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Max-Planck-Institut fur Virusforschung, Tubingen, Germliany
STRUCTURE OF THE MODEL VIRUSES ............................................................ 1
Maus-Elberfeld Virus ......................................................................... 1
Fowl Plague Virus ........................................................................... 2
Newcastle Disease Virus.......................................................................3
SIGNIFICANCE OF VIRAL COMPONENTS ................. .......................................... 4
Biological Investigation of Isolated Viral Components ...........................................
Investigation of Viral Reproduction ............................................................
Reproduction of Maus-Elberfeld virus ........................................................
Reproduction of fowl plague virus and Newcastle disease virus ................................
Summary.................................................................................... 12
CONCLUDING REMARKS ......................................................................... 14

INTRODUCTION tioned that "concentration is the prudence of

The invitation to deliver an opening lecture to life," and we have tried to follow his advice, con-
this audience has placed me in a somewhat centrating our attention mainly on three animal
difficult situation. Such a lecture is commonly viruses. The general plan was to purify first the
expected to present a panorama-like view over a infective particles, the viria, and to study their
field of research. This task, however, is for animal composition. Valuable information has been ob-
virology at present hardly realizable. tained by dissecting the viria and studying the
One feels in this case like a historian who was composition of their components. Viral anatomy
assigned the task of integrating recent events was followed by viral physiological studies in
into valid historical form. He will certainly run order to clarify the significance of the various
into the danger of overestimating some and structural elements.
underestimating others in importance. His tract I hope that I can convince you that such con-
will become therefore more or less speculative or centrated action in a rather narrow field of
even misleading. Hence an honest historian will virology can lead to important findings and
postpone such an integration to a later time, when stimulate new ideas. In presenting the results I
the chaff has already been separated from the function only as the speaker of our group, since
wheat by the winds of critical examinations and no appreciable success would be achieved with-
comparisons. At the moment he will prefer to out the tireless efforts of all members of the
restrict himself to an exact description and group.
critical evaluation of those events which he
could follow himself. STRUCTURE OF THE MODEL VIRUSES
In virology so many new findings have been Maus-Elberfeld Virus
published during the last few years that it is The three viruses investigated all belong to the
impossible to study all of them critically. To ribonucleic acid (RNA)-containing viruses. The
avoid in this situation mistakes like those just smallest is the Maus-Elberfeld (ME) virus, a
mentioned I would like to focus attention only on member of the Columbia-SK group (9, 14). In
those results which have been achieved by our purified preparations the shadowed ME virion
group. It was, I believe, Emerson who men- seems to have a spherical appearance (Fig. 1).
' Based on ONR lecture, presented at the Electron micrographs of replicas of viral crystals
Annual Meeting of the American Society for suggested, however, that they were like other
Microbiology, Kansas City, Mo., 6 May 1962. dwarf or nani-viruses polyhedrons, having a size
of about 24 mAu (Fig. 2). Crystallized prepara-
tions contained, in addition to protein, more than
20% RNA. It is suggested that the viral RNA
is surrounded in some manner by protein, since
ribonuclease does not destroy the biological ac-
tivity of the virion. Further information on the
structural details of the ME virion were obtained
by investigations with the negative staining
technique. In electron micrographs (Fig. 3, 4),
the polyhedral shape of the virion is clearly
recognizable. Subunits of the polyhedron having a FIG. 3. ME virus. Negatively stained with phos-
diameter of 40 to 50 A were observed, although photungstic acid (PTA).
no details of the subunits as contained in the
virion have yet been resolved. It may be, how-
ever, that the tiny particles observed in electron
micrographs of virus samples purified by density
gradient centrifugation (Fig. 4) are released sub-
units, although their diameter is larger than
would be expected. They apparently contain a
hole in the center.
More information is now available on the
nucleic acid component of ME virus, which was
extracted with phenol from a crystallized virus
preparation. The fraction obtained was rather
pure as indicated by the high E 260/E 280 ratio
of 2.05. In an analytical ultracentrifuge the bulk
of extracted RNA sedimented with a velocity
similar to that of infectious tobacco mosaic virus
(TMV) RNA (Fig. 5); thus one can assume that
its molecular weight, like that of TMV RNA, is
FIG. 1. ME virus, shadowed. about 2,000,000.
Fowl Plague Virus
Much more complex in composition than the
ME virus is our next model, the fowl plague virus,
which belongs to the influenza A viruses (3, 20,
27-33, 37-41, 52, 55). The size of this virus
ranges between 70 and 80 m1A (Fig. 6). Chemi-
cally, in addition to RNA and protein, carbohy-
drate and lipids are found. Furthermore, a
neuraminidase is present. Assuming that all
particles contain an equal amount of nucleic acid,
the RNA content per fowl plague virion corre-
sponds to a molecular weight of about 2,000,000.
The structural appearance of the fowl plague
virion as revealed by negative staining reflects its
complex chemical composition (Fig. 7). Like
other viruses of the influenza group, it possesses
an envelope armed with tiny spikes, which are
30 to 40 A thick and 100 to 120 A long. The
envelope seems to enclose a coiled filamentous
FIG. 2. ME virus, replica of a crystal. structure.

Subunits ?

FIG. 4. ME virus. PTA.

(UV-optic) S2 _J28 S tion to protein it contains the viral RNA. One

gains the impression from investigations of nega-
tively stained s antigen with the electron micro-
scope that the RNA strand is surrounded by
loosely arranged protein subunits (Fig. 10).
Experiments to isolate the viral RNA by phenol
treatment of intact viria led to preparations with
sedimentation constants not higher than 17 S
(Cook, unpublished data), whereas the RNA of
ME virus exhibits a sedimentation constant of
about 30 S. The reason for this difference is not
yet understood. It is conceivable that to fowl
FIG. 5. Sedimentation pattern of ME virus RNA. plague virion are attached larger amounts of
ribonuclease, the action of which cannot be pro-
When the lipids of the virion are removed by hibited during the isolation procedure.
ether, two types of subunits can be isolated. One Newcastle Disease Virus
is called "hemagglutinin," since it possesses
hemagglutinating activity; it looks like a tiny The largest model investigated is the New-
star (Fig. 8), with the spikes on the surface. Its castle disease virus (NDV), having a diameter of
over-all diamiter is 30 to 35 m/i. It is suggested 120 to 180 mjA (23-26, 35, 42). There are some
that after removal of the lipid the viral envelope indications that this virus belongs in the same
disintegrates into small pieces which roll together group as the mumps and parainfluenza viruses.
like a hedgehog and form thereby the structure The general building principle of this model re-
just described. This consists of protein and sembles that of fowl plague virus. An electron-
carbohydrate and contains the neuraminidase dense center, and a halo with much lower density
activity. As in the case of the hemagglutinin which sometimes detaches from the center, can be
envelope, we have also been unable to obtain the observed on electron micrographs of unshadowed
inner viral component, called "g or internal s preparations (Fig. 11). The chemical composition
antigen" in an intact state after ether treatment of NDV has not yet been thoroughly explored but
of the virion. The filament, which is about 15 m1A seems to be comparable to that of fowl plague
thick, apparently degrades into pieces of various virus. It is certain that it contains lipids as well as
length from 15 to 100 mI.t (Fig. 9). Chemical protein and RNA. Furthermore, it also possesses
investigation of this fraction showed that in addi- a neuraminidase. Electron microscopically, Home
et al. (16) observed with the negative staining
technique an analogous spike-armed envelope
and an inner component reminiscent of the inter-
nal s antigen filament of fowl plague virus but
with appreciable differences in fine structure (Fig.
12). Somewhat earlier we had isolated this inner
component from NDV by ether treatment (Fig.
13) and found it, like internal s antigen of fowl
plague virus, to contain RNA. The fine structure
of the NDV inner component (Fig. 13) resembles
that of TMV. It is about 170 A thick and con-
tains a central channel 50 A in diameter. There is
no doubt that the subunits of this structure are
much more tightly packed than those of fowl
FIG. 6. Fowl plague virus, shadowed. plague s antigen. The isolated hemagglutinin of
NDV, however, is morphologically nearly indis-
tinguishable from that of fowl plague virus (Fig.
The results presented with respect to the struc-
ture of the three viruses are not yet sufficient for
construction of exact models. But I think that
the presentation of very schematic drawings can
be risked in order to impress upon our minds
those structural principles which are important
for our further considerations (Fig. 15).
The significance of the various components of
the viria can be evaluated by various methods.
Some information can be obtained by biological
investigation of the isolated viral components.
Important knowledge was gained by using intact
FIG. 7. Fowl plague virus, PTA. viria and following the behavior of their compo-

FIG. 8. Hemagglutinin of fowl plague virus, PTA.


- X extracted Ai
B E phenol
| g; b E with were investigated. The
RNA extracted from ME virus, like that of
many other viruses, was able to induce host
cells to form new viria (9, 13). By following
the sedimentation of such active fractions by
infectivity tests after ultracentrifugation in a
separation cell, it could be demonstrated that the
biological activity is coupled to RNA with a
sedimentation constant of about 30 5 (Table 1).
With the 17 5 RNA pieces of fowl plague virus,
no biological activity was detectable (Cook,
unpublished data). Thus, one might suggest that
the total complement of RNA contained in a
virion is needed to transfer to a host cell the
information required for the production of new
Of the two types of subunits obtained from fowl
plague virus by ether treatment, neither the
FIG. 9. The g or internal s antigen of fowl plague
virus, shadowed.

w~~~I is

'lE. 1
FIG 11 Newcastle disease virus, unshadowed.

FIG. 10. The g or internal s antigen of fowl plague

virus, PTA.

nents during the reproduction process inside the

host cell. Fluorescent antibody and isotopic
labeling techniques proved to be very useful for
this purpose.
Biological Investigation of Isolated Viral
Components Hone et at.
Impressive results were achieved, by the first FIG. 12 ANewcastle disease virus, PTA4 (Hor ne
type of approach, when the viral nucleic acids et al. Virology 11:79).

FIG. 13. The g or internal s antigen of NDV. Shadowed and PTA.

Examinations of both types of fowl plague split

products revealed furthermore that they do not
possess interfering activity (51). Isolated internal
s antigen can be recognized as virus-specific ma-

terial only by serological methods. Its serological

specificity is relatively low. Only very slight
serological differences could be observed between
s antigens of various influenza A strains (3, 20).
....M. T Thus s antigen possesses a group antigen. How-
ever, the antigen of the hemagglutinin subunit,
_ the surface component of the virion, is highly
specific. It has apparently no antigenic component
in common with the s antigen. In immunization
experiments in animals, only the hemagglutinin
proved to be fully capable of inducing immunity
and neutralizing antibody. Hemagglutinin is, as
mentioned earlier, that component of fowl plague
FIG. 14. Hemagglutinin of NDV, PTA. virion which contains its hemagglutinating
Hemogglulinin Corresponding information is not yet available
RAPoNeuramiinidasr for the isolated subunits of NDV. But in the few
R~~~~~~iD~ Antige areas already examined the situation is similar to
that of fowl plague virus.
Fowl plague-v. TABLE 1. Biological determination of sedimentation
velocity of ME virus RNA extracted from
crystallized virus*
FIG. 15. Scheme of ME, fowl plague, and NDV
viruses. Infectivity of Infectivity of S
controlt supernatant
(LD6o) (LD50)
internal s antigen nor the hemagglutinin was 34S
infectious (3, 20, 29-32, 39). From the hemagglu- 7.8X104 1.3X103
3.1 X 102 1.0 X 102 21 S
tinin, infectivity could hardly be expected since it
does not contain nucleic acid. The RNA amount * Centrifugation was for 20 min at 44,770
present in the isolated s antigen pieces is appar- rev/min.
ently not sufficient for induction of the host cell. t Control not centrifuged.
Investigation of Viral Reproduction
The second approach to clarify the significance-
of viral components was the investigation of the
multiplication process.
Reproduction of ME virus. The concept ob-
tained with respect to the multiplication mecha-
nism of ME virus is still rather incomplete.
Nevertheless, the results available from studies
with the L-cell system allow us to recognize some
important features and problems (11-13). Like
other viruses, the ME virus goes into an eclipse
phase. Shortly after infection, the ratio of in-
fective virus particles to infected cells falls to FIG. 17. ME-infected L cells, 8.5 hr after in-
about 0.001. The events leading to the loss of fection. Stained with ME-specific fluorescent
infectivity of the penetrating ME virus have not antibody.
yet been studied. Following the production
kinetics of new viral material in synchronously
infected L cells, it emerged (Fig. 16) that the x.4
phenol-extractable infectious RNA starts to in- * 1.1
crease between 1 and 2 hr after infection. The
production of viral protein, as studied by the
complement-fixation test and by pulse-labeling 0.
the viral protein with C14-leucine, seems to follow
the same time course as that of RNA. There is
reason to believe that the subunits of the viral 0,6
protein shell are synthesized in the cytoplasm;
by fluorescent antibody, viral antigen is first de- 042
tectable at about 3 hr after infection and is found 0,2
exclusively in this region of the cell (Fig. 17). The
nucleus remains free of detectable fluorescence 1 2 3 4 5 6 7
during all stages of infection. The curve repre- hours pi. -.
senting the appearance of new infective particles FI G. 18. Incorporation of C'4-leucine into total
increases 1 to 1.5 hr after that of viral RNA and protein of ME-infected L cells as compared with
protein. The particles are apparently assembled normal L cells, ratios of radioactivity indicated on
the ordinate. The intervals on the abscissa indicate
the times when the 15-mmn pulses of the C'4 leucine
were given. At the end of the pulse the radioactivity
I of the total protein of infected and normal cells was
g to
Eo [ inside the cytoplasm, because ME virus cannot be
neutralized as long as it is associated with the cell,
whereas other viruses which are formed at the
membrane are neutralized.
The protein and nucleic acid metabolism of the
ME-infected system undergoes radical changes at
early stages of the cycle. Pulse experiments with
C14-leucine suggested (Fig. 18) that the rate of
FIG. 16. Multiplication of ME virus in L cells. total protein synthesis begins to decrease shortly
Synthesis of ME viria (X), viral RNA (0), and after infection (Hausen, unpublished data). Most
viral protein (A, CF-test; 0, incorporation of interesting are the changes with respect to RNA
C'4-leucine) . synthesis (12). They were detected autoradio-

graphically, using 5-min pulses of tritium-labeled unpublished data). Adding FPA up to 1 hr after
uridine. Normal L cells show, under these condi- infection inhibits the decrease of nuclear RNA
tions, a strong labeling of the nucleus, especially synthesis as well as the increase of cytoplasmic
of the nucleoli (Fig. 19), whereas the cytoplasm RNA synthesis. After addition at 3 hr postinfec-
is practically free of label. In infected cells, how- tion inhibition of RNA synthesis in the nucleus.
ever, nuclear RNA synthesis begins to decrease 2 persists but no synthesis of RNA in the cytoplasm
hr after infection. After 3 to 3.5 hr, only a negligi- occurs. Thus, special virus-induced proteins seem
ble number of cells showed nuclear tritium- to be necessary before the RNA metabolic
uridine incorporation. Instead, increasing changes observed in connection with ME virus
amounts of labeled compound were found in the multiplication can proceed.
cytoplasm starting at about 3 hr after infection Correlating these findings with the observa-
(Fig. 19). Thus, in the RNA metabolism of ME- tions described earlier, it seems reasonable to
infected L cells, at least two different processes believe that the cytoplasmic RNA is mostly viral
seem to occur: first, an inhibition of nuclear RNA RNA. This became somewhat improbable, how-
synthesis, and then a new, rapid RNA synthesis ever, following the chemical characterization of
in the cytoplasm. Corresponding controls insured the RNA synthesized later (4 to 6 hr) in the
that RNA and not an acid-soluble compound was multiplication cycle (Scholtissek, unpublished
concerned. data). The method used was developed by
By using small amounts of p-fluorophenylala- Scholtissek (44-46) and is hereafter referred to
nine (FPA), which still inhibit viral production, simply as "chemical RNA characterization." The
it can be demonstrated that an FPA-sensitive newly synthesized RNA is labeled by P32. After
phase precedes the changes observed (Hausen, digestion of the labeled RNA with ribonuclease
and separation of the various oligonucleotides by
chromatography, the oligonucleotide pattern of
the labeled RNA is determined. This pattern is
much more characteristic than is the simple base
ratio. Examination with this method showed that
the total complement of RNA synthesized at 4
to 6 hr after infection in the cell is not identical
with viral RNA. Nevertheless, the rapidly syn-
thesized cytoplasmic RNA seems to participate in
Ask A M"
* some manner in the synthesis of viral protein be-
cause the latter is produced at nearly the same
time at the same cell site. In view of the recently
advanced hypothesis of protein synthesis (2), one
Contra i might speculate that the particular cytoplasmic
RNA has a messenger function. At the present
time, however, this is nothing more than pure
F g t' o ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

By It:..AL speculation.
The ME virus seems to us to be a very inter-
esting model with respect to its reproduction
i,. r, 3.
mechanism. The problems here are based on very
impressive and characteristic changes, and are
therefore relatively easy to attack.
Reproduction of fowl plague virus and New-
castle disease virus. Somewhat more information
is available with regard to the multiplication
process of fowl plague virus (1, 4-6, 17, 19, 22,
ME, 5h P-F
30, 31, 33, 34, 41, 43, 47-49, 53, 54, 56). The fea-
FIG. 19. Incorporation of H3-uridine in L cells.
tures evaluated here are integrated into a sche-
(top) normal cells; (bottom) liE-infected cells, 5 matic picture. I am fully aware that in presenting
hr after infection. this scheme I must neglect somewhat the cate-
gorical imperative postulated initially. To bring
the scheme into a reasonable form, I must sub-
stitute some missing links by speculation.
As demonstrated in the scheme (Fig. 20), the
fowl plague virion attaches with the spikes of its
shell onto the cell surface. The possible function of
the neuraminidase probably contained in the
spikes during the penetration of viral material into
the cell is still open to discussion. After adsorption
onto the cell, the fowl plague virion goes into
eclipse. The ratio of infective virus to the number
of cells acting as infective centers falls to a value
smaller than 0.01. Investigations with P32-labeled
virus indicated that eclipsing of the virion is paral-
leled by liberation of s antigen from which the 0 2 C 6 0 10
RNA seems to have been released. In brief, these rime of adding FPA
investigations showed that shortly after infection (hours p.i.)
homogenized suspensions of the cells contained a FIG. 21. Influence of FPA ($00 jig/ml) on viral
considerable amount of P32 carriers which could production capacity of chick embryo cells, when
be precipitated by highly specific s antigen anti- added at different times after infection.
serum. This serum does not react with intact
viria. After precipitation of s antigen, labeled the appearance of viral RNA and other viral com-
RNA was still contained in the supernatant. This ponents, as far as they can be identified by the
seems to be viral RNA, since no significant trans- available tests. When FPA was added after the
fer of viral p32 to cell substances was observed. No critical time of about 1 hr postinfectien, a ma-
convincing experimental evidence is available at terial behaving serologically like s antigen and
present as to how the hemagglutinin shell of the containing viral RNA was produced in amounts
virion behaves during the degradation procedure. comparable to those occurring under normal
Before the production of detectable amounts conditions. Further effects of FPA will be con-
of new viral material begins, a substance has to sidered later on.
be formed the occurrence of which could be With regard to the functions of the early oc-
demonstrated only indirectly using FPA as curring FPA-sensitive material, which is very
inhibitor, as in the case of ME virus (Fig. 21). probably protein, no fully convincing explanation
The dose of FPA used did not disturb markedly can be offered at present even in this somewhat
the metabolism of cell RNA, the incorporation more intensively investigated case. Since, how-
of C'4-leucine into cell protein, or the eclipsing of ever, FPA does not markedly disturb synthesis of
fowl plague virus after infection. When added early normal cell RNA and since fowl plague viral
in the multiplication cycle, however, it inhibited RNA does not contain abnormal nucleotides, the
proteins in question may not be particular en-
zymes needed for viral RNA synthesis. Moreover,
the hypothesis comes to mind that in the case of
fowl plague virus the particular protein works in
stabilizing the viral genetic material; once this
has occurred, synthesis of viral RNA and of pro-
tein behaving serologically like s antigen protein
can no longer be stopped by FPA, as the normal
cell RNA metabolism might not be stopped by
FPA, since corresponding stabilized templates
are already established in the cell.
During the normal viral multiplication cycle,
new viral RNA and s-antigen protein can be first
FIG. 20. Scheme of fowl plague virus reproduc- detected 2 to 3 hr after infection, just after estab-
tion. lishment of the FPA-sensitive factor. The over-

whelming amount of initially appearing viral

RNA can be precipitated by s antigen antiserum.
This implies that either both components of the
viral inner component are assembled immediately
after their synthesis or they are formed as a unit.
That the s antigen appearing inside the cell is
indeed identical with the inner component of the
virion can be shown by isolating this component
from cell homogenates and comparing it with s
antigen liberated from viria (Fig. 22). According
to our investigations with specific fluorescent
antibody, which have already been reported on
numerous occasions, s antigen is produced in the C.
cell nucleus and seems to be transported later
into the cytoplasm. The alternative sometimes
discussed, that the later appearance of s antigen
in the cytoplasm occurs by the creation of new
cytoplasmic s antigen synthetic centers, appears
rather improbable in light of the results of the
experiments performed with FPA. After adding _
this compound 2 hr after infection, s antigen
accumulated in the nucleus and caused remark-
able swelling of this cell organelle (Fig. 23). This
finding not only supports the first theory but also
indicates that an FPA-sensitive compound
participates in the transport of the s antigen
from the nucleus to the cytoplasm.
Somewhat after the appearance of s antigen, FIG. 23. Localization of s antigen by fluorescent
hemagglutinin can be detected. In contrast to antibody in chick embryo lung cells. a, c, e, g: no
a antigen, hemagglutinin antigen can be found FPA; b, d, f, h: FPA 2 hr after infection. Time of
with fluorescent antibody exclusively in the staining; a and b, 4 hr after infection; c and d, 7 hr;
cytoplasm. Attempts to isolate the respective e and f, 10.5 hr; g and h, 13 hr.
component from cell homogenates by adsorption
on and elution from red cells led to preparations
which contained structures that were quite
S X 1 Ha 9from those we expected to find (22, 317
43). Although they possessed hemagglutinating,
neuraminidase, and virus-specific antigenic ac-
Sw ^ tivity and were lacking infectivity, as was to be
expected from the hemagglutinin component,
they resembled in structure and chemical com-
position microsomes of normal cells (Fig. 24).
The microsomal nature of these elements was
further stressed by the finding of glucose-6-
phosphatase activity and by isolation of ribo-
some-like particles from them with fluorocarbon
(Fig. 25). No convincing evidence was obtained
that these ribosomes contain virus-specific
material. Such material was isolated, however,
_ quite recently by another method used to de-
-- grade the lipoprotein membrane of the micro-
FIG. 22. The s antigen of fowl plague isolated somes and seems to behave biologically and
from infected tissue. physically like the hemagglutinin spikes of the

Comparing the over-all RNA metabolism of

the virus-chick embryo cell system with that of
corresponding normal cell cultures, it emerged
(49) that the rate of total RNA synthesis in-
creases at first in the infected system, reaching a
maximum at 3 hr, and declines later (Fig. 26).
Chemical characterization of the nucleic acid
produced showed that the early increase co-
incides with and is caused by the additional

FIG. 24. "Viromicrosomes" isolated from fowl

plague-infected cells.

virion (Maes, unpublished data). The microsomal

elements containing this viral compound are now
called "viromicrosomes." It was convincingly
shown that other hemagglutinating nonin-
fectious particles, known as "incomplete forms
of v. Magnus-type" have another origin and are
really incomplete viria lacking internal s antigen
(21, 22). The fact that hemagglutinin-containing
microsomes can be isolated from infected cells
lends further support to the concept that the
surface component of the fowl plague virus is
completed in the cytoplasm.
The sequence of appearance of s antigen and
hemagglutinin and their different localization
inside the cell suggested that both are completed
separately. The experiments with FPA strength- FIG. 25. Ribosomes isolated from "viromicro-
ened this suggestion. By adding FPA 2 hr after somes."p
infection, it is possible to achieve the production
of s antigen without corresponding appearance of ,34N/No xw00
hemagglutinating and infectious material (Fig.
21). With later addition of FPA, the inhibition 120
of hemagglutinin and infectious virus production
declines gradually. 110
During the further course of the normal
multiplication cycle, both viral subunits are 100

transferred to the cell periphery where they are 90

assembled into new viria near the cell membrane
or mostly in protrusions of it. Electron micro- 801
scopic studies suggested that they are coated at
this occasion with lipid originating from the cell
wall. Cellular origin of the viral phospholipids
was indeed demonstrated, using an isotopic tracer
(53). The assembly of s antigen and hemag-
glutinin seems to be a rather selective process, 1 2 3 4 5 6 7 a 9
Slundn pi. -
since even s antigen which accumulates in the FIG. 26. Incorporation of p32 into total RNA of
presence of FPA is not incorporated to an chick embryo cells infected with fowl plague virus
appreciable extent into virus that is formed (N) compared with normal cells (No). The hori-
after release of the inhibition. zontal lines indicate time of p32 pulses.

synthesis of viral RNA, which is, according to the myxoviruses, lipids-probably of cellular
earlier experiments of Wecker (53), produced origin-serve to hold the viral components
de novo. Be&sides viral RNA and normal cell together in the completed virion.
RNA, no further RNA was observed to be pro- More thorough investigation of the viral
duced during the infectious cycle, not even during multiplication process led to the detection of some
that period when hemagglutinin appears. Thus factors which do not belong to the equipment of
it is not very probable that a RNA different from the virion itself. They seem to be, however,
viral RNA is engaged in the production of essential for establishing the virus-synthesizing
fowl plague viral protein. Unfortunately, chick capacity inside the host cell. First, there may be
embryo cells were unsuitable for autoradio- one particular substance of the host cell which
graphic studies which we would like to perform undresses the virion initially in order to liberate
to settle this topic somewhat more. its nucleic acid. That an undressing of the virion
The contribution of our group to the under- precedes its reproduction process could be rather
standing of NDV reproduction is still of a pre- convincingly shown with fowl plague virus.
liminary nature. It may be mentioned briefly Furthermore, virus systems as different as ME
that a material resembling the viral inner com- and fowl plague produce, apparently early in
ponent (24), and something like viromicrosomes the reproduction cycle, particular proteins which
(Rott and Reda, unpublished data), have been are reminiscent of the "early protein" of phages.
isolated. Therefore, one might suggest that at According to Levintow and co-workers (18), such
least some parallels exist in the reproduction early protein seems to occur also in the polio
mechanism of fowl plague and ND viruses. system. With regard to the function of these
proteins in the animal viral systems, no more is
Summary known at present than that they are necessary
The observations described with respect to for the normal progression of viral reproduction.
the significance of the various virus components A more thorough study of them might, however,
support the concept, already widely accepted, shed some light on the early steps which lead to
that the viral nucleic acid which is situated in the realization of viral information inside the
the interior of the viria possesses all the informa- cell.
tion the host cell needs to produce new virus. A most interesting problem is posed, finally,
With ME virus, this could be proved rather by the drastic RNA metabolic changes observed
convincingly. With fowl plague virus, at least by our group in ME virus-infected cells and by
some indication was obtained in this direction. Franklin and Rosner (7) in Mengo virus-infected
The main tasks of the materials surrounding cells. Of special interest is the establishment of
the nucleic acid are to protect this component an efficient RNA-synthesizing system in the
and to facilitate its penetration into the cell. cytoplasm at a time when viral protein appears
Thereby the "foreign gene" viral nucleic acid there. It may well be that the rapidly synthesized
becomes a "strolling gene" and therewith an cytoplasmic RNA is concerned with the produc-
infectious agent. The surface material contains, tion of virus protein.
on the other hand, those components of the virus
which induce immunity in the organism, and the APPLICATION OF SOME FEATURES
sites where neutralizing antibodies attack. EVALUATED TO PROBLEMS OF PRACTICAL
During the multiplication procedure, the viral IMPORTANCE
nucleic acid or the nucleic acid containing Although the survey presented offers many
inner component and the coating material seems more problems than well-established facts, some
to be generally produced separately and to be of the latter may lead to implications of practical
assembled later to form the new virion. importance.
This principle is well-established now in the The finding that the immunizing capacity of
case of fowl plague virus and can also be sug- the fowl plague virus resides in its hemagglutinin
gested from the results with ME virus. Further shell material led to the proposal to use, instead
evidence for such a concept is delivered by the of inactivated total virus, the isolated hemag-
genetic studies of Hirst (15), who found "pheno- glutinins of influenza and possibly other myxo-
typic mixing" with several animal viruses. In viruses as vaccines. The potency of such a

vaccine may be demonstrated only by one result TABLE 2. Behavior of NH20H-treated fowl plague
(3). An injection of 0.003 mg of purified swine virus in the plaque test*
influenza hemagglutinin, prepared with Freund Dilutiont
adjuvant (10), initiated in a rabbit after 6 Time
weeks a hemagglutination-inhibition titer of of
treat- 10-1 102- 10 10-4 10- 10-6 10| 10"
1:32,000. With such vaccines, the occurrence mnent (1.1 (1.1 (1.1 (1.1 (1.1 (1.1 (1.1 (1.1)
x x x x x x x
of undesirable reactions would certainly fall to a 107) 106) 105) 104) 10') 102) 10)
minimum, since in contrast to intact virus iso- hr
lated hemagglutinin is practically free of normal o 110 18 3
host-cell components. 1.5 35 4 0 0
Where the isolation of the immunizing viral 3 64 2 0 0
shell material is not yet possible, one should 6 48 0 0
inactivate the respective viria for vaccine pro- 7.5 159 3 0
duction with a chemical which does not change 9 251 4 0
the antigenic material but acts exclusively on * Treatment was with 0.2 M NH20H. Results
the viral genetic substance, the nucleic acid. In are expressed as the number of plaques formed
this respect, hydroxylamine is clearly superior per culture.
to the still commonly used formaldehyde. NH20H t The numbers in parentheses correspond to
seems to react exclusively with the nucleic acid, the number of virus particles (original PFU's)
where, according to Schuster (50), it changes present in each dilution. Number of cells per cul-
the pyrimidine bases as indicated in Fig. 27. ture: -106.
Small RNA-containing viruses, like iIE,
polio, and EE, as well as the larger influenza
viruses, could be inactivated by this compound.
The deoxyribonucleic acid-containing Herpes
RNA chain (wnh cytidytic- and uridylec acid) -
Purines do not react wiih NH2 OH

NH2 0
NI L I I~~~~~~
- --Phosphate
Ribose - Phoqpate- Ribose
~~~~~~I hsat
Phosphate - -
0 2 4t 6' 06'
Hours of treatsnt
I 2MolsNH2 OH
FIG. 28. Inactivation of M1E virus by NH2OH. 0,
0.1 mu; 0, 0.5 .if 2H20H, pH 7.0, 20 C.
HIt7 ~CH2 NH2 0CN N,.
virus and, surprisingly, the NDV and mumps
N~~~~N virus strains examined proved to be resistant
-- -phosphate- Ribose Phosphate- Ribose - Phosphate- - -
(8, 36), although the latter viruses are similar in
their over-all structure to the influenza agents. In
this connection it was of some interest that TMV
probable further rea-uin
2 H20
was also resistant, but extracted TMIV-RNA
sensitive to NH20H. Thus, differences in coating
O *NH3
of viral RNA may play a role in causing differ-
HMt CH2 NH2 ences in their behavior to NH20H.
The inactivation kinetics (36) of ME virus with
O \N' S~ ( Urea)
NH20H were strictly exponential over the course
- - -Phosphate- Ribose - Pate- Ribose - Phosphate - - - examined (Fig. 28). An irregularity was observed
FIG. 27. Reaction of NH20H with RNA with fowl plague virus. After inoculating tissue
(Schuster). cultures with multiplicities higher than one of
* -------
incompletely inactivated fowl plague virus -a

i -0 - T Em
samples, more plaques appeared than were to be I/0 E m ..... 0 - 0
m |
expected from the number of those appearing /a0
,/nw *
at higher dilutions (Table 2). In this connection, ;/v *E Om0
multiplicity is referred to the plaque-forming *-
m mmmo

units originally present. This phenomenon is * ma ao

a 0 00 00 0 3 a0 o 0o o o
caused by multiplicity reactivation (Cook, /.W
0 - -0 -
00 00000 0 0o 0 3 00o o o o o
unpublished data). In preparing vaccines, the
o o o o o o
difficulty resulting therefrom can be overcome Am. lnnnnnnn

by an intensive treatment with NH20H, that is,

by causing many hits in the nucleic acid strand. FIG. 30. Immunization experiments with in-
Even after 4 days of incubation at 20 C with 1 fluenza virus vaccines in mice. 0, mouse without
M NH20H, neither influenza nor fowl plague virus
pathological changes in the lung; *, mouse died.
Preparation of hydroxylamine vaccine: see text.
lost antigenic potency (Fig. 29). Respective Preparation of formaldehyde vaccine: 0.018%
vaccines were at least as potent, usually even formaldehyde, 2 days, 20 C. For experimental details,
somewhat more potent, in the animal than see Schafer and Rott (36).
corresponding cautiously prepared formalde-
hyde-treated vaccines (Fig. 30). Thus NH2OH solid foundation. The simple differentiation by
might be used in future to prepare safe and NH20H, for example, can be of some value in
potent vaccines of various viruses. this sense.
Finally, it may be stressed briefly that each
advance in knowledge of the structural and CONCLUDING REMARKS
chemical behavior of viruses is strongly needed I hope I have been able to demonstrate that
in order to place viral taxonomy onto a more the concentrated action of our group on a very
small sector of animal virology was not quite
unsuccessful but has created at least some ideas
of possibly a more general importance for the
fight against virus diseases. At the present time,
when concentrated scientific activity is often
directed toward the destruction and not toward
the preservation of life, one might fear that the
phrase "concentration is the prudence of life"
will some day be inverted into "concentration is
the stupidity of life." To avoid such an occur-
rence, scientific workers all over the world would
do well to return to the ethics of the stoicist
Seneca, who noted in his Naturales Quaestiones
that "by exploration of the facts natural sciences
base the moral life of man on a solid foundation;
they liberate him from fear and let him recognize
the glory and magnitude of divine creation."
I wish to thank R. Rueckert for assistance in
the translation of the manuscript.
Most of the work presented was supported by
the Deutsche Forschungsgemeinschaft.
Antigen-dilutions LITERATURE CITED
FIG. 29. Results of chessboard complement- 1. BREITENFELD, P. M., AND W. SCHAFER. 1957.
fixation tests with fowl plague virus. 0, untreated The formation of fowl plague virus antigens
control virus; *, formaldehyde-treated virus; A, in infected cells, as studied with fluorescent
NH20H-treated (1 M, 20 C, 4 days) virus. antibodies. Virology 4.328-345.
2. BRENNER, S., F. JACOB, AND M. MESELSON, AND A. E. FARNHAM. 1960. The structure
1961. An unstable intermediate carrying and composition of myxoviruses. Virology
information from genes to ribosomes for 11:79-98.
protein synthesis. Nature 190:576-581. 17. HOTZ, G., AND W. SCHAFER. 1955. Ultrahisto-
3. DAVENPORT, F. M., R. RoTT, AND W. SCHAFER. logische Studie uber die Vermehrung des
1960. Physical and biological properties of Virus der Klassischen Geflugelpest. Z.
influenza virus components obtained after Naturforsch. lOb:1-5.
ether treatment. J. Exptl. Med. 112:765-782. 18. LEVINTOW, L., M. M. THOREN, J. E. DARNELL,
4. FRANKLIN, R. M. 1958. The growth of fowl AND J. L. HOOPER. 1962. Effect of p-fluoro-
plague virus in tissue cultures of chicken phenylalanine and puromycin on the repli-
macrophages and giant cells. Virology 6: cation of poliovirus. Virology 16:220-229.
81-95. 19. MCTLLER, G. C., S. v. ZAHN-ULLMANN, AND
5. FRANKLIN, R. M. 1958. The synthesis of fowl W. SCHAFER. 1960. Incorporation of leucine-
plague virus products in a proflavine-inhib- C14 into a virus-specific protein in homog-
ited tissue culture system. Virology 6:525- enates of chick embryo cells. J. Biol. Chem.
539. 235:660-664.
6. FRANKLIN, R. M., AND P. M. BREITENFELD. 20. ROTT, R., AND W. SCHAFER. 1960. Physik-
1959. The abortive infection of Earle's alisch-chemische und biologische Eigen-
L-cells by fowl plague virus. Virology 8:293- schaften des Virus N und seine Beziehungen
307. zur Influenza A-Untergruppe der Myxo-
7. FRANKLIN, R. M., AND J. ROSNER. 1962. viren. Zentr. Vet. Med. 7:237-248.
Localization of ribonucleic acid synthesis 21. RoTr, R., AND W. SCHAFER. 1960. Untersuch-
in mengo-virus-infected L-cells. Biochim. ungen uber die hfimagglutinierenden-nicht-
Biophys. Acta 55:240-241. infektiosen Teilchen der Influenza-Viren. I.
8. FRANKLIN, R. M., AND E. WECKER. 1959. Die Erzeugung von 'Inkompletten Formen'
Inactivation of some animal viruses by beim Virus der Klassischen Geflugelpest
hydroxylamine and the structure of ribo- (v. Magnus Phanomen). Z. Naturforsch.
nucleic acid. Nature 184:343-345. 15b :691-693.
9. FRANKLIN, R. M., E. WECKER, AND C. HENRY. 22. RoTT, R., AND W. SCHAFER. 1961. Unter-
1959. Some properties of an infectious ribo- suchungen uber die haimagglutinierenden-
nucleic acid from mouse encephalomyelitis nichtinfektiosen Teilchen der Influenza-
virus. Virology 7:220-235. Viren. II. Vergleichende Untersuchungen
10. FREUND, J. 1956. The mode of action of im- uber die physikalisch-chemischen und
munologic adjuvants. Advan. Tuberc. Res., biologischen Eigenschaften der Teilchen.
p. 130-148. Z. Naturforsch. 16b:310-321.
11. HENRY, C., AND R. M. FRANKLIN. 1959. 23. RoTT, R., AND W. SCHAFER. 1961. Fine struc-
Growth of mouse encephalomyelitis virus ture of subunits isolated from Newcastle
in Earle's L-cells. Virology 9:84-95. disease Virus (NDV). Virology 14:298-299.
12. HAUSEN, H. 1962. Cytologische Studien uber 24. Rorr, R., AND I. M. REDA. 1963. Nachweis
die Vermehrung des ME-Virus in L-Zellen. und Reinigung eines loslichen Antigens aus
Lokalisierung der RNS- und Virusprotein- Newcastle disease-infiziertem Gewebe.
Synthese mit autoradiographischen und Zentr. Vet. Med. 1OB:57-66.
fluoreszenzoptischen Methoden. Z. Natur- 25. RoTT, R., H. FRANK, AND W. SCHAFER. 1961.
forsch. 17b:158-160. Isolierung und Eigenschaften der hamag-
13. HAUSEN, P., AND W. SCHAFER. 1961. Produk- glutinierenden Komponente des Virus der
tion von Virus-RNS und -Antigen bei der Newcastle disease. Z. Naturforsch. 16b :625-
Vermehrung des MiLuse-Encephalomyelitis- 626.
(ME)-Virus. Z. Naturforsch. 16b :72-73. 26. RoTT, R., I. M. REDA, AND W. SCHXFER. 1962.
14. HAUSEN, P., AND W. SCHAFER. 1962. Unter- Isolation and characterization of hemag-
suchungen uber ein Mause-Encephalo- glutinating noninfectious particles produced
myelitis-Virus. Reinigung und physikalisch- during multiplication of Newcastle disease
chemische Eigenschaften des Virus. Z. virus (NDV). Virology 16:207-209.
Naturforsch. 17b:15-22. 27. SCHXFER, W. 1951. Eigenschaften tierischer
15. HIRST, G. K. 1959. Virus-host cell relation, Virusarten, untersucht an den Geflugel-
p. 96-144. In T. M. Rivers and F. L. Horsfall pestviren als Modell. I. Die komplement-
[ed.), Viral and rickettsial infections of man. bindenden Antigene bei der Klassischen
J. B. Lippincott Co., Philadelphia. Geflugelpest. Z. Naturforsch. 6b:207-212.
16. HORNE, R. W., A. P. WATERSON, P. WILDY, 28. SCHXFER, W. 1955. Vergleichende sero-im-
munologische Untersuchungen uber die 1952. Eigenschaften tierischer Virusarten,
Viren der Influenza und Klassischen Gefli- untersucht an den Geflugelpestviren als
gelpest. Z. Naturforsch. lOb:81-91. Modell. III. Weitere Untersuchungen uber
29. SCHAFER, W. 1957. Units isolated after die physiko-chemischen und morpholo-
splitting fowl plague virus, p. 91-103. In gischen Eigenschaften der Geflugelpest-
G. E. W. Wolstenholm [ed.], The nature of viren. Z. Naturforsch. 7b:29-33.
viruses. E. C. P. Millar-Churchill Ltd., 41. SCHAFER, W., K. MUNK, AND M. MUSSGAY.
London. 1956. Physikalisch-chemische und bio-
30. SCHAFER, W. 1959. Some aspects of animal logische Eigenschaften des gereinigten
virus multiplication, p. 20-39. In M. Pollard 'loslichen Antigens' der Klassischen Gefli-
[ed.], Perspectives in virology. John Wiley gelpest. Z. Naturforsch. llb:330-338.
& Sons, Inc., New York. 42. SCHAFER, W., G. SCHRAMM, AND E. TRAUB.
31. SCHAFER, W. 1959. Some observations con- 1949. Untersuchungen fiber das Virus der
cerning the reproduction of RNA-contain- atypischen Geflugelpest. Z. Naturforsch.
ing animal viruses, p. 61-81. In A. Isaacs 4b :157-167.
and B. W. Lacey Fed.], Virus growth and 43. SCHAFER, W., W. ZILLIG, AND K. MUNK. 1954.
variation. Cambridge University Press, Isolierung und Charakterisierung hAmag-
Cambridge, England. glutinierender, nicht-infektioser Einheiten
32. SCHAFER, W. 1959. The comparative chemistry bei Klassischer Geflugelpest. Ein Beitrag
of infective virus particles and other virus- zur Kenntnis der 'Inkompletten Virusfor-
specific products: animal viruses. In F. M. men.' Z. Naturforsch. 9b:329-340.
Burnet and W. M. Stanley [ed.], The viruses, 44. SCHOLTISSE, C. 1959. Charakterisierung von
vol. 1. Academic Press, Inc., New York. Ribonucleinsauren verschiedener Herkunft,
33. SCHAFER, W., AND K. MUNK, 1952. Reinigung die gleiche BasenverhAltnisse besitzen,
und Eigenschaften eines loslichen Antigens durch enzymatischen Abbau. Biochem. Z.
der Klassischen Geflugelpest. Z. Natur- 331:138-143.
forsch. 7b:573-574. 45. SCHOLTISSEK, C. 1959. Beziehungen zwischen
34. SCHAFER, W., AND K. MUNK. 1952. Eigen- der Ribonucleinsaure-Synthese im Zellkern
schaften tierischer Virusarten untersucht und in verschiedenen Cytoplasmafraktionen
an den Geflugelpestviren als Modell. IV. der Rattenleber in vivo. Biochem. Z. 331:
Untersuchungen uber den Ablauf der Vermeh- 365-374.
rung beim Virus der Klassischen Gefiuigel- 46. SCHOLTISSEK, C. 1960. Verwandtschaftsbe-
pest. Z. Naturforsch. 7b:608-619. ziehungen zwischen den Ribonucleinsauren
35. SCHAFER, W., AND R. RoTT. 1959. Unterein- des Zellkerns, der Mitochondrien, Mikro-
heiten des Newcastle disease- und Mumps- somen und des Zellsaftes bei Rattenleber,
Virus. Z. Naturforsch. 14b:629-631. Milz und Nieren. Biochem. Z. 332:458-466.
36. SCHAFER, W., AND R. ROTT. 1962. Herstellung 47. SCHOLTISSEK, C., AND R. ROTT. 1961. Unter-
von Virusvaccinen mit Hydroxylamin. suchungen uber die Vermehrung des Virus
Verlauf der Inaktivierung und Wirkung des der Klassischen Gefhlugelpest. Die Synthese
Hydroxylamins auf verschiedene biolo- der virusspezifischen Ribonucleinsaure
gische Eigenschaften einiger Viren. Z. Hyg. (RNS) in infizierten Gewebekulturen em-
148:256-268. bryonaler Huhnerzellen. Z. Naturforsch.
37. SCHAFER, W., AND G. SCHRAMM. 1950. Uber die 16b :109-115.
Isolierung und Charakterisierung des Virus 48. SCHOLTISSEK, C., AND R. ROTT. 1961. Influence
der Klassischen Geflugelpest. Z. Natur- of p-fluorophenylalanine on the production
forsch. 5b:91-102. of viral ribonucleic acid and on the utiliz-
38. SCHAFER, W., AND E. WECKER. 1958. Uber die ability of viral protein during multiplica-
Wirkung von Ribonuklease auf 32P-mar- tion of fowl plague virus. Nature 191:1023-
kiertes 'gebundenes Antigen' des Virus der 1024.
Klassischen Geflugelpest. Arch. Exptl. 49. SCHOLTISSEK, C., AND R. ROTT. 1961. Zusam-
Vet. Med. 12:418-422. menhange zwischen der Synthese von
39. SCHAFER, W., AND W. ZILLIG, 1954. Uber den Ribonucleinsaure und Protein bei der
Aufbau des Virus-Elementarteilchens der Vrermehrung eines Virus der Influenza-
Klassischen Geflugelpest. I. Gewinnung, Gruppe (Virus der Klassischen Gefltigel-
physikalisch-chemische und biologische Ei- pest). Z. Naturforsch. 16b:663-673.
genschaften einiger Spaltprodukte. Z. 50. SCHUSTER, H. 1961. The reaction of tobacco
Naturforsch. 9b:779:788. mosaic virus ribonucleic acid with hydroxyl -
40. SCHAFER, W., K. MUNK, AND 0. ARMBRUSTER. amine. J. Mol. Biol. 3:447-457.
51. TOKUMARU, T., AND W. SCHAFER. 1958. Inter- 54. WECKER, E., AND W. SCHAFER. 1957. Studien
ferenz beim Virus der Klassischen Geflugel- mit 32P-markiertem Virus der Klassischen
pest. I. Die Wirkung von UV-inaktiviertem Geflugelpest. Z. Naturforsch. 12b :483-492.
Virus, von Virusspaltprodukten und RDE 55. ZILLIG, W., W. SCHXFER, AND S. ULLMANN.
auf die Vermehrung des Geflugelpest-Virus 1955. Uber den Aufbau des Virus-Elementar-
in der Gewebekultur. Z. Naturforsch. 13b:
704-713. teilchens der Klassischen Geflugelpest. II.
52. WATERSON, A. P., R. RowT, AND W. SCHAFER. Chemische Eigenschaften des Elementar-
1961. The structure of fowl plague virus and teilchens und seiner Spaltprodukte. Z.
virus N. Z. Naturforsch. 16b:154-156. Naturforsch. 10b:199-206.
5.3. WECKER, E. 1957. Die Verteilung von 32p im 56. ZIMMERMANN, T., AND W. SCHAFER. 1960.
Virus der Klassischen Geflugelpest bei Effect of p-fluorophenylalanine on fowl
verschiedenen Markierungsverfahren. Z. plague virus multiplication. Virology 11:676-
Naturforsch. 12b :208-210. 698.