(CANCER
RESEARCH
26, 995-1008, May 1966]
The Fine Structure
of the Yaba Monkey
Tumor
Poxvirus
ETIENNE de HARVEN1 AND
DAVID
S. YOHN2
Division of Cytology, Sloan-Ketlering
New York
Institute for Cancer Research, New York, New York, and Roswell Park Memorial Institute,
Summary
The fine structure of the Yaba monkey tumor poxvirus has
been described by electron microscopy of thin sections of the
tumors and of infected tissue cultures. The intracytoplasmic
maturation of the virus has been analyzed and described in 6
sequential phases. The Yaba virus resembles other previously
described viruses of the pox group. Regular subunits have been
demonstrated on the surface of immature particles. The dumb
bell-shaped core of the mature elements seems to appear inside
the immature particles. The tissue culture findings are consistent
with the hypothesis that the cycle of the Yaba virus may be
slower than that of other poxviruses. The observations are com
pared with the results obtained in the study of other viruses of
the pox group in view of their role in the initiation of neoplasms
tin several animal species, including man.
Introduction
In 1958, Bearcroft and Jamieson (5) reported an outbreak of
subcutaneous tumors among a colony of rhesus monkeys housed
in Yaba, Nigeria. Soon afterward, Andrews et al. (2) demon
strated that these tumors were transmissible by cell-free filtrates
and identified the oncogenic agent as a member of the pox group.
This classification has been confirmed by Niven et al. (37), who
were the 1st to study the Yaba virus under the electron micro
scope. Immunologie studies showed that this virus is unrelated
to other viruses of the pox group, although it is morphologically
similar to the Shope fibroma, the molluscum contagiosum, and
the vaccinia viruses.
Sproul et al. (47) studied the pathogenesis of the tumors and
classified them as histiocytomas. These tumors always regress,
apparently because the virus has a cytopathic effect in vivo.
After subcutaneous inoculation of the virus in susceptible mon
keys, histiocytes proliferate in the infected area. This growth is
considered to be neoplastic, although permanent malignant trans
formation has yet to be observed. The virus is not oncogenic in
mice, rats, hamsters, rabbits, dogs, or cats (1). So far, only mon
key and man (24) have been found to be susceptible.
The virus has been further characterized in vitro in simian and
human cells (32, 50), and its localization within the cells has been
demonstrated by the use of immunofluorescent technics.
1Aided by a Career Scientist Award of the Health Research
Council of the City of New York, Contract 1-325.
2Supported in part by Research Grant CA-07998 from the
USPHS.
Received for publication August 9, 1965; revised December 17,
1965.
Buffalo,
The present investigation was undertaken to obtain additional
information of the fine structure of the virus at various phases of
its intracytoplasmic development in biopsies of the tumors and
in infected tissue cultures.
Material
and Methods
Production of the Tumors
A 3-year-old rhesus monkey (Macaca mulatta) was inoculated
with IO6tumor-inducing doses of Yaba virus. The virus was ob
tained by extraction of tumors with saline and Genetron as de
scribed previously (50). Within 14 days subcutaneous nodules
appeared and biopsies of these nodules were taken 7 days later.
Fixation of Tumor Tissue
The tissue samples were fixed in 6.5% glutaraldehyde (43)
buffered at pH 7.5 with 0.1 M phosphate buffer at 0°Cfor about
4 hr. The samples were then washed for 1 day in cold 0.1 Mphos
phate buffer containing 0.33 M sucrose, postfixed in 2% Os04 in
0.1 M phosphate buffer for 2 hr, dehydrated in ethanol, and em
bedded in a mixture of epoxy resin recently proposed by Erlandson (19). Polymerization was carried out overnight at 60°C.
Sections were prepared with a Porter-Blum microtome equipped
with a diamond knife, mounted on bare grids (12), stained with
uranyl acetate followed by lead hydroxide (31), and coated with
a single thin carbon film.
Tissue Culture Technics
Monolayer cultures of the continuous cell line of cercopithecus
kidney, BSC-1 (50), cultivated in T-30 flasks in Medium 199
supplemented with 10% agamma calf serum, were inoculated
with IO7tissue culture infectious doses of Yaba viruses and in
cubated at 35°C.The titration of the inoculum was performed as
described previously (50). These cultures were prepared for elec
tron microscopy 1, 2, 3, 4, and 5 days after inoculation with the
virus. The cells were detached from the glass with a rubber "po
liceman" and centrifuged at low speed. Cell pellets were fixed
for about 10 min at 0°Cin 1% glutaraldehyde adjusted at pH
7.5 with 0.1 M phosphate buffer, briefly rinsed with the buffer
supplemented with 0.33 M sucrose, postfixed in 2% osmium tetroxide for 1 hr, and embedded in Epon (33).
Electron Microscopy
Sections of both tumor tissue and infected tissue cultures were
examined with a Siemens Elmiskop I electron microscope, using
the double condenser system, 80-kv accelerating voltage, and
MAY 1966
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995
Etienne de Harven and David S. Yohn
50-/i molybdenum objective apertures. Original magnifications
ranged from 8000 to 40,000 times. Ilford N. 60 "photo-mechani
cal" plates were used throughout this study.
TABLE 1
DIMENSIONSOFTHEVIRUSES
Structure
Observations
Measurements
Viral unit-membrane
2 dense
Tumor Cells
Under the electron microscope, many virus particles were seen
within the cytoplasm of the tumor cells. Although these particles
resemble previously described viruses of the pox group (14, 37,
40), both the immature and mature Yaba viruses are character
ized by structural features which have not been previously re
ported and which will be described here in detail.
A frequent aspect of the tumor cells can be seen in Fig. 1. A
large part of the cytoplasm is occupied by an inclusion body con
taining numerous virus particles. Several characteristics of ma
ture viruses are readily apparent at low magnification: their
elongated or bricklike shape, their high electron density, and
their accumulation in a circumscribed cytoplasmic area showing
vacuoles and membranous material of presumably degenerative
nature. The viral inclusion is not in contact with the plasma or
the nuclear membrane. Particulate glycogen, present throughout
the cytoplasm of the cells, is especially abundant between the
viruses of the inclusion body. The nucleus appears normal and
shows no detectable sign of involvement in the virus proliferation.
Most of the particles are fully mature viruses, as judged by com
parison with previously described agents of the same group.
Similar intracytoplasmic accumulations of mature viruses have
been observed in a large percentage of the cells of the Yaba tumor
samples. They were seen only in the tumor cells, not in occasional
connective tissue cells.
The observation within the cytoplasm of tumor cells of areas
where the viruses multiply, areas which will be referred to as
"viral factories" (8, 9), was more instructive for the analysis of
the complex process of viral morphogenesis.
The complexity of the viral development as seen within the
cytoplasm of a tumor cell is illustrated in Fig. 2. Multifocal fac
tories are surrounded by zones where mature particles accumu
late. Seven round masses of medium dense material are in the
immediate vicinity of the maturing viruses. Their fine structure
is better demonstrated in Fig. 6. Many cylindrical forms are seen
in various orientations. They show a tendency to cluster, forming
groups of 2-6 parallel elements in close contact with each other.
They contain material of low electron density, and their fine
structure is shown in Fig. 8.
A typical factory area is seen in Fig. 3. Between the nuclear
membrane and the plasma membrane normal cytoplasmic com
ponents have practically disappeared and immature forms of the
virus are scattered through a fine fibrillar matrix. To facilitate
the description of the various forms of the virus, the develop
mental sequence of the virus will be divided arbitrarily into 6
stages (Table 1). The earliest recognizable aspect of the immature
particle will be referred to as Type 1, and the fully mature virus,
shown in Fig. 1, as Type 6.
Several examples of Type 1 are easily identified in Fig. 3. They
appear as "crescents" of various lengths but of similar curvature
and membranous structure. In 3-dimensional reconstruction,
such crescents probably correspond to cupule-shaped structures,
having a concave and a convex surface. The convex surface is
99(5
lines of 30-35 A sepa
rated
by
25-30 A
Immature viruses
Types 1 and 2
Type3
Types 4, 5, and 6
a
2800-3200 A
2500-2700 A
3500-3900 A
1500-2000
Virus associated structures
Long cylinders
900-1000
Subunits on immature particles
Subunits in the inclusions, •
Subunits in long cylinders
Dense paracrystal inclusions
clear
in diameter
in diameter
in diameter;
A thick
A in diameter;
long
150 A long
space
1-2M
'
150 A long; 75 A wide
150 A long; 75 A wide
Fibers 30-35 A thick
always covered by short, radially oriented spines, "crew-cut"like (Figs. 4, 5, 10). The spines are already seen on the convex
surface of the shortest crescents, as if they participated in the
genesis of the viral membrane. A homogeneous material, of
greater density than the matrix of the factory area, accumulates
in the concavity of Type 1 viral cupules.
Type 2 are completed spheroidal particles limited by a mem
brane identical to that of Type 1. The outer surface is covered
by regular, hairy spines. The particle is filled with an electrondense material similar to what was already observed in the con
cavity of Type 1.
A group of much denser particles is seen in the upper left part
of Fig. 3. Slightly elongated, they always show a characteristic
inner core which has the shape of a biconcave disc or a dumbbell.
Depending on the number of membranes surrounding these parti
cles, they will be referred to as Type 5 or 6. It is difficult to visual
ize the transition between the immature Types 1 and 2, on one
hand, and the mature Types 5 and 6, on the other. The succeed
ing micrographs, however, demonstrate how such a transition
could occur. But before leaving Fig. 3, an additional detail should
be pinpointed. Free ribosomes are not evenly distributed through
out the factory area. They are grouped in clusters in the close
vicinity of Type 5 viruses. They do not form typical "polysome"
patterns, but show occasional double row linear arrangements
("zipper"-like).
The sharp boundary between a viral factory area and a periph
eral rim of normal cytoplasm is shown in Fig. 4. Particulate gly
cogen and short double membranes of rough endoplasmic reticulum are evident in the normal cytoplasm. The distribution of
these cytoplasmic components seems to stop abruptly at the
edge of the factory area where Types 1 and 2 viruses are easily
recognized. One particle shows an eccentric nucleoid of extremely
high electron density, and 2 other particles, of a diameter some
what smaller than that of Type 2, are completely filled with
material of a similar high density. Whether or not the small nu
cleoid will participate in the formation of completely dense parti
cles remains to be seen. Meanwhile, it seems appropriate to label
these forms Type 3, since they retain the shape of Type 2 but
show the considerable electron density of more mature viruses.
CANCER RESEARCH VOL. 26
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Fine Structure of Yaba Monkey
Two types of inclusions are seen in close proximity to develop
ing viruses. One is a large accumulation of a material of medium
density (/) which will be better analyzed in Fig. 6 and which
seems to be similar to the inclusion seen in Figs. 2 and 3. The
others are small crystal-like structures (CR) of an electron density
similar to that of Type 3 virus nucleoids and showing 10-11 dense
lines for 100 m/n, each dense line being 30-35 A thick. As in Fig.
3, a cluster of free ribosomes is seen in the vicinity of maturing
viruses.
Fig. 5 shows, at high magnification, the structure of a Type 2
immature particle seen in a tumor cell. A unit-membrane en
circles the entire particle, which is coated by short spines that
average 150 A in length and are responsible for its characteristic
"crew-cut" profile.
Fig. 6 illustrates the fine structure of the masses of medium
density seen in Figs. 2-4. Far from being homogeneous, these
masses are made of small subunits which are probably cylindrical
in shape. Careful examination of this material reveals indeed a
number of circular profiles, approximately 75 A in diameter,
mixed with short parallel double lines 75 A apart and 150 A long,
which are consistent with the cylindrical shape of the subunits
forming the inclusion. It is interesting to note that the dimen
sions of these small cylindrical subunits are very close to those of
the spiny coat of the immature particles.
Fig. 7 shows, at higher magnification, the periodic structure of
the other type of virus-associated inclusions shown in Fig. 4. The
dense lines, corresponding to filamentous or membranous struc
tures, are approximately 30-35 A thick.
Also present within the viral factories are long cylindrical
structures (C). The number and grouping of the cylinders are
illustrated in Fig. 2, and 1 isolated example is seen in the itpptr
part of Fig. 3. Finally, Fig. 8 illustrates in almost tangential sec
tion the structure of 2 such cylinders. Their surface is made of
regularly organized circular subunits, in a honeycomb pattern.
There is again a striking resemblance between these subunits and
those shown in Fig. 6. Tangential sections of immature particles
also show a honeycomb pattern of circular profiles of the same
diameter. The principal differences between the architecture of
the long cylinders (Fig. 8) and that of the immature viruses
(Fig. 5) are that no unit-membrane is seen in the cylinders and
that their lumina are filled with a material of a texture and a
density similar to that of the surrounding background, whereas
a denser and finer substance soon accumulates inside the imma
ture viruses.
In Figs. 9-14 the hypottietical phases of the maturation of the
Yaba virus are presented. Although all but 1 of these forms were
seen on the previous plates, still it is difficult to understand the
transition from Form 3 to Form 5. Intensive examination of
factory areas, however, reveals the existence of a rare particle,
shown here in Fig. 12 and which will be referred to as Stage 4.
These forms might bridge the gap between Forms 3 and 5, since
they still have the spiny coated membrane and the increased
electron density of Type 3 but already have the elongated shape
of Types 5 and 6. In addition, they show internal membrane
structures which might represent the future dumbbell-shaped
internal core of the mature forms.
The sequential presentation of Figs. 9-14 represents o hypothe
sis of maturation of the Yaba virus, which does not necessarily
imply a general scheme of the morphogenesis of other poxviruses.
MAY 1960
Tumor Poxvirus
This maturation process is, however, quite similar to that of vac
cinia virus for which a more chronologic study was made easier
by a precise knowledge of the duration of the intracellular cycle.
All the developmental forms seen in Figs. 9-14 have been de
scribed in the previous figures, with the exception of Type 4, seen
in Fig. 12. This particle still has a spiny coat and a visible unitmembrane. It has, however, the elongated shape of the more
mature elements. Inner membranous structures have appeared
exactly in its long axis and might correspond to a flat sac.
Type 6 is the most frequently observed form. It accumulates
in large cytoplasmic inclusions (Fig. 1). Type 5 is seen less fre
quently at the periphery of factory areas. Types 1-4 are all ob
served in factory areas but not in comparable numbers. Types 1
and 2 are very easy to find, Type 3 is less common, and Type 4 is
extremely rare. This might suggest a very rapid transformation
from Type 3 to Type 5.
Tissue Culture
Immature particles were first observed 3 days following infec
tion. Thereafter, greater numbers of immature particles accumu
lated. Mature particles, Types 5 and 6, were not seen until the
5th day following infection. A typical BSC-1 cell infected with
the Yaba virus 5 days before fixation is illustrated in Fig. 15.
Several stages (Types 1, 2, 3, and 5) in the viral differentiation
process are recognizable. Fully mature particles (Type 6) were
rarely seen in the infected tissue cultures. Whether this reflects
the low rate of the virus development or is a function of tissue
culture conditions will be discussed later.
The diameter of Type 3 is definitely smaller than that of Type
2, and this difference is not caused by the level at which the par
ticles have been sectioned. When particles are sectioned too far
away from an equatorial plane, the details of the viral membrane
and of their ¡••piny
coat are blurred, which is not the case for the
2 particles of Type 3 seen here. While Types 3 and 5 particles are
of comparable size, Type 5 lacks the spiny coat and has the elon
gated shape obviously resulting from the formation of the inter
nal core.
Discussion
The fine structure of the Yaba virus as seen in thin sections of
both tumor cells and infected tissue cultures is strongly reminis
cent of several other previously described viruses of the pox
group, namely vaccinia (9-11, 22, 30, 35, 41), Shope fibroma (7),
and molluscum contagiosum (4, 15, 23). In all these cases a cyto
plasmic viral development characterized by the progressive
maturation of incomplete forms has been described, and the
dumbbell-shaped core as well as the numerous membranes form
ing the viral envelopes has been observed.
Nuclear participation has not generally been noted in poxvirus
cycles with the exception of the Shope fibroma, where Bernhard
et al. (7) described hypertrophie nucleoli, and of the molluscum
contagiosum, where Dourmashkin et al. (15) noticed intranuclear
condensations. Hypertrophie nucleoli have been observed in
BSC-1 cells by means of histochemical staining (50). In the final
stages of Yaba virus synthesis in BSC-1 the chromatin clumps
and the nucleolus are quite prominent. However, this seems
characteristic of a dying or dead cell and cannot be interpreted as
indicative of nuclear participation. In the present electron mi997
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Etienne de Harven and David S. Yohn
croscope study no significant abnormalities were observed in the
nucleoli and nuclei of BSC-1 and of tumor cells.
As with other poxviruses, the Yaba virus develops in a wellcircumscribed area of the cytoplasm referred to as "factories."
(One hesitates to give to these areas the name of "viroplasm,"
since we do not know how much of the background material of
the factories represents raw material for incorporation into future
viruses.) The immature viruses are spherical in shape and of an
electron density only slightly greater than that of the surrounding
background. The mature viruses are elongated, brick-shaped, of
extremely high electron density, and contain a dumbbell-shaped
core. This seems to summarize the morphogenesis of the Yaba
virus as well as that of other agents of the pox group.
The membrane of the immature Yaba virus has a typical unitmembrane structure (42), as previously demonstrated by Dales
and associates (10, 11) for the vaccinia virus. This membrane
seems to originate de novo in factory areas, without any connec
tion with preexisting membranous structures. Type 1, the cres
cents, represent, therefore, an interesting model for the stud}' of
membranogenesis in general, since the thickness of the 2 dense
lines (30-35 A each, separated by a clear space 25-30 A wide) is
similar to what has been described for a large variety of cellular
membranes (42). It is interesting to note that the assembly of
viral material in crescent, or cupule-shaped, structures, has also
been observed in viruses unrelated to the pox group. In early
phases of the development of the Friend murine leukemia virus,
"crescents" were observed (13) under the bulging plasma mem
branes of the infected cells.
One feature of the early forms of the Yaba virus, which has
not been clearly demonstrated in other poxviruses, is the presence
on their surface of radially oriented spines. Nagington and Home
(36), however, described "radially arranged spicules" at the sur
face of early forms of vaccinia virus seen by means of negative
staining. These spicules and the surface subunits described in the
present study are probably of a similar nature. They appear in
the very beginning of virus differentiation, on the shortest visible
crescent, and seem to vanish later, during final phases in the
maturation process. Tangential sections have revealed them to
have a very regular, honeycomb arrangement and probably a
cylindrical structure. It is suggested that the assembly of these
subunits plays a role in the initial phase of viral membranogene
sis. It is also suggested that the large inclusions such as those
seen at low magnification in Figs. 2-4, and at high magnification
in Fig. 6, represent accumulation of similar subunits available
for the construction of future viral membranes, or synthetized
in excess as a result of some imbalance or asynchronism in the
various synthetic processes triggered by the presence of infecting
viral DNA in factory areas.
When immature particles are "closed" (Type 2), a very dense
material starts to accumulate inside them, forming the charac
teristic "nucleoid" or completely filling the particle (Type 3).
Dales (9) has shown that in vaccinia virus, the nucleoid is com
posed of filaments measuring 15-50 A in width, and he has sug
gested that these filaments represent the viral DNA, supporting
the previously expressed view of Epstein (18). Such filaments
were not observed within the maturing particles in our material,
perhaps because in Yaba viruses the dense material lacks a regu
lar orientation. Completely dense particles (Type 3) are believed
to be a further step in the maturation of the Yaba virus. One
998
should remember that "electron density" in thin-sectioned tissue
corresponds mainly to the presence of uranium and lead ions
fixed on various structures during the "staining" procedure. Al
though nonspecific, uranium is known to have a great affinity
for DNA (26). Recently described technics (34) may facilitate
analysis of the part played by osmium oxides in the high electron
contrast of viral nucleoids.
The increase in density of the immature particles (passage from
Type 2 to Type 3) possibly corresponds to the synthesis of viral
DNA. It occurs at a moment when the viral membrane is com
pleted ("closed") and might, therefore, indicate an in situ syn
thesis of DNA. It might also result from the permeability of the
membrane, which is still coated with radially oriented spines, to
viral DNA precursors.
Occasionally, dense polygonal inclusions were seen in the vicin
ity of maturing viruses (Fig. 4 at low, and Fig. 7 at high magnifi
cation). The density of these inclusions compares only with that
of the viral nucleoids. In addition, these inclusions have a paracrystalline periodicity, the dense lines being similar to what Dales
(9) observed in vaccinia virus nucleoids. It is suggested, therefore,
that these inclusions might represent accumulated viral nucleopròteins.
The final phase in the maturation of the Yaba virus includes
the formation of a dumbbell-shaped core (Types 5 and 6) and
the addition of an outer envelope. Our findings suggest that the
core is formed by the opening of a flat sac located INSIDE the
dense immature particle (Type 4). Further studies will perhaps
show whether this is a unique property of the Yaba virus, or
whether it occurs also during the maturation of other pox viruses.
As indicated by Tamm and Bablanian (48), and by Dales and
Kajioka (10), host RNA or a new species of RNA probably plays
a role in the synthesis of viral proteins during the multiplication
of DNA virus. Salzman et al. (44, 45) have recently shown that
RNA similar in base composition to viral DNA is formed in the
cytoplasm of HeLa cells infected with vaccinia virus. It may be
assumed that a parallel situation exists in cells infected by the
Yaba virus. Groups of ribosomes have been observed in our mate
rial very close to maturing viruses. It is suggested that such ribo
somes might be the depositories of a "D-RNA" (6) which par
ticipates in specific viral protein synthesis. Ribosomes have been
described in close association with viral factories, in the case of
Shope fibroma virus (20), and of vaccinia virus (30).
Intriguing long cylinders (Figs. 2 and 3 at low, and Fig. 8 at
high magnification) are frequently associated with Yaba virus
factories. The only arguments suggesting their possible relation
ship with the virus are based upon their topographical distribu
tion and also upon the close resemblance between the surface
subunits of these cylinders (Fig. 15) and those covering the im
mature viruses. It might be that these cylinders represent some
aberrant forms of the virus. One should remember, however, that
the fine structure of monkey cells has not been very extensively
studied so far, and that it is hazardous to claim that such struc
tures are not present in noninfected monkey cells.
The present study has been ba.sed on the observation of thinsectioned infected cells. Several authors have used negative stain
ing to study the morphology of vaccinia viruses (9, 36, 38, 41),
fowlpox virus (27), or Yaba virus (39). Apparently, negative
staining adds little to the information obtainable by the thinsection method. Purified extracts of infected cells will invariably
CANCER RESEARCH VOL. 26
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Fine Structure of Yaba Monkey Tumor Poxvirus
contain a mixture of the various developmental forms of the
virus. Identification of these forms in negatively stained material
will depend on how much phosphotungstic acid penetrates the
particles so that their inner components are visible. If little or no
phosphotungstic acid penetrates the viruses, only exterior surface
structures will be demonstrated. Surface tubules or threads have
been described by this method (9, 36, 38). The membrane on
which these structures appear is difficult to identify, and desicca
tion artifacts are hard to evaluate. It seems that the structure
of the poxvirus is too complex to be fruitfully analyzed only by
the negative staining technic.
Tumor cells are not a suitable material to study the timing of
virus development. Infected tissue cultures provide a much
better basis for such studies. Our tissue culture material was in
fected with the Yaba virus 1-5 days prior to fixation for electron
microscopy. Although a few immature virus particles were ob
served after 3 days' incubation, viral synthetic activity became
readily apparent 4 and 5 days after infection. Even at that time,
most of the particles were of the immature type (Fig. 15). These
results indicate that the Yaba virus development occurs at a com
paratively slow rate in BSC-1 cells at 35°C.Whether a more rapid
cycle would occur in other cell cultures is not known. A prelimi
nary comparison of Yaba and vaccinia viruses synthesis in BSC-1
cells at 35°Cindicates that the difference in rate is a function of
the viral genome; the vaccinia infectious cycle is 4-6 times more
rapid (Yohn, I). S., Haendiges, V. A., and Grace, J. T., Jr., un
published information).
The cycle of other viruses of the pox group has been extensively
investigated. In the case of vaccinia virus, the 1st infective par
ticles appear 10 hr after infection (8, 11, 29). Tracing the com
plete intracytoplasmic cycle of vaccinia virus in L cells by electron
microscopy radioautography, Dales (9, 10) was able to confirm
that the complete cycle of the virus takes approximately 8-10 hr.
More recently, Appleyard and Westwood (3) have shown that
rabbit poxvirus begins to appear as soon as 5 hr after seeding
HeLa (ERK) cells. Febvre (20) found that the production of the
Shope fibroma virus by a line of sensitive rabbit testis cells starts
8 hr after infection, and Israeli and Sachs (28) observed a high
percentage of rabbit cells with viral intracytoplasmic inclusions
24 hr after infection with the Shope fibroma virus.
The chronology seems different for the Yaba virus. According
to Levinthal and Shein (32), typical immunofluorescence in
rhesus monkey kidney or in human embryonic kidney cells ap
pears only 7 days after inoculation. Yohn and de Harven (50),
studying the cytopathic effect of the Yaba virus in BSC-1 cells,
observed microscopic evidence of infection 5 days after inocula
tion with high doses of the virus. By immunofluorescenee and
histochemical methods, viral antigen synthesis first appeared 48
hr after inoculation of BSC-1 cells (49, 51).
These findings suggest that the late maturation of Yaba virus
observed by electron microscopy reflects the slow development
of Yaba virus in BSC-1 cells. Although this protracted synthetic
cycle appears to be a most striking difference between the Yaba
virus and other agents of the pox group, Dourmashkin (16) ob
served that the intracytoplasmic development of molluscum
contagiosum virus in HeLa cells also required 4-6 days.
In conclusion, it should be stressed that poxviruses illustrate
especially well the complex role of viruses in cancer. Indeed, poxviruses have been demonstrated as oncolytic (46), cocareinogenic
(17), or oncogenic. Oncogenic poxviruses have been demonstrated
in rabbits (21), in man (4, 24), and in monkeys (2). Typical pox
viruses have been also observed in a benign tumor of a bird (25).
In most of these cases the tumors produced are benign and spon
taneously regress. It is hoped that morphologic studies will make
possible a more precise classification of the poxviruses. Such in
vestigations may be valuable for further analysis of the intracyto
plasmic maturation of poxviruses, and of the complex role of
poxvirus in neoplastic growth.
Acknowledgmen
ts
We wish to express our gratitude to Dr. G. E. Palade, in whose
laboratory part of this work was done when one of us (E. de Har
ven) was a guest investigator at The Rockefeller Institute.
The skillful assistance of Mrs. C. Jamieson and Miss N. Lampen
in this electron microscope study is gratefully acknowleded, as
well as the assistance of Miss P. Baldwin in the preparation of the
manuscript.
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7. Bernhard, W., Bauer, A., Harel, J., and Oberling, C. Les
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Strain L Cells Followed with Labeled Viral Deoxyribonucleic
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11. Dales, S., and Siminovitch, L. The Development of Vaccinia
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13. de Harven, E., and Friend, C. Further Electron Microscope
Studies of a Mouse Leukemia Induced by Cell-free Filtrates.
Ibid., 7: 747-52, 1960.
14. de Harven, E., and Yohn, D. Electron Microscopy of the Yaba
Tumor Virus. J. Appi. Phys., 35: 3082, 1964.
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Etienne de Harven and David 8. Yohn
15. Dourmashkin,
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cope électronique du virus du "molluscum contagiosum."
Compt. Rend. Acad. Sci., 246: 3133-36, 1958.
16. Dourmashkin, 11., and Febvre, H. L. Culture in vitro sur des
cellules de la sourche HeLa et identification
au microscope
électronique du virus du molluscum contagiosum. Ibid., %lß:
2308-10, 1958.
17. Duran-Reynals,
M. L. Carcinogenesis in Cortisone-treated
Mice following Vaccinia Dermal Infection and Application of
Methylcholanthrene.
J. Nati. Cancer Inst., 29: 635-65, 1962.
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Mature Vaccinia Virus. Nature, 181: 784-85, 1958.
19. Erlandson, R. A. A New Maraglas, D. E. R. 732, Embedment
for Electron Microscopy. J. Cell Biol., 22: 704-709, 1964.
20. Febvre, H. The Shope Fibroma Virus of Habbits. In: A. J.
Dalton and F. Haguenau (eds.), Tumors Induced by Viruses;
Ultrastructural
Studies. New York: Academic Press, 1962.
21. Fenner, F. II. Classification of Myxoma and Fibroma Viruses.
Nature, 171: 562-63, 1953.
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of Vaccinia Virus. Science, 117:10-13, 1953.
23. — —. Intracellular Forms of Pox Viruses as Shown by the
Electron Microscope (Vaccinia, Ectromelia, Molluscum Con
tagiosum). J. Exptl. Med., 98: 157-72, 1953.
24. Grace, J. T., Jr., and Mirand, E. A. Human Susceptibility to
a Simian Tumor Virus. Ann. N. Y. Acad. Sci., 108: 1123-28,
1963.
25. Grégoire,A., L'Hardy, J.-P., and de Harven, E. Etude au
microscope électronique d'une tumeur
cutanée bénigne
associéeà un virus du groupe Pox chez un Passereau (Prunella
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26. Huxley, H. E., and Zubay, G. Preferential Staining of Nucleic
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1959.
30. Kajioka, R., Siminovitch, L., and Dales, S. The Cycle of
Multiplication of Vaccinia Virus in Earle's Strain L Cells. II.
Initiation of DNA Synthesis and Morphogenesis. Ibid., 24:
29,5-309, 1964.
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at High pH in Electron Microscopy. J. Biophys. Biochem.
Cytol., 11: 729-32, 1961.
32. Levinthal, J. M., and Shein, H. M. Propagation of a Simian
Tumor Agent (Yaba Virus) in Cultures of Human and Simian
Renal Cells as Detected by Immunofluorescence. Virology, 23:
268-70, 1964.
1000
33. Luft, J. H. Improvements in Epoxy Resin Embedding Meth
ods. J. Biophys. Biochem. Cytol., 9: 409-14, 1961.
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tron Microscope. II. Vaccinia and Fowl Pox Viruses. J. Exptl.
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36. Nagington, J., and Hörne, R. W. Morphological Studies of
Orf and Vaccinia Viruses. Virology, 16: 248-60, 1962.
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38. Noyes, W. F. Further Studies on the Structure of Vaccinia
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39.
. Observations on Two Pox-Tumor Viruses. Ibid., 25:
666-69, 1965.
40. Owens, G., Metzgar, R., and Grace, J. T., Jr. Ultrastructure
and Related Immunologie Characteristics
of the Yaba Virus.
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Their Derivatives. Biochem. Soc. Symp., No. 16, pp. 3-43,
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and Electron Microscopy; The Preservation of Cellular Ultrastructure and Enzymatic Activity by Aldehyde Fixation. J.
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45. Shatkin, A. J., Sebring, E. D., and Salzman, N. P. Vaccinia
Virus Directed RNA: Its Fate in the Presence of Actinomycin.
Science, 148: 87-90, 1965.
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49. Yohn, D. S., Grace, J. T., Jr., and Haendiges, V. A. Immunofluorescent and Histochemical Study of Yaba Tumor Virus
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50. - —. A Quantitative
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51. Yohn, D. S., and de Harven, E. Yaba Tumor Poxvirus Synthe
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CANCER RESEARCH VOL. 26
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Fine Structure of Yaba Monkey Tumor Poxvirus
"'
•
- v•
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FIG. 1.—Tumor cell with a large intracytoplasmic
viral inclusion (MF). PM, Plasma membrane;
A", nucleus. X 22,500.
MAY 19GG
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1966 American Association for Cancer Research.
1001
Etienne de Harven and David S. Yohn
FIG. 2.—Tumorcell showing accumulations of mature particles (MV) and "factories" (F). Cylinders are seen in longitudinal section
in C, and arrows indicate the same cylinders transversely or obliquely cut. Six inclusions of medium density are observed (7). R, ribosomes. X 24,000.
Fio. 3.—Tumorcell demonstrating various phases of viral development. Arrows indicate the presence of dense material in the con
cavity of crescent, like Type 1. PM, Plasma membrane; MV, accumulations of mature viruses; C, cylinders associated with viral fac
tories; K, ribosomes; /, inclusions associated with viral factories; /, Type 1; è,Type 2; 5, Type 5; 6, Tyoe 6. X 32,000.
Flo. 4.—Tumorcell showing the abrupt transition between normal cytoplasm containing endoplasmic reticulum (Eli) and the factory
area (F). Type 3 particles are of a somewhat smaller diameter than Type 2. Note the periodicity of the dense crystalloid inclusions
(CR) associated with the factory. The arrow indicates a nucleoid within a Type 3 particle. This nucleoid has the same electron density
as that of the crystalloid inclusions (CR). R, Ribosomes; 7, inclusions associated with viral factories; PM, plasma membrane; /, Type
1; g, Type 2; 6, Type 0. X 50,000.
FIG. 5.—Highmagnification of a Type 2 particle. The arrow points at the typical unit-membrane, coated with radially oriented subunits (SS). X 155,000.
FIG. 6.—Highmagnification of an inclusion (7) similar to those seen in Figs. 2-4, and composed of numerous circular and parallel
profiles suggesting the cylindrical form of the subunits. X 104,000.
FK¡.7.—Highmagnification of a crystalloid inclusion (CR) similar to those seen in Fig. 4. X 350,000.
FIG. 8.—Highmagnification of a longitudinal section of 2 cylinders (C) similar to those seen in Figs. 2 and 3. The wall of these struc
tures is made up of subunits, which are regularly disposed in honeycomb pattern (upper part of the picture), while the arrow indicates
longitudinally cut subunits resembling the surface subunits (SS) of Fig. 5. X 220,000.
FKÕS.9-14.—Hypothetical reconstruction of the complete intracytoplasmic maturation of the Yaba virus within tumor cells.
FIG. 9.—AType 1 crescent, a 2-dimensional image of a cupule-shaped immature virus. The unit-membrane structure of the viral
membrane is clearly seen. The convex surface is covered by spiny projections, and material denser than the background accumulates
in the concavity of the cupule. X 110,000.
FIG. 10.—Type2. It is a spheroidal structure which corresponds to the completion of Type 1. The unit-membrane is well defined.
The particle is coated with spiny projections on its entire surface and is filled with material of finer texture and higher density than
the surrounding matrix. X 110,000.
FIG. 11.—Type3. The diameter of the particle is smaller than that of Type 2 particles. The spiny coat is still present, although less
distinct than in Types 1 and 2. A finely granular material of particularly great electron density fills the entire virus. This material is
still denser at the periphery, against the inner face of the viral membrane (arrow). A group of ribosomes (R) is seen in the tipper left
corner. X 124,000.
FIG. 12.—Theparticle shown, representing Type 4, still has a spiny coat (SS) and a visible unit-membrane. A line of higher density
(arrow), comparable to the one seen in Fig. 6, is apparent. The particle has an elongated shape, and inner membranous structures have
appeared (crossed arrows) exactly in its long axis. Although these structures are not well defined at this stage, they seem to correspond
to a flat sac with a maximum density at its inner surface. X 140,000.
FIG. 13. Type 5. The general shape is more elongated than Type 4, and the spiny coat has vanished. The inner sac is opening up,
giving its typical dumbbell shape to the inner core and displacing the dense matrix of the particle which now fills in 2 lenticular spaces
on both sides of the dumbbell-shaped core. The arrow points at the dense line already seen in Figs. 11 and 12. X 130,000.
FIG. 14.—Type6, the fully mature particle like those seen in Fig. 1. The only difference between Types 5 and 6 is the addition of
an external envelope which is again double. The origin of this envelope was not apparent in our material. G, Glycogen. X 180,000.
FIG. 15.—BSC-1tissue culture cell, 5 days after infection with the Yaba poxvirus. Types 1, 2, 3, and 5 particles are scattered through
the cytoplasm. Ar, Nucleus. X 57,000.
1002
CANCER RESEARCH VOL. 26
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VOL. 26
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VOL. 26
Fine Structure of Yaba Monkey
MAY 1966
Tumor Poxvirus
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Etienne de Harven and David S. Yohn
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CANCER RESEARCH VOL. 26
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The Fine Structure of the Yaba Monkey Tumor Poxvirus
Etienne de Harven and David S. Yohn
Cancer Res 1966;26:995-1008.
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