AN ANALYSLS OF THE HISTORY OF
INFECTLOUS NUCLEIC ACIDS
by

Mark Weidenbaum,
University of Connecticut

This work is based on a

Directed Studies Summer Program
carried out at Stanford University
in the Summer of 1975 under the
direction of Dr. Joshua Lederberg.
 

TABLE OF. CONTENTS

Abstract
Part | - History
Preliminary ILdeas
Quantétative Studies
Proof of Infectious Nucleic Acids
Part Ll - Analysis
Francesco Sanfelice
Inclusion Bodies
Cytochemical Staining of Inclusion Bodies
A Mistake in Interpretation
Problems in Communication
Part [Ll - The Graphical Citation Index
- [nt roduct ion
Chronological Perspective
Clustering |
Combination of Techniques
Problems
Summary
Bibliography
(To be used in conjunction with accompanying
chart entitled, ‘Citation Index for Infectious

Nucleic Acids.'')

Acknowledgments

20
ABSTRACT

This review traces the significant developments in virus chemistry
from Beijerinck's (1898) recognition of viruses as distinct bodies to
the demonstration of their infectious nucleic acid nature by Gierer and
Schramm (1956). The study demonstrates how cytochemical staining methods
applied to inclusion bodies served asa useful method for investigating
the early years of virus chemistry. The investigation also analyzes the
_effects of Stanley's misinterpretation of proteins as infectious agents
and ‘his dismissal of nucleic acids as being important in the viral infec-
tion process. The study reveals that Sanfelice's (1918) observations
were lost from central thought but anticipated many later developments.
The review includes a discussion of graphical citation indexing as
well as a graphical citation index surveying the history of infectious

nucleic acids from 1873 to 1960.

~
§
 

PART ! - HISTORY

Preliminary ideas

In 1892 D. lwanowski 4 recognized that the juice of Turkish Tobacco
plants having the tobacco mosaic disease remained active after being
passed through a Chamberland filter (a standard microorganism filter).
Via this work, Iwanowski had demonstrated the existence of what is now
known to be a virus. Yet, he chose to regard the infectious agent as
bacterial in nature.

Six years later M. Beijerinck! repeated Iwanowski's work but inter-
preted the results in terms of a ''contagious living fluid." Therefore,
he was the first to recognize the fundamental difference between the
"Filter passing agent!" and ordinary bacteria.

After Beijerinck's work several diseases were determined to be
caused by “filter passing agents," which had been termed viruses. In
1898" Loeffler and Frosch’? first discovered an animal virus ,°2 that
causing hoof-and-mouth disease in cattle. In 1911 Walter Reed”? deter-
mined that yellow fever was ‘also a viral disease.

The identification of another major family of viruses, namely

82

those affecting bacteria, followed when Twort and d'Herelle?? recog-

nized the "bacteriophages.!'
:
Although viruses were thus recognized as organisms distinct in
themselves, their structure and mechanism remained unknown at this time.

76

As W. Stanley’ stated, ''...the general nature of the viruses was unknown
and they had been regarded variously as invisible forms of ordinary
bacteria, as a new kind of invisible living organism, as protozoa, as

unusual products of cellular metabolism, as enzymes, and as different

kinds of inanimate chemical* substances.!!
In general, the problems involved in isolating pure virus samples
and obtaining conclusive data prevented significant progress. As the

83

reviews by Roux®?, Wolbacke®, Twort’”, and Bayon? demonstrate no particu-
lar theory predominated.
Quantitative Studies

Until 1935 the ideas concerning the nature of viruses were largely
based on conjecture. However, in that year W. Stanley’ successfully
crystallized Tobacco Mosaic Virus (TMV) protein via ammonium sulfate
precipitation. He then confirmed that this virus multiplied only within
the cells of certain definite hosts, thereby differentiating from normal
pathogenic bacteria. By thus isolating a crystalline "protein'' having
TMV activity, Stanley facilitated direct measurements of infectivity by
correlating the amounts of TMV protein present with the degree of virus
activity.

* After measuring chemical and physical constants of the crystallized
"protein," Stanley concluded that ''...the high molecular weight proteins
carrying virus activity are characteristic of virus-diseased activity."
Hence, his evidence pointed towards the existence of "infectious proteins.!'/"

Although Stanley had assumed that his TMV protein preparations were
sufficiently pure, Bawden and Pirie? showed that liquid crystals of TMV
contained 0.5% phosphorus and 2.5% carbohydrates. They noted that these
materials were nucleic acids of the "ribose type.'7 They also postulated
that the TMV proteins consisted of long fiber-like particles that aggre-
gated to form longer threads.

Despite this work, Stanley maintained that his preparations were

good enough and that the phosphorous 'impurities'' to which Baden and

Pirie referred were of little significance. He based this conclusion
on his earlier findings that phosphorus was unnécessary for virus activity.
Thus, he maintained that different viruses led to the synthesis of
different proteins and that nucleic acids were of minor importance.

? had determined that RNA particles had

By 1942 Cohen and Stanley!
an average molecular weight of 300,000 and were highly assymetrical. More
importantly, this time Stanley also recognized that Bawden and Pirie's
“nucleic acids'' were far more important than he had earlier suspected.
Cohen and Stanley concluded from their data that viral nucleic acids
existed as threadlike molecules, the length of which corresponded to that
of the intact virus molecule.

7 During the mid-1940's emphasis shifted away. from proteins and
towards nucleic acids as the possible infectious elements of viral in-
fection. Avery then contributed his finding that in Pneumococcus the
sub§ tance which induced transformation of one bacterial type to another
“appéared to be...sodium deoxyribonucleate, inducing the synthesis of non-
nitrogenous polysaccharide composed of glucose-glucuronic acid linked
in glycosidic union!"

Although Avery was working with bacteria and not viruses, his
discovery of DNA as the "Transforming Principle" stimulated interest in
nucleic acids as vectors of infectivity. He had verified that the
inducing substance (DNA) and the subs tances it induced (high molecular
weight proteins) were chemically distinct and biologically specific. These
Findings heightened the possibility that nucleic acids themselves were
responsible for infectivity.

In 1952 Hershey and Chase’! employed radioactive sulfur and phos-
phorus tracers to prove that the viral] protein shel] attaches to the cell

+

-3-
wall of its target but does not enter the cell itself. Instead, it
injects its nucleic acid core into the cell. Despite these findings
Hershey and Chase could not explain how the virus replicated once inside
the “infected cell.

In an attempt to explain nucleic acid structure, Pauling and

a7 postulated that the nucleic acids might form an alpha-helix.

Corey
Although this prediction was wrong, the idea of a helix contributed to
Watson and Crick's °° demonstration in 1953 that DNA was composed of a
double helix held together by hydrogen bonding and van der Waals forces.

Later that year Watson and Crick?! proposed that the double helix
contained a pair of complementary template strands which could pull
apart. Each strand could. then form S new complementary chain. They
suspected that the sequence of bases attached to the sugar~phosphate
backbone making up the strands was the code that carried genetic infor-
mation.

The Watson and Crick model resulted in the interpretation that
viral nucleic acids carried their own replication instructions. Once
inside their host, they could replicate and take control of the cell
machinery for protein synthesis. The viral nucleic acids could then
use this machinery for the synthesis of the high molecular weight
proteins necessary for their protective shells. Thus, the viral
nucleic acid could itself be the infectious agent responsible for
disrupting cell metabolism.

Proof of Infectious Nucleic Acids
The final proof that nucleic acids were themselves capable of

32

infection came in 1956 when Gierer and Schramm showed that bare

‘

~h-
RNA, purified from TMV, was itself capable of inducing infection in the
tobacco plant. They stated: We are thus led to conclude that the in-
fectivity is due to the nucleic acid itself 3?

Gierer and Schramm's work stimulated subsequent investigation for
infectious nucleic acids in animal and bacterial Viruses. In 1957
Spizizen’? attempted to establish that T2 bacteriophage DNA could infect
E. coli bacteria protoplasts, but his preparations were impure and his

. . 0
results inconclusive. Later that year Fraser et 13

performed the same
experiment and presented more evidence that naked bacteriophage DNA could
induce infection. However, it remained for Guthrie and Sinsheimer to
prove conclusively in 1960 that “protoplats of E. coli can be infected
with the DNA of ax174 37. (GX174 is another bacteriophage.)

| Concurrent with this work on bacteriophages, Colter et a./8
demonstrated that infectious nucleic acids existed in animal viruses.
He stated: "Ribonucleic acid isolated from Ehrlich ascites tumour
cells infected with Mengo encephalitis virus is infectious, and the
ribonucleic acid component, rather than residual intact virus particles,

a

is responsible for this activity 18

PART I! - ANALYSIS
Francesco Sanfelice
While studying Epithelioma Contagiosum (a viral skin disease
observed mainly in birds) in 1914, Francesco Sanfelice®? noted that
"it is most interesting to see how a disease can be produced with the

nucleoproteide which was extracted from the diseased tissue.!' {In 1928

Bronfenbrenner’ ! that Sanfelice had extracted a substance which per-

*

-5-
petuated disease as if it ‘were a living virus!!! Unfortunately, the
limitations of optical, physical, and chemical techniques prevented
Sanfelice from obtaining more than speculative evidence concerning the
nature of the Epithelioma Contagiosum virus mechanism.

Nevertheless, Sanfelice was apparently the first to suggest that
Viral infection was due to something other than attack by the intact
virus. Rather, he speculated that the ''nucleoproteide'',not the complete
virus particle, was the infectious element. Thus, he anticipated much
of what has since been determined concerning the mechanism of viral
attack.
Inclusion Bodies

One of the earliest clues concerning the nature of viruses
centered on the observation that some diseases induced development of
cellular inclusions, referred to as inclusion bodies. In 188] Rivolta’!
observed such inclusion bodies in the cells of chickens having fowl pox.
Bollinger’ made similar observations while working with fowl diseased
with Moluscum Contagiosum. In 1894 Guarnieri 2° discovered typical in-
clusions in cells having vaccinia (small pox virus).

At first these inclusion bodies were mistaken for protozoa. This

59

mistake gave rise to the term "Chlamydozoa! During the period of

approximately 1910 through 1930 this term was used to describe inclusion
bodies.

Although inclusion bodies had been recognized since 1873, their
specific connection with virus activity was not demonstrated unti]
Paschen?© did this in 1917. Subsequently, two fundamental theories arose

concerning the relationship of inclusion bodies to viruses. One theory

4

-6-
ee

proposed that the inclusions were products of the reaction of the
infected cell to the virus. The other theory, which has since been
Proven, was that inclusion bodies were virus colonies themselves.
Cytochemical Staining of Inclusion Bodies

Once inclusion bodies were associated with viruses, the former
were tested cytochemically for a variety of substances, including nucleic
acids. Consequently, it has been possible to use cytochemical staining
methods as a means of exploring the early years of virus chemistry.

The cytochemical test most useful in studying these years has been
the Feulgen Reaction, developed by Feulgen?° in 1924, {tt is still the
single most definitive test for DNA. It involves: hydrolysis of the alde-
hydes “tn the nitrogenous bases of DNA which is then followed by rosaniline
Staining.

:. Another method for testing nucleic acids (usually RNA) involved
differential staining. In 1940 Brachet!° discussed the use of ribo-
nuclease to cleave ''pentosenucleic acid"! into soluble mononucleotides as
a specific test for RNA. . The material to be tested was stained via the
Feulgen reaction before and after the action of the ribonuclease in order
to make sure that no DNA had contributed to the observed results.

Cowdry?! was apparently the first to apply the Feulgen test directly
to inclusion bodies. In 1928 he stated, "'...the Feulgen reaction showed
both types of inclusions contain little or no thymonucleic acid.1!

38

Haagen and Kodama used the Feulgen reaction in 1937, getting positive
results (indicating the presence of DNA) for "inclusion bodies" and
negative results for "elementary bodies ,1'38 (There is some confusion

today about the precise meaning of this statement as the terms "inclusion

-]-
body" and "elementary body" are now considered virtually synonymous.
The first proof that inclusion bodies contained DNA came in 1940

71

when Smadel and Hoagland used a positive Feulgen reaction to prove the
presence of DNA in vaccinia-induced inclusion bodies.
A Mistake in Interpretation

Since little was possible before the crystallization of TMV,

W. Stanley's work was a major step in turning virology into a disciplined
and quantitative science.

However Stanley erred seriously in insisting that viruses were
pure protein and in initially dismissing Bawden and Pirie's emphasis on
"impurities.' tronically, the very "impurities"! to which they referred
had, in fact, accounted for the infectious activity which Stanley had
measured and mistakenly attributed to viral proteins. Due to his prowess
at the time, Stanley's misinterpretation of the possible role of nucleic
“ acids directed virus research in the late thirties and mid-forties toward
the study of proteins and away from investigation of nucleic acids.

Prob lems in Communications )

). With regard to Sanfelice, why was his original and provocative work,
done in 1914, lost from the focus of the scientific world? Since no ideas
during his time could be substantiated, why were his ideas ignored while
others flourished? A possible hypothesis is that his use of scientific
German was very poor. Thus, his contemporaries probably had so many
problems with his ill-constructed sentences that they did not seriously
consider the content of his work. Hence, Sanfelice's valuable insights

did not fluorish.
 

2. it has often been necessary to use critical reviews in order to
determine how well given ideas were accepted in the scientific community.
In particular, considerable controversy: arose concerning the distribution
of credit between Spizizen, Fraser, and Guthrie and Sinsheimer for first
proving the existence of infectious phage DNA.

The critical reviews on this topic highlight some differences in
approach among investigators. While Ravine? and Schramm? gave Fraser
credit for proving infectious bacteriophage DNA, Kozloff © and Colter
and Elle? did not agree. While Kozloff and Colter demanded more proof,
Ravin and Schramm accepted Fraser's evidence as sufficient proof. It
_ appears that Ravin's and Schramm's interpretations were incorrect since
they were based on premature assumptions. Ravin's statement ''these
findings, albeit preliminary, on the infection of protoplasts by viral
DNA raise enormous possibilities for the future...'' implies that infectious
DNA had already been proven. Actually, the first accepted proof of
* infectious DNA came with DiMayorca2# in 1959, a year after Ravin wrote
his review.
°3. The need for improved communication has clearly been demonstrated
in reviewing the literature. An example of this is in the article by
Bland and Robi now" wherein they state, ''So far as we are aware, Haagen?®
(1937) is the only investigator who has applied this reaction (Feulgen
reaction) to a virus. He stated that the inclusion bodies of vaccinia
gave a positive Felgen reaction, but that the elementary bodies are
negative .!"© Indeed, Bland and Robinow were not aware that Cowdry had
applied the Feulgen test to inclusion bodies in 1928.

From the above, it is apparent that as the volume of work grows,
new and more effective means of communication are needed. One useful

approach which will be described is the Graphical Citation Index.

—9-
Part III - The Graphical Citation Index

A. Introduction

The accompanying citation index provides a visual means for
tracing developments in virus chemistry from 1873-1960. Continuing
analysis of the history of virus chemistry is particularly warranted
in view of the close relationship between molecular genetics and the
study of infectious nucleic acids. This study centers on the events
leading up to the demonstration of the infectious nucleic acid nature
of viruses.

Such analysis clarifies and readily exposes historically
significant developments by pointing out changes of ideas in addition
to new experimental proceedings. Moreover, such interpretation serves
as a working tool for re-evaluating early insights and possibly
minimizing repetitive experimental work.

The index has several features which are briefly outlined below.
B. Chronological Perspective

-A broad overview of the index clearly shows periods of high
activity during 1935-1942 and 1953-1960. These time periods, which
show much higher “publication density" than the periods 1873-1935 and
1943-1953, generally follow some critical investigation that made
availdble new materials or concepts. For example, Stanley's 1935
TV crystallization (reference 74) essentially sparked the high
“publication density" that ensued from 1935-1942 since it provided
a previously unavailable material, the crystallized Tobacco Mosaic
virus. Similarly, Hershey and Chase's labelling experiments in 1952
(reference 41) together with Fraenkel-Conrat's work on TMV structure
in 1955 (reference 28) stimulated the high “publication density" from

1953-1960 by providing new empirical and theoretical input concerning
_.the mechanism of viral attack. @)
| Although there may be some omissions, this graphical method
Physically displays the general periods of activity as well as

those of relative passivity.

C. - Clustering

The central power of this index lies in that it reveals which
authors commonly cited the same references and thereby determines
the common-reference-clustering-pattern for each article. Such
clustering patterns may then be used to determine the relatedness
of different articles according to their degree of common citation.
If two articles cite a common reference, they are probably related
to each other. Otherwise, they would not have cited the same work.
If two or more articles cite two common references, they are
almost certain to be closely related. Hence, as their number of
common citations increases, the probable relatedness of two (or
more) articles also increases.

- The determination of relatedness by use of the graphical display
is of great use in searching the literature. Using the index one first
determines the clustering pattern for a given article. Having then
found several articles with at least one reference common to that
initially given (and probably many more) one can directly examine
these and bypass much of the mass of unrelated material that usually
accompanies a literature search.

This method clearly depends on the completeness of the citation
index used for the search. The full value of this technique thus
increases directly relative to the completeness of the index.
Although not exhaustive, the accompanying index provides a starting

point for surveying the literature on infectious nucleic acids.

It is recommended that this work be more fully expanded.
A third service provided by the index is that it highlights

D. Combination of Techniques

where different fields overlap and physically shows where isolated
techniques have been combined. New applications of existing techniques
has visibly been critical to many of the investigations reviewed here.
Some examples include (1) Smadel and Hoagland's demonstration of viral
DNA by application of the Feulgen stain to inclusion bodies (reference
71) and (2) Stanley's use of ammonium sulfate precipitation with
globulins to precipitate TMV (reference 74).

Thus, by following the techniques cited on the chart, it is
possible to decipher where and how different methods came together.
This process confirms that important results often follow when two
isolated findings are pooled and also brings out the more common

instances of lack of communication between investigators.

E. Problems

5 Presently, there are very few graphical citation indices
available. This limits the amount of investigation that can be
done with them. Therefore, an important task now is to develop and
provide more complete graphical citation systems.

In addition, there are some inherent difficulties which result
from the failure by some authors to directly cite original references.
Instead, some authors cite secondary references or even none at all
if the tectmique which they involve is very common. This introduces
the possibility that the references upon which the index is built
may themselves have incomplete dibliographies.

One solution to this problem would be to adopt the convention

whereby a formal bibliographic listing would not be necessary to

warrant an index connecticn. Rather, simply mentioning a method
)

‘or concept in the body of the Paper would suffice for indexing purposes,
‘Another problem arises from the possibility that authors may cite
the same reference for different purposes. As a result, the relatedness
of citing articles would not be guaranteed simply by their clustering
patterns.
This difficulty mainly affects those articles commonly citing
only one reference. As long as one or two additional common citations

exist this problem is insignificant.

F. Summary
Despite its problems listed above, the graphical citation index

isa powerful tool for analyzing developments in their proper
historical framework and for facilitating rapid and accurate
literature searches. .

This method is universally applicable to all areas of study and
provides an immediate Picture of how past events have shaped a
given field. “Hence, it is an excellent teaching tool .

A complete catalogue of citation indices covering specific
topics and sub-topics in well defined disciplines could be one of
the most useful investigative tools available.

As all branches of investigation become increasingly complex,
corresponding problems arise concerning how to maintain the necessary
levels of communication. In such light, the citation system outlined
above becomes increasingly important since it highlights particular
developments, places them in proper perspective, and facilitates

rapid information transfer between different sources.

wt
Note:

BIBLIOGRAPHY

The following sources are graphically displayed in the accompany-

ing Citation Index. The key is as follows:

© denotes general reference
(J denotes review article or text

ZN denotes key (i.e., highly significant)
article

Those articles listed in the Bibliography with an * are not included on
the Citation Index.

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GOPH

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Bawden, F. C., N. W. Pirie, J. D. Bernal and ft. Fankuchen,
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Bayon, H. P., “Virus Diseases of Bacteria, Plants, and Verte-
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Beijerinck, M. W., Verd. Akad. Wetensch. Amsterd. 11, 6,
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Bland, J. W. W., and C. F. Robinow, "Application of the Feulgen
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Bland, J. 0. W., and C. F. Robinow, ''The Inclusion Bodies of
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Bollinger, 0., "Ueber Epithilioma Contagiosum beim Haushuhun und
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Borrel, A., "Sur Les Inclusions de 1'Epithelioma Contagieux des
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Brachet, J., "La Detection Histochemique Des Acids Pentosenucleiques.!'!
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Bronfenbrenner, (Jordan and Falk), The Newer Knowledge of Bacteriology
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*

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Ol OQ Bs

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Burnet, F. M., ''Contribution a ]'Etude de T'Epithelioma Contagieux
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Burnet, F. and W. M. Stanley, The Viruses, Vol. J (General Virology),
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Caspersson, T. and J. Schultz, 'Pentose Nucleotides in the Cy to-
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589 1942, —

Colter, J. S., H. H. Bird and R. A. Brown, "Infectivity of Ribo-
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ACKNOWLEDGEMENTS

L would like to express my appreciation to Dr. Joshua Lederberg,
Chairman, Department of Genetics, Stanford University, for his guidance.
1 would also like to thank Ruth Redse for translating several articles

from German.

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