Did Thomas Francis Jr. Isolate Influenza A in 1937?

In 1937, a few years after Smith, Andrewes, and Laidlaw supposedly isolated influenza A in 1933 and after Thomas Francis Jr. supposedly isolated it in 1934, Francis Jr. once again claimed to have the direct isolation of influenza A “virus,” this time in tissue culture medium and on an egg membrane. Or at least that is what he titled his study. Does this claim hold up?

Direct Isolation of Human Influenza Virus in Tissue Culture Medium and on Egg Membrane.

“Throat washings in Tyrode’s solution were obtained on the second day of illness from a patient acutely ill with influenza. The material was centrifugalized at 2500 revolutions per minute for 30 minutes. The supernatant fiuid was then filtered through a graded collodion membrane of 500 mp average pore size.’ Flasks containing 4.5 CC. of chick embryo Tyrode medium* were inoculated with 2.5 CC. amounts of the filtrate (shown by ferret inoculation to contain active virus). Transfers of 0.5 cc. amounts of the culture material to 4.5 CC. of fresh medium were made at 48-hour intervals. Mice were inoculated intranasally with culture fluid of the 5th transfer. Mouse passages were made at 4-day intervals. NO significant lesions were seen in the lungs of mice of the first 3 passage groups. In mice of the 4th passage, however, sugestive lesions were seen ; these were more definite in the 5th passage, and in the 6th passage death of the mice with extensive pulmonary involvement occurred. The virus was identified as human influenza virus by means of neutralization tests with known immune serum.

These results indicate that virus was recovered directly from the throat of a human influenza patient by the introduction of the filtered throat washings of the patient into tissue culture medium. The virus not only survived but probably multiplied, since the final dilution of the original material was approximately 1:3OO,OOO at the time the culture material was first given to mice. The behavior of the virus after its inoculation into mice was very similar to that of virus established directly in mice from human throat washing.

Burnet has reported in detail the behavior of human influenza virus introduced after passage through ferrets, onto the chorio-allantoic membrane of the developing chick. The direct cultivation of the virus of common cold on the chorio-allantoic membrane has also been reported. Attempts were therefore made to utilize this procedure in the isolztion of virus directly from the human patient. Throat washings in broth were obtained from two patients acutely ill with influenza. By means of ferret inoculation and subsequent adaptation to mice, virus was shown to be present in the material. Portions of the throat washings of these patients were pooled, centrifugalized at 2500 r.p.m. for 30 minutes and filtered through a graded collodion membrane of 500 mtL average pore size. The chorio-allantoic membrane of a 12-day chick embryo was inoculated with 0.05 cc. of the filtrate. Passages were made at 4-day intervals by inoculating additional embryonic membranes with finely ground emulsions of the previous passage membranes. Thickening of the membranes was observed and white plaque formations were seen. A suspension of the 3rd passage membrane was inoculated into a ferret and into mice. The ferret developed typical experimental influenza and its convalescent serum neutralized human influenza virus. No lesions were seen in the lungs of mice of the first 3 passages. In those of the 4th, 5th, and 6th passages small pulmonary lesions were seen, but they have not been sufficiently extensive to permit of conclusive neutralization tests.

After the 3rd transfer on egg membranes the virus was placed in storage for several days. Since that time the changes in the passage
membrane have been less pronounced. These brief observations indicate, however, that human influenza can be isolated directly and maintained for a time, at least, upon the embryonic membranes of developing chick.

The practicability of both the procedures described above is being studied on a larger scale. Certain disadvantages obtain. These
consist in the inability to demonstrate the presence of influenza virus in tissue culture medium except by establishing the virus in animals and in the fact that the lesions produced by the virus in the early passages on embryonic membranes are too irregular to enable one to identify the virus. It seems possible that future studies will furnish means of eliminating such difficulties.”

https://doi.org/10.3181%2F00379727-36-9144P

In Summary:

• Throat washings in Tyrode’s solution were obtained on the second day of illness from a patient acutely ill with influenza
• Quick note: Tyrode’s solution is said to be made up of sodium chloride (salt), potassium chloride, calcium chloride, (all three are in ice melt and toxic to animals) magnesium chloride, glucose, and Polyvinylpyrrolidone – a synthetic water-soluble polymer made from the monomer N-vinylpyrrolidone https://www.aatbio.com/resources/buffer-preparations-and-recipes/tyrode-s-solution-acidic
• The material was centrifugalized at 2500 revolutions per minute for 30 minutes
• The supernatant fiuid was then filtered through a graded collodion membrane of 500 mp average pore size
• Flasks containing 4.5 CC. of chick embryo Tyrode medium were inoculated with 2.5 CC. amounts of the filtrate (shown by ferret inoculation to contain active “virus”)
• Transfers of 0.5 cc. amounts of the culture material to 4.5 CC. of fresh medium were made at 48-hour intervals
• Mice were inoculated intranasally with culture fluid of the 5th transfer
• Mouse passages were made at 4-day intervals
• NO significant lesions were seen in the lungs of mice of the first 3 passage groups
• In mice of the 4th passage, however, suggestive lesions were seen which were more definite in the 5th passage, and in the 6th passage death of the mice with extensive pulmonary involvement occurred
• In other words, he failed with the first 3 attempts of toxic chick embryo goo injected directly into the nose of mice and progressively made it more toxic in each passage until he got the desired results
• The “virus” was identified as human influenza “virus” by means of neutralization tests with known immune serum (impossible to know without having a purified/isolated “virus” to begin with)
• Francis Jr. claims the “virus” not only survived but probably multiplied, since the final dilution of the original material was approximately 1:3OO,OOO at the time the culture material was first given to mice
• He decides to try to grow the invisible “virus” on chick embryos:
  1. Throat washings in broth (no mention of what the broth contained) were obtained from two patients acutely ill with influenza
  2. By means of ferret inoculation and subsequent adaptation to mice, “virus” was shown to be present in the material (i.e. Francis Jr. kept passing diseased tissue goo between ferrets and mice and once he finally made them sick, he decided a “virus” was present)
  3. Portions of the throat washings of these patients were pooled (i.e. mixed together), centrifugalized at 2500 r.p.m. for 30 minutes and filtered through a graded collodion membrane of 500 mtL average pore size
  4. The chorio-allantoic membrane of a 12-day chick embryo was inoculated with 0.05 cc. of the filtrate
  5. Passages were made at 4-day intervals by inoculating additional embryonic membranes with finely ground emulsions of the previous passage membranes (i.e. every 4 days they ground up chick embryos and injected this goo into other chick embryos)
• Inoculations of 3rd passage was done in mice and a ferret
• The ferret developed typical experimental influenza and its convalescent serum neutralized human influenza “virus”
• No lesions were seen in the lungs of mice of the first 3 passages
• In those of the 4th, 5th, and 6th passages small pulmonary lesions were seen, but they were not sufficiently extensive to permit of conclusive neutralization tests (useless antibody test that is nonspecific and can not be used to show presence of “virus” without ever purifying/isolating one first)
• Francis Jr. provides two challenges to his findings:
  1. The inability to demonstrate the presence of influenza “virus” in tissue culture medium except by establishing the “virus” in animals
  2. The fact that the lesions produced by the “virus” in the early passages on embryonic membranes are too irregular to enable one to identify the “virus”
• As virologists tend to do, he passes the buck as it seemed possible that future studies wiould furnish means of eliminating such difficulties

Francis Jr. concluded his own study with the two glaring admissions of being unable to identify a “virus” using tissue culture medium and egg membrane yet somehow he felt compelled to title the study:

DIRECT ISOLATION OF A “VIRUS” IN TISSUE CULTURE MEDIUM AND ON EGG MEMBRANE.

Claiming something in the title of the study which is not backed up by the actual evidence presented in it is just par for course in virology. It is a long-held tradition that they have carried on up to today as virologists constantly claim “isolation” of a “SARS-COV-2 virus” while consistently failing to produce this evidence in every single paper. Of course, as Francis Jr. himself stated, it is always left to future studies to overcome the challenges and lack of definitive evidence. The promises of the work being reproduced, replicated, and completed is the dangling fruit left hanging for the hungry, waiting for it to fall while starving to death when it never does. The titles of the studies are always misleading and are rarely backed up by the findings in the paper itself.

This is why you always read the fine print.