Hamre’s “Coronavirus” 229E Paper (1966)

In 1966, Dorothy Hamre “isolated” what she termed specimen 229E from tissue cultures using specimens from college students. She noticed what she said was distinct cytopathic changes (CPE) from known “viruses” and the results she obtained from indirect antibody testing led her to conclude it was a new respiratory “virus.” The 229E “virus” is now considered one of the main causes of the common cold and even though Hamre is often overlooked in the history books, she is considered the person who  truly “isolated” the first “coronavirus:”

Finding Dorothy Hamre, the First Person to Isolate a Strain of a Coronavirus

“In seeking an answer to this apparently simple question, I chanced upon the work of Dorothy Hamre, a virologist and infectious disease researcher at the University of Chicago’s department of medicine. In a paper co-authored with John J. Procknow, Hamre describes a new virus “possibly associated with mild upper respiratory illnesses of man.”

Hamre was the first person to isolate a strain of a coronavirus, which she did from samples taken from students at the university’s medical school. This strain was designated as 229E. Almost at the same time, in Britain’s Common Cold Research Unit in Salisbury, David Tyrrell and Malcolm Bynoe were isolating what appeared to be a new class of viruses from organ cultures, with the strain B814 appearing to resemble an avian bronchitis virus. In 1967, June Almeida, working in Tyrrell’s laboratory, produced the first image of the virus.”

Finding Dorothy Hamre, the First Person to Isolate a Strain of a Coronavirus

There are quite a few problems with declaring Hamre the first person to “isolate” a “coronavirus” let alone any “virus” at all. As can be seen from the above article, the “coronavirus” was not imaged until 1967 by June Almeida, a year after Hamre supposedly “isolated” 229E and a full 2 years after D.A. Tyrrell “isolated” B814 in 1965. Thus, there was zero visual evidence of any “virus” supplied by Hamre (or Tyrrell).

Another major issue is that the evidence supplied by Hamre for the existence of 229E consisted entirely of indirect evidence taken from antibody results rather than direct evidence of any purified/isolated “virus.” Direct evidence, which is entirely lacking in virology, is that which directly proves the issue at hand. Indirect evidence, on the other hand, is a fact or set of facts that, if they are true, allow a person to infer the fact in question. Hamre’s indirect evidence consisted of tests such as complement fixation, neutralization tests, hemagglutination tests, and egg inoculations. Looking at both complement fixation and neutralization tests should provide a clear picture as to why this evidence is inadequate.

Complement Fixation

Complement fixation is an indirect serological test which is said to detect the presence of specific antibodies or antigens by a reaction that causes fixation of complement resulting in membrane lysis. It was commonly used to determine antibodies in the past but has fallen out of favor for more modern methods due to several issues addressed below:

Complement Fixation Test- Steps, Advantages and Disadvantages

“It is a classic method for demonstrating the presence of antibody in patient serum. It is based on the principle that antigen-antibody complex fixes the complement. As coupling of complement has no visible effects or changes, it is necessary to use an indicator system consisting of sheep RBC and coated with anti-sheep RBC antibody. Complement lyses antibody coated RBC.

The complement fixation test consists of two components.

The first component is an indicator system that uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of complement usually guinea pig serum. When these elements are mixed in optimum conditions, the anti-sheep antibody binds on the surface of red blood cells. Complement subsequently binds to this antigen -antibody complex formed and will cause the red blood cells to lyse.

The second component is Test System (A known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement). These two components of the complement fixation method are tested in sequence. Patient serum is first added to the known antigen, and complement is added to the solution. If the serum contains antibody to the antigen, the resulting antigen-antibody complexes will bind all of the complement. Sheep red blood cells and the anti-sheep antibody are then added. If complement has not been bound by an antigen-antibody complex formed from the patient serum and known antigens, it is available to bind to the indicator system of sheep cells and anti-sheep antibody. Lysis of the indicator sheep red blood cells signifies both a lack of antibody in patient serum and a negative complement fixation test. If the patient’s serum does contain a complement-fixing antibody, a positive result will be indicated by the lack of red blood cell lysis.”

Disadvantages of Complement Fixation Test

1. Not sensitive – cannot be used for immunity screening.
2. Time-consuming.
3. Often non-specific e.g. cross-reactivity between Herpes Simplex Virus and Voricella Zoster Virus.

Complement Fixation Test- Steps, Advantages and Disadvantages

As can be seen, this test (which utilizes human, sheep, and Guinea pig blood) is not sensitive nor is it specific. These are two essential requirements needed in order to gain accurate results, especially when dealing with an unseen “novel virus.” Any results gained from such a test should be considered extremely questionable in not entirely unreliable.

Neutralization Tests

Another indirect method used by Hamre to determine she had a new “virus” is known as neutralization tests. This is another serologic test which is based on the binding of the theoretical antibodies to the theoretical pathogen or to toxins, resulting in the inhibition of their biological properties. In the case of 229E, this included the testing of antisera from ferrets and Guinea pigs to determine that 229E did not react to any of the “known viruses.” Besides the glaring problem of not being able to accurately determine theoretical antibody neutralization due to the lack of purification/isolation of any of the theoretical “viruses” studied, there are known issues with this test as highlighted from the below three sources:

“A phenomenon of fundamental medical and biological importance is the neutralization of viruses by antibodies.¹⁴ Although neutralization is defined as the elimination or reduction of the virus’s ability to replicate, it does not imply a particular mechanism of interference with the process of replication. Moreover, the measurement of neutralization can depend on the choice of host cell. Thus the neutralizing activity of a given antibody for a given virus is not an intrinsic property of the antibody but is a property of the relationship between the antibody and the virus, under defined conditions. Consequently, neutralization titers in serum do not always correlate perfectly with protection from infection or disease in vivo.”

“Not all antibodies that bind to molecules on the virion surface will neutralize the virus in all conditions. For a given virus-encoded gene product, such as the influenza virus hemagglutinin, binding of antibodies to some sites, but not others, will effect neutralization. Some gene products on the virion surface may fail to routinely support viral neutralization (e.g., influenza neuraminidase).”

https://doi.org/10.1016/B978-0-7020-6896-6.00015-6

“An issue with this assay that has recently been identified is that the neutralization ability of the antibodies is dependent on the virion maturation state and the cell-type used in the assay.^[6] Therefore, if the wrong cell line is used for the assay it may seem that the antibodies have neutralization ability when they actually do not, or vice versa they may seem ineffective when they actually possess neutralization ability.”

https://en.m.wikipedia.org/wiki/Plaque_reduction_neutralization_test

“Although the PRNT typically provide the greatest specificity for Zika and other viruses, serological assays tend to be subject to cross-reactivity especially in patients with prior flavivirus infection or immunization history. In addition, the test is relatively cumbersome and time intensive (few days), compared to other EIA tests.”

https://www.labce.com/spg1050613_plaque_reduction_neutralization_tests_prnt.aspx

Neutralization tests are dependent on the type of host cell used, do not always correlate with protection against disease, are affected by the binding or lack thereof by antibodies, and can be subject to cross-reactivity. Since cross-reactivity and false-positive results are concerns for these tests, it is clear that they are not as sensitive nor as specific as they are claimed to be and that they should not be used in order to discover a “novel virus.”

Hamre relied on inaccurate results from indirect serologic tests in order to claim the discovery of a “virus.” It should go without saying that unseen theoretical antibody reactions can not be used in order to claim that an unseen theoretical “virus” exists, yet that is exactly what Hamre did. Presented below is the full paper from 1965 containing Hamre’s treasure trove of indirect serologic evidence:

A New Virus Isolated from the Human Respiratory Tract.

“In the winter of 1962, five agents were isolated in secondary human kidney tissue cultures which presented a cytopathic effect (CPE) quite distinct from that customarily produced by known viruses associated with respiratory illnesses. This report presents the evidence for considering these agents as strains of a new respiratory virus, possibly associated with mild upper respiratory illnesses of man.

Materials and methods. The sampling procedure employed in the study of respiratory illnesses among medical students enrolled in the program at the University of Chicago as well as methods for virus isolation in tissue cultures have been described (1,2).

Serologic methods. Antiserum to the prototype strain of the new virus was prepared in guinea pigs following the procedure used for rhinovirus antisera( 2). Neutralization tests were carried out by the same method employed for rhinoviruses. The method for complement fixation (CF) tests with respiratory syncytial virus was used (3). CF antigen was prepared from WI-38 cells inoculated with high multiplicity of virus and harvested at the time of maximum CPE. The crude pool was frozen and thawed 3 times and centrifuged to remove the cells and debris.

Results. Isolation and growth in tissue culture. This virus was recovered from 5 specimens, 4 obtained from individuals with minor upper respiratory illnesses and one from a well individual during the winter of 1962 (Table I). All of these specimens yielded virus only after a second blind passage in human kidney cells. No virus was recovered in the secondary monkey kidney cultures or H.Ep. 2 cell cultures inoculated with these specimens. These viruses produced CPE in human diploid cell strains and these cultures, HEL(1) or WI-38(4), were used for all further experiments. The CPE in HEL or WI-38 was slow, with first changes noted after 6 days’ incubation at 33°C on roller drums. The cell monolayer became “stringy” in appearance but this developed generally rather than focally. Inclusion bodies were not found in cells stained by H and E. Many small vacuoles in the cytoplasm of cells were the first changes noted in stained cells.

The virus isolated from student specimen 229E was chosen as the prototype strain and purified by 3 serial selections at limiting dilutions in WI-38 cells. Guinea pig antiserum prepared with purified virus had a homologous titer of 1:1200 in neutralization tests with 20 TCID50 of 229E virus and also neutralized the other 4 strains shown in Table I.

Attempts to reisolate these viruses from the frozen original specimens were successful in 3 out of the 5 cases. The reisolations were accomplished by the use of the WD human diploid cell strain, obtained from Dr. L. Hayflick of the Wistar Institute. None of the WI-38 cultures showed any evidence of CPE even though as many as 4 blind passages were carried out. Obvious CPE developed in WD cultures by the second passage.

Characterization of the new virus, 229E. Hemagglutination tests. High titered pools of 229E grown in WI-38 cell cultures were inoculated into secondary monkey kidney cultures and H.Ep. 2 cell cultures and 2 blind passages carried out. There was no evidence of CPE in either cell culture nor was there hemadsorption of guinea pig red blood cells on the monkey kidney cultures. WI-38 cell cultures infected with this virus also did not hemadsorb guinea pig cells. High titer pools of 229E virus were tested for hemagglutinin with both guinea pig and chicken red blood cells at 4OC room temperature and 37°C. No hemagglutinin was demonstrated. Plaques were produced by 229E virus on WI-38 cultures under a methyl cellulose overlay after 7 days’ incubation at 33°C.

Ether sensitivity. Two strains of the new respiratory virus (229E and 2996) have been tested and shown to be inactivated by treatment with 20% ether at 4°C overnight.

Nucleic acid determination. The 229E virus has been tested with both S-fluorodeoxyuridine (FUDR) and 5-iododeoxyuridine (IUDR) (Table 11). Neither of these compounds inhibited the multiplication of 229E in WI-38 cultures. Another strain, 299G, was also tested and found not to be inhibited by IUDR. These results indicate that the nucleic acid of these viruses is RNA. Controls for these experiments included vaccinia virus which was inhibited by both these compounds and poliovirus type 1 which was not inhibited by either compound. The inhibition of vaccinia virus by IUDR could be reversed by addition of thymidine.

Stability. Preliminary tests indicated that 229E is a relatively stabIe virus. There was no loss in titer after storage for 10 days at
4OC nor after 2 hours at 37OC. Infectivity was destroyed after incubation at 56°C for 10 minutes.

Size. The size of 229E was determined by filtration through gradocol membranes. It passed through a 170 mp membrane but not through a 110 nip. Employing Black’s factor (S), the size of the virus is 89 mp.

Egg inoculation. A pool of 229E having a titer of 10-5.2 was inoculated intra-amniotically, 0.1 ml, to each of eight 10-day-old chick embryos. After 5 days’ incubation at 37°C the amniotic and allantoic fluids from these embryos were tested individually for hemagglutinin with chicken red blood cells at room temperature and at 4OC. All were negative. There were no deaths among the embryos inoculated and no gross lesions in embryos or membranes at time of harvest.

Cultures for Mycoplasma. Cell cultures employed in our laboratories for isolation of viruses frequently contain Mycoplasma. After treatment of WI-38 cultures with 50 ug/ml of aureomycin, no Mycoplasma could be detected by culture either anaerobically or aerobically on PPLO agar plates, nor were they isolated from pools of 229E virus grown in such WI-38 cultures.

Serologic tests. The 229E virus was tested for neutralization by antisera for some of the known myxoviruses shown in Table III. None of the antisera neutralized 229E virus. Recently 229E CF antigen prepared in our laboratories was tested by Dr. Robert Chanock of NIAID with the antisera prepared in his laboratory to the known myxoviruses. These were ferret antisera for respiratory syncytial virus and measles; guinea pig antisera for parainfluenza 1 (HA-2 and Sendai) , parainfluenza 2 (CA and SVS), parainfluenza 3 (HA-1 and SF-4), parainfluenza 4, mumps, influenza A?, influenza B, influenza C, and NDV. Homologous titers ranged from 1:40 to 1:1280. There were no cross reactions by
complement fixation test with these sera and 229E antigen.

Antibody in human sera. The results of neutralization tests and complement fixation tests on the sera of students from whom 229E virus was isolated are summarized in Table IV. A 4-fold or greater rise in neutralizing antibody was detected in sera from all of the students from whom the virus had been isolated, and 4 out of 5 showed a rise by CF test. However, CF titers with one exception (243E,F) were low.

Discussion. The ether sensitive RNA virus described above was isolated during the second year of a 5-year study of URIs among medical students at the University of Chicago. No further isolations were made in the succeeding years. However, human kidney cultures were replaced by WI-38 cultures for virus isolation the fourth and fifth years of the study. Although this virus could be adapted to grow in WI-38 cells, limited experience with reisolation indicated that WI-38 cultures might not be optimal for isolation. The WD diploid cell was apparently more sensitive. Unfortunately, this cell strain has not been recovered from frozen storage at the Wistar Institute.

Demonstration of an antibody rise to 229E virus by 2 testing methods in sera of students from whom the virus was isolated provides evidence for the human origin of this virus. Four of the five isolations were made from specimens from URIs suggesting that this virus may be etiologically associated with these illnesses. Further serologic testing is in progress to determine the frequency of infection with this agent.

Summnry. A new ether sensitive RNA virus was isolated during surveillance of URI among medical students in the winter of 1962. This virus is antigenically unrelated to all known human myxoviruses.

https://doi.org/10.3181%2F00379727-121-30734

In Summary:

• Dorothy Hamre is considered the first person to isolate a “coronavirus” in 1966 with 229E
• However, she relied entirely on indirect serological evidence obtained from cell cultures as the first images of particles assumed to be “coronaviruses” were not obtained until 1967 by another researcher
• Hamre used complement fixation, neutralization, and other serologic tests to claim an invisible “virus” was present
• Complement fixation is based on the principle (i.e. a basic truth or theory: an idea that forms the basis of something) that antigen-antibody complex fixes the complement
• It is necessary to use an indicator system consisting of sheep red blood cells  (RBC) and coated with anti-sheep RBC antibody
• The first component is an indicator system that uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of complement usually guinea pig serum
• The second component is Test System (A known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement)
• Patient serum is first added to the known antigen, and complement is added to the solution
• It is assumed that if the serum contains antibody to the antigen, the resulting antigen-antibody complexes will bind all of the complement and if this occurs, sheep red blood cells and the anti-sheep antibody are then added
• It is also assumed that if the patient’s serum does contain a complement-fixing antibody, a positive result will be indicated by the lack of red blood cell lysis
• Disadvantages of the Complement Fixation test include:
  1. Not sensitive – cannot be used for immunity screening.
  2. Time-consuming.
  3. Often non-specific e.g. cross-reactivity between Herpes Simplex “Virus” and Voricella Zoster “Virus.”
• Neutralization tests were also used by Hamre to determine her new “virus”
• The measurement of neutralization can depend on the choice of host cell
• Thus the neutralizing activity of a given antibody for a given “virus” is not an intrinsic property of the antibody but is a property of the relationship between the antibody and the virus, under defined conditions
• Consequently, neutralization titers in serum do not always correlate perfectly with protection from infection or disease in vivo
• Not all antibodies that bind to molecules on the “virion” surface will neutralize the “virus” in all conditions
• For a given “virus-encoded” gene product, such as the influenza “virus” hemagglutinin, binding of antibodies to some sites, but not others, will effect neutralization
• Some gene products on the “virion” surface may fail to routinely support “viral” neutralization
• If the wrong cell line is used for the assay it may seem that the antibodies have neutralization ability when they actually do not, or vice versa they may seem ineffective when they actually possess neutralization ability
• Although the PRNT typically provide the greatest specificity for Zika and other “viruses,” serological assays tend to be subject to cross-reactivity (i.e. they are not specific)

• In the winter of 1962, five agents were “isolated” in secondary human kidney tissue cultures (i.e. not purified) which presented a cytopathic effect (CPE) quite distinct from that customarily produced by known “viruses” associated with respiratory illnesses
• Hamre stated that her report presented the evidence for considering these agents as strains of a new respiratory “virus,” possibly associated with mild upper respiratory illnesses of man
• The methods for the “isolation” of the “virus” were not described here but in previous papers

Quick Detour on Hamre’s “Virus Isolation” Methods:

From the listed citations, this is all I could find about the methods of “isolation:”

Viruses Associated With the Common Cold

“No attempt was made to evaluate
the illnesses clinically. If a student said he had a cold, the specimen was considered an acute one. For isolation of viruses, nose and throat swabs were extracted in broth containing bovine serum albumin and this material was inoculated directly into tissue cultures without prior freezing.”

doi: 10.1080/00325481.1964.11695113.

As can be seen, the samples were immediately subjected to a broth contraining bovine serum albumin. There is no way of knowing what else was added in the broth. This matrial was directly inoculated into tissue culture. In other words, this “virus” was not isolated nor purified directly from a human.

End Detour

• Antiserum to the prototype strain of the new “virus” was prepared in guinea pigs following the procedure used for rhinovirus antisera
• Neutralization tests were carried out by the same method employed for rhinoviruses
• The method for complement fixation (CF) tests with respiratory syncytial “virus” was used
• CF antigen was prepared from WI-38 cells inoculated with high multiplicity of “virus” and harvested at the time of maximum CPE
• WI-38 cells are diploid human cell line composed of fibroblasts derived from lung tissue of a 3-month-gestation female fetus which came from the elective abortion of a Swedish woman in 1962, and was used without her knowledge or permission https://en.m.wikipedia.org/wiki/WI-38
• The “virus” was recovered from 5 specimens, 4 obtained from individuals with minor upper respiratory illnesses and one from a healthy individual during the winter of 1962
• All of these specimens yielded “virus” only after a second blind passage in human kidney cells
• No “virus” was recovered in the secondary monkey kidney cultures or H.Ep. 2 cell cultures inoculated with these specimens
• These “viruses” produced CPE in human diploid cell strains and these cultures, HEL or WI-38, were used for all further experiments
• The “virus” isolated from student specimen 229E was chosen as the prototype strain and purified by 3 serial selections at limiting dilutions in WI-38 cells
• In other words, Hamre is claiming to have purified an already unpurified mixture of human throat/nasal washings mixed with broth containing bovine albumin which had been added to fetal diploid cells by using multiple serial passages of limiting dilution into the same fetal diploid cells
• This “purified” toxic mixture was injected into Guinea pigs to create antiserum for neutralization tests
• Attempts to reisolate these “viruses” from the frozen original specimens were successful in 3 out of the 5 cases
• None of the WI-38 cultures showed any evidence of CPE even though as many as 4 blind passages were carried out
• Obvious CPE developed in WD cultures by the second passage
• In other words, Hamre could not create the cytopathogenic effect (CPE) she was looking for in the WI-38 cells even though she claimed successful isolation of “virus” in these cells so Hamre used a different cell line to create the effect she wanted to see
• High titered pools (i.e. mixtures of cultures) of 229E grown in WI-38 cell cultures were inoculated into secondary monkey kidney cultures and H.Ep. 2 cell cultures and 2 blind passages carried out
• There was no evidence of CPE in either cell culture nor was there hemadsorption of guinea pig red blood cells on the monkey kidney cultures
• WI-38 cell cultures infected with this “virus” also did not hemadsorb guinea pig cells
• High titer pools of 229E “virus” were tested for hemagglutinin with both guinea pig and chicken red blood cells at 4OC room temperature and 37°C
• No hemagglutinin was demonstrated
• The size of 229E was determined by filtration through gradocol membranes as the invisible “virus” was assumed to  pass through a 170 mp membrane but not through a 110 mp
• Employing Black’s factor (S), she estimated the size of the unseen “virus” as 89 mp
• A pool of 229E having a titer of 10-5.2 was inoculated intra-amniotically, 0.1 ml, to each of eight 10-day-old chick embryos
• All tests for hemagglutinin with chicken red blood cells were negative and there were no deaths among the embryos inoculated and no gross lesions in embryos or membranes at time of harvest
• Cell cultures employed in Hamre’s laboratories for isolation of “viruses” were frequently contaminated with Mycoplasma so the WI-38 cultures were treated with 50 ug/ml of aureomycin
• The 229E “virus” was tested for neutralization by antisera for some of the known myxoviruses and none of the antisera neutralized 229E “virus”
• There were no cross reactions by complement fixation test with these sera and 229E antigen
• 4 out of 5  samples showed a rise by CF test however, CF titers with one exception (243E,F) were low
• The ether sensitive RNA “virus” was “isolated” during the second year of a 5-year study of URIs among medical students at the University of Chicago
• No further isolations were made in the succeeding years
• Hamre claimed the demonstration of an antibody rise to 229E “virus” by 2 testing methods in sera of students from whom the “virus” was isolated provided evidence for the human origin of this “virus”
• Four of the five isolations were made from specimens from URIs (one was from a healthy individual…but apparently this isn’t important…) suggesting that this “virus” may be etiologically associated with these illnesses
• Due to all of the negative indirect antibody results, Hamre concluded her “virus” was antigenically unrelated to all known human myxoviruses, thus it was a new “virus”

As is seemingly always the case with these “virus” papers, assumptions are made based on small sample sizes and inaccurate tests while conflicting evidence is ignored in order to claim a new “virus” has been discovered. Nowhere in this paper did Dorothy Hamre ever purify and isolate a “virus” directly from a sick human. In fact, she apparently isolated a “virus” from the sample taken from a healthy patient which should have been a dead giveaway that her results were fatally flawed. However, Hamre persisted and continued to perform indirect serologic tests for known “viruses” in order to claim that she had a new “virus” once the results for each of the known “viruses” failed. She never even attempted to prove pathogenicity with her specimen 229E. The cytopathogenic effect (CPE) virologists attempt to claim as specific to “viruses” was absent in Hamre’s WI-38 cultures from which she had claimed to isolate her “viruses” from. No EM images were taken of any particles claimed to be the “virus.” In fact, the first “coronavirus” images would not come until a year later by an entirely different researcher.

Hamre’s entire basis for a new “virus” rested solely on the negative serological results she obtained from her experiments. The problem, however, is that antibodies themselves are an entirely theoretical concept never proven scientifically. Before the presence of these unseen antibodies can be taken as evidence, their form and function must also be proven and the results shown to be entirely specific to the “virus” being studied. Even if antibodies had been scientifically proven, it would be impossible to identify specific antibody reactions to an unseen “virus” that had not been purified/isolated. This step would logically need to happen first before any antibody result could be considered reliable. It is absolutely absurd to believe that one theoretical concept (i.e. antibodies) can be used to claim the existence of another theoretical concept (i.e. “viruses”), yet virologists have made a career out of doing exactly that. It is time to stop being fooled by the indirect evidence presented (cell culture CPE, antibodies, genomes, etc.) and demand that direct evidence (purified/isolated particles taken directly from sick humans and proven pathogenic naturally) of any “virus” is presented.

Until then, it is safe to say that, like all other “viruses,” 229E has never been scientifically proven to exist.