D.A. Tyrrell’s “Coronavirus” Discovery Paper (1965)

If you want to truly understand the insane leaps in logic, the numerous assumptions, and the lack of evidence regarding any “virus,” you must always trace things back to the original source for the discovery of said “virus.” If the paper claiming the isolation of a novel “virus” is unable to actually show purification/isolation of the particles assumed to be a “virus” directly from a human as well as prove the particles pathogenic in a natural way, it is game over from the very start. There can be no “future studies” to expand and elucidate on an unproven theoretical concept being passed around as fact. The original researchers and the original materials must be where this proof comes from in order for future researchers to utilize the same methods and materials so that they can attempt to reproduce and replicate the same results. This never happens.

The paper presented below is the 1965 paper by D.A. Tyrrell which is considered the first evidence of human “coronaviruses.” What you will notice upon reading it are the various assumptions that are made as well as the extent to which the researchers go to create the effect that they are looking for. The evidence itself (if you can call it that) stems from a small sample size of one nine-year-old boy and a limited number of volunteers. You will notice that different chemicals and additives are mixed together with the sample which negates any claims for both purification and isolation. You will see that no “virus” was ever identified here:

Cultivation of a Novel Type of Common-cold Virus in Organ Cultures

In recent years it has become evident that the common cold and similar minor upper respiratory diseases are due to infec- tion with viruses belonging to a number of different groups, including adenoviruses, myxoviruses-such as the influenza, para-influenza, and respiratory syncytial viruses-enteroviruses, and rhinoviruses. When tests adequate to detect all these are used a virus or a 83-haemolytic streptococcus can be isolated from about one-third of patients suffering from colds and related diseases (Working Party, 1965). The failures might occur because no virus or bacteria were present in the respiratory secretions tested, but in one study (Kendall et al., 1962) two out of four such specimens which apparently contained no virus were administered to volunteers and produced colds; it is therefore likely that some failures are due to the presence of viruses which cannot be cultivated by present methods.

In the past four years efforts have therefore been made to discover something of the nature of such viruses and to devise methods of cultivating them in the laboratory. Some success has been achieved and is reported in this paper.

Materials and Methods

New Viruses.-The primary sources of these were nasal washings in phosphate-buffered saline which were collected at the height of a cold, mixed with an equal volume of bacteriological nutrient broth, and stored at -70′ C.

Other viruses were propagated in chick embryos or in tissue cultures as appropriate, and were handled by standard methods.

Organ cultures were prepared mainly from the tracheas of 14- to 22-week-old human embryos obtained at hysterotomy from cases in which there was no clinical suspicion of an infection in the mother or foetus. Four to six tissue fragments were planted with the ciliated surface uppermost on the scratched surface of a 6-cm. plastic Petri dish. (Falcon), and 1.25 ml. of Medium 199 (Glaxo) containing 0.035 g. of sodium bicarbonate per litre was added. The dish was incubated at 33′ C. in a humidified box, and the medium was changed daily for two days. Cultures were then inoculated by dripping 0.3 ml. of inoculum on to the fragments ; thereafter the medium removed each day was mixed with broth and stored at – 70° C. After about 10 days some cultures were fixed in Bouin’s solution, embedded, sectioned, and stained with haematoxylin and eosin.

Haemagglutination-inhibition tests were performed, using four agglutinating doses of virus and sera treated with cholera filtrate (Philips); tests were done in 0.025-ml. volumes, using the Takatsy apparatus as modified by Sever (1962). Human group 0 cells were used in almost all tests.

Volunteers.-The methods of obtaining, housing, and observing volunteers have been described elsewhere (Andrewes, 1949). Sera were collected from some volunteers at the time of developing symptoms of a cold, and again about two weeks later after leaving the unit.

Results

Most work has been done with a nasal swab and washing number B814, obtained from a boy with a typical common cold in 1960 (Kendall et al., 1962). Further infectious secretions were obtained from volunteers who developed colds after intranasal inoculation of the original specimen. In this way three serial passages of the cold-producing agent were made in man, and it was concluded that it must be self-propagating. In over 20 experiments washings were tested by inoculation into a variety of test systems for known viruses. These are outlined in Table I, and should have revealed the presence of influenza A, B, or C, para-influenza 1, 2, 3, or 4, respiratory syncytial viruses, herpes simplex virus, and adenovivruses, cytopathic enteroviruses or rhinoviruses, or mycoplasma, particularly Mycoplasma pneumoniae. None was found. Further tests with a limited number of techniques showed that there was no evidence that the cold-producing agent was propagated even for a few days to a sufficient extent to produce the small amount of virus usually needed to cause a cold in a volunteer (Table II).

We therefore attempted to determine by experiments in volunteers a few basic properties which would confirm that we were indeed dealing with a virus. These experiments, also shown in Table II, indicate that the infectivity of B814 can pass a bacteria-tight filter, is inactivated by ether, and can induce colds in volunteers given sufficient antibiotics to cure a fully developed infection with the Eaton agent (M. pneumoniae). These results showed that B814 is a virus, not a mycoplasma, and that it is not an adenovirus, enterovirus, or rhinovirus because it is ether-labile. Another uncultivable agent produced colds in two out of six volunteers after ether treatment. This was the agent recovered from the subject H. G. P. on 26 July 1957 (Tyrrell and Bynoe, 1961). It was concluded that there must be at least two biologically different viruses among these “uncultivable” viruses.

Cultivation of B814 in Organ Cultures

It was thought that the failure to cultivate the virus in tissue culture probably arose because the virus was unable to propagate in the highly modified dedifferentiated cells used so far. It had been shown that human nasal or tracheal ciliated epithelium maintained in organ culture would support the growth of representative strains of influenza A and herpes virus (Hoorn, 1963), adenovirus, poliovirus, coxsackievirus A21, echovirus 11, H and M rhinoviruses (Hoorn and Tyrrell, 1965), and also myxoviruses such as influenza B and C, para-influenza virus types 1, 2, 3, and 4, and respiratory syncytial virus (Tyrrell and Hoorn, 1965). Several viruses were successfully grown from nasal washings without passage in any other culture system. It was therefore thought reasonable to attempt to propagate the B814 virus in cultures of this type.

The experiments done so far are summarized in Table III. There was some trouble with bacterial and fungal contamination at first, but later the technique outlined under “Methods” was found to be trouble-free. It was regularly possible to produce colds in volunteers who were given culture fluids from the first or later passages. For several reasons it is believed that the B814 virus was multiplying and causing these colds.
Firstly, no colds were produced by fluids from “dummy” cultures containing no tissue and inoculated one or two days before with nasal washings. Similarly, no colds were produced by fluids from numerous uninoculated parallel cultures set up from the same embryos, changes at the same time in the same cabinet, and using the same medium as those used for the virus-infected cultures; no colds were produced by medium from inoculated cultures in which ferret trachea was used instead of human tissue. On the other hand, colds were produced with fluids coming from cultures which had been changed up to eight times after inoculation, involving a lapse of at least a week in culture and a probable dilution of the order of 108 of the original inoculum.

Colds were also produced after four serial passages in which the fluids collected daily between one (sometimes three) and 10 days after inoculation were pooled and used to inoculate further batches of cultures. This method of serial passage was adopted in order to ensure that some infectious virus was passed, because in some experiments with rhinoviruses and respiratory syncytial virus the viruses had been shed into the medium for only a few days and at rather unpredictable intervals after the inoculation of the cultures. Fluid for this fourth serial passage was also inoculated after overnight treatment with ether; it caused colds in none of six volunteers. Another aliquot was filtered through a membrane of A.P.D. 0.59 pu and produced colds in three out of six volunteers who were treated with demethylchlortetracycline. It was concluded that these colds were due to the presence of an ether-labile virus, as were those produced by the washings used to initiate the serial passage. Similar culture fluids failed to cause disease when inoculated intramuscularly and intranasally into adult white mice, intracerebrally and intraperitoneally into suckling white mice, and intracerebrally and intranasally into guinea-pigs; and no virus was isolated by amniotic inoculation of 10-day-old chick embryos.

Studies of Volunteers’ Sera

A number of paired sera were available from volunteers who had developed colds after receiving the B814 virus either as washings or as tissue-culture fluid. It was thought possible that the sera might manifest a rise in antibody titre against
some known myxovirus because the B814 virus might be either a myxovirus of a known serotype which was more difficult to cultivate than any previously encountered, or a myxovirus of a new serotype with a distant antigenic relationship to one or more known types. The sera were therefore titrated by haemagglutination-inhibition test in this laboratory, and by complement-fixation tests by Dr. L. Hatch in the Portsmouth Laboratory of the Public Health Laboratory Service. The results are summarized in Table IV. A few rising titres were detected against influenza C and para-influenza viruses; the rises detected by complement-fixation were observed in two pairs with two or three antigens. It was concluded that B814 did not belong to any of the serotypes of myxovirus used, but might be distantly related to influenza C or Sendai viruses. Paired sera from six volunteers were tested for their ability to fix complement with culture fluids collected from the fifth serial passage and concentrated fifteenfold with polyethylene glycol. No fixation was observed.

Clinical Features of Colds Produced by B814 Virus

The frequency of some symptoms, clinical signs, and certain other data regarding volunteers who developed colds after inoculation with B814 material are listed in Table V. This shows that the illnesses were slightly more frequent, were more severe, and had a shorter incubation period when they followed the inoculation of organ-culture fluid than after the inoculation of nasal washings. This could all have been the result of the administration of a larger dose of virus. In both groups the illness was a typical common cold. Fever was rare, but there was often considerable malaise, and the nose often streamed with watery secretion-one volunteer used 120 paper handkerchiefs in one day-but there was little cough and no sputum, and on the average the disease cleared up in less than a week. The clinical picture in 213 volunteers infected with an M rhinovirus is also shown in Table V; this was significantly different in that malaise and constitutional upset were less common, and sore throat, cough, and sputum were more so-the respiratory symptoms were less strictly confined to the nose and lasted longer, but there was less nasal discharge at the height of the illness.

Preliminary Attempts to Propagate More Otherwise Uncultivable Viruses in Organ Cultures

A number of other nasal washings were collected from patients with colds and tested for respiratory viruses. From certain of these no virus was recovered, and so they were inoculated into organ cultures. Some results obtained are outlined in Table VI. This shows that the viruses contained in these specimens were apparently propagated in organ cultures, because culture fluids caused colds. The organ-culture fluids were tested further by inoculating them into a range of tissue cultures. In the case of the M.T. strain the virus apparently grew, as judged by inoculation of volunteers, but was obviously different from B814, because tests in volunteers showed that it was ether-stable. When organ-culture fluids were inoculated into other cultures they produced a cytopathic effect which resembled that due to rhinoviruses.

The specimens from another organ-culture experiment with strain G.T. were titrated in human diploid cells and the data are plotted on Fig. 1, which shows that a rhinovirus had grown freely and could be readily detected after three passages in organ culture. The original nasal washing had produced some doubtful cytopathic effect when tested in four batches of human-embryo-kidney cells, and a more definite effect in one strain of human-embryo fibroblasts, although the latter was not successfully passaged. Finally, the virus F.T. was apparently a rhinovirus that failed to produce any cytopathic effect in the form in which it was present in the nasal secretions and in the first passage in organ cultures, although it could produce colds. It nevertheless multiplied in organ culture, and became adapted so that it rapidly damaged the cilia (see below), and also was able to produce a cytopathic effect in cells of a human fibroblast strain. We report these experiments in order to illustrate that organ cultures may be of value in cultivating and recognizing other “difficult” respiratory viruses, and also to emphasize that because a virus is grown in organ cultures it is not safe to conclude that a new type of virus has been cultivated.

Detection of Infection in Organ Cultures

It has been shown earlier that the ciliary activity of infected cultures may be reduced or absent compared with that in uninoculated cultures. Fixed and stained sections may also show degeneration or shedding of some or all of the surface epithelium (Hoorn, 1963; Hoorn and Tyrrell, 1965). Attempts were therefore made to detect these effects in the cultures used in the experiments described above. The results are outlined in Table VI. The activity of the cilia on each tissue fragment was examined by reflected light daily or on alternate days. In experiments in which ciliary activity continued vigorously in the controls it was sometimes noted that the ciliary activity of all or most of the virus-infected fragments had ceased. This is recorded (as +) in Table VI, as is also the day on which a reduction in ciliary activity was first recognized. It can be seen that this effect was observed in only the later passages of B814 and was never clear-cut; possible or definite degenerative changes, particularly in the tracheal ciliated epithelial cells, were seen in fixed and stained tissue in which during life the cilia were thought to be beating less well than in the control.

We also attempted to detect the presence of B814 by using the phenomenon of virus interference as we had done in earlier studies on rhinoviruses (Hitchcock and Tyrrell, 1960). A set of human-embryo-trachea organ cultures was inoculated with fluid from the fourth passage of the virus (see Table III). The cultures were incubated as usual for five days, and then, although the cilia were beating freely, they were challenged with Sendai virus, the U strain of echovirus 11, or a human strain of para-influenza 3. The media were harvested daily and titrated for infectivity by inoculation of serial dilutions into monkey-kidney-cell cultures. The results were generally the same with each virus, and may be illustrated by those obtained with Sendai virus (Fig. 2). The titre was tenfold or more lower in cultures previously infected with B814 than in those previously inoculated with media from uninfected organ cultures. This was noted in all eight comparative titrations, using the three challenge viruses mentioned. It was concluded that infected cultures could be definitely detected by virus interference or reduction in ciliary activity.

Discussion

These experiments seem to show that organ cultures of human tracheal epithelium can support the growth of at least one respiratory virus which we have been unable to grow by any other laboratory technique. After considerable initial doubts we now believe that the B814 strain is a virus virtually unrelated to any other known virus of the human respiratory tract, although, since it is ether-labile, it may be a myxovirus. It is disappointing that so far no satisfactory serological test is available, but there are many possibilities still to be explored for instance, we have not yet tried to use the fluorescent antibody technique with sections of organ cultures, because model experiments with cultures infected with influenza virus were not encouraging. Further, we are testing in organ cultures specimens from other patients with respiratory disease in order to find out how frequently hitherto unrecognized viruses can be isolated. These results will be reported later, but it seems likely that some of these may be fastidious rhinoviruses. However, it also seems possible that organ cultures of other tissues might be useful in propagating other viruses which have not so far been grown in dedifferentiated cells-for example, the viruses of molluscum contagiosum, and gastro-enteritis. In order to increase the chances of success in such experiments it is also necessary to look for further improvements in the techniques of organ culture, in particular a substitute for human foetal cells and improvements in the medium and other conditions used.

Summary

Volunteers developed colds after the intranasal inoculation of secretions derived from a boy with a common cold. Colds developed, although the secretions were passed through a filter of A.P.D. 0.59 pu and the volunteers were treated with demethylchlortetracycline. No colds developed if the washings were treated with ether.

The virus thus demonstrated would not grow in tissue cultures and eggs which would support the multiplication of known viruses of the upper respiratory tract. It multiplied and was serially propagated in organ cultures of human foetal tracheal epithelium. The colds produced by washings and tissue cultures were clinically similar, and in the aggregate distinct, from those produced by M rhinoviruses.

Sera of infected volunteers were tested by haemagglutination-inhibition and complement-fixation tests; a small proportion showed slight rises against influenza C and Sendai viruses.

Infection of organ cultures with B814 was detected with difficulty by a decline in ciliary activity and by degenerative changes in sections of the tissue, but there was a tenfold to a hundredfold reduction in titre on challenge of the cultures with other viruses-virus interference.

Other viruses distinct from B814 have been recognized and similarly cultivated, including uncharacterized ether-stable viruses which may be rhinoviruses.”

doi: 10.1136/bmj.1.5448.1467.

In Summary:

• When testing sick patients, a “virus” or a 83-haemolytic streptococcus could be “isolated” from about one-third of patients suffering from colds and related diseases
• The failures might occur because no “virus” or bacteria were present in the respiratory secretions tested
• In one study (Kendall et al., 1962) two out of four specimens which apparently contained no “virus” were administered to volunteers and produced colds
• Tyrrell assumes that it is therefore likely that some failures are due to the presence of “viruses” which cannot be cultivated by present methods
• He states that for four years, efforts had been made to discover something of the nature of these assumed, non- cultivable “viruses” and to devise methods of cultivating them in the laboratory
• In other words, they needed new lab techniques in order to create these “viruses”
• Nasal washings were stored in phosphate-buffered saline which were collected at the height of a cold and mixed with an equal volume of bacteriological nutrient broth
• Other “viruses” were propagated in chick embryos or in tissue cultures as appropriate, and were handled by standard methods
• Organ cultures were prepared mainly from the tracheas of 14- to 22-week-old human embryos obtained at hysterotomy from cases in which there was no clinical suspicion of an infection in the mother or foetus
• Four to six tissue fragments were planted with the ciliated surface uppermost on the scratched surface of a 6-cm. plastic Petri dish and 1.25 ml. of Medium 199 containing 0.035 g. of sodium bicarbonate per litre was added
• What is in Medium 199?
  • “The liquid growth medium on top is known as Medium 199, a salt solution that includes vitamins, amino acids, and fetal bovine serum, plus sucrose, phosphate, glutamate, neomycin, and recombinant human albumin”
  • https://www.mentalfloss.com/article/61502/whats-measles-vaccine
• The dish was incubated at 33′ C. in a humidified box, and the medium was changed daily for two days
• Cultures were then inoculated by dripping 0.3 ml. of inoculum on to the fragments; thereafter the medium removed each day was mixed with broth and stored at – 700 C
• After about 10 days some cultures were fixed in Bouin’s solution, embedded, sectioned, and stained with haematoxylin and eosin
• Haemagglutination-inhibition tests were performed, using four agglutinating doses of (assumed) “virus” and sera treated with cholera filtrate
• Most work was done with a nasal swab and washing number B814, obtained from a 9-year-old boy with a typical common cold in 1960
• Further infectious secretions were obtained from volunteers who developed colds after intranasal inoculation of the original specimen (which had already been mixed with additives)
• In this way, Tyrrell claims three serial passages of the cold-producing agent were made in man, and it was concluded that it must be self-propagating
• Test for numerous “viruses” were done and none were found
• Further tests with a limited number of techniques showed that there was no evidence that the cold-producing agent was propagated even for a few days to a sufficient extent to produce the small amount of “virus” usually needed to cause a cold in a volunteer
• Experiments indicated that the infectivity of B814 could pass a bacteria-tight filter, was inactivated by ether, and could induce colds in volunteers given sufficient antibiotics to cure a fully developed infection with the Eaton agent (M. pneumoniae)
• Another uncultivable agent produced colds in two out of six volunteers after ether treatment
• This was the agent recovered from the subject H. G. P. on 26 July 1957
• It was concluded (assumed again) that there must be at least two biologically different “viruses” among these “uncultivable viruses”
• It was thought that the failure to cultivate the “virus” in tissue culture probably arose because the “virus” was unable to propagate in the highly modified dedifferentiated cells used
• It had been shown that human nasal or tracheal ciliated epithelium maintained in organ culture would support the growth of other “viruses” and since several “viruses” were successfully grown (i.e. not isolated, they were created) from nasal washings without passage in any other culture system, it was therefore thought reasonable to attempt to propagate the B814 “virus” in cultures of this type
• There was some trouble with bacterial and fungal contamination at first
• It was regularly possible to produce colds in volunteers who were given culture fluids (i.e. not purified/isolated “virus”) from the first or later passages
• Tyrrell believed B814 was a “virus” for several reasons:
  1. Firstly, no colds were produced by fluids from “dummy” cultures containing no tissue and inoculated one or two days before with nasal washings (i.e. they could not produce colds with direct nasal washings, only cultured ones…pretty much game over right here)
  2. Similarly, no colds were produced by fluids from numerous uninoculated parallel cultures set up from the same embryos, changes at the same time in the same cabinet, and using the same medium as those used for the “virus-infected” cultures
  3. No colds were produced by medium from inoculated cultures in which ferret trachea was used instead of human tissue
  4. On the other hand, colds were produced with fluids coming from cultures which had been changed up to eight times after inoculation, involving a lapse of at least a week in culture and a probable dilution of the order of 108 of the original inoculum
  5. Colds were also produced after four serial passages in which the fluids collected daily between one (sometimes three) and 10 days after inoculation were pooled (i.e. mixed together) and used to inoculate further batches of cultures
• Fluid for this fourth serial passage was also inoculated after overnight treatment with ether; it caused colds in none of six volunteers
• Another aliquot was filtered through a membrane of A.P.D. 0.59 pu and produced colds in three out of six volunteers who were treated with demethylchlortetracycline
• Quick side note, demethylchlortetracycline adverse effects include:
  1. Sores or swelling in your rectal or genital area
  2. Rectal discomfort
  3. Nausea
  4. Vomiting
  5. Diarrhea
  6. Loss of appetite
  7. White patches or sores in your mouth or on your lips
  8. Swollen tongue
  9. Trouble swallowing
  10. Headache
  11. Dizziness
• https://www.rxlist.com/declomycin-side-effects-drug-center.htm
• It was concluded that these colds were due to the presence of an ether-labile “virus,” as were those produced by the washings used to initiate the serial passage (however, they did not see, purify, nor isolate any “virus”)
• Similar culture fluids failed to cause disease when inoculated intramuscularly and intranasally into adult white mice, intracerebrally and intraperitoneally into suckling white mice, and intracerebrally and intranasally into guinea-pigs
• No “virus” was isolated by amniotic inoculation of 10-day-old chick embryos
• A few rising titres were detected against influenza C and para-influenza “viruses;” the rises detected by complement-fixation were observed in two pairs with two or three antigens
• It was concluded that B814 did not belong to any of the serotypes of myxovirus used, but might be distantly related to influenza C or Sendai “viruses”
• Paired sera from six volunteers were tested for their ability to fix complement with culture fluids collected from the fifth serial passage and concentrated fifteenfold with polyethylene glycol yet no fixation was observed
• The illnesses were slightly more frequent, were more severe, and had a shorter incubation period when they followed the inoculation of organ-culture fluid than after the inoculation of nasal washings
• Tyrrell assumes this could all have been the result of the administration of a larger dose of “virus” from the organ-culture fluid (rather than admitting this fluid full of additives was more toxic)
• The clinical symptoms were of the common cold and consisted of:
  • No fever as this was rare, but there was often considerable malaise
  • The nose (of which the culture fluid was injected) often streamed with watery secretion-one volunteer used 120 paper handkerchiefs in one day
  • There was little cough and no sputum
  • On the average the disease cleared up in less than a week
• In other words, the clinical signs were fatigue and a watery nose
• A number of other nasal washings were collected from patients with colds and tested for respiratory “viruses” and from certain of these no “virus” was recovered
• The “virus”-negative specimens were subsequently inoculated into organ cultures and it was then assumed that the “viruses” contained in these specimens were apparently propagated in organ cultures, because culture fluids caused colds
• The organ-culture fluids were tested further by inoculating them into a range of tissue cultures
• In the case of the M.T. strain the “virus” apparently grew, as judged by inoculation of volunteers, but was obviously different from B814, because tests in volunteers showed that it was ether-stable
• When organ-culture fluids were inoculated into other cultures they produced a cytopathic effect which resembled that due to rhinoviruses
• In other words, culturing nasal washings that did not cause colds in organs mixed with additives did eventually cause colds and it was assumed a “virus” grew yet it was a different “virus” than B814 as it was ether-stable and produced CPE resembling rhinovirus
• Keep in mind CPE patterns are supposed to be specific to a particular “virus” yet this example obviously shows that this is not the case
• The original nasal washing had produced some doubtful cytopathic effect when tested in four batches of human-embryo-kidney cells, and a more definite effect in one strain of human-embryo fibroblasts, although the latter was not successfully passaged
• The “virus” F.T. was apparently a rhinovirus that failed to produce any cytopathic effect in the form in which it was present in the nasal secretions and in the first passage in organ cultures, although it could produce colds
• Tyrrell wanted to emphasize that because a “virus” is grown in organ cultures it is not safe to conclude that a new type of “virus” has been cultivated
• While trying to detect infection by way of ciliary activity, this effect was observed in only the later passages of B814 and was never clear-cut
• Possible or definite degenerative changes, particularly in the tracheal ciliated epithelial cells, were seen in fixed and stained tissue in which during life the cilia were thought to be beating less well than in the control
• Tyrrell also attempted to detect the presence of B814 by using the phenomenon of “virus” interference
• “Virus” interference was an indirect method used in the 1930’s to the 1960’s and is defined as the host resistance to a superinfection caused by a pathogenic “virus” causing obvious signs of disease and/or mortality due to the action of an interfering “virus” abrogating the replication of the former “virus” https://www.frontiersin.org/articles/10.3389/fimmu.2021.674216/full
• In other words, they claimed one invisible “virus” was reducing the effect of the other invisible “virus”
• Tyrrell claimed that these experiments seemed to show that organ cultures of human tracheal epithelium could support the growth of at least one respiratory “virus” which he had been unable to grow by any other laboratory technique
• After considerable initial doubts, he now believed that the B814 strain was a “virus” virtually unrelated to any other known “virus” of the human respiratory tract, although, since it was ether-labile, it may have been a myxovirus
• Tyrrell claimed they were testing in organ cultures specimens from other patients with respiratory disease in order to find out how frequently unrecognized “viruses” could be isolated
• It also seemed possible that organ cultures of other tissues might be useful in propagating other “viruses” which had not been grown in dedifferentiated cells
• In order to increase the chances of success in such experiments, he stated it was necessary to look for further improvements in the techniques of organ culture, in particular a substitute for human foetal cells and improvements in the medium and other conditions used
• Volunteers developed colds after the intranasal inoculation of secretions derived from one boy with a common cold
• Colds developed, although the secretions were passed through a filter of A.P.D. 0.59 pu and the volunteers were treated with demethylchlortetracycline
• No colds developed if the washings were treated with ether
• In other words, colds developed in those given toxic antibiotics but not in those not given one
• The “virus” demonstrated would not grow in tissue cultures and eggs which would support the multiplication of known “viruses” of the upper respiratory tract
• It multiplied and was serially propagated in organ cultures of human foetal tracheal epithelium
• Supposedly “virus-specific” antibody tests showed slight rises against influenza C and Sendai “viruses”
• Infection of organ cultures with B814 was detected with difficulty by a decline in ciliary activity and by degenerative changes in sections of the tissue
• There was a tenfold to a hundredfold reduction in titre on challenge of the cultures with other “viruses-virus interference”
• Other “viruses “distinct from B814 were recognized (i.e. indirectly, not visually) and similarly cultivated, including uncharacterized ether-stable “viruses” which may have been rhinoviruses

Did you notice that there was no purification/isolation of any “virus” nor were there any accompanying EM images of any “corona-like” particles? This whole study consisted of assumptions about invisible imaginary “viruses” created from unpurified tissue cultures from fetal organs that sometimes were associated with people having runny noses after they were subjected to intranasal inoculations and antibiotics. They could not even produce any illness when direct inoculations of nasal washings were used, only with the addition of demethylchlortetracycline. The clinical symptoms consisted of fatigue and a watery, runny nose yet was absent any fever, cough, sputum, etc. Tyrrell and Co. could not even produce any valid antibody reactions (as if there are any) as they experienced cross-reactions with other “viruses.” They also admitted that just because a “virus” was grown in tissue culture, it did not mean it was a “novel virus.”

If you look at this paper both critically and logically, it is abundantly clear that the researchers went in assuming that uncultivable “viruses” could be grown in fetal tissues and then proceeded to claim success if there was a small hint of illness which they could associate with their toxic tissue-culture/antibiotic nasal stew. If there were contradictory or negative findings, it was assumed to be either due to the presence of a different invisible “virus” and/or that more consistent results would be generated in the future with better technology, materials, and methods. Did that evidence ever materialize? If you know anything about B814, you will know that it did not as it is the forgotten red-headed step child in the “coronaviridae” family.

This paper is supposed to be THE evidence for the existence of “corinaviruses.” Did you see any?