Was Influenza A Isolated in 1933?

Two years after Richard Shope “discovered” that an invisible “virus” was the assumed cause of the swine flu in 1931, three other researchers decided to determine the “viral” cause of human influenza. These researchers were Wilson Smith, Christopher Andrewes, and Patrick Laidlaw. In order to determine the cause of influenza in humans, these brave researchers set out to do so by attempting to create experimental disease…in ferrets. Here is a brief bit of background information before we delve into their paper:

80 years ago today: MRC researchers discover viral cause of flu

“In the spring of 1933 a team of Medical Research Council (MRC) staff gathered nasal fluids and throat garglings from a sick researcher, filtered them, and dripped them into ferrets. Within forty-eight hours the ferrets would start sneezing and displaying signs of an influenza-like disease. This research formed the basis of an extraordinarily important Lancet paper by Wilson Smith, Christopher H Andrewes and Patrick Laidlaw, published on 8 July 1933, identifying a ‘virus’ as the primary causative agent for influenza.”

“Walter Morley Fletcher, Secretary of the MRC, suggested to the War Office and Army Medical Services that attention should be turned to the possible role of a so-called ‘filter-passing virus’, and in November 1918 the search for the virus began. The first British investigations into the role of a virus in influenza were carried out by two teams in France and within weeks both claimed they had identified a filterable agent from sick servicemen.

These findings were controversial – there was no test for a virus, so its presence had to be inferred: it could not be seen with light microscopes, retained by bacterial filters or studied using culture methods. Only the presence of symptoms, and traces in serological tests suggested any ‘thing’ was present in infected people (and animals).”

“New interest in applying this approach to influenza was sparked in 1931, when the American researcher, Richard E. Shope announced that a combination of a bacillus and a filterable virus produced a disease in pigs – ‘hog flu’ – analogous to human influenza.

Ferret Flu

In late 1932 the MRC decided to concentrate on influenza, placing the research under Laidlaw’s control, with experienced virus researchers Smith and Andrewes. At the time, the main obstacle for virus researchers was that they lacked a viable experimental animal to study influenza. Solving this problem was the team’s first task.

Through January 1933 they tested nasal and lung material taken from influenza patients on rats, mice, guinea pigs, monkeys, pigs and horses. These efforts failed. They then turned to the ferrets at Mill Hill. In early February 1933, Smith dripped (“instilled”) filtered nasal and throat garglings taken from Andrewes, who had himself caught influenza, into the noses of two ferrets. Unfortunately, before Smith could isolate the virus a distemper out-break destroyed the experiment. By chance, Smith himself caught influenza on 4 March, and this time Andrewes used his throat garglings and his instillation method to infect some ferrets.

They quickly identified the infecting agent as a virus on the basis that it was filterable, invisible, and not cultivable, but still produced disease in the animal. They named it “W.S.” and it became the NIMR’s master strain. Through spring 1933, they traced “the full course of [the] illness” in 64 ferrets, noting the analogies of “ferret flu” to human influenza. They were now able to develop a serological test showing that recovered ferrets had antibodies which inhibited the disease, an important piece of evidence in establishing the identity and role of the virus. With this test it was possible to trace neutralising antibodies in sick and healthy Londoners, and thus determine the presence of the virus in the human population. At the same time, they compared their virus antibodies with those identified by Shope in hog flu, to show that their virus specifically produced human influenza.

Smith, Andrewes and Laidlaw made a cautious claim in their report to the Lancet that,

the evidence strongly suggests that there is a virus element in epidemic influenza, and we believe that the virus is of great importance in the aetiology of the human disease.

The report caused a minor media sensation. The Lancet editorialized that it “offered almost conclusive evidence that the primary cause of human influenza is a filterable virus.”

https://www.google.com/amp/s/amp.theguardian.com/science/the-h-word/2013/jul/08/influenza-virus-discovery-mrc-nimr

It seems the criteria for success for proving a flu “virus” was to fail at trying to infect nearly every animal with an invisible “virus.” The “virus” had to be non-cultivable and assumed to pass through a filter. The also invisible antibodies needed to react to the invisible “virus” to back up the original assumed claim. Makes plenty of sense, right? Let’s see what else we can learn from their research. The full paper and a summary are presented below:

A VIRUS OBTAINED FROM INFLUENZA PATIENTS

The epidemic of influenza at the beginning of 1933 afforded an opportunity of making an experimental study of this disease, the results of which are here embodied in a preliminary communication. Throat-washings were obtained from a number of patients as early as possible after the onset of definite symptoms. On the assumption that the aetiological agent of influenza was probably a filtrable virus the throat-washings were filtered before use through a membrane impermeable to bacteria. The filtrates, proved to be bacteriologically sterile, were used in attempts to infect many different species. All such attempts were entirely unsuccessful until the ferret was used and the first success was only secured towards the close of the epidemic.

The initial successful experiment was made with two ferrets, both of which received a filtrate of human throat-washings, both subcutaneously and by intranasal instillation. Both animals became obviously ill on the third day after infection and exhibited symptoms of the characteristic disease which is described below. It was found that the disease could be transmitted either by contact or by direct transference of nasal washings from a sick to a healthy ferret. At this point therefore the work was transferred to the Institute’s farm laboratories at Mill Hill, where it could be carried out under the conditions of rigid isolation of individual experimental animals evolved and used by Dunkin and Laidlaw 1 in their work on dog distemper * (1926).

The Disease in Ferrets

The ferret disease is characterised by a two-day incubation period, a diphasic temperature response, symptoms of nasal catarrh and variable systemic disturbances. In the infected animal the temperature † rises abruptly about 48 hours after infection, often exceeding 105° F. or even 106° F. It subsides on the third or fourth day only to rise again on the fourth or fifth day. In the course of the next day or two the temperature gradually returns to normal, and in most cases remains thereafter within normal limits.

Coincidently with the primary rise of temperature the ferret looks ill, is quiet and lethargic, often refuses food, and may show signs of muscular weakness. The catarrhal symptoms usually begin on the third day. The eyes become watery and there is a variable amount of watery discharge from the nose. This nasal discharge at times becomes sticky and may be mucopurulent, thus causing matting of the fur along the edges and at the corners of the nostrils. The animal sneezes frequently, yawns repeatedly, and in many cases breathes partly through the mouth with wheezy or stertorous sounds which clearly indicate a considerable degree of nasal obstruction. Such obstruction rarely accompanies a copious nasal discharge. The tip of the nose is often very pale. The signs of illness may last for only a few days but sometimes continue for ten days, after which the ferret again becomes perfectly normal. There is considerable variation as regards both the temperature response and the intensity and time of appearance of the local symptoms. In a few ferrets a typical diphasic temperature response has occurred without any nasal symptoms, and in one case well-marked symptoms were noted without any elevation of temperature. These animals when tested later were found to be immune. Very occasionally a ferret, a short time after recovery, has had a relapse in which the temperature curve and the symptoms have been similar to those of the primary illness. The disease has never been fatal in the 64 cases observed throughout the full course of the illness. Fig. 1 illustrates the temperature response of -a ferret which had a typical attack of the disease, with a relapse.

In ferrets killed during the first and second febrile periods the mucous membrane of the nasal passages shows acute inflammation. Sections across the turbinate bones show, in the soft parts, acute vascular congestion, dilated lymph channels, numerous leucocytes passing out through the epithelium, and serious derangement of this structure. There is almost invariably complete disappearance of ciliated cells, and occasionally patchy necrosis of the whole thickness of the epithelium may be observed. No histological feature, such as an inclusion body, has as yet been discovered which can be called characteristic of the disease.

Passage of the Virus

The disease has frequently been transmitted by placing a normal ferret in the same cage as a sick one for 24 hours. The majority of virus passages, however, have been made by the following technique.

The infected animal is killed when showing symptoms, often at the beginning of the second temperature rise. The turbinates are scraped out, ground up with sand, and emulsified in about 20 c.cm. of equal parts of broth and saline. The emulsion is lightly centrifuged, and about 1 c.cm. of the supernatant fluid is dropped into the nostrils of another ferret. In this way 26 serial passages of one strain of virus have been made, and every animal of the series has shown the typical temperature response together with definite symptoms of the disease. A hundredfold dilution of the usual preparation has also been found to be regularly infective.

The method we are forced to employ has the serious disadvantage that it is impossible to make accurate quantitative experiments. The concentration of virus in the emulsion is unknown and it is impossible to determine what proportion of the amount instilled into the nose is retained, but no other route of inoculation has yet proved successful and other tissues tested—e.g., spleen, lymph glands, and blood-have been uniformly non-infective.

Throat-washings from eight human cases diagnosed as influenza have been inoculated into ferrets: five of these produced the ferret disease described above, although four were tested before the importance of utilising temperature records was recognised. From one of these cases throat-washings on the first and second day, and nasal discharge on the third day, were infective for ferrets, but on the sixth day no virus was recoverable from the nasal discharges, and on this day there was considerable improvement in the patient’s catarrhal symptoms. A filtrate prepared from an emulsion of lung tissue from a fatal case of influenzal pneumonia likewise produced the ferret disease.

Throat-washings from four human subjects not suffering from influenza were non-infective. Of these, two were taken from men who had recovered from influenza and who at the time of their illness had supplied washings which were the genesis of the
transmissible strains of virus with which we have done most work.

The nasal secretions of a man who was suffering from a severe common cold were also non-infective.

Filtrability of the Virus

Most of the human throat-washings were filtered before use through membranes having an average pore size of 0.6µ. The membranes were Gradocol membranes
made and supplied by Mr. W. J. Elford, Ph.D. 2 (1931), to whom we are greatly indebted. The filtrability of the virus after ferret passage was tested repeatedly. Invariably filtrates of an emulsion of the nasal mucosa from a sick ferret through membranes having an average pore size of 0-6 µ were found to produce the typical disease. A tight membrane (a.p.s. 0.25 µ) was used on one occasion only; the resultant filtrate was infective. It is probable therefore that the virus of ferret influenza is no larger than the viruses of vaccinia or herpes febrilis.

The infectivity of the filtrates, coupled with the fact that we failed to grow anything from the filtrate on a variety of media under aerobic or anaerobic conditions, has convinced us that we are dealing with a true virus disease. We have examined a number of bacteria from ferrets and human beings and so far we have failed to discover any microorganism which will mimic the disease when cultures are instilled into a ferret’s nose. Haemophilus influenzœ, Haemophilus canis, and Haemophilus influenzae (suis) administered along with virus produced at most only minor variations in symptoms.

Active Immunity

Ferrets which have recovered from the disease are invariably found to be immune to subsequent infection with the same strain of virus. This holds true whether the immunity test is done a few days after the disappearance of symptoms or five or six weeks later. One of two ferrets tested after the lapse of three months proved to be solidly immune and the other had a very mild attack of the disease with prompt recovery. At the present time it remains uncertain whether viruses from different human sources cross-immunise completely. No means of securing an active immunity apart from giving the disease itself have yet been found.

Virus Neutralisation

The serum of a ferret which has recovered from the disease will neutralise strong emulsions of the virus, provided that the serum and virus are mixed together before being inoculated intranasally into the test animal. Normal ferret serum has no such power of virus neutralisation, even when dilute virus is used in the test. Fig. 2 illustrates a virus neutralisation experiment.

Many human sera are capable of neutralising dilute ferret virus. Sera obtained from ten patients after their recovery from influenza were all found to have neutralising antibodies, but their demonstration was not constant, for two of the sera when retested failed to protect the experimental animals. Such irregularity is probably due to our inability to measure the dosage of virus employed, and it is quite possible, under the conditions of the only test at present available, that an overwhelming dose of virus is given in one case and not in another. Control ferrets inoculated with virus alone or with virus mixed with normal serum were always included in these neutralisation tests; they invariably developed the disease.

Three human sera from individuals with no history of a recent attack of influenza were also tested: one showed neutralising properties; the other two were inactive. Such a result would be expected in tests on a population shortly after an epidemic of influenza.

Relationship to Swine Influenza Virus

A disease of swine, which arose spontaneously at the time of an influenza epidemic in America-has been described by Shope 3-(1931, 1932). We are indebted to him for samples of the swine influenza virus, and also for cultures of Haemophilus influenzae (suis), an organism which plays an important role in the serious and fatal cases of the swine disease. The virus when inoculated intranasally into ferrets gave rise to a disease with diphasic temperature response, and all the symptoms described above-in fact a disease indistinguishable from the ferret disease caused by virus of human origin. The swine influenza virus was also readily transmissible serially through ferrets. In striking contrast to swine influenza the ferret disease was not modified in character when cultures of Haemophilus influenzae (suis) were
inoculated together with the virus.

Cross-immunity tests have shown that this swine influenza virus bears a close antigenic relationship to the virus strain
of human origin which has been chiefly used in our work. Ferrets after recovery from disease caused by the swine virus proved to be solidly immune to the human strain of virus. Ferrets convalescent from the human virus disease were not completely immune to the pig strain of virus.

Summary and Discussion

A disease of ferrets, produced by the intranasal instillation of filtrates of throat-washings obtained from influenza patients, is described.
The disease is transmissible serially in ferrets either by contact or by the intranasal instillation of virus-containing material.
The infective agent has, so far, only been recovered from the nasal passages of sick ferrets.
The disease was produced by five of the eight throat-washings obtained from influenza patients in the early stages of the disease.
Throat-washings from healthy persons and influenza convalescents caused no illness in ferrets.
The nasal secretions from a subject with a severe common cold caused no illness in ferrets.
Human sera, particularly those from influenza convalescents, were found to contain antibodies capable of neutralising the virus of the ferret disease.
Swine influenza virus caused a disease in ferrets which was indistinguishable from that produced by virus of human origin, and the pig and human viruses have close antigenic relationships.

We consider that the evidence given above strongly suggests that there is a virus element in epidemic influenza, and we believe that the virus is of great importance in the setiology of the human disease. This view receives considerable indirect support from the fact that Shope found that the pig virus was the essential factor in swine influenza. The epizootic disease could only be produced by combining two separate agents: (1) a virus ; (2) Haemophilus influenzae (suis). The virus alone produced a disease so mild that it was difficult to recognise, and the bacillus alone appeared to be harmless. Our results with ferrets, so far as they have gone, are consistent with the view that epidemic influenza in man is caused primarily by a virus infection. It is probable that in certain cases this infection facilitates the invasion of the body by visible bacteria giving rise to various complications. Analogous examples of this type of double infection are seen in swine influenza and dog distemper epizootics. Decisive evidence on this point, and indeed on the importance of the virus we have described, can, we feel, only be secured by intensive study during an influenza epidemic, since direct experiments on man are fraught with difficulties. We are led to the publication of this preliminary note by the hope that our findings may be of assistance to those, wherever they may be situated, whose fate it may be to study the next epidemic of influenza.

We desire to thank the various practitioners through whose kindness we obtained throat-washings from influenza patients.”

https://doi.org/10.1016/S0140-6736(00)78541-2

In Summary:

• In the spring of 1933, Smith, Andrewes, and Laidlow gathered nasal fluids and throat garglings from a sick researcher, filtered them, and dripped them into ferrets
• Within forty-eight hours the ferrets would start sneezing and displaying signs of an influenza-like disease
• They published their results on July 8th, 1933, identifying a ‘virus’ (their quote marks, not mine) as the primary causative agent for influenza
• Prior to this, in 1918, the first British investigations into the role of a “virus” in influenza were carried out by two teams in France and within weeks both claimed they had identified a filterable agent from sick servicemen
• These findings were controversial for a few reasons:
  1. There was no test for a “virus” so its presence had to be inferred
  2. It could not be seen with light microscope
  3. It could not be retained by bacterial filters or studied using culture methods
  4. Only the presence of symptoms, and traces in serological tests suggested any ‘thing’ was present in infected people and animals (not sure how this differs from today…)
• Richard E. Shope announced that a combination of a bacillus and a filterable “virus” produced a disease in pigs which lead to increased interest in finding a “virus” in humans
• At the time, the main obstacle for “virus” researchers was that they lacked a viable experimental animal to study influenza
• Through January 1933 they tested nasal and lung material taken from influenza patients on rats, mice, guinea pigs, monkeys, pigs and horses but these efforts failed
• They quickly identified the infecting agent as a “virus” on the basis that it was filterable, invisible, and not cultivable, but still produced disease in the animal
• Through spring 1933, they traced “the full course of [the] illness” in 64 ferrets, noting the analogies of “ferret flu” to human influenza
• They were now able to develop a serological test showing that recovered ferrets had antibodies which inhibited the disease, an important piece of evidence in establishing the identity and role of the “virus”
• Smith, Andrewes and Laidlaw made a cautious claim in their report to the Lancet that the evidence strongly suggests that there is a “virus” element in epidemic influenza
• The Lancet editorialized that it “offered almost conclusive” evidence that the primary cause of human influenza is a filterable “virus”

• On the assumption that the aetiological agent of influenza was probably a filtrable “virus” the throat-washings were filtered before use through a membrane impermeable to bacteria
• All attempts to infect animals were entirely unsuccessful until the ferret was used and the first success was only secured towards the close of the epidemic
• The initial successful experiment was made with two ferrets, both of which received a filtrate of human throat-washings, both subcutaneously and by intranasal instillation
• The ferret disease was characterised by:
  1. A two-day incubation period
  2. A diphasic temperature response
  3. Symptoms of nasal catarrh
  4. Variable systemic disturbances
• Coincidently with the primary rise of temperature the ferret:
  1. Looks ill
  2. Is quiet and lethargic
  3. Often refuses food
  4. May show signs of muscular weakness
  5. Eyes become watery
  6. Has a variable amount of watery discharge from the nose
  7. Sneezes frequently
  8. Yawns repeatedly
  9. Breathes partly through the mouth with wheezy or stertorous sounds
• There was considerable variation as regards both the temperature response and the intensity and time of appearance of the local symptoms
• Some ferrets seemed “immune” while others were not and had relapses
• The disease had never been fatal in the 64 cases observed throughout the full course of the illness
• In ferrets killed during the first and second febrile periods the mucous membrane of the nasal passages showed acute inflammation (perhaps the intranasal inoculation had something to do with this…?)
• No histological feature, such as an inclusion body, had been discovered which could be called characteristic of the disease
• The majority of “virus” passages were made by the following technique:
  • The infected animal is killed when showing symptoms; often at the beginning of the second temperature rise
  • The turbinates are scraped out, ground up with sand, and emulsified in about 20 c.cm. of equal parts of broth and saline
  • The emulsion is lightly centrifuged
  • About 1 c.cm. of the supernatant fluid is dropped into the nostrils of another ferret
• In this way 26 serial passages of one strain of “virus” were made
• The method they used had the serious disadvantage that it was impossible to make accurate quantitative experiments
• The concentration of “virus” in the emulsion is unknown and it is impossible to determine what proportion of the amount instilled into the nose is retained
• No other route of inoculation proved successful and other tissues tested (spleen, lymph glands, and blood) were uniformly non-infective
• Throat-washings from eight human cases diagnosed as influenza were inoculated into ferrets: only five of these produced the ferret disease and four were tested before the importance of utilising temperature records was recognised
• The nasal secretions of a man who was suffering from a severe common cold were also non-infective
• Invariably filtrates of an emulsion of the nasal mucosa from a sick ferret through membranes having an average pore size of 0-6 µ were found to produce the typical disease
• Thus they conclude it is probable that the “virus” of ferret influenza is no larger than the “viruses” of vaccinia or herpes febrilis (none of which they had ever seen with their own eyes)
• The infectivity of the filtrates, coupled with the fact that they failed to grow anything from the filtrate on a variety of media under aerobic or anaerobic conditions, convinced the researchers that they were dealing with a true “virus” disease
• They tested various bacteria from humans and animals such as Haemophilus influenzœ, Haemophilus canis, and Haemophilus influenzae (suis) administered along with “virus” and produced at most only minor variations in symptoms (seems to destroy Shope’s theory)
• One of two ferrets tested after the lapse of three months proved to be solidly immune and the other had a very mild attack of the disease with prompt recovery
• It remained uncertain whether “viruses” from different human sources cross-immunise completely
• No means of securing an active immunity apart from giving the disease itself had been found
• The serum of a ferret which recovered from the disease will neutralise strong emulsions of the “virus,” provided that the serum and “virus” are mixed together before being inoculated intranasally into the test animal
• Sera obtained from ten patients after their recovery from influenza were all found to have neutralising antibodies, but their demonstration was not constant, for two of the sera when retested failed to protect the experimental animals
• They assume such irregularity was probably due to their inability to measure the dosage of “virus” employed, and that it was possible, under the conditions of the only test at present available, that an overwhelming dose of “virus” was given in one case and not in another
• Three human sera from individuals with no history of a recent attack of influenza were also tested: one showed neutralising properties; the other two were inactive
• They were indebted to Richard Shope for samples of the swine influenza “virus,” and also for cultures of Haemophilus influenzae (suis)
• In striking contrast to swine influenza the ferret disease was not modified in character when cultures of Haemophilus influenzae (suis) were inoculated together with the “virus”
• Cross-immunity tests showed that this swine influenza “virus” beared a close antigenic relationship to the “virus” strain of human origin
• However, ferrets convalescent from the human “virus” disease were not completely immune to the pig strain of “virus“
• The infective agent had only been recovered from the nasal passages of sick ferrets (coincidentally, also where they were inoculated…)
• The disease was produced by five of the eight throat-washings obtained from influenza patients in the early stages of the disease
• Swine influenza “virus” caused a disease in ferrets which was indistinguishable from that produced by “virus” of human origin
• The researchers consider that the evidence given strongly suggests that there is a “virus” element in epidemic influenza, and they believe that the “virus” was of great importance in the setiology of the human disease
• Their view received indirect support from the fact that Shope found that the pig “virus” was the essential factor in swine influenza
• Shope’s epizootic disease could only be produced by combining two separate agents: (1) a “virus;” (2) Haemophilus influenzae (suis)
• The “virus” alone produced a disease so mild that it was difficult to recognise, and the bacillus alone appeared to be harmless
• Their results with ferrets were consistent with the view that epidemic influenza in man is caused primarily by a “virus” infection
• Decisive evidence on the importance of the “virus” they described could only be secured by intensive study during an influenza epidemic, since direct experiments on man are fraught with difficulties (leaving it to future studies as is always the case)

It is clear to see that once again a “virus” is claimed without any DIRECT visible proof of said “virus.” There was no attempt at purification nor isolation of any kind and it was admitted that the amount of “virus” could not be quantified. Smith, Andrewes, and Laidlaw failed in numerous attempts to “infect” animals with their assumed “virus” right from the start. Once they finally found that the ferrets became somewhat sick from their human throat-washings, they decided to kill the sick ferrets, grind up their nasal turbinates wirh sand, combine the minced nasal tissues with equal parts broth and saline, and shove this goo up the nose of other ferrets. Once the ferrets displayed a rise in body temperature, they were killed and this disgusting process was started all over again at least 26 times.

These researchers credit the work of Richard Shope and his discovery of the swine flu “virus” for their own success. They even claim that the swine flu “virus” given to them from Shope produced the same disease in ferrets as seen in pigs and man. This raises the question: if they knew pigs carried either the same or a similar “virus,” why did they keep testing other animals and eventually decide on ferrets instead of testing and confirming Shope’s findings on pigs? Were they unable to reproduce these results? According to the Guardian article above, pigs were one of the many animals they failed to infect.

As always seems to be the case in these papers, the researchers were cautious with their claims and left up the “DECISIVE” work to determine the significance of their findings to other future researchers in order to see if their results had any merit. This is hardly the convincing “proof” of an influenza A “virus” and nowhere near the “almost conclusive evidence” that the primary cause of human influenza is a filterable virus” as the Lancet proclaimed it to be. It’s just another fictional link in the long pseudoscientific chain of virology.