,
but were not serotyped. All the viruses recognised by direct inoculation of standard cultures were also recognised by testing the media of organ cultures inoculated with the same washing. However, additional rhinoviruses were cultivated in organ cultures and were then readily recognised by their effect on diploid cells, although the original specimens were negative when tested in these cells. Some of these viruses were difficult to propagate and identify, because they grew to low titres in human diploid fibroblast cells. In addition, six further cold-producing agents were detected because organ culture fluids caused colds in volunteers. The tests are incomplete, but no reduction in ciliary activity was noted in cultures in which these agents were growing. Altogether, therefore, twenty-five viruses or other agents were recovered from 33 specimens (75%) and nineteen of these (57%) were recognised in the laboratory. Of the 8 specimens from which no virus was isolated, 6 were still available and were inoculated into volunteers. 3 of these produced colds; hence it is clear that there are a few cold-producing agents which cannot at present be propagated in organ cultures. We conclude, nevertheless, that these organ cultures were an efficient method of propagating viruses from patients’ colds. The combination of organ culture with diploid fibroblast cell cultures revealed the presence of nine more rhinoviruses than could be found by standard techniques. But we obviously need to devise simple methods of detecting some of the other new agents in organ culture medium and then to characterise them. Some at least may resemble the B814 virus described earlier.

DISCUSSION

Against the possibility that the viruses might originate from the organ cultures is the finding that a cold developed in only 1 out of 113 volunteers who were given fluids from uninoculated cultures made from embryos used in these experiments and that no viruses were isolated by inoculating the same fluids into “standard” tissue cultures. Washings collected from 11 persons who had recovered from their colds were also tested; a rhinovirus was isolated in organ culture from 1 woman who had carried a similar virus two weeks before, when she had a cold; and one culture fluid caused a cold in a volunteer. Thus there were two apparent isolations from 11 specimens; this is a significantly lower rate than the rate obtained by all methods from all patients with colds (p < 0’01) and the rate from the 23 patients from whom viruses were not isolated by standard methods (p < 0-05). These figures and the typical clinical picture produced in volunteers show that the agents isolated are the genuine cause of the colds.

SUMMARY

A virus or uncharacterised cold-producing agent was cultivated from 25 of 33 nasal washings by inoculating them into organ cultures of human-embryo nasal or tracheal epithelium.”

doi: 10.1016/s0140-6736(66)92364-6.

In Summary:

• Tyrrell stated it was usually possible to cultivate a “virus” from about a quarter to a third of adult patients with common colds
• He claimed organ cultures from human fetal tracheal or nasal epithelium were used to cultivate some apparently “new viruses:”
  1. Two rhinoviruses which produced a cytopathic effect in human diploid-cell strains only after passage in organ culture
  2. A rhinovirus which would not grow at all in such strains
  3. An ether-labile “virus” apparently unrelated to any of the known ether-labile “viruses” of the human respiratory tract
• They tested nasal washings including  four pools (mixtures of samples of washings from multiple people) taken from volunteers infected with strains of “virus” which they had repeatedly failed to cultivate
• The washings were made with isotonic phosphate-buffered saline, pH 7-1, and were mixed with an equal volume of nutrient broth and stored at -70°C until tested
• All specimens were tested in “standard” tissue cultures of:
  1. Monkey kidneys
  2. HeLa cells
  3. Human diploid cell strains
  4. Human fetal kidney
• The specimens were also inoculated into organ cultures; medium was collected daily from these between the second and tenth day and stored with broth as for nasal washings
• The fluids were then combined, and the pool was retested in standard cultures; if negative, it was inoculated intranasally into 6 or more volunteers
• If 1 or more volunteers got colds the specimen (a mixture of tissue and organ cultures from many subjects) was taken to contain a “virus”
• In most cases two passages were made in organ cultures
• The rhinoviruses were identified by their biological properties, (i.e. not visualized directly) but were not serotyped
• Additional rhinoviruses were cultivated in organ cultures and were then readily recognised by their effect on diploid cells, (again, not directly visualized) although the original specimens were negative when tested in these cells
• Some of these “viruses” were difficult to propagate and identify, because they grew to low titres in human diploid fibroblast cells
• Six further cold-producing agents were detected because organ culture fluids caused colds in volunteers
• Twenty-five “viruses” or other agents were recovered from 33 specimens (75%) and nineteen of these (57%) were recognised in the laboratory
• Of the 8 specimens from which no “virus” was isolated, 6 were still available and were inoculated into volunteers
• 3 of these produced colds; thus it was clear to Tyrrell that there are a few cold-producing agents which could not be propagated in organ cultures
• The combination of organ culture with diploid fibroblast cell cultures revealed the presence of nine more rhinoviruses than could be found by standard techniques
• In other words, through multiple mixed cultures, they produced varying results and any that were recognizable were attributed to known “viruses” whereas the others were attributed to new “viruses” even though tests were negative and no “virus” was “isolated”
• Tyrrell stated that they obviously needed to devise simple methods of detecting some of the other new agents in organ culture medium and then to characterise them (i.e. they could not characterize the “virus” so they assumed the “virus” was there until they could find a way to “see” it)
• He felt some at least may resemble the B814 “virus” described earlier
• Against the possibility that the “viruses” might originate from the organ cultures was the finding that a cold developed in only 1 out of 113 volunteers who were given fluids from uninoculated cultures made from embryos used in these experiments (what caused the cold in that one volunteer then?)
• There were two apparent isolations from 11 specimens which was considered a significantly lower rate than the rate obtained by all methods from all patients with colds (p < 0-01) and the rate from the 23 patients from whom “viruses” were not isolated by standard methods (p < 0-05)
• Tyrrell concluded that a “virus” or uncharacterised cold-producing agent was cultivated from 25 of 33 nasal washings by inoculating them into organ cultures of human-embryo nasal or tracheal epithelium

All of the results presented by Tyrrell pertain to cell and tissue cultures. There were no attempts to purify and isolate particles assumed to be “viruses” directly from the samples. In fact, there was no mention of purification methods of any kind even for the cultured goo. It should be apparent that the exact opposite of purification/isolation of any “virus” occurred here. The researchers starved cells and mixed together nasal washings with various chemicals/nutrients along with foreign animal/human DNA and looked for different kinds of indirect evidence such as the cytopathogenic effect to claim a ‘virus” was present. However, this effect can be created from the culturing conditions as well as from any of the numerous contaminants. The clinical symptoms could be explained by the unnatural inoculation method and/or the various other substances mixed together with the original sample. No “virus” is necessary to explain the results and any “virus” they found was based on nothing but assumptions as no particles were ever EM imaged, characterized, nor proven pathogenic. Tyrrell even stated within his paper that 25 out of 33 samples contained a “virus” or other uncharacterized cold-producing agent. He could not conclude his paper by confidently claiming the discovery of any “virus” as he knew that he did no such thing.