[CANCER RESEARCH 32, 1340-1350,
June 1972]
Morphological Studies on Herpes virus sylvilagus in Rabbit
Kidney Cell Cultures1
Ursula Heine and Harry C. Hinze2
National Cancer Institute, Bethesda, Maryland 20014 [U. H.J, and Department of Medical Microbiology, University of Wisconsin, Madison,
Wisconsin 53706 [H. C. H.J
SUMMARY
Electron microscopic studies have revealed early stages of
Herpesvirus sylvilagus development
in the nuclei of the
infected
cells. Immature
virions are similar in their
morphology to known herpesviruses. The development of the
naked particles occurs either at nuclear or at preformed
cytoplasmic membranes, giving rise to two kinds of mature
virions characterized by morphologically different outer coats.
Cellular response to the infection is expressed by the
appearance of nuclear inclusion bodies and a few lysosomes in
the cytoplasm.
INTRODUCTION
H. sylvilagus was found recently as an indigenous agent in
cottontail rabbits (12). Studies on its physical, chemical, and
biological properties indicate that it belongs in the herpesvirus
group; due to its host specificity and antigenic characteristics
it was recognized as a new member of this group (14). It
stimulates the growth of lymphoid organs in cottontail rabbits,
resulting mainly in a generalized hyperplasia of lymphoid
elements, but in some instances malignant lymphomas have
been observed (13).
The maturation
of the virus in its host cells and
morphological changes in these cells due to the infection with
the virus have not been reported thus far. In the present study
we give a detailed report describing the life cycle of the virus
in its host cells and the morphological alterations that are
caused by infection with H. sylvilagus.
from the kidney cell culture of a naturally infected cottontail
rabbit was used for infection. The virus was inoculated into
monolayers of DRK-3 cells in a concentration of 100 to 200
plaque-forming units/tissue culture dish, and the cells were
incubated for 4 to 6 days. The virus was identical with the
original isolate (CHV-1) by cross-neutralization, fluorescent
antibody staining, growth in culture, and the pathology
produced in experimentally infected cottontail rabbits.
Electron Microscopy. The monolayers containing foci of
infected and rounded cells were fixed in situ with 3% buffered
glutaraldehyde (20), rinsed with and stored in Sorensen's
buffer, postfixed with chrome-osmium (5), and embedded as
monolayers in Epon-Araldite (17). To obtain ultrathin
sections, selected areas containing foci of infected cells were
cut with an LKB Ultrotome, double stained, and examined
either in a Siemens Elmiskop 101 or 1A electron microscope,
equipped with double condenser and 50-jum objective ap
erture; an accelerating voltage of 60 or 80 kV was used.
RESULTS
The monolayers of DRK-3 cells consist of a homogeneous
sheet of epithelioid cells in which numerous small foci
(microplaques) of usually not more than 30 to 100 round,
highly refractile cells are present. These round cells easily lose
contact with the plastic tissue culture dishes thus giving rise to
small holes in the monolayers which first can be detected in
the center of the foci.
The majority of the cells studied and described here are
those round cells found adjacent to the lacunae.
Nucleus. As usually observed in herpesvirus-infected cells
MATERIALS AND METHODS
the chromatin aggregates as an irregular layer of high electron
density adjacent to the nuclear membrane. The nucleolus is
Cell Cultures. A cloned subline of diploid New Zealand rather small, composed of a spherical body of granular
White rabbit kidney cells (DRK-3) was used for infection. The material also with high electron density (Fig. 1). In contrast,
cells were grown as monolayers in Falcon plastic tissue culture
the interior of the nucleus is relatively homogeneous, only
dishes (60 x 15 mm) and maintained as previously described
lightly stained, except for the presence of an inclusion body of
(14).
low to medium density (Fig. 1). The matrix of these inclusion
Virus. The 2nd isolate of H. sylvilagus (CHV-2) obtained
bodies is made up of fine fibrils. In addition, small spherical
shells of about 40 nm in diameter (as can be seen in Fig. 7) are
1Part of this work was sponsored by the Fogarty International
numerous throughout the inclusion bodies and throughout the
Center and Institute de la Santéet de la Recherche Médicale,
and was matrix of the nuclei. These shells have been found previously
carried out at the Collège de France, Laboratoire de Médecine in different cells infected with herpesvirus (9, 10, 18). Their
Experimentale.
importance is still obscure.
3Whose portion of this work was supported by USPHS Grant
Early forms of virus assembly are infrequent. These are
CA-10395 from the National Cancer Institute.
Received February 15, 1972; accepted March 15, 1972.
partially bilaminar structures characterized by an incomplete
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Ultramorphology ofH. sylvilagus in Cultured Cells
shell surrounding a shell of low density (Fig. 1, inset). Three
types of immature virions are always present in the inclusion
bodies. One form consists of a shell of low density, often
containing a tiny dot of osmiophilic material, surrounded by
another more osmiophilic shell. These particles are present in
different, mostly large amounts in the inclusion bodies (Fig.
2). The 2nd type of immature particles, shells containing dense
cores (Fig. 2, short arrow) are rarely seen. The same is true for
the 3rd type^-comprising incomplete virus particles consisting
of only 1 single shell (Fig. 2, long arrow). Naked particles are
often so numerous in the nucleus that they are arranged in
paracrystalline arrays.
Although the majority of the inclusion bodies in infected
nuclei are composed of very fine fibrillar material we observed
a few consisting of relatively dense, thick, and long fibers, as
can be seen in Fig. 3. The diameter of these fibers is about 15
nm. In some areas of the inclusion bodies and frequently in
close contact with naked virions, tubules, or rods of different
length are present (Fig. 3 and inset). Often they reach a length
of approximately 3 urn. Their diameter is the same as that of
the immature virions, 100 to 110 nm, and they are composed
of 2 electron-dense layers, one surrounding the other. In
addition,
tubules with only 1 envelope were found
infrequently. They contain stacks of a material with an
appearance similar to that of the inner virion shells (Fig. 4).
Immature virions lacking the outer envelope accumulate
often near the nuclear membrane and are dispersed between
the dense aggregates of chromatin (Fig. 5). At this stage
bundles of dense fibrils are frequent in the nucleus and in the
cytoplasm (Fig. 5). Unlike other herpes-type virus-infected
cells, the nuclear membranes do not form extensive
proliferations,
sheets, and tubules, protruding into the
cytoplasm but, as can be seen in Fig. 6, the nuclear membrane
buds into the nucleus, giving rise to numerous vacuole-like
inclusions containing small amounts of cytoplasm including
ribosomes. Sometimes mature virions have been found in these
vacuoles (Fig. 6, inset).
Incomplete virions, composed of cores and capsids, may
obtain their envelope by budding through the nuclear
membrane. Infrequently, enveloped particles can be found in
clusters in vacuoles inside the nucleus (Fig. 7). These particles
possess electron-dense cores of a diameter of about 65 nm, and
they exhibit a distinct capsid with a diameter of 100 nm,
which is recognizable due to the electron-lucent area between
the capsid and the envelope (Fig. 6, inset). The envelope is
distinct, and the enveloped particle is 130 nm in diameter.
Cytoplasm. In late stages of the infection morphological
changes also take place in the cytoplasm. As can be seen in
Fig. 8, in the area of the cytocentrum, thick membranes
arranged in repetitious, onion-like layers are common. At
higher magnification (Fig. 9), it is evident that these
membranes are formed by the "back to back" fusion of parts
of the smooth endoplasmic reticulum and, possibly, parts of
the Golgi apparatus. In rare instances, a similar membrane
convergence can be seen at the nuclear membrane (Fig. 8,
arrow), where folds of the inner leaflet of this membrane form
a comparable structure.
Lysosomal bodies are frequent during late stages of
infection. The appearance of large poly ribosomes as shown in
Fig. 10 was frequently observed in late stages of the infection
(7).
Virions, those of the immature type, are found in close
contact with arrays of dense membranes in the cytoplasm
(Figs. 11 and 12), and infrequently the budding of an
immature particle through these membranes was observed
(Fig. 11, arrow). Due to this process naked particles obtain a
dense outer coat and can be distinguished easily from those
formed at the nuclear envelope (compare Figs. 11 and 12 with
Figs. 6 and 7). Virions enveloped at cytoplasmic membranes
are larger than those found in the nucleus. The core has a
diameter of approximately 90 nm, the capsid has one of 125
nm, and the enveloped virion has a diameter of about 200 nm.
In addition, immature, unenveloped particles are often seen in
close contact with lysosomal bodies (Fig. 12).
Fine Structure of the Mature Virions. The mature virions
possess a large, electron-dense core; a capsid of medium
electron density; and an outer envelope consisting of a unit
membrane. Two forms of the mature virions must be
distinguished. As shown above, particles maturing at the
nuclear membrane contain an electron-lucent space between
capsid and envelope. These particles have been observed only
inside the nucleus. The 2nd group, which is the larger one,
comprises those particles that are released from cytoplasmic
membranes. They possess a dense layer between the capsid and
the enveloping unit membrane. The width of this layer may
vary considerably in different areas of the virions (Fig. 13). In
addition, a fuzzy coat is frequently present on the surface of
the virions (Fig. 13). In the extracellular spaces as shown in
Figs. 13 and 14, only particles of the 2nd group can be found.
On rare occasions, virions enveloped by 2 onion-like
membrane layers could be seen (Fig. 14). The possibility exists
that these double envelopes may be formed from cytoplasmic
membranes which underwent "back to back" convergence in
the cytoplasm of the infected cells.
Virions in extracellular spaces are not only pleomorphic but
often appear to be damaged and exhibit broken coats. As a
result, a loss of cores was frequently observed in these virions
(Fig. 14).
DISCUSSION
As shown by Hinze (14) the virus under study belongs to
the herpesvirus group because of its physical, chemical, and
biological properties. Many of the morphological criteria, i.e.,
the mode of multiplication, the size and structure, confirm the
virological results; but it is necessary to emphasize that this
virus in its relationship to the host cell possesses morphological
characteristics and induces host cell reactions which make it
possible to distinguish it easily from other known viruses in
the herpesvirus group.
As is known for other herpesviruses (2, 4, 6, 10, 18, 19, 21,
22), early stages of H. sylvilagus development are found in the
nuclei of the infected cells, and a comparison of the
intranuclear forms among different herpesviruses with those
described here for H. sylvilagus shows their similarity.
Immature virions accumulate in areas designated inclusion
bodies. In cells infected with H. sylvilagus these bodies are
similar to those found in Herpesvirus saimirÃ-virus-infected
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1341
Ursula Heine and Harry C. Hinze
cells (10). Their morphology at the level of the electron
microscope is distinctly different from the classical inclusion
body known to be present in cytomegalovirus-infected cells.
The latter consists of electron-dense fibrillar material of
characteristic skein-like formation (11, 16). The inclusion
body provoked by H. sylvilagus is less electron dense.
Immature cytomegalovirions are always found at the periphery
of the skein-like inclusions, but virus particles in H.
sylvilagus-infecled
cells are distributed
throughout
the
inclusion body, sometimes even in paracrystalline arrays.
In addition, we observed tubules or rods in close association
with immature virions visible in the nuclei, and it is
emphasized that their diameter corresponds with the size of
the naked virions. Similar tubules have been seen in cells
infected with herpes simplex virus (19). The composition of
these structures is unknown; but, due to their appearance, it is
reasonable to conclude that they may be composed of the
same material as the naked virions. Possibly, by some
unknown factors, the assembly of part of the viral precursor
material may be malfunctioning, giving rise to these aberrant
structures.
In herpes simplex-infected cells, the proliferation of the
nuclear membrane is extremely pronounced resulting in large
sheets and tubules of membranous structures throughout the
cytoplasm (19). Reduplication of nuclear membranes is also
common in varicella-zoster-infected cells and this membrane
material as well as the nuclear envelope proper might be used
to produce the outer envelope of the virus (4). Similar sheets
of membranes were not observed in H. saimiri-infected cells
(10). Also in the material studied here this form of
proliferation of the nuclear membranes only rarely takes place.
One of the unique differences of this virus in comparison to
the well-known herpes simplex virus is the budding of the
nuclear membranes into the nuclear matrix thus giving rise to
numerous vacuole-like bodies inside the nucleus.
These vacuoles may contain a single enveloped virion in any
given section and it is possible that the virus envelopment
takes place during the formation of the buds. However, here,
as already described for herpes simplex (19), the envelopment
probably occurs so fast that we have been unable to
demonstrate the process. In addition, large vacuoles containing
mature virions as well as cytoplasmic blebs are common in the
infected nuclei. Also here, the nuclear membrane seems to be
actively involved in producing viral envelopes.
In previous reports, it has already been pointed out that the
outer coat of the immature virions in the herpes group can also
be acquired at cytoplasmic membranes (3, 8, 19). We observed
the same in our material. Budding through fused cytoplasmic
membranes of the smooth endoplasmic reticulum or, possibly,
the Golgi zone was seen infrequently; and in contrast to
virions enveloped at the nuclear membrane, the virus particles
released from these cytoplasmic structures have a heavy outer
coat. They are also considerably larger than the particles found
inside the nuclei. Only these particles have been observed in
extracellular spaces.
Cook
and Stevens
(4) have already
emphasized
morphological differences between mature intranuclear and
cytoplasmic varicella-zoster virions and conclude that the
morphologically different and damaged particles found outside
1342
the nuclei are vulnerable to extranuclear degradation, thus
rendering them noninfectious. Likewise, Nazerian et al. (18),
in their studies on a herpesvirus in turkeys, and Ahmed and
Schidlovsky
(2), in observations
on Marek's disease
virus-infected cells, described 2 morphologically distinct types
of enveloped virions. They too emphasize that the difference
in appearance of the mature virions may be due to the
different origin of the envelope which may be either
cytoplasmic or nuclear.
Stackpole (22), in discussing the herpes-type virus of the
frog renal carcinoma, raises the possibility of a relatively
different infectivity depending on the source of the viral
envelope.
Ablashi et al. (1) recently increased the titer of//, saimirÃ-by
a factor of IO5-5; they did this by freezing and thawing the
host cells. Under the same circumstances cytomegalovirus
shows a reduction in titer, whereas herpes simplex virus was
not affected at all. Previously, we have shown (10) that H.
sa/m/ri-infected cells also contain 2 morphologically distinct
mature kinds of virus particles: contained within the nucleus
are those particles with an electron-lucent area around the
core; in the cytoplasm and in extracellular spaces are those
with electron-dense and heavy outer coats. Also here, it is
reasonable to consider that 2 morphologically distinct viral
entities may exhibit different biological activities.
It has been shown that H. sylvilagus is highly cell bound
(15) and less than 1% of the replicating virus is released into
the supernatant medium. The data on different herpesviruses
are consistent with the suggestion that the H. sylvilagus virions
with heavy coats may be noninfectious, whereas those mature
particles found in the nucleus may be the replicating and
infectious entities.
At present, the composition of the dense viral coat is
unknown. The material might be of proteinaceous nature since
it is found in close association with the endoplasmic reticulum,
however, it might either represent a virus-specific antibody or,
more likely, it might be composed of material also present in
lysosomal bodies. Its morphological similarity to material
accumulating in lysosomes as shown in ultrathin sections
supports the latter view. As discussed previously (22) this
material might then represent a more general cellular defense
mechanism against the virus. Either view could represent an
explanation for the possible inactivity of the cytoplasmic and
extracellular virions, which are coated with this material.
In 1965, Becker et al. (3) stated, "The fact that the
infective group B herpes viruses are avidly cell-associated does
not appear to have a simple morphologic solution." In some
respects, this statement still appears valid. However, the recent
morphological studies of the cell-associated, tumor-producing
DNA viruses have produced evidence which has contributed to
the better understanding of the complicated events that govern
the infectivity process.
ACKNOWLEDGMENTS
The authors are deeply indebted to Dr. F. Haguenau, College de
France, for her generous cooperation and support which greatly
facilitated this work.
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Ultramorphology of H. sylvilagus in Cultured Cells
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'•
"
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1343
SLTj
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-->
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-
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V
Fig. 1. DRK-3 cells infected with H. sylvilagus. Early stage of infection with inclusion body (IB) containing few immature virions (arrow).
Nucleolus (N) is small and of high electron density. X 17,500. Inset, immature and partially formed virions are present in the inclusion body during
early stage of infection. The virions consist of 2 shells, the outer one often not yet completed (arrow), and frequently contain a minute
electron-dense dot. X 70,000.
Fig. 2. Inclusion body with numerous naked virions. Three different types are visible: double shells, single shells (longarrow), single shells with
dense center (short arrow). X 38,000.
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.
AST
Fig. 3. Part of an inclusion body consisting of relatively thick, long, and dense fibrils. Double-shelled virions are in close proximity to the fibrils.
Chromatin (CH) aggregates at lower left. A short rod (arrow) is near the naked virions. X 84,000. Inset, long rods composed of 2 electron-dense
layers are frequently present in the inclusion bodies. Their length may reach 3 pm and their diameter may reach 100 to 110 nm, corresponding to
that of naked virions. X 35,000.
Fig. 4. Short tubules with a single envelope are filled with material resembling the inner virion shells cut tangentially. These structures are
frequently observed in different areas of the inclusion bodies in the presence of numerous naked virions. X 75,000.
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1972
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1345
P >-,.->.
",...• •¿'«
•¿â€¢*;VX..-
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Fig. 5. Naked virions are amassed between the dense chromatin near the nuclear membrane (arrows). More immature virions are present at the
edge of the large inclusion body (IB) that fills the major part of the nucleus. Dense aggregates of fibrils (F) are in the cytoplasm and in the nucleus.
X 21,000.
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;M 7
•¿rraüss
Fig. 6. Intensive budding of the nuclear membrane into the nucleus gives rise to numerous vacuoles in the nuclear matrix. Many of these
vacuoles contain cytoplasmic blebs including ribosomes. Few contain mature virions. This process starts at certain areas of the nuclear membrane
which are recognizable due to the thickening and slight invagination of the membrane (arrows). X 28,000. In the inset, the 3 components of the
mature virions (C, core;/, capsid;£, envelope) are clearly distinguishable due to the electron-lucent space between envelope and core. X 85,000.
Fig. 7. and inset. Accumulation of cytoplasmic blebs and mature virions (arrows) in vacuoles inside the nucleus. Virion envelopes are probably
supplied by the nuclear membrane. Small shells of about 40 nm diameter (heavy arrow) are present. X 48,000.
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1347
nub
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:
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Fig. 8. The presence of thick membranes (arrows) in the area of the cytocentrum and the appearance of lysosomes (L) are the most outstanding
changes in the cytoplasm during the later part of the infection. X 16,000.
Fig. 9. Higher magnification of part of Fig. 8. The electron-dense membranes are formed by "back to back" fusion (arrows) of lamellae of the
endoplasmic reticulum and, possibly, the Golgi apparatus. X 49,000.
Fig. 10. Cytoplasm of a DRK-3 cell infected with H. sylvilagus. Polyribosomes of unusual length (arrows) are common in these cells. X 50,000
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Fig. 11. Budding of immature virions at the thickened endoplasmic lamellae in the cytoplasm (long arrow). Thus the virions acquire a heavy
outer coat (short arrow). X 63,000.
Fig. 12. Immature virions are in close contact with a lysosome (short arrows) and adjacent to a thick cytoplasmic membrane (long arrow). Two
apparently mature virions (V) are inside the cytoplasm. X 63,000.
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1349
Fig 13. and inset. Mature virions in the extracellular space. Electron-dense cores (O, slightly stained capsids (/), and the outer envelopes (E) are
recognizable. The space between capsid and envelope is filled with a homogeneous, electron-dense matrix. A fuzzy coat is present on the surface of
some of the virions (arrows). X 80,000; inset, X 100,000.
Fig. 14. Plcomorphic virions in the extracellular space. One particle is enveloped by 2 onion-like membrane layers (V). Other virions have
broken envelopes and have lost their internal components (arrows). X 70,000.
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Morphological Studies on Herpesvirus sylvilagus in Rabbit
Kidney Cell Cultures
Ursula Heine and Harry C. Hinze
Cancer Res 1972;32:1340-1350.
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