It is a pleasure to see so many of you here this morning after the very pleasant evening that we all enjoyed last night.
I want to take this opportunity to express my thanks to the group at Lindau
for inviting me and Mrs. Stanley to attend this meeting.
Fortunately it fitted in with a trip through Europe in company with our three daughters.
They too are enjoying this bit of hospitality.
I can only say that it reminds me very much of an earlier pleasant experience in Stockholm which Mrs. Stanley and I enjoyed.
Lindau it seems to me is for the time being just a little bit of Sweden.
Now, to the topic of viruses.
I have considerable difficulty in determining the nature of the talk this morning
because we range here in the audience from experts in a variety of specialities to, as I understand, beginning students.
This makes it somewhat difficult.
I shall attempt therefore to give a sort of cross section of the virus work
carried out in our laboratory at the University of California, in Berkeley.
First I shall attempt just a little bit of background material of philosophy if you please.
Then a little bit of chemistry, then some current work on poliomyelitis virus and vaccine.
And hope that you will find something of interest in one or another of these divisions.
Now, viruses represent a comparatively new field since they were discovered just at the turn of the century.
The first virus to be discovered was that of tobacco mosaic, a plant virus.
The next was an animal virus, that of the foot and mouth disease of cattle.
The third was the virus of yellow fever discovered by Reed and co-workers in 1901.
For about 30 years the work on viruses stemmed largely on medical aspects, upon the disease producing characteristics of viruses.
Very little was known about the true characters of these infectious disease producing agents.
Now, perhaps a definition of a virus at the outset.
They are small, extremely small, self duplicating mechanisms
- I’ll come later as to whether or not they are molecules or organisms –
which multiply or reproduce only within the living cells of certain specific hosts.
During their multiplication or their growth or their reproduction in these living cells they occasionally change or mutate.
And when they do this, they produce a new kind of a disease.
Hence the viruses provide an unusually fruitful pathway to the area that Professor Muller will speak about later.
Because of the ability to reproduce or to grow, because of the ability of viruses to mutate or to change,
they have for years been regarded as examples of life.
Now, in order to understand what one speaks about when one talks about a living entity,
it is necessary to indulge in a bit of philosophy with respect to the nature of life.
You have no difficulty in seeing the persons surrounding you as examples of living entities.
And yet the metal of the microphone you have as an admitted example of a non living structure.
Yet somewhere in between there is a borderline, a line of the unknown so to speak,
a boundary between living things and non-living things.
It is of some interest that an early philosopher, Aristotle, thought about this boundary line
and about 2,000 years ago suggested that the boundary line between living things and non-living things
was doubtful and perhaps non-existent.
So now you may ask where do the viruses fit in to this size of structures which exists in the world?
And I have prepared a slide which I hope will give you a better idea of this borderline area.
By 1930 chemists had worked up to the large molecules, some of which you heard Professor Staudinger discuss yesterday.
The large macromolecules of the proteins which on this scale will come to somewhere in this range.
In other words, chemists working with larger and larger and larger molecules worked up to protein molecules,
for example those of the hemocyanin molecule.
And beyond this there was the gap of the unknown.
The biologists on the other hand working downwards from larger and larger animals
had come down to the second line from the top, that is bacillus prodigiosus, for example.
The bacteria, which on this scale are in the neighbourhood of 450 or so millimicrons,
and between the bacteria or the smallest of the living organisms of the biologist
and the largest of the chemical molecules of the chemists, there was this unknown area.
This is the area I think that Aristotle was speaking,
that the boundary line in between the two was doubtful and perhaps non-existent.
Prior to the discovery of the viruses, this area was an unknown area.
But, as you can see, many, many, many entities have been filled in here,
so that if you look at the size of the structure, going from the smallest here to the largest there,
there is almost a continuum with respect to structures.
So that in truth there is no boundary line now between the accepted living organisms of the biologist,
such as bacillus prodigiosus and the molecules of the chemists at this level.
And the viruses, these infectious disease producing agents have been the structures
which serve to fill in at long last this gap which has existed for years and years.
Well, now I indicated that from the time of the discovery of viruses, around the turn of the century,
up until about 1935, the basic nature of these structures in this area was unknown.
It was not known whether they were still smaller ordinary living organisms like bacillus prodigiosus.
Some new kind of a chemical molecule or simply a specialised grouping attached to a chemical molecule.
And it became very important to determine the exact nature of one of the viruses.
And for this, the virus in the middle here, tobacco mosaic virus was selected for chemical study.
And to make a long story quite short, this virus was isolated in the form of a crystallisable nucleoprotein.
And after a long series of tests designed to prove whether or not the biological activity
was a part and parcel of the protein, the nucleoprotein molecule,
it was concluded that beyond a reasonable doubt the virus activity was a specific property of the nucleoprotein.
Then came of course the business of finding out whether tobacco mosaic virus was a representative virus
or whether it was something unusual.
So studies on a variety of viruses were made.
And for example another virus was obtained in the form of the beautiful dodecahedral crystals that you see here.
This is another plant virus, that of the tomato bushy stunt virus.
It can be isolated in the form of the crystals that you see here.
These crystals are composed of very tiny macromolecules about 30 or so millimicrons in diameter.
And then, to give you a cross section now of the variety of structures that have been isolated
in the form of purified viruses, I’d like to show this slide,
which in effect covers almost the entire range of sizes of the viruses that I showed you on the first chart of sizes.
Starting at the top upper left, we have the vaccinia, elementary bodies of vaccinia,
the vaccine which is used to protect you against smallpox, you will probably all have had the little scratch on your arm.
Probably didn’t realise that they were rubbing into your arm the large redshaped objects that you see on the upper right hand.
At the upper right we have an example of a smaller virus, that of influenza virus.
An extremely interesting virus, one which could serve as the subject of an entire lecture,
because this virus in 1918 caused the deaths of more people than had died on the battle fields of two great world wars.
150 million people had influenza in the winter of 1918 and of these 50 million died.
In our own country of the United States we lost over 400,000 persons within a 4 month period
and activities of that virus are on the upper right hand side.
And in 1918 influenza was not even recognised as a virus disease, for it was not so discovered until 1931,
first by the English workers and a similar virus in swine by Doctor Shope in our laboratory.
Here is an example of a group of viruses known as bacteria viruses, the viruses which attack bacteria.
Here is a T2 bacterial virus with an unusual structure, such as you see, with the head and the sperm shape tail.
On the right hand side here is an extremely interesting virus because this is a cancer producing virus.
A virus which produces cancer in rabbits in the United States.
It is of course of extreme interest
because this provides one of the avenues that approached to the solution of the cancer problem.
This is another example of a bacteria virus or a bacterial virus.
This one has the very short stubby tail, this tail organ is very important
because it is believed that this provides a mechanism by means of which the infective process is carried out.
And for some time it was not recognised that this particular bacteria virus had a tail.
Here is an example of the little macromolecules which go to make up the tomato bushy stunt virus,
which gives the beautiful crystals that you saw just a moment ago.
Here is a familiar tobacco mosaic virus about which I shall say just a bit more in a moment.
And here are some molecules, macromolecules perhaps is a better word, of a virus which affects orchids.
You ladies perhaps have not realised that the flowers that you wear on your dresses in the evening,
the orchids are subject also to virus infection.
And when you isolate the virus which causes a disease of orchids, you obtain the material on the right.
Now, if you go from the small spherical virus through the range of sizes that you see here,
you cover almost the entire range from about 20 millimicrons up to 300 millimicrons.
So that here you have a birds eye view of real structures
which have been found to exist in this borderline which existed between the living and the non-living.
Now, for just a little bit of chemistry, needless to say my background and my training is in chemistry.
Having isolated some of these materials such as tobacco mosaic viruses,
it was only natural to subject purified preparations of this virus to the normal procedures of characterisation,
analysis and so forth.
And the next slide will give you an example of the building blocks which go to make up the tobacco mosaic virus.
Now, for those of you who are not chemists, don’t worry too much, I’ll point out in gross terms the significance of this slide.
But first this row or columns give the amounts of the various amino acids which go to make up the tobacco mosaic virus.
You can see them here.
These amounts are very characteristic,
whether you isolate the tobacco mosaic virus in Sweden, in the United States, in Australia,
whether you isolate the virus from Turkish tobacco plants, from tomato plants, from spinach plants.
In other words, there is a characteristic composition which exists throughout the world
and regardless of the host in which you grow this material.
Now, I indicated at the beginning of this talk that one of the characteristics of viruses is that they can mutate.
Needless to say as chemists we wondered what would happen when a virus mutates and causes a slightly different kind of a disease.
We therefore isolated it and purified a variety of strains of tobacco mosaic virus.
And the results of these analysis are also on this slide.
We have the masked strain of the virus, the JD141 strain of the virus,
the green aucuba, the yellow aucuba, the ribgrass virus and two cucumber viruses.
These for a time have been regarded as strains of tobacco mosaic virus.
Now, whenever differences in composition exist, differences in composition from the tobacco mosaic virus,
the figure has been imposed in a little block.
So as I have indicated, those of you who are not chemists,
you need only to determine the number of little figures enclosed in a heavy line
to get some idea of the nature and extent of the changes which exist.
For example, there are only two changes in the JD141 strain, here and here.
On the other hand, in the case of the Holmes ribgrass virus, there are many, many changes in the nature of the amino acids.
And as you can see, there is an example here of the introduction of a new amino acid into the strain.
In other words, the amino acid or the building block, if you please, histidine does not exist in the tobacco mosaic virus.
Yet when this strain mutated and formed the Holmes ribgrass strain,
it was accompanied by the introduction of histidine, it was accompanied by the introduction of a new amino acid.
This data provided therefore the first information concerning the nature of mutation in the field of viruses.
And I hope this may also serve as an indication of the nature
of the changes which take place in the mutation of genes of higher organisms.
And if so, this provides an experimental approach to the nature of the changes
that Professor Muller has been interested in these many years.
Now, we have recently been interested in the detailed structure of tobacco mosaic virus.
You can make a variety of kinds of studies on this virus
and one of the useful approaches from a standpoint of technique
is the newly developed spray drop technique of Professor Williams in our laboratory.
In which a solution of virus is mixed in known proportions with a polystyrene latex solution.
The polystyrene latex particles are shown here and the tobacco mosaic virus particles are here.
And you see a small segment at the right which has been enlarged, so that you can see the particles somewhere on the plain.
By mixing the two, you get a relationship between the number of particles of polystyrene latex
which you know by virtue of having prepared the solution and the number of particles of tobacco mosaic virus.
You get a micro drop and hence a representative sample of this mixture by means of a spraying technique from an atomiser.
And the spray drop is showing an outline here, shows quite clearly.
Hence, this micro drop gives you a representative sample and enables you to get a relationship
between the number of particles of tobacco mosaic virus and the number of particles of the polystyrene latex.
And thus you can carry out much work on the relationship of biological activity to the little rods which go to make up the virus.
Now, our recent work on the structure, the detailed structure of these rods, has given us some rather unusual results.
This work was stimulated primarily by the results obtained in our laboratory and in other laboratories
on the bacteriophage particles.
And work, stemming also from the workers here in Germany on a protein component
which can be obtained by degradation of tobacco mosaic virus.
The next slide shows in outline some of the electron micrographs which can be obtained from such mixtures.
At the upper left in high magnification is the intact tobacco mosaic virus particle.
And if you break this particle, for example by means of high sound treatment,
you can simply slice it across as you would slice a piece of sausage.
If you do that, you get the two particles, or you get particles such as those that you see in the upper right hand corner.
These have been found to be hexagons so it would appear that the cross section of this rod is that of a hexagon.
Within the past few months it has been possible to devise a technique
by means of which an individual macromolecule can be partially denatured.
If you subject a solution of tobacco mosaic virus to mild temperature treatment, to mild heat, the one end will tend to ball up.
And when this is then treated with a detergent such as sodium dodecyl sulphate,
the part of the protein which has balled up will disappear and leaving the fine thread that you see here.
We now believe that this represents for the first time proof of the location of the nucleic acid portion of the nucleoprotein.
In other words that the nucleic acid is centrally located in the virus rod.
Now, another bit of information which indicates a similar conclusion is when you take the so called X protein
that can be obtained from diseased plants, or if you take a degradation product,
as Dr. Klauser has shown in this country, and cause it to re-aggregate,
you can retain once again the hexagonal shaped cross section material.
But as you can see, there is a hole down the centre of the tube.
Well, needless to say the re- aggregation or the re-synthesis, partial re-synthesis
of this unusual virus rock provides a very great challenge to the chemist.
Theoretically it should be possible to bring together the protein components
which go to make up this biologically active material and the nucleic acid components
and hopefully be able to re-synthesise the biologically active particle.
And I’m sure that there is very active work going on in Germany
and I know that there is very active work going on in our laboratory in this direction.
This then gives you the beginning of the intimate detailed structure of a biologically active structure.
The biological activity consisting of the ability to reproduce itself under certain specific conditions.
I believe that with time the chemist should be able to synthesise the building blocks which go to make up a virus,
such as for example tobacco mosaic virus with the ultimate building blocks being protein units of only 17,000 molecular weight.
Already synthetic approaches in connection with insulin and with some of the other hormones are well on the way.
And I believe that within the next few years the biochemists should be able to synthesise these small building blocks,
the structural units of a virus.
And then, through a special technique, cause these to assemble around the nucleic acid component and regain their activity.
This may be a little bit on the utopian side, but I believe that it is a real possibility within the next few years.
And with this of course you see because of the genetic characteristic of viruses
one can then gaze just a bit further over the horizon
and see that the chemist should be able eventually to determine the nature of the germ bioism of the world.
And this of course would give the chemists a type of power which currently seems to rest only in the hands of the atom physicist.
(laughter).
Now, having gone this far with the structure of tobacco mosaic virus,
I know that I dare not stop here, particularly with Professor Butenandt in the audience, or at least I believe I saw him here.
I’d like to provide at least for his benefit our perhaps most recent ideas on the ultimate structure of tobacco mosaic virus.
And I must say that this next structure is based,
not entirely on work in our own laboratory but on work carried out in Germany and in England and in many other laboratories.
And it is just a sort of a trial balloon with respect to the intimate and detailed structure
of this very interesting nuclear protein rod.
We believe that it’s, we know I should say, perhaps it’s a matter of fact that it is 150 Å across.
And that it is 1,500 Å in length and it consists of a progression of spirals
in which there are probably 12 1/3 of little subunits per turn.
And that viewed in cross section, the nucleic acid which composed entirely of ribonucleic acid
is located in the central area of the nucleoprotein molecule.
This of course we have now fairly definite experimental methods.
There is reason to believe, based primarily on x-ray work in England,
that the protein consists of little sub units of 17,000 molecular weight and that these are divided in half.
And that probably the protein chain runs along in this direction.
And that there is very probably a hole, a completely empty hole in the centre of the structure.
Why should nature select this type of structure for this unusual biological activity of the viruses?
I’m sure that I do not know.
But I believe that this may be characteristic of the virus structure,
something similar to this seems to occur in the case of the bacterial viruses.
We have a similar pattern in three or four other viruses about which we know partially.
So that this may represent an unusual type of structure which gives this type of genetic material.
It is believed that the infectious process consists of the entrance of the virus particle
followed by a dissolution or disappearance of the protein overcoat.
The reproduction of the nucleic acid portion of this followed by the assembly of a protein overcoat
around each individual particle subsequent to that.
And this protein overcoat apparently is the means
by which nature has provided this important genetic material with the ability to withstand the rigours of the world.
And that this is the reason that the viruses are able to exist as infectious disease producing agents.
Now, if I have the lights please.
I’d like to divert now and speak just a few moments about the work on poliomyelitis virus.
The lantern operator has very kindly given you a preview in the form of a slide
which you just saw there of purified poliomyelitis virus.
Perhaps we’ll return to it.
We have had a job as chemists working for the national foundation for infantile paralysis
of studying the biochemical properties of poliomyelitis virus.
We initially used the cotton wrap cord and brain material as starting material.
More recently, as a result of the very important discovery of Dr. Enders of Harvard university,
showing that poliomyelitis virus could be grown in non-nervous tissue,
we turned to tissue culture for the production of poliomyelitis virus.
And as you know used monkey kidneys as the tissue which was subjected to tissued culture.
This gave us an unusually good starting material and we were able to obtain
what we believe to be completely pure poliomyelitis virus.
In brief, this virus is a small spherical particle, 28 millimicrons in cross section,
and most unexpectedly has been found to contain about 30% of ribose nucleic acid.
I must say we had anticipated that there would be deoxyribose nucleic acid.
But this has not been found to be the case.
It is so far as we can tell entirely ribose nucleic acid.
One need only to give thought that if this 30%, a very high amount of nucleic acid
- for example tobacco mosaic virus has only 6% nucleic acid, poliomyelitis virus has 30% nucleic acid –
if this nucleic acid is centrally located, the attempt to make a vaccine by means of treatment with formaldehyde,
which acts primarily on the peripheral protein component, may be wrong, may be in error,
because the antigenic characteristics of the virus probably are located on the exterior.
And the reproductive capacity probably is located on the nucleic acid, on the interior.
Hence the approach of workers in Chicago for example to inactivate by means of ultraviolet light, may be a better approach.
We developed over the course of the years a procedure for purifying poliomyelitis virus on a commercial scale.
Unfortunately this has not yet been put into practice in the United States, although there is some reason now to believe,
as a result of what generally has been characterised in our newspapers as a mess, it is now possible that there will be a change.
It is perhaps worth knowing from the standpoint of science generally
that we in the United States have made some very serious errors in our program of poliomyelitis vaccine.
This program has been entirely self contained within the National Foundation for Infantile Paralysis
which is a voluntary organisation headed by a layman, Mr. Basil O’Connor.
Decisions with respect to the program have been made almost entirely
either through closed committees consisting of a few individuals.
As a result the progress has not been subjected to the ordinary procedures and techniques of science.
I need not tell this audience that science has progressed by virtue of a simple stepwise procedure of discovery,
of publication, so that your fellow scientists can check upon your results
and that you go on stepwise with discovery, publication, verification or confirmation and so forth.
This has not been possible in connection with the polio program because publication has lagged far behind.
And that the important decisions have been made with small closed groups
which have not subjected themselves to the criticism of their colleagues.
For example, in accordance with Dr. Salk’s initial recommendation,
the committee of quite eminent virologists incorporated a strain known as the Mahoney strain into the vaccine.
Over very vigorous protests by numerous scientists,
this strain is remarkable in its paralytic propensities, its ability to cause paralysis.
And you may have seen in the newspapers
that vaccination in some cases in the United States was followed by outbreaks of poliomyelitis.
Serological checks have proved this to be due to the Mahoney strain and just the day before I sailed for Europe,
at a congressional hearing there was unanimous agreement by a group of experts, including Dr. Salk,
that the Mahoney strain should be removed and replaced by a less virulent strain.
Had this been subjected to the criticism generally,
I think the Mahoney strain would never have been put into the vaccine in the first place.
Another example, the use of formaldehyde as a means of enactivating the virus.
The tissue culture fluid consists of about a 10,000th of a milligram of virus per cc.
In a medium containing approximately 1,000 to 10,000 times more extraneous protein than virus.
The interaction therefore between formaldehyde and the virus is conditioned largely by this large amount of impurity.
And those of you who are chemists know very well that all chemical reactions are subject to equilibrium constants
and that these can vary.
And hence that the assumption that the interaction between formaldehyde and poliomyelitis virus went to completion,
to get a completely inactive material, was actually in error and it was against the best known chemical principles.
And yet here again, this was not subjected to the wide spread criticism,
which again I think would have resulted in another or perhaps more severe testing procedure
which would have eliminated this particular error.
It is not generally known, but I happen to know and it’s not a secret I think, that the manufacturers,
the six manufacturers in our country have had extreme difficulty in obtaining material,
obtaining vaccine which is totally inactive.
The general run of most of them, they obtain about 30% of the vaccine which contains fully active virus.
The testing procedures have gradually been improved so that the material which is now being released is relatively safe.
But it is uncertain.
Information which is now available, just been released,
has shown that strains of pools of strains of the viruses when completely inactive by all known tests
have become active when they are mixed to form the try valid pool.
In other words, this vaccine consists of equal mixtures of three different poliomyelitis strains.
You can take completely inactive, by any tests yet devised, individual strains, mix them together,
test them again and then find active material.
This was a great surprise to Dr. Salk and to the manufacturers.
But again, if one had realised that the ratio between the biologically active virus
and the inert monkey kidney protein ranges from 1 to 1,000, to 1 to 10,000,
that you can have a redistribution of the formaldehyde and thus account for this reactivation.
This has resulted of course in the discarding of an additional 30% of the vaccine made by the manufacturers.
So that I can assure you that the experience of the United States
has not been a very profitable one so far as the manufacturers have been concerned.
Then lastly I’ve indicated that we have developed a procedure for the large scale purification of a virus.
One doesn’t know what will happen on repeated injections of a vaccine containing so much organ protein, monkey kidney protein.
There are some eminent immunologists who believe
that repeated vaccination with a vaccine containing large amounts of an organ protein
will eventually sensitise a substantial number of individuals to this organ protein.
And hence cause very disastrous results.
I think again that it’s quite probable that now as a result of things being thrown out in the open
that a purification procedure will be introduced into the production of the vaccine.
I need only comment that if this is done, the safety of the vaccine can be increased immeasurably,
perhaps 100 to 1,000 fold by virtue of working with concentrated material in concentrated form and then diluting back again.
It’s quite possible that ultraviolet light may enter, as I’ve already indicated, the vaccine program.
I think this is an example of where haste makes waste.
The great urgency to get a vaccine that caused Mr. O’Connor to push his scientists very, very hard
and I’m sure that he did it with the best of motives.
But he is unfamiliar with science.
He does not know the true pathways of science.
And I think we as scientists made a mistake in not preventing him from distorting this normal pathway,
because it is possible that we have suffered a rather severe setback in medicine in the United States as a result of this,
shall I say mess, which we hope is well on the way towards being clarified.
Fortunately this has not been repeated in many other countries.
England for example has called off their entire program and I think it has not been started to my knowledge here in Germany.
Permit me now to close with just a few remarks on the place of viruses in the world.
I have indicated that they are in this borderline region between the living and the non-living.
I indicated that certain viruses can cause cancer in plants and in animals.
We believe that the virus approach offers a most fruitful approach to the human cancer problem.
We have just within the past month in our laboratory isolated a normal human cell which can be grown in large quantities,
which is readily available throughout the world as a matter of fact, because it consists of the human amnion cell,
which is the little membrane which surrounds the baby at birth.
And as in the United States you have a great many babies in Germany and wherever a baby is born, if you select the amnion,
the chorion membrane, you have an equivalent amount of cells to that which exist in a monkey kidney for example.
We have also found just recently that this human amnion cell can be used to support the growth of poliomyelitis virus.
Hence very probably, again the production of polio vaccine will be switched from the monkey kidney,
which is extremely difficult to obtain as those of you who are in medical research well know, to the human amnion cell.
One only has to rule out the probability or the possibility of carrier viruses and this may not be too difficult to do.
This human amnion cell therefore offers great potentialities,
not only for poliomyelitis which was not in our minds when this discovery was made, but for the human cancer problem,
because this offers now the possibility of making extracts from various human cancers
and determining the effect of such extracts in normal human cells.
The viruses therefore provide I believe an excellent approach to the human cancer problem.
They always will interest us of course as to the nature of life itself
because you have here in the structures that you saw on the screen examples of living,
duplicating mechanisms which certainly must represent the most elemental form of life itself.
And then in this day when we think so much in terms of what may happen to the world, I think the viruses,
despite the fact that they are disease producing agents, probably hold many, many important secrets.
Because as we learn more about the structure of the viruses
we should be able to eliminate the infectious diseases and possibly we should be able to eliminate cancer.
I believe that the latter disease is one of the most devastating diseases with which to deal.
And that through the viruses we have that approach.
So that the viruses should provide a means for better health for the people of the world.
And I’m sure that if we can overcome the difficulties which surround our war-like attributes and gain a peaceful world,
then we still cannot enjoy that world if we are subject to infectious diseases and to organic diseases
and to disease such as cancer.
But given the peaceful world, then mankind will have the opportunity to enjoy that world
if he can make good use of the knowledge which I’m sure can be gleaned from these little microscopic entities, the viruses.
Thank you very much.
(Applause).