Detection of Viruses by Chemical and Biological Means
SVENGARD
( Department
of Virus Research, Karolinska Institutet
The discovery of the polyoma virus ( 80 ) pro
vides the strongest incentive yet for continued
serious efforts to find a human cancer virus. Ac
cording to previous experience in the field of ani
mal viruses, the polyoma virus should prove to be
one member of a group of related viruses with
different species specificities, causing similar dis
eases. When attempts to isolate human viruses
have failed, we have reason to question the suit
ability of the materials and methods used. This
attitude served as a basis for the present discus
sion of virus detection by biochemical and bio
logical means. An attempt was made to envisage
situations in which a virus would not be readily
detected and to analyze the reasons for this,
rather than summing up the various current tech
nics for virus detection and their limitations. For
this purpose the discussion was grouped under
three headings, corresponding to the known
stages in the developmental cycle of viruses:
provirus, vegetative virus, and mature virus.1
I. PROVIRUS
The evidence of the existence of a provirus
stage is conclusive only for bacteriophages. In
this case, however, prophage may be regarded as
the "natural" form of the virus and lysogeny as a
mechanism safeguarding the survival of both
host and virus. For a complete review of the ly
sogeny problems reference is made to Lwoff
( 50 ). In this connection only some salient points
will be summarized.
The fate of an infecting temperate phage
seems to be determined at the moment of pene
tration or very soon thereafter. The factor decid
ing between the alternatives reduction to pro
phage or production of vegetative phage appears
to be the metabolic state of the host cell. Reduc
tion does not automatically lead to establishment
of lysogeny; the prophage first has to become at
tached to and apparently interact with a specific
1 The term mature virus, referring only to properties of
the virus particle itself, is preferred to infective virus with
its implication of host-virus interaction.
Medicai School, Stockholm, Sweden )
intracellular site, to all appearances a specific
chromosomal locus. By this interaction both the
respective locus and the prophage seem to be
stabilized.
The infection represented by the lysogenic
state seems, in most cases, to remain completely
silent, with no morphologic or metabolic charac
teristics to distinguish a lysogenic from an uninfected cell. The two important features revealing
the true status of the lysogenic bacterium are ( a )
"immunity," i.e., refractoriness to lysis by related
(incompatible) virulent phages, and (fo) sus
ceptibility to spontaneous or sometimes also to
artificial "induction."
The phenomenon called immunity suggests
that a specific chromosomal locus is also involved
in infection with virulent phages. If a lysogenic
bacterium is exposed to a related virulent phage,
the latter is adsorbed and its nucleic acid in
jected. However, as long as the prophage blocks
the specific locus, no multiplication of virulent
phage takes place, although its nucleic acid may
remain potentially active for considerable peri
ods of time. If the prophage is activated by in
duction, synthesis of both the temperate and the
virulent phage is initiated.
In the situation as here described the genetic
information contained in the prophage DNA
seems not to find any demonstrable expression.
This is not always the case, however, the most
notable example being the toxogenic C. diphteriae. The capacity of toxin production is invar
iably associated with lysogeny, and lysogenization by the temperate phage from a toxogenic
bacterium invariably confers toxogenic capacity
to a nontoxogenic variant ( 26 ). Thus, if the bac
terial gene determining toxogenicity is not iden
tical with the prophage DNA it must be linked
to it with an unusual firmness. It would seem a
more plausible explanation that the same DNA
molecule which in a "resting" state controls the
synthesis of toxin, when activated is command
ing synthesis of phage. A quantitative and quali
tative analysis of the phage DNA might provide
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GARD—Chemical and Biological Studies of Virimeli
an answer to this question. As another example
of "phenotypic" prophage manifestation the mucoid growth of certain lysogenic enteric bacteria
may be mentioned.
Whether or not animal proviruses exist is a
matter of discussion. The majority of the inapparent, persistent infections with animal viruses
have been clearly shown to represent a carrier
state as principally distinct from virogeny. In a
tissue culture system a carrier state is established
if the rate of growth of the cell population and
the rate of spread of infection are in balance.
This can be artificially achieved with susceptible
cells and virulent vims by addition to the me
dium of neutralizing antibodies in the proper
concentration, as first described by Ackermann
and Kurtz (1). A similar equilibrium may exist
in a relatively resistant cell population even with
out antibodies in the medium ( 15, 60). In a typi
cal carrier culture only a fraction of the total cell
population is infected at any given time. Persist
ence of infection depends upon extracellular
transfer of virus as shown by the facts that the
culture can be "cured" by addition to the me
dium of antibody in high concentrations as well
as by cloning.
In some Rous virus-carrying tissue cultures
each individual cell may actually be infected
( 83 ). However, since virus seems to be continu
ously released, the situation can hardly be analo
gous to lysogeny in bacteria. Most probably the
intracellular virus is in a vegetative phase
throughout, although the rate of virus synthesis
is sufficiently low not to interfere significantly
with the growth of the host cell. The same ex
planation seems to fit in the persistently infected,
resistant cell line, described by Puck and Cieciura (60).
Persistent infections in the macro-organism are
usually more difficult to analyze. Inapparent in
fections in higher animals, with one exception,
seem to be associated with continuous produc
tion of antibodies, an indication that mature vi
rus is released at least intermittently. The excep
tion is LCM virus-carrying mice which, accord
ing to Traub (86), do not develop serologie im
munity. As the infection in this case is initiated
in utero, Burnet (14) suggested that immuno
logie tolerance might be involved, which indeed
seems to be the case (34).
A slow or intermittent release of mature virus
is by no means inconsistent with the assumption
of virogeny. As a matter of fact the most charac
teristic property of a lysogenic culture is the con
tinuous, spontaneous release of mature phage.
Therefore, the demonstration in persistently in
fected tissue of small amounts of virus cannot
serve to distinguish between carrier state and
virogeny. If, on the other hand, systematic at
tempts consistently fail to demonstrate the pres
ence of mature virus during clinically silent peri
ods, the assumption of a true virogenic state
gains in probability. For such reasons the recur
ring herpes simplex and the swine influenza vi
rus in the intermediate lungworm host (72) ap
pear to be likely candidates, even if positive evi
dence is lacking.
The only definitely established provirus, the
prophage, is of the DNA type and somehow as
sociated with the genome of the host cell. A pri
ori there is no obvious reason not to expect a
similar pattern of infection within other groups
of viruses as well, and then primarily among in
tranuclear DNA agents, i.e., a category to which
the herpes virus belongs. No RNA viruses have
yet been found in a provirus stage, and the whole
question must here be left wide open.
Detection of a provirus may be extremely dif
ficult. The most promising approach should be
attempts to induce formation of vegetative and
mature virus, for instance, by application of ion
izing irradiation or carcinogenic and mutagenic
chemicals. Some prophages, however, have re
sisted all attempts at artificial induction. It is pos
sible that systematic studies of such systems will
reveal additional physical or chemical agents
with inducing capacity. Prolonged cultivation in
vitro to improve the chances of spontaneous in
duction should also be tried.
The only possibility to detect and identify a
resting provirus would seem to be by chemical
means. In all probability provirus consists of nu
cleic acid only and can hardly be expected to
carry any immunological markers. Thus, the for
midable task of the biochemist would be to single
out and identify as foreign a nucleic acid that
can be expected to appear in the pool in a ratio
of one molecule per cell. This will obviously not
be theoretically possible, unless the provirus has
some unique constituent.
Until recently nucleic acids were supposed to
contain no more than four bases: in DNA, adenine, guanine, cytosine, and thymine; in RNA
the same set, except that uracil replaces thymine.
The structural specificity is presumably ac
counted for entirely by the sequential order of
the base pairs or bases in the chain. Virus nucleic
acids generally seem to have no particular chem
ical characteristics setting them apart from those
of the host cell. However, in 1953 Wyatt and
Cohen ( 95 ) isolated from DNA of T-even phages
a previously unknown base, 5-hydroxymethyl-
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cytosine ( HMC ), so far not found anywhere else
in nature. These phages contain no cytosine,
which is thus completely replaced by HMC. As
another unique feature glucose enters into the
molecule, probably in glucosidic linkage with the
methyl group of HMC (76, 88). It might also be
mentioned that Tessman et al. (84, 85) recently
reported an abnormally high irradiation sensitiv
ity of the small phages S 13 and <j>X 174, which
was interpreted as an indication of a singlestranded DNA instead of the double helix of the
Watson-Crick model (89).
In recent years new bases, primarily methyl
ated purines and pyrimidines, have been identi
fied as minor constituents of nucleic acids (3, 18,
48 ). The significance of these observations is still
obscure; sometimes the appearance of such bases
or an increase in their concentration is observed
under abnormal metabolic conditions together
with deviations from the base ratios required by
the Watson-Crick theory. It would thus seem
that the chemical composition of nucleic acids
may be less monotonous than hitherto assumed,
which means that chemical analysis might be
come a practicable means of identification of in
dividual nucleic acids.
II. VEGETATIVE VIRUS
Methods devised for the detection of vegeta
tive virus will assume particular practical impor
tance in cases of slowly growing viruses where
relatively small amounts of mature virus are re
leased and extracellular virus is continually neu
tralized by specific antibodies or inactivated by
other, nonspecific agents present in the tissues.
This type of infection should be expected prin
cipally in species and tissues with a high degree
of resistance to the infectious agent, which, in
turn, would mean that demonstration of small
amounts of mature virus by means of infection
experiments would be almost impossible if no
other more sensitive test systems were available.
A complete analysis of the vegetative phase
should include both viral and nonviral substances
produced in the course of the infection. To be
considered are (a) virus nucleic acid or nucleoprotein, (b) one or more proteins forming inte
gral parts of the mature virus particle, (c) spe
cific byproducts of the virus synthesis, regularly
appearing as "soluble antigens," etc., but appar
ently not incorporated in the virus particle, and,
finally (d) substances of a nonspecific nature.
A. NUCLEICAcros
Synthesis of virus DNA may take place in the
nucleus as seems to be true of papilloma, herpes,
Vol. 20, June, 1960
adeno, and several insect viruses, or in the cyto
plasm. So far only two groups of cytoplasmic
DNA viruses are recognized, the pox viruses and
the capsular insect viruses. The members of these
groups can be characterized as structurally com
plex viruses, probably more autonomous than
others. Whether or not they should be classified
as true viruses is debatable. As pointed out by
Burnet (14) the replication pattern of the pox
viruses resembles that of the psittacosis group.
The fact that the fibroma-myxoma virus appar
ently belongs among the pox viruses indicates
that cytoplasmic DNA viruses cannot be disre
garded as possible tumor agents. However, the
size and morphological peculiarities to be ex
pected from viruses of this category makes it
seem unlikely that they should remain unno
ticed and escape identification even if attempts
to demonstrate infectivity were unsuccessful. For
this reason cytoplasmic DNA agents shall not be
made the subject of further deliberations in the
present connection.
The intranuclear replication of animal DNA
viruses shows some features resembling those ob
served in the synthesis of the T'-even phages:
margination—"precipitation"—of the chromatin,
appearance of a structureless "pool" of viral
DNA, and subsequent "crystallization" with for
mation of spatially oriented virus "nuclei" (43,
44, 57). To this writer's knowledge no attempts
have been made to clarify the processes leading
to the breakdown of the chromosomes of the host
cell. It would be of interest to know what chem
ical relations, if any, exist between such extreme
ly radical alterations of the structure and the dis
turbances of the nuclear activity observed in ma
lignant tumor cells.
Also, RNA virus replication seems to be possi
ble both in the nucleus and in the cytoplasm. In
tranuclear synthesis of fowl plague G-antigen
(9) has been definitely established; electron mi
crographie evidence of formation of poliovirus
"nuclei" in the nucleolar and perinucleolar region
has been presented (64). The most convincing
evidence to date of cytoplasmic virus RNA syn
thesis is the recent report of crystalline structures
in the cytoplasm of cells infected with one insect
virus (94) and Coxsackie B 5 virus (56), respec
tively.
The problem of detection by chemical means
of vegetative virus nucleic acid is less formidable
than the task of finding a pro virus. At present,
the success of such attempts will depend entirely
upon the presence of specific constituents, pri
marily purines and pyrimidines, possibly sugars.
The base ratios determined on the pooled nucleic
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GARD—Chemical and Biological Studies of Viruses
acid extracted from a tissue represent, of course,
averages. Supposedly such mixtures are made up
of thousands of individual molecular species with
quite possibly widely differing ratios. The virus
particles, on the other hand, contain only one or
very few molecules of nucleic acid, and the puri
fied preparations, therefore, possess a chemical
homogeneity entirely lacking in the first type of
preparation. It is hardly justifiable to base con
clusions concerning structural characteristics on
a comparison of two such incongruous materials.
For a further elucidation of this question meth
ods will have to be developed by which a sep
aration of individual nucleic acids in a mixture
is feasible. It does not seem entirely Utopian to
hope that structural specificity might be made to
work for such purposes. Even methods by which
the degree of homogeneity could be estimated
without a complete separation of molecular spe
cies would be of great help.
It is usually assumed that the pure nucleic acid
is devoid of antigenic activity, and immune sera
prepared against whole virus are reported to
have no neutralizing effect on the infectivity of
phenol-extracted virus (30). However, in stud
ies on poliovirus-infected cells with the fluores
cent antibody technic Buckley (11) found a very
faint but apparently specific fluorescence of the
nucleolus and the perinucleolar region, i.e., that
part of the nucleus which probably serves as the
site of virus RNA synthesis. The nucleus re
mained otherwise unstained, while the cytoplasm
was gradually filled with staining material. The
significance of this observation is somewhat ob
scure. Two interpretations are possible: the faint
fluorescence of the nucleolus may indicate either
the presence of minute amounts of a specific pro
tein or else that the RNA as such has a certain
immunologie specificity. A further study of the
phenomenon appears justifiable.
The vegetative phase corresponds largely to
the "eclipse" in the cycle of infection, character
ized by a loss of infectivity which was reported
not to return until morphologically typical virus
particles reappeared
during the maturation
phase. In view of the now established fact that
the pure nucleic acid of many viruses is by itself
infective, the current explanation of the eclipse
phenomenon is hardly satisfactory. Virus nucleic
acid is being synthesized throughout the whole
period, and failure to demonstrate its presence in
infection experiments can apparently not be at
tributed solely to the disintegrated state of the
virus. As perhaps the most likely explanation of
the failures it may be assumed that earlier meth
ods used for extraction of nucleic acid from the
731
cells were unsuitable. In particular, it should be
essential during preparation to protect the nu
cleic acid from tissue nucleases in order to pre
vent inactivation.
There is some evidence to indicate that these
assumptions might be correct. Wecker and
Schäfer(91) isolated infectious EEE and WEE
RNA from infected cells by treatment with cold
phenol, whereas extraction of purified virus with
the same technic yielded negative results.
Wecker (90) later found that the virus protein
was soluble in hot phenol and that treatment of
the virus at temperatures of 40°-60°
C. gave nu
cleic acids of an activity comparable to that pre
viously found in RNA prepared from cells. He
concluded that the cellular RNA most probably
represented vegetative virus. Phenol treatment
undoubtedly
inactivates all tissue enzymes,
which might account for the positive results.
There are thus good reasons to hope that de
tection of vegetative virus with biological meth
ods will prove to have a more general applicabil
ity. The low yields of activity obtained with the
phenol technic as it is now practiced are an ob
vious disadvantage. The reason may be partial
inactivation in the process of extraction, or a low
avidity of the pure nucleic acid for the cell, or
both. However, improvements in the technics of
preparation as well as infectivity testing can
probably be achieved. Eventually tests on pure
nucleic acid rather than mature virus may prove
to offer great advantages. It was recently re
ported ( 52, 58 ) that poliovirus RNA could cause
infection in cells completely resistant to mature
virus. Infection was followed by one cycle of vi
rus replication only. Apparently the mature virus
released from the first infected cells was incapa
ble of initiating a second cycle. Similar results
were obtained with phage when the receptor
mechanism was sidetracked by simultaneous re
moval of the bacterial membrane (protoplast
formation) and liberation of the phage DNA
core by osmotic shock (77). Even if self-propa
gating infections could not be established in
these experiments, the possibility by such means
to widen the host range of a virus must be con
sidered as a valuable addition to the diagnostic
arsenal.
B. VIRUSPROTEINS
The chemical properties of a number of plant
virus proteins have been studied in detail, includ
ing amino acid analyses and preliminary se
quence determinations (46). In general the pro
tein of the virus particle seems to be made up of
a number of identical subunits linked together
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Cancer Research
by relatively weak secondary bonds. Dissociation
and reassociation of the subunits of rod-shaped
plant viruses are easily achieved, whereas spheri
cal viruses are more resistant. The number of
subunits may vary considerably, from about 20
to more than 2000 per virus particle, with mo
lecular weights from 18,000 and upwards.
So far no exotic or unusual amino acids have
been found in plant virus proteins. Although the
amino acid composition is specific enough to dis
tinguish between variants of the same virus, it
does not show any peculiarities, setting virus pro
teins clearly apart from those of the host cell.
Some enzyme-resistant proteins are supposed to
have a hemicyclic structure without free N-terminal groups ( 25 ).
The highly specialized proteins of phages shall
be mentioned only in passing. The anatomy of
the bacterial cell calls for a particular functional
differentiation of the virus particle which can
hardly be expected to be copied in any animal
viruses.
In comparison, information on animal virus
proteins is meager. Among the best studied are
the relatively complex myxoviruses. As shown by
Hoyle (37) the virus particle can be disrupted
by treatment with ether with release of two dis
tinct types of subunits : hemagglutinin and group
antigen. Schäferand co-workers (9, 35, 66-68,
92), studying fowl plague virus, describe the
hemagglutinin as a spherical particle, 30 m/j. in
diameter, and composed of protein and polysaccharide. It carries the hemagglutinating,
enzymic, and antigenic capacity (type specific V
antigen) of the intact virus. The group antigen
(S-antigen, or G-antigen in Schäfer'sterminol
ogy) appears in the shape of 12-m^. particles
made up of nucleoprotein. The hexagonal discs
obtained by Valentine and Isaacs (87) by tryptic digestion of the virus represent presumably
the intact nucleoprotein subunit. In the virus
particle it is supposed to be arranged in one turn
of a spiral; when liberated by enzyme treatment
this structure collapses, forming the hexagonal
discs; in the course of ether treatment it appar
ently breaks down into smaller fragments. Each
virus particle is supposed to contain six hemag
glutinin units in close packing, the nucleoprotein
occupying the central space between them and
lipides filling out the rest. The lipides are pre
sumably derived mainly from the host cell sur
face.
So far the chemical characteristics of animal
virus proteins have hardly been systematically
studied. Ion exchange chromatography used for
purification purposes has revealed certain inter
Vol. 20, June, 1960
esting adsorption-elution patterns (36), which
presumably must be attributed to the protein
component. Present data do not permit any gen
eral conclusions and very little speculation be
yond the fact that the technic itself looks prom
ising.
The enzyme activity of certain virus proteins,
notably those found in the myxovirus group,
might appear to offer possibilities of detection
and identification of such proteins by purely
chemical methods. However, even after many
years of systematic studies the chemistry of in
fluenza virus enzyme-substrate reactions is not
sufficiently clarified to make a chemical identi
fication feasible. The highly specific affinities to
be expected would also make a search for a sub
strate for an unknown virus an extremely hap
hazard undertaking. Less specific enzymes like
the ATPase reported to enter into some fowl leukosis viruses (54) cannot be distinguished from
normal tissue components and would thus have
no diagnostic potentialities.
Therefore semibiologic methods—hemagglutination-elution, hemadsorption, and immunologie
analysis—are the most valuable of those at pres
ent available. The capacity of certain viruses or
purified proteins of agglutinating red cells is
based on the presence in the cell surface of a re
ceptor substance of sufficient specificity. As an
equivalent of this phenomenon the in vitro reac
tion between purified influenza virus and puri
fied receptor substance leads to formation of a
precipitate (82). Poliovirus, with which no hemagglutination has been obtained, nevertheless
seems to react specifically with a cellular lipoprotein (33), presumably lacking in erythrocytes. This observation, further supported by the
apparently specific capacity of susceptible cells
to adsorb virus (42), indicates that virus-recep
tor mechanisms may have to be expected in most
virus-host cell systems. Detection by direct pre
cipitation of virus and receptor substance re
quires a preparatory purification of both com
ponents which also have to be used in high con
centrations. A possibility that might be worth
trying, when naturally agglutinable cells cannot
be found, is coating of native or tannic acidtreated red cells with receptor substance in an
attempt to develop a more sensitive and less ex
acting test system. The receptors so far identi
fied seem to belong in the class of surface lipoglycoprotein antigens.
The simplest and by far most sensitive method
of detecting the protein component in the cell is
with the aid of specific antibodies, particularly
by means of Coons's fluorescent antibody technic.
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GARD—Chemicaland Biological Studies of Viruses
Applying specific antibodies against the G-antigen and the hemagglutinin, respectively, Schäfer
and his group found that the former first ap
peared in the nucleus of cells infected with fowl
plague virus, later probably migrating out into
the cytoplasm. The synthesis of the hemagglu
tinin seemed to be initiated later than the G-antigen, and it was found only in the cytoplasm. The
synthesis of several other virus proteins has been
followed by the same technic (11, 49, 59, 93).
Generally the amount of stainable material in the
cells is remarkable. In the later stages of infec
tion the entire cytoplasm appears fluorescent, an
indication that much more specific protein is syn
thesized than is needed for incorporation in the
comparatively small amount of mature virus that
is eventually released ( representing a fraction of
about 10~5 of the cell mass).
C. NONVIHAL,
SPECIFICSUBSTANCES
Some of the nonviral material produced in and
released from infected cells might simply be re
garded as components of the virus particle, syn
thesized in excess of what is finally incorporated
into the mature virus. These substances differ in
no respect from the counterparts that can be ex
tracted from the virus particles. The intracellular
myxovirus hemagglutinin and soluble antigens
exemplify the principle.
Often, however, other nonviral substances ap
pear, specific in the sense that they are foreign to
the host cell and are synthesized only in the
course of a specific infection. A particularly in
teresting example is provided by the so-called in
complete influenza virus (51). The incomplete
virus forms particles of a somewhat varying size
but of the same order as that of the classical vi
rus. They seem to contain normal hemagglutinin
and large amounts of lipides. According to Ada
( 2 ) they may be free of nucleic acid or contain
reduced amounts of this constituent. In view of
the fact that the mature particle is supposed to
contain one single RNA molecule, Ada's inter
pretation of the analytical data would imply that
only a fraction of a molecule were incorporated
in the incomplete virus particle, which in turn
would indicate aberrations in the RNA synthesis.
However, the results obtained by Ada could be
equally well explained by the assumption that
his preparations represented mixtures of com
plete and incomplete virus, a likely explanation
considering the technical difficulties associated
with the preparative separation of the two. In
complete virus has the capacity of interfering
with mature virus in a peculiar way. Thus, after
exposure of the cells to a mixture of mature and
733
a large proportion of incomplete virus at high
multiplicities, the amount of hemagglutinin re
leased from the cells remains at normal levels but
is accounted for mainly by incomplete virus. The
proportion of RNA-containing mature virus is re
duced, sometimes to 10~6 of a normal yield. The
incomplete virus alone seems not to be able to
incite any infection or induce the cells to produc
tion of hemagglutinin.
It is tempting to speculate on the mechanism
underlying these phenomena. The RNA content
of the virus particle corresponds, as already men
tioned, to a single molecule of 2 X IO8 mol. wt.
One molecule is supposed to carry sufficient
coded information for synthesis of one specific
protein only. If this is so, the myxovirus RNA
could provide the pattern for the synthesis of ei
ther the G-antigen protein or the hemagglutinin
but not both. The information needed for pro
duction of one of the proteins would have to be
supplied from some other source. Most likely, the
protein itself would serve this purpose. Accord
ing to Schäferthe complete G-antigen enters the
cell, whereas the hemagglutinin is supposed to
be largely retained on the cell surface. However,
it would be difficult to explain the interference
phenomenon without assuming penetration into
the cell of the interfering agent. It seems reason
able, therefore, to assume that the hemagglutinin
is capable of entering and that this is indeed a
sine qua non for the synthesis of virus.
According to this line of thought, then, at least
one of the six hemagglutinin subunits of the virus
particle would have to enter the cell together
with the nucleoprotein. The RNA of the latter is
presumably self-replicating, provides the infor
mation necessary for synthesis of the G-antigen
protein, and somehow activates the cell in such a
way that the hemagglutinin can serve as a primer
for the synthesis of the identical enzyme in the
cytoplasm. If more than one hemagglutinin unit
enters, presumably inducing an excessive hemag
glutinin synthesis, the RNA synthesis would seem
to be inhibited, and the end-product consists
largely of incomplete virus.
This phenomenon, infection ending in dissolu
tion and destruction of the cell with release of
considerable amounts of a specific, noninfective
material (but none or only insignificant quanti
ties of mature virus), resembles the production
of bactericines, lytic but noninfective substances,
identical with or closely related to phage lysin
(75). Both phenomena provide examples of sit
uations in which the true nature of the processes
as induced by a virus infection might be almost
impossible to assess.
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In the formation of incomplete virus the syn
thesis of the basic components of the virus may
be considered to proceed in a regular fashion—
unless Ada is right, in which case an aberration
of the nucleoprotein synthesis would have to be
postulated. The main abnormality, however, is in
the maturation process. To what extent this situ
ation is replicated in other virus-host systems is
at present not known. Obviously, the above rea
soning is applicable only to more complex viruses.
In the small viruses, containing a single protein,
all information needed for the virus synthesis can
be carried by the nucleic acid. Nevertheless, ma
terial morphologically slightly resembling incom
plete virus seems to appear regularly in poliovirus-infected cells. Schwerdt and Schaffer (70)
reported the presence in purified virus prepara
tions of a nucleic acid-free protein component.
Immunologically, this component is distinct from
the virus particles (47), in electron micrographs
it looks like an empty sphere of approximately the
same size as the virus particle ( 69 ). The signif
icance of these observations is obscure. The noninfective soluble antigen might represent a reg
ular byproduct of the virus synthesis or the result
of an erratic synthesis.
Substances discussed in this section, being proteinaceous, are antigenic. For their detection in
the interior of the cell the fluorescent antibody
technic should be particularly suitable. Cell ex
tracts may preferably be analyzed with the aid of
any of the gel precipitation technics or immunoelectrophoresis.
D. NONSPECIFICSUBSTANCES
The phenomenon of interference has generally
been regarded as an expression of a competition
between viruses for certain key enzyme systems
in the cell. However, in a series of papers Isaacs
and co-workers (12, 13, 38-40) have presented
evidence of an alternative mechanism of a pre
sumably entirely different nature. Cells exposed
to inactivated myxoviruses or to active virus of
low or moderate virulence were found to produce
a substance capable of conferring resistance to
other cells against virulent strains of a variety of
related as well as unrelated viruses. This sub
stance, presumably a protein, was called interferon.
Interferon does not interact with virus in vitro;
it does not prevent adsorption of virus on the
cell surface, but it does inhibit synthesis of
(myxovirus) nucleoprotein. Resistance induced
by treatment of the cells with interferon requires
12-24 hours to reach maximum levels. Induced
Vol. 20, June, 1960
cells do not themselves release interferon, unless
they are challenged with active virus. In the chick
embryo chorioallantoic membrane infected with
influenza virus, no interferon seems to be released
as long as virus is produced at a high rate. When
virus synthesis has passed its peak, however, in
terferon appears in the allantoic fluid (39).
Isaacs has hazarded a guess that interferon
might represent an aberration of the normal virus
synthesis at some intermediate stage and that the
presence of interferon in the cell would serve to
redirect the synthesis to production of more inter
feron instead of the normal intermediate. Con
sidering the proteinaceous nature of interferon
this could hardly explain the inhibition of nucleic
acid synthesis that is apparently established. As
another possibility it might be suggested that
interferon has a place in a normal regulatory
mechanism in the cell, serving the purpose of
controlling the nucleic acid synthesis. If so, inter
feron would be expected to show a certain degree
of species specificity, which indeed seems to be
the case (39).
Apparently interferon might become an im
portant means of detection of infections with
moderate viruses in which histologie lesions are
absent and the amounts of virus or nonviral spe
cific substances are below the threshold of demonstrability. The chemistry of interferon is not
yet known to such an extent as to permit a sug
gestion of chemical diagnostic methods. For the
time being only biological demonstration is pos
sible, including determination of resistance of
infected cells to challenge with selected indicator
viruses and induction of resistance with material
released from infected cells.
III. MATURE VIRUS
The mature virus would by definition possess
infectivity as its most characteristic marker. If,
for various reasons to be discussed below, infec
tion tests are unsuccessful, other methods of de
tection might be tried. In vitro demonstration by
chemical or immunological methods would—in
most cases—require a concentration and partial
purification of the test material. Occasionally, as
found by Beard's group ( 20, 21, 55 ) with certain
strains of fowl leukosis virus, enormous amounts
of the agent may appear free in the blood or
other body fluids, from which it is easily obtained
simply by centrifugation. More often virus will
be scarce and found only in tissues. Without
methods by which results of fractionation experi
ments can be evaluated, purification will be ex
tremely difficult. Beyond a statement to the effect
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GARD—Chemical and Biological Studies of Viruses
that differential centrifugation probably has a
wider applicability than any other method tested,
few general recommendations can be made.
Whatever the outcome of other attempts to
demonstrate the existence of a virus, the crucial
test will always be infection experiments in vol
unteers, in test animals, or in tissue cultures. Pro
vided a virus exists, infection tests may yet yield
negative results for any one or more of the fol
lowing reasons.
A. VIRUSCONCENTRATION
The virus concentration in the test material
may be too low for detection by biological means.
This again may be accounted for in several ways.
1. Only small quantities of virus may be re
leased from the cells and at a low rate. In such
cases a concentration of the test material might
produce positive results. It is, of course, impor
tant to select material of initially highest possible
virus content.
2. The virus may be continuously inactivated
as it is released from the cell. A very labile virus
may simply undergo spontaneous inactivation;
it may be sensitive to tissue enzymes; or it may
interact with specific or nonspecific inhibitors,
including antibodies. Accordingly, attention must
be paid to the methods for preparation of inocula.
Rapid preparation at low temperatures may be
essential; the use of stabilizers may be tried, such
as cysteine, reducing the oxidation rate. It has
been shown that some viruses are inactivated by
enzymes and that the enzyme sensitivity spec
trum to a certain extent serves to characterize
individual viruses (53). The effect of tissue en
zymes on the yield of active virus seems not to
have been studied, however, and enzyme inhib
itors have not been used in attempts to improve
yields. Antibodies and inhibitors can be removed
by perfusion, extensive washing, electrophoresis,
etc., before the cells are disrupted and intracellular virus is set free.
B. VIRULENCEOF THEVIRUS
Viruses of very low virulence may fail to pro
duce manifest infections. In principle, two differ
ent situations may be envisaged: the virus may
be regularly infective but the infection estab
lished remain inapparent; or the infection rate
may be low. The latter case will be discussed in
a following section in connection with host re
sistance.
At least theoretically a state of virogeny might
account for an inapparent infection. As already
mentioned, no unequivocal evidence of the exist
735
ence of this state in the field of animal virology
has been presented. Since lack of evidence proves
nothing, it would be unwise to disregard the
possibility, however. Detection of virogeny might
be possible either by means of induction and
identification of subsequently appearing vegeta
tive or mature virus, or it may express itself as
a change in some particular property of the host
cell.
Artificial induction of prophage can be
achieved with agents apparently affecting the
chromosomal structures in a manner as yet un
known. According to Lwoff (50) induction is
possible only under certain conditions; the cells
have to be metabolically preconditioned. In some
cases all attempts at artificial induction have
failed. What is known about provirus and its in
duction refers exclusively to DNA viruses, pre
sumably attached to or incorporated in the chro
mosomes. It would be futile now to discuss the
hypothetical case of an RNA provirus, its possible
point of attachment in the cell, and the mech
anisms of its induction.
So far no successful attempts to induce cyto
logie manifestations in inapparently infected tis
sue cultures have been reported. The enhanced
susceptibility of irradiated cells reported by Puck
and co-workers ( 61 ) obviously is a phenomenon
of a different nature. Systematic studies of the
effects of conditioning and induction on such
carrier cultures as those described by Puck and
Cieciura (60) and by Deinhardt et al. (15)
would seem to be of great interest.
Even in host-prophage systems where attempts
at artificial induction have failed to produce re
sults, spontaneous induction occurs. This would
have to be expected also of an animal provirus.
Spontaneous induction is a comparatively rare
event even in inducible prophage systems. Evi
dence of an activation, therefore, can be expected
only in a small fraction of a population of in
fected host cells. A recent report by Dmochowski
et al. ( 17 ) is of a certain interest in this connec
tion. The authors describe virus-like structures in
human leukemic tissues as well as in cells of such
origin cultivated in vitro. Rounded particles of a
uniform size in a double-contoured membrane
appeared either in cytoplasmic inclusions or extracellularly. A characteristic feature was the
sporadic occurrence of this phenomenon. In dis
eased tissues large areas showed no reaction at
all of this type; in limited areas affected cells
were observed in perhaps one out of 30 sections.
In tissue cultures 2-12 weeks of culturing and up
to ten subcultures were needed to bring out the
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736
Cancer Research
phenomenon. This observation thus conforms
with what could be expected from a virogenic
cell population. Needless to say, many other in
terpretations are possible. In any event, the
technic used was exactly what can be generally
recommended for detection of a provirus, i.e.,
prolonged cultivation of presumably infected
cells in vitro.
It was already pointed out that prophage at
least sometimes confers an easily demonstrable
new hereditary property to the host cell, e.g., toxogenic capacity. In cases of suspected virogeny it
may therefore be worth while to look for charac
teristics distinguishing supposedly infected from
normal cells. Tumor biologists have of course
paid much attention to the genetic constitution
of tumor cells and listed large numbers of charac
teristics, distinguishing them from normal tissue
cells as well as one tumor from the other, the
tendency being to regard each individual tumor
as an equivalent of a separate biological species.
Such an analysis includes all the innumerable
genes stemming from the parent cell from which
the tumor took its origin, whereas in the present
connection the interest should be centered
around one single "gene" that would probably be
Vol. 20, June, 1960
host cell, at a rate proportionate to the rate of
excess assimilation, was observed. Eagle (19)
and others have also shown that certain metab
olites may be indispensable for the production of
virus. Together these observations suggest cer
tain approaches to the study of inapparent in
fections in tissue cultures.
In a stable system the establishment of an inapparent infection could be expected to cause
some changes in growth and metabolism of the
tissue. Deinhardt et al. (15) observed that their
NDV-carrying culture had a higher rate of res
piration but a lower rate of growth than an uninfected culture of the same cell line. In the
present writer's laboratory another system is cur
rently being studied where the only manifesta
tion of a presumed virus infection is a consistent
reduction in the metabolic rate of the tissue cul
tures.
As the "masked" state of infection probably is
a function of the ratio virus/host synthesis, it
should be possible to affect the manifestations of
infection by selective nutritional regimentation.
Thus, for unmasking, optimal conditions for virus
synthesis should be established, while at the
same time the normal metabolic activity of the
the same in a large proportion of the various tu
cell would have to be restricted. There are al
mors observed. In other words, we should look ready many indications that conditions of nutri
tion of tissue cultures may have a decisive influ
for common, not for distinguishing, characteris
ence upon virus yields and incubation periods.
tics of tumors. One such common feature of hu
man cancers has indeed been reported. Björklund Systematic studies of this problem on inappar(7), using immune cytolysis, and Zilber (96), ently infected cultures should yield much valu
able information.
using active anaphylaxis as an aid in immuno
logie analysis of cancer cells, have found a com
C. HOSTRESISTANCE
mon antigen in all tumors examined which seems
Failure to produce infection after transfer of
to be absent in normal tissues. According to
virus-containing material to a new host may
Björklundet al. (8) the antigen is a phospholiposimply be a failure to infect, accounted for by
protein localized to the cell surface.
resistance of the intended host organism or host
Even with virus in the vegetative phase, con
cell. In principle, host resistance may be either
tinuously evolving into maturation and being re
leased, an infection may remain inapparent, as in constitutional or conditioned.
the system described by Puck and Cieciura ( 60 ).
1. It is hardly to be doubted that resistance
In that case infected cells could be cloned and and susceptibility to infection may be genetically
give rise to infected populations. Whether or not determined. This is true for the macro-organism
an infected cell will be able to grow and divide as well as at the cellular level. As examples may
probably depends upon the rate at which virus is be mentioned that Gross's agent of mouse leuke
being synthesized. In Jacob's (41) elegant ex
mia (31) goes in the inbred mouse lines C3H
periments on phage it was shown that virus syn
and C57BL but not in DBA or the heterozygous
thesis had a priority over the synthesis of host Swiss, whereas the reverse is true of Friend's vi
material. In bacteria kept on a strict metabolic
rus ( 28 ). Hardly any two lines of HeLa cells are
regimen only phage was synthesized, if the rate identical with regard to susceptibility to viruses;
of assimilation was kept below a certain threshold
Puck and Cieciura ( 60 ) described a method for
value. With assimilation in excess of what was selection of resistant lines. As illustrated by the
needed for virus synthesis at maximum rates, mouse leukemia viruses, resistance and suscepti
metabolites and energy for synthesis of host ma
bility may be strictly specific and therefore large
terial became available; a residual growth of the ly unpredictable characters. For such reasons it
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GARD—Chemicaland Biological Studies of Viruses
is important that a sufficient number of genet
ically distinct host organisms be used in tests for
unknown viruses.
Resistance is seldom absolute; it can often be
overcome by massive infection. Papilloma virus
in domestic rabbits may serve as an illustration;
one ID50 corresponds in this case to about 80
million physical particles (5). Thus, in many sit
uations the problem of detection can be reduced
to that discussed on page 735 of this section: pos
itive results might be obtained, if a sufficient vi
rus concentration in the test material can be es
tablished.
As a particular type of constitutional resistance
the examples mentioned on page 731 should be
remembered. In those systems—chick embryo or
chick tissue culture cells/poliovirus RNA and
bacterial protoplasts/osmotically shocked phage
—resistance depends upon the barriers against
penetration of virus into the cell. If the process
can be shunted around these barriers, a manifest
infection might follow.
2. A naturally susceptible cell may be condi
tioned into resistance by interference with the
receptor mechanism. Thus, the lining of the
chorioallantoic cavity of the chick embryo ac
quires a temporary resistance to influenza virus
infection after treatment with receptor destroying
enzyme or periodate ( 23, 81 ).
In most cases the mechanism of conditioning is
less obvious. A possible effect of nutrional factors
upon the relative rates of synthesis of virus and
host material was discussed in a previous section.
Similar mechanisms operating on the macroorganism level might be responsible for changes
in resistance observed in connection with avita
minosis or amino acid deficiencies. In the present
connection the effect of hormones is of special
interest. Shwartzman (73, 74), studying poliovirus infection in the hamster, found that corti
sone treatment enhanced the susceptibility of the
animals, whereas testosterone reduced it. In this
case the hormones probably act on the brown fat
tissue. Cortisone stimulates the brown fat, the
cells grow in size, and assimilation of lipides in
creases. Under these conditions virus multiplies
in the tissue to high concentration levels. Testos
terone has the opposite effect. The analogy to the
well known, less well understood conditioning
effect of hormonal stimulation in the develop
ment of the Bittner tumor in mice is apparent.
Another example of growth stimulation as a
conditioning factor has been described by Friedewald (27). Methylcholanthrene applied on the
rabbit epidermis acts as an irritant, producing
proliferation and hyperkeratinization. A skin area
737
so treated may be found as much as 10,000 times
more susceptible to papilloma virus infection
than is normal skin.
In the bacteriophage field, lysogeny is one of
the most common and most important causes of
resistance to infection. In metazoans immunity
after a previous exposure probably plays a great
er role. However, inapparent infection can also
produce resistance through interference (cf. p.
734). This possibility may be particularly impor
tant to keep in mind in a discussion of cancer
and viruses. Apparently, some of the known tu
mor viruses are widely disseminated, almost ubiq
uitous; the animals seem to become infected very
soon after birth or maybe already in utero or in
ovo; they carry the infection in a clinically inapparent form for considerable periods of time.
If the etiology of human cancer is a virus (or
viruses), the epidemiology of the disease can
hardly be explained without assumption of a
similar near ubiquity of the causative agent.
Therefore, inapparent infection might be ex
pected to be of common occurrence, causing
resistance of the macro-organism as well as of
cells in tissue culture.
In this connection a phenomenon, familiar to
most workers handling tissue cultures, assumes
special significance. Trypsinized tissue usually
grows rapidly during a period of about a week
after explantation. Sooner or later growth slows
down, and after three to five subcultures the cell
population remains stationary or decreases. Some
times one or more islands of morphologically
changed elements appear in the sheet of cells.
This metamorphosis is usually associated with
increased growth capacity, and the cells now can
be established in permament culture. The mor
phology of such cells resembles that of estab
lished tumor cells; capacity of giving rise to
malignant tumors has been observed; and the
immunological character of such cells has been
found indistinguishable from that of cancer cells.
Obviously, such observations have to be evalu
ated with great caution. This is not the place for
an exhaustive discussion of the various possible
interpretations; it shall only be emphasized that
the possibility of an induction of inapparent virus
infections should not be neglected. If that is the
explanation of the phenomenon, resistance be
cause of inapparent infection might make detec
tion of tumor viruses by means of tissue cultures
difficult, depending upon how widely virus is dis
seminated. Dmochowski et al. (17) reported that
the agent found in human leukemic tissue pro
duced suggestive changes in monkey but not in
human tissue culture. Without access to more
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738
Cancer Research
detailed data it is difficult to evaluate this piece
of information; it may prove to be significant,
however.
IV. SOME CHEMICAL AND BIOLOGICAL
CHARACTERISTICS OF KNOWN
TUMOR VIRUSES
By a rough classification four groups of tumorinducing viruses may be distinguished, producing
papillomas, leukosis, sarcomas, and cancerous
tumors, respectively. Each one of these groups
contains several members with different host
specificities or immunological properties. It must
be emphasized, however, that a classification at
present can be only tentative, as the true inter
relationships between the various tumor viruses
are unknown.
Only two tumor viruses have been prepared in
a sufficiently pure state to permit conclusions
concerning their chemical characteristics. Rabbit
papilloma virus (10, 45, 65) contains DNA and
protein and is synthesized in the cell nucleus.
One fowl leukosis virus ( 4 ) was found to contain
RNA; the site of replication is apparently in the
cytoplasm. The fact that ATPase activity was
found to be invariably associated with this virus
(55) suggests that virus synthesis takes place in
or in the immediate vicinity of mitochondria.
Most tumor viruses can be regarded as mod
erate. The incubation periods are often remark
ably long, and manifestation of the infection
sometimes requires some conditioning stimulus.
Rous sarcoma virus retains its moderate charac
ter in tissue cultures; it does modify the morphol
ogy of the host cell (83), but has so far not
acquired cytocidal properties on subcultivation.
Others, like avian lymphomatosis virus (24, 71)
and polyoma virus (79), produce degenerative
changes in tissue cultures and may become
adapted in the course of serial passages. The
viruses so far isolated have a narrow host range
in vivo. They may be less specific in tissue cul
ture.
Polyoma virus is of special interest because of
its versatility. It propagates and produces symp
toms in several breeds of mice and in hamsters.
Large inocula produce degenerative lesions in
mice, particularly in the kidneys; otherwise tu
mor formation is the main symptom, involving
many different organs and tissues ( 78 ). The virus
is antigenic, and both neutralizing and CF anti
bodies are produced (62, 63). It possesses hemagglutinating capacity (22). With fluorescent
antibody, stainable material appears first in the
nuclei of infected cells; later a migration to the
Vol. 20, June, 1960
cytoplasm occurs, and the nuclei lose the capacity
to fix antibody. At this stage the cells begin to
show signs of degeneration, and hemagglutinin
appears ( 32 ). The similarities with myxovirus in
fection are considerable. Early in the course of
infection the cells develop resistance to superinfection with vesicular stomatitis virus, often
used as an indicator virus in studies on inter
ference.
Some tumor viruses appear as physical, appar
ently mature particles in the interior of the cell—
papilloma virus in the nucleus, the milk factor in
the cytoplasm ( 16 ). Avian leukosis virus is found
in cytoplasmic inclusions (6). No intracellular
particles are demonstrable in Rous virus-infected
cells; maturation in this case seems to take place
at the surface of the cell as in myxovirus infec
tions (29).
It would appear from this brief summary that
tumor viruses by no means form a uniform group.
So far there are few indications of any character
istics setting them apart from other animal virus
es. However, the behavior of Rous virus in tissue
culture is somewhat unusual. The infection is
apparent, the manifestation being a distinct
change in the morphology of the host cell. Virus
seems to be continuously synthesized and re
leased. Yet the viability of the host cell seems not
to be impaired.
The strict host specificity of the mouse leukosis
viruses is another unusual feature. Genetically
determined resistance to infection is usually not
an all-or-none phenomenon; the term resistance
has to be applied in a relative sense. It is possible
that future studies with improved technic will
prove this to be true also of resistance to the
agents causing leukemia. On the basis of present
evidence, however, the possibility of an "immu
nity" analogous to that found in lysogenic bac
teria should be seriously considered.
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Detection of Viruses by Chemical and Biological Means
Sven Gard
Cancer Res 1960;20:728-741.
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