Microbiol.
Immunol.
Vol. 21 (10), 593-600, 1977
Electron
Microscopic Study of Hemadsorption on Vaccinia Virus Infected Cells
Yasuo SABURI,1Kenji OKUDA,2Toyozoh TAKAHASHI, and Ichiro TADOKORO YokohamaMunicipal Institute of Health, Yokohama,and Departmentof Bacteriology, YokohamaCity UniversitySchoolof Medicine, Yokohama (Received for publication, November 26, 1976)
Abstract Hemadsorption (HAD) induced in HEp-2 cells infected with vaccinia virus was observed. In ultrathin sections, binding of 36 red blood cells (RBCs) was examined in detail and 3 types of HAD were observed : (1) direct and close binding of RBCs to infected HEp-2 cells (cyto-HAD) was observed in cross sections of 27 RBCs, (2) binding of RBCs through microvilli of infected cells was found in 11 RBCs, and (3) five RBCs were distorted to form tentacle-like projections by which they were bound to the HEp-2 cell surface. Scanning electron microscopy revealed that more than 30% of the RBCs were bound to microvilli of vaccinia virus-infected HEp-2 cells, and that the number of microvilli twined round each RBC was over ten. RBCs were attached to certain microvilli through swollen sucker-like tips which were not observable in non-infected HEp-2 cells. RBCs sometimes revealed a polygonal shape at regions of binding to microvilli. Virion-mediated RBC-HEp-2 cell binding could not be observed.
Since hemadsorption (HAD) phenomenon was described by Vogel et al (14), several authors observed the ultrastructure of binding of erythrocytes to virus-infected cells (4-6, 13). The development of HAD was shown to be a dynamic process, with binding of erythrocytes mediated through the microvilli of host cells (11). The purpose of the present study, utilizing light and transmission and scanning electron microscopy, is to elucidate the ultrastructure of the binding between vaccinia virus-infected cells and adsorbed red blood cells (RBSs), with special reference to the role of microvilli in HAD. MATERIALS
AND
METHODS
Cells and virus. HEp-2 cells were cultured in Earl's balanced salt solution containing 0.5% lactalbumin hydrolysate, 0.1% yeast extract, 100 units/ml penicillin and 0.1 mg/ml streptomycin (hereafter abbreviated as YLE medium) supplemented 1 Present address: Department of Virology , Okayama University Medical School, Shikatacho, Okayama 700, Japan. 2 Present address: Department of Genetics , Washington University School of Medicine, Missouri 63110, U.S.A. 593
594
Y.
SABURI
ET AL
with 10% calf serum. After virus inoculation the medium was changed to YLE medium supplemented with 2% calf serum (maintenance medium). DIE-25 strain of vaccinia virus, which was kindly donated by Dr. T. Kitamura, the National Institute of Health, Tokyo, Japan, was used throughout these experiments. The virus stocks were prepared by inoculating chorioallantoic membranes of 10-day-old embryonated hen's eggs with seed virus diluted to 10-4. Three days later the membranes wereh arvested, homogenized with glass powder in a mortar, and 30 ml of phosphate-buffered saline (PBS) (m/100 phosphate, 0.8% NaCl, pH 7.2) was added before centrifuging at 590 g for 20 min. The supernatant fluid was diluted 1:20 with YLE medium and stored in a freezer at -70 C. The 50% tissue culture infective dose for such stocks was (TCD50) =107.5/ml when titrated in HEp-2 cell cultures. HAD. added
For
to
paration in
of
a
at
culture
was
PBS.
The
layers
RBC
to
the
adsorbed
remove
cells
were
to to
stock
per
PBS make
and
of
up
then
examined
3
37
pre-
for ml
hr
at
per 37
C.
suspension,
the
volume
C.
The
of maincell
with
light
cells
10.0
a 0.5%
times by
was
For HEp-2
17-20
into
at
virus
incubation and
about
made
1 hr
washed
of
After
one-tenth
were
stock tube.
tube,
for and
for
the
culture
virus.
Leighton
adsorb
erythrocytes
ml
a Leighton
reincubated
added
RBCs
of
with
allowed
unattached
ml ml
3 times was
RBCs
in
a monolayer
0.4 (1.0
washed
0.1
prepared
microscopy, with
the
microscopy,
slips
medium
suspension
and
containing
medium
electron
were
medium
cover
inoculated
added,
erythrocytes
tenance
for
was
electron
on
maintenance
bottle)
Chicken in
C,
scanning
cultures
sections
bottle
37
and
cell
thin
culture
1 hr
light
HEp-2
mono-
maintenance
and
electron
mic-
roscopy. For
studying
removed
from
(1 :3,000
dilution).
was
hemadsorption a
culture The
resuspended
RBC
in
suspension
incubated
for
mounted
1 hr
with
fixed drated
2.5%
coated
graded by
with
carbon
and
HEp-2
The finally
formed and scopes
electron
cell
5 min. and
studied
of
the light
gold,
and
infected
for
5 min;
milliliter
cells
cover
in
1.0%
alcohol
and
was
placed
slips
in
osmium
which
drying
observed
x g
of
were in
PBS
the
pellet
the
0.5%
the
mixture
on
a
was
glass
slide,
microscopy. on
of point
590
mixture
cultures
post-fixed
critical
cells acid
One-half
of
by
Cell
the
at
medium.
drop
virus-infected
was
method. with
a
Leighton
tubes
tetroxide
and
followed
The
by
scanning
specimens
were dehyisoamyl
were
electron
then
microscope
HSA-2A).
Transmission an
and
spun
suspension
One
vaccinia
ethylenediaminetetraacetic
was
concentrations
prepared
(Hitachi,
C.
the
microscopy.
and
cells, of
maintenance
glutaraldehyde,
through
acetate,
single 5 ml
suspension
to
slip
electron
with
37
a cover
Scanning
of
added at
with
cell
5 ml
was
on
bottle
pellet
a
citrate,
(Hitachi,
microscopy.
was
scraped
of cells
embedded
with lead
sheet
in
was an
Porter-Blum the HU-12A
After off
from
dehydrated Epon-araldite
microtome. specimens and
were HS-6).
fixing a
by
culture
the bottle
through
graded
mixture.
method and
double
observed
with
at
concentrations
Ultrathin
After
described pelleted
staining transmission
of
sectioning with
above, 590 •~
g for
alcohol
was
per-
uranyl
acetate
electron
micro-
HEMADSORPTION
ON VACCINIA-INFECTED
CELLS
595
RESULTS
HAD Observedwith a Light Microscope Unstained preparations of RBCs adsorbed by infected HEp-2 cells were examined with a light microscope. About one-half of the cells adsorbed numerous RBCs i.e., were HAD+, while the other half was HAD- (Fig. 1B). Each HAD cell contained from a few to 50 or more RBCs. When adsorbed RBCs were widely scattered on the cell, binding occurred predominantly in parallel to the long axis of the RBC (Fig. 1A). When RBCs were tightly packed at the cell periphery, binding was perpendicular to the long axis of the RBC (Fig. 1B). It seemed that there was few marked deformation of RBCs in our experimental conditions. HAD Observedwith a ScanningElectronMicroscope That HAD was mediated by microvilli appeared likely from the results of scanning electron microscopic study (Fig. 2). In some fields, RBCs were found to be attached to sucker-shaped tips of microvilli (Fig. 3), while in other fields they were attached to non-swollen ones (Fig. 4). Also, some deformed RBCs, which were probably caused artificially, were often pointed at the site of attachment to microvilli (Fig. 5). The opposite side of the view shown in Fig. 5 was inadvertently obtained in which we observed the surface of an RBC covered by more than 10 microvilli that had emerged from a projection of the host cell (Fig. 6). A similar observation was recorded in Fig. 7, for one of two RBCs adsorbed to a projection of an infected HEp-2 cell. About 11 microvilli were twined round the RBC surface. HAD Observedwith a TransmissionElectronMicroscope Adsorbed RBCs were observed in ultrathin section. A number of mature virions were seen in the cytoplasm. We did not observe RBC binding to virions budding from the cytoplasmic membrane or microvilli as is characteristic for HAD observed in cells infected with para- or ortho-myxoviruses (3, 6). Our observations revealed that RBCs were adsorbed most frequently to a single process from the cell, but in some instances they attached through several binding processes (Figs. 8, 9, 10, and 11). RBCs were closely attached to host cell membranes in 27 of the 35 RBCs examined at a high resolution, with an electron-lucent gap of 10-40 nm between the membranes of the HEp-2 cell and RBC (Fig. 12). No significant morphological changes were recognized on the cytoplasmic membrane of the host cell. The contour of the RBC was smooth, while that of the HEp-2 cell tended to be wavy (Fig. 12). Typical microvillus-erythrocyte binding was observed (Figs. 8 and 9) in which RBCs were attached to numerous long microvilli of the cell, and in agreement with Duc-Nguyen (5) HAD mediated by tentacle-like processes was also seen (Fig. 11).
596
Y. SABURI
1-A
FT AI
1-B
2
4
3 Figs.
1-4
HEMADSORPTION
ON VACCINIA-INFECTED
CELLS
597
DISCUSSION
Our data demonstrate that vaccinia virus-induced HAD, which occurs through either microvilli or "cyto-HAD," is similar to myxo- and para-myxovirus-induced HAD as already reported (6, 11). It remains obscure at present what is meant biologically by the relation of the long axis of adsorbed RBCs to the infected cell as observed by light microscopy (Figs. 1A and 1B). We could not observe "virus-HAD" of the type described by Hotchin et al (6) for influenza virus-infected cells. This negative finding is in keeping with the maturation process of vaccinia virus, namely that the virion does not contain hemagglutinin (2) and hence does not produce any HAD+ virions protruding from the cell surface as is the case with myxovirus HAD (6). However, we did observe "cyto-HAD" (6) in most fields of the ultrathin section preparation. The presence of less electrondense gap between the RBC and host cell had been reported (1, 5, 8). Although the low electron-dense gap was observed in several fields, a series of dense lines bridging between the cell membrane and RBC reported by Compans et al (4) was not found. It was already reported that adsorption of a single RBC might involve binding through the tips of about 3 or 4 HAD+ microvilli containing mature Newcastle virions (11). In vaccinia-infected cells we observed that about 10 microvilli were twined each RBC, not only through their tips, but also throughout their projected length. In addition, the tips of microvilli sometimes swelled, producing a suckerlike attachment to the RBC (Fig. 3). Stokes (12) recently reported that vaccinia viruses were observed to escape from the host cell individually from the tips of microvilli and sometimes appeared to be enclosed by the host membrane-like sheath. These observations might suggest the mechanism of sucker-like swelling of the tips of microvilli at the attaching site of RBCs. Mannweiler and Rutter (10) reported Fig.
1A. with in
Fig.
their Fig.
Light
1B,
1B.
are
Light
RBSs
2.
attach
RBCs.
Bar
3. rovilli
their
:
the
cell.
Note
degree RBCs
that
the
of HAD
the
of a HEp-2
cells
(RBCs),
RBCs
attach
of infected
end virus.
to
with the
cell
infected
contrasting to
of hemadsorption
cell
microvillus-erythrocyte
and
HEp-2
those
HEp-2
Unstained.
micrograph
HEp-2
(HAD)
blood
as compared
longitudinal with
red
the
to
HEp-2
those cell
by
: 20 ƒÊm.
of a high
electron to
in
cell.
Bar
cells. Fig.
One
1A.
Another
HEp-2
Note
HEp-2
cell
that cell
most (upper
: 20 ƒÊm.
of an
infected bindings
HEp-2
cell.
About
are
seen
at
least
that
the
tips
of
the
tips.
30 in
magnification like
a sucker
of the and
area
indicated
in
RBC-HEp-2
cell
binding
Fig.
2.
Note
is mediated
through
2 micBar
1 ƒÊm. Fig.
4. of an in
Fig.
Scanning infected 3.
11
10 ƒÊm.
Higher swell
Bar
infected
of hemadsorption
of chicken
HEp-2
numerous with
be
Scanning attach
the
Unstained.
micrograph
not
degree
number
to
adsorbed
might
RBCs
of a low
A small
adsorbed
sides.
right)
of the left)
Fig.
virus.
lateral
(lower
Fig.
micrograph
vaccinia
electron host
Bar
cell
: 2 ,ƒÊm.
micrograph attach
onto
of HAD the
surface
of an of an
infected RBC.
HEp-2 Their
tips
cell. are
Several not
swollen
microvilli as those
:
598
Y. SABURI
ET Al
5
6
7
8
Fig.
5.
RBCs
Scanning are
pulled
electron
micrograph
through
microvilli
of HAD
of an
in several
infected
areas,
HEp-2
resulting
cell
in polygonal
monolayer. shapes.
The Bar:
10
μm. Figs.
6 and
7.
projection Fig.
6.
Ten
Fig.
7.
About
RBC
Scanning
of
(upper),
an
or
electron
infected
more 10
microvilli
microvilli
however,
micrographs
HEp-2 are are
of the
reverse
side
of the
RBCs
adsorbed
on
a
cell. visible
seen
attaches
twined directly
on
the round
to
surface the
of surface
a projection
the
RBC.
of the of
an
Bar: RBC
infected
5 ƒÊm. (lower). HEp-2
The cell.Bar:
other 2
μm. Figs. Fig.
8-12. 8.
Electron Note
infected
micrographs HEp-2
of cells
ultrathin
adsorbing
sections. an
RBC
by
6 microvilli.
Bar:
1 ƒÊm.
that erythrocytes were bound to HeLa cells infected with mumps virus by either short cell processes or by several small protuberances. Uno et al (13) also showed that several microvilli of monkey cells infected with influenza virus twined round chicken RBCs, using a scanning electron microscope. The results are similar to Fig. 4 of this paper. In vaccinia-infected cells RBCs were most frequently adsorbed by two modes; cyto-HAD and microvillus-erthrocyte binding. If some external force is exerted on RBC adsorption represented by these two modes of binding, the stronger of the two
HEMADSORPTION
ON
VACCINIA-INFECTED
9
10
11
12
Fig.
9.
Two
HEp-2
cell,
Fig.
10. of
11. seen
but
The
brane Fig.
RBCs
the
(lower are
HEp-2 host
cell cell
Two
RBCs
(arrows).
Bar:
right)
do
by
many
bound has
2 RBCs
Bar:
and
attach
closely
to
Bar:
2 ƒÊm.
microvilli.
adsorbed
(arrows). (left
not
which
CELLS
the
attach
cytoplasmic
closely
to
599
membrane
the
of the
cytoplasmic
mem-
2 ƒÊm.
right)
are
moderately
distorted
and
tentacle-like
portions
are
2 ƒÊm.
Fig. 12. High magnification of an attachment zone between the cytoplasmic membranes of an HEp-2 cell and RBC. An electron-lucent gap of 10-40 nm in width is shown (arrows). While the contour of the RBC is smooth, that of the HEp-2 cell is irregular. Around the nucleus of the RBC a perinuclear cisterna is seen. Bar: 100 nm. HEp-2, HEp-2 cell; RBC, chicken red blood cell; N, nucleus. should
remain
intact.
erythrocyte polygonal between
the
mode
of
was
plays
We
had
binding
et
al
and
a
electron
and
in the
microscope
1,900 •~ is
a
useful
(13 •~ g
for tool
a
scanning
the
producing
binding
remains
105
was
10
min to
shown
from
the
disease fluid
found
(unpublished
microscope
that
move-
HEp-2 and
examine
which
Newcastle
force
mm),
forces
obscure
vaccinia-infected
Mannweiler electron
microvillus-
RBC,
with
shearing to
fields, the
it
infected
structures. used
it
connection,
cells
tube
three-dimensional authors
this
adsorbed
to
some
measure
cells,
considerable
centrifuge up
to
HEp-2 In
a
in
of
difficult
HeLa
RBCs
centrifugations
areas (13)
role.
withstand
in
is
that
deformation
infected
RBCs to
noted
in it
major
centrifuged
cultured
scanning surface
and
have
withstood A
RBCs the
we
resulted Because
enough
been
regarded
5).
adsorbed
strong
(11).
which
Uno
adsorbed
between
ment
large
(Fig.
binding
binding
this
binding
shapes
virus
In
membrane
cells that
the
data).
and
characterize
and
Rutter to
analyse
(10), the
600
Y. SABURI
ET AL
ultrastructure of HAD induced by mumps, influenza and vaccinia viruses, respectively, coming to a conclusion that the processes, protuberances or microvilli of the host cell played an important role in the adsorption of erythrocytes to the infected cell surface. Support for the assumption might be found in the following two reports. Kumon (9), studying HAD with scanning immunoelectron microscopy, demonstrated the presence of abundant virus-specific antigens along slender and long protrusions binding the erythrocytes. Ichihashi and Dale (7) also showed, using a ferritin-antibody technique, that the hemagglutinin appeared throughout the cell surface including microvilli of HeLa cells infected with vaccinia virus. With these techniques, it may be possible to reveal the correlations between the viral antigens at the cell surface and the erythrocytes adsorbed. The script. School Center
authors We also
are very wish to
grateful to Dr. P.I. Marcus for his advice and critical thank Dr. Y. Nagano, Department of Microbiology,
of Hygiene, for his interest, and Dr. K. Ishihara, Hospital, for his skillful technique on transmission
Department electron
reading of the manuKitasato University
of Dermatology, microscopy.
National
Cancer
REFERENCES
1) 2) 3)
4) 5) 6) 7) 8) 9) 10)
11) 12) 13)
14)
Baker, R.F., Gordon, I., and Stevenson, D. 1965. Electron microscope study of hemadsorption in measles virus infection. Virology 27: 441-445. Chu, C.M. 1948. Studies on vaccinia hemagglutinin. I. Some physicochemical properties. J. Hyg. 46: 42-48. Compans, R.W., Harter, D.H., and Choppin, P.W. 1967. Studies on pneumonia virus of mice (PVM) in cell culture. II. Structure and morphogenesis of the virus particle. J. Exp. Med. 126: 267-276. Compans, R.W., and Dimmock, N.J. 1969. An electron microscopic study of single-cycle infection of chick embryo fibroblasts by influenza virus. Virology 39: 499-515. Due-Nguyen, H. 1968. Hemadsorption of mumps virus examined by light and electron microscopy. J. Virol. 2: 494-506. Hotchin, J.E., Cohen, S.M., Ruska, H., and Ruska, C. 1958. Electron microscopical aspects of hemadsorption in tissue cultures infected with influenza virus. Virology 6: 689-701. Ichihashi, Y., and Dale, S. 1971. Biogenesis of poxviruses : Role of A-type inclusions and cell membranes in virus dissemination. Virology 46: 507-532. Ichihashi, Y., and Dale, S. 1971. Biogenesis of poxviruses : Interrelationship between hemagglutinin production and polykaryocytosis. Virology 46: 533-543. Kumon, H. 1976. Morphologically recognizable markers for scanning immunoelectron microscopy. II. An indirect method using T4 and TMV. Virology 74: 93-103. Mannweiler, K., and Rutter, K. 1975. High resolution investigations with the scanning and transmission electron microscope of hemadsorption binding sites of mumps virus-infected HeLa cells. J. Gen. Virol. 28: 99-109. Marcus, P.I. 1962. Dynamics of surface modification in myxovirus-infected cells. Cold Spring Harbor Symp. Quant. Biol. 27: 351-365. Stokes, G.V. 1976. High-voltage electron microscope study of the release of vaccinia virus from whole cells. J. Virol. 18: 636-643. Uno, F., Ueba, 0., Kumon, H., Tsutsui, K., Tawara, J., Kaneshige, T., and Kaneshige, H. 1975. Observations of the hemadsorption on influenza virus-infected cells. Igaku no Ayumi 95: A-577578 (in Japanese). Vogel, J., and Shelokov, A. 1957. Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture. Science 126: 358-359.
Requests for reprints should be addressed to Dr. Yasuo Sakuri, Department Okayama University Medical School, Shikatacho, Okayama 700, Japan.
of Virology,
Immunol.
Vol. 21 (10), 593-600, 1977
Electron
Microscopic Study of Hemadsorption on Vaccinia Virus Infected Cells
Yasuo SABURI,1Kenji OKUDA,2Toyozoh TAKAHASHI, and Ichiro TADOKORO YokohamaMunicipal Institute of Health, Yokohama,and Departmentof Bacteriology, YokohamaCity UniversitySchoolof Medicine, Yokohama (Received for publication, November 26, 1976)
Abstract Hemadsorption (HAD) induced in HEp-2 cells infected with vaccinia virus was observed. In ultrathin sections, binding of 36 red blood cells (RBCs) was examined in detail and 3 types of HAD were observed : (1) direct and close binding of RBCs to infected HEp-2 cells (cyto-HAD) was observed in cross sections of 27 RBCs, (2) binding of RBCs through microvilli of infected cells was found in 11 RBCs, and (3) five RBCs were distorted to form tentacle-like projections by which they were bound to the HEp-2 cell surface. Scanning electron microscopy revealed that more than 30% of the RBCs were bound to microvilli of vaccinia virus-infected HEp-2 cells, and that the number of microvilli twined round each RBC was over ten. RBCs were attached to certain microvilli through swollen sucker-like tips which were not observable in non-infected HEp-2 cells. RBCs sometimes revealed a polygonal shape at regions of binding to microvilli. Virion-mediated RBC-HEp-2 cell binding could not be observed.
Since hemadsorption (HAD) phenomenon was described by Vogel et al (14), several authors observed the ultrastructure of binding of erythrocytes to virus-infected cells (4-6, 13). The development of HAD was shown to be a dynamic process, with binding of erythrocytes mediated through the microvilli of host cells (11). The purpose of the present study, utilizing light and transmission and scanning electron microscopy, is to elucidate the ultrastructure of the binding between vaccinia virus-infected cells and adsorbed red blood cells (RBSs), with special reference to the role of microvilli in HAD. MATERIALS
AND
METHODS
Cells and virus. HEp-2 cells were cultured in Earl's balanced salt solution containing 0.5% lactalbumin hydrolysate, 0.1% yeast extract, 100 units/ml penicillin and 0.1 mg/ml streptomycin (hereafter abbreviated as YLE medium) supplemented 1 Present address: Department of Virology , Okayama University Medical School, Shikatacho, Okayama 700, Japan. 2 Present address: Department of Genetics , Washington University School of Medicine, Missouri 63110, U.S.A. 593
594
Y.
SABURI
ET AL
with 10% calf serum. After virus inoculation the medium was changed to YLE medium supplemented with 2% calf serum (maintenance medium). DIE-25 strain of vaccinia virus, which was kindly donated by Dr. T. Kitamura, the National Institute of Health, Tokyo, Japan, was used throughout these experiments. The virus stocks were prepared by inoculating chorioallantoic membranes of 10-day-old embryonated hen's eggs with seed virus diluted to 10-4. Three days later the membranes wereh arvested, homogenized with glass powder in a mortar, and 30 ml of phosphate-buffered saline (PBS) (m/100 phosphate, 0.8% NaCl, pH 7.2) was added before centrifuging at 590 g for 20 min. The supernatant fluid was diluted 1:20 with YLE medium and stored in a freezer at -70 C. The 50% tissue culture infective dose for such stocks was (TCD50) =107.5/ml when titrated in HEp-2 cell cultures. HAD. added
For
to
paration in
of
a
at
culture
was
PBS.
The
layers
RBC
to
the
adsorbed
remove
cells
were
to to
stock
per
PBS make
and
of
up
then
examined
3
37
pre-
for ml
hr
at
per 37
C.
suspension,
the
volume
C.
The
of maincell
with
light
cells
10.0
a 0.5%
times by
was
For HEp-2
17-20
into
at
virus
incubation and
about
made
1 hr
washed
of
After
one-tenth
were
stock tube.
tube,
for and
for
the
culture
virus.
Leighton
adsorb
erythrocytes
ml
a Leighton
reincubated
added
RBCs
of
with
allowed
unattached
ml ml
3 times was
RBCs
in
a monolayer
0.4 (1.0
washed
0.1
prepared
microscopy, with
the
microscopy,
slips
medium
suspension
and
containing
medium
electron
were
medium
cover
inoculated
added,
erythrocytes
tenance
for
was
electron
on
maintenance
bottle)
Chicken in
C,
scanning
cultures
sections
bottle
37
and
cell
thin
culture
1 hr
light
HEp-2
mono-
maintenance
and
electron
mic-
roscopy. For
studying
removed
from
(1 :3,000
dilution).
was
hemadsorption a
culture The
resuspended
RBC
in
suspension
incubated
for
mounted
1 hr
with
fixed drated
2.5%
coated
graded by
with
carbon
and
HEp-2
The finally
formed and scopes
electron
cell
5 min. and
studied
of
the light
gold,
and
infected
for
5 min;
milliliter
cells
cover
in
1.0%
alcohol
and
was
placed
slips
in
osmium
which
drying
observed
x g
of
were in
PBS
the
pellet
the
0.5%
the
mixture
on
a
was
glass
slide,
microscopy. on
of point
590
mixture
cultures
post-fixed
critical
cells acid
One-half
of
by
Cell
the
at
medium.
drop
virus-infected
was
method. with
a
Leighton
tubes
tetroxide
and
followed
The
by
scanning
specimens
were dehyisoamyl
were
electron
then
microscope
HSA-2A).
Transmission an
and
spun
suspension
One
vaccinia
ethylenediaminetetraacetic
was
concentrations
prepared
(Hitachi,
C.
the
microscopy.
and
cells, of
maintenance
glutaraldehyde,
through
acetate,
single 5 ml
suspension
to
slip
electron
with
37
a cover
Scanning
of
added at
with
cell
5 ml
was
on
bottle
pellet
a
citrate,
(Hitachi,
microscopy.
was
scraped
of cells
embedded
with lead
sheet
in
was an
Porter-Blum the HU-12A
After off
from
dehydrated Epon-araldite
microtome. specimens and
were HS-6).
fixing a
by
culture
the bottle
through
graded
mixture.
method and
double
observed
with
at
concentrations
Ultrathin
After
described pelleted
staining transmission
of
sectioning with
above, 590 •~
g for
alcohol
was
per-
uranyl
acetate
electron
micro-
HEMADSORPTION
ON VACCINIA-INFECTED
CELLS
595
RESULTS
HAD Observedwith a Light Microscope Unstained preparations of RBCs adsorbed by infected HEp-2 cells were examined with a light microscope. About one-half of the cells adsorbed numerous RBCs i.e., were HAD+, while the other half was HAD- (Fig. 1B). Each HAD cell contained from a few to 50 or more RBCs. When adsorbed RBCs were widely scattered on the cell, binding occurred predominantly in parallel to the long axis of the RBC (Fig. 1A). When RBCs were tightly packed at the cell periphery, binding was perpendicular to the long axis of the RBC (Fig. 1B). It seemed that there was few marked deformation of RBCs in our experimental conditions. HAD Observedwith a ScanningElectronMicroscope That HAD was mediated by microvilli appeared likely from the results of scanning electron microscopic study (Fig. 2). In some fields, RBCs were found to be attached to sucker-shaped tips of microvilli (Fig. 3), while in other fields they were attached to non-swollen ones (Fig. 4). Also, some deformed RBCs, which were probably caused artificially, were often pointed at the site of attachment to microvilli (Fig. 5). The opposite side of the view shown in Fig. 5 was inadvertently obtained in which we observed the surface of an RBC covered by more than 10 microvilli that had emerged from a projection of the host cell (Fig. 6). A similar observation was recorded in Fig. 7, for one of two RBCs adsorbed to a projection of an infected HEp-2 cell. About 11 microvilli were twined round the RBC surface. HAD Observedwith a TransmissionElectronMicroscope Adsorbed RBCs were observed in ultrathin section. A number of mature virions were seen in the cytoplasm. We did not observe RBC binding to virions budding from the cytoplasmic membrane or microvilli as is characteristic for HAD observed in cells infected with para- or ortho-myxoviruses (3, 6). Our observations revealed that RBCs were adsorbed most frequently to a single process from the cell, but in some instances they attached through several binding processes (Figs. 8, 9, 10, and 11). RBCs were closely attached to host cell membranes in 27 of the 35 RBCs examined at a high resolution, with an electron-lucent gap of 10-40 nm between the membranes of the HEp-2 cell and RBC (Fig. 12). No significant morphological changes were recognized on the cytoplasmic membrane of the host cell. The contour of the RBC was smooth, while that of the HEp-2 cell tended to be wavy (Fig. 12). Typical microvillus-erythrocyte binding was observed (Figs. 8 and 9) in which RBCs were attached to numerous long microvilli of the cell, and in agreement with Duc-Nguyen (5) HAD mediated by tentacle-like processes was also seen (Fig. 11).
596
Y. SABURI
1-A
FT AI
1-B
2
4
3 Figs.
1-4
HEMADSORPTION
ON VACCINIA-INFECTED
CELLS
597
DISCUSSION
Our data demonstrate that vaccinia virus-induced HAD, which occurs through either microvilli or "cyto-HAD," is similar to myxo- and para-myxovirus-induced HAD as already reported (6, 11). It remains obscure at present what is meant biologically by the relation of the long axis of adsorbed RBCs to the infected cell as observed by light microscopy (Figs. 1A and 1B). We could not observe "virus-HAD" of the type described by Hotchin et al (6) for influenza virus-infected cells. This negative finding is in keeping with the maturation process of vaccinia virus, namely that the virion does not contain hemagglutinin (2) and hence does not produce any HAD+ virions protruding from the cell surface as is the case with myxovirus HAD (6). However, we did observe "cyto-HAD" (6) in most fields of the ultrathin section preparation. The presence of less electrondense gap between the RBC and host cell had been reported (1, 5, 8). Although the low electron-dense gap was observed in several fields, a series of dense lines bridging between the cell membrane and RBC reported by Compans et al (4) was not found. It was already reported that adsorption of a single RBC might involve binding through the tips of about 3 or 4 HAD+ microvilli containing mature Newcastle virions (11). In vaccinia-infected cells we observed that about 10 microvilli were twined each RBC, not only through their tips, but also throughout their projected length. In addition, the tips of microvilli sometimes swelled, producing a suckerlike attachment to the RBC (Fig. 3). Stokes (12) recently reported that vaccinia viruses were observed to escape from the host cell individually from the tips of microvilli and sometimes appeared to be enclosed by the host membrane-like sheath. These observations might suggest the mechanism of sucker-like swelling of the tips of microvilli at the attaching site of RBCs. Mannweiler and Rutter (10) reported Fig.
1A. with in
Fig.
their Fig.
Light
1B,
1B.
are
Light
RBSs
2.
attach
RBCs.
Bar
3. rovilli
their
:
the
cell.
Note
degree RBCs
that
the
of HAD
the
of a HEp-2
cells
(RBCs),
RBCs
attach
of infected
end virus.
to
with the
cell
infected
contrasting to
of hemadsorption
cell
microvillus-erythrocyte
and
HEp-2
those
HEp-2
Unstained.
micrograph
HEp-2
(HAD)
blood
as compared
longitudinal with
red
the
to
HEp-2
those cell
by
: 20 ƒÊm.
of a high
electron to
in
cell.
Bar
cells. Fig.
One
1A.
Another
HEp-2
Note
HEp-2
cell
that cell
most (upper
: 20 ƒÊm.
of an
infected bindings
HEp-2
cell.
About
are
seen
at
least
that
the
tips
of
the
tips.
30 in
magnification like
a sucker
of the and
area
indicated
in
RBC-HEp-2
cell
binding
Fig.
2.
Note
is mediated
through
2 micBar
1 ƒÊm. Fig.
4. of an in
Fig.
Scanning infected 3.
11
10 ƒÊm.
Higher swell
Bar
infected
of hemadsorption
of chicken
HEp-2
numerous with
be
Scanning attach
the
Unstained.
micrograph
not
degree
number
to
adsorbed
might
RBCs
of a low
A small
adsorbed
sides.
right)
of the left)
Fig.
virus.
lateral
(lower
Fig.
micrograph
vaccinia
electron host
Bar
cell
: 2 ,ƒÊm.
micrograph attach
onto
of HAD the
surface
of an of an
infected RBC.
HEp-2 Their
tips
cell. are
Several not
swollen
microvilli as those
:
598
Y. SABURI
ET Al
5
6
7
8
Fig.
5.
RBCs
Scanning are
pulled
electron
micrograph
through
microvilli
of HAD
of an
in several
infected
areas,
HEp-2
resulting
cell
in polygonal
monolayer. shapes.
The Bar:
10
μm. Figs.
6 and
7.
projection Fig.
6.
Ten
Fig.
7.
About
RBC
Scanning
of
(upper),
an
or
electron
infected
more 10
microvilli
microvilli
however,
micrographs
HEp-2 are are
of the
reverse
side
of the
RBCs
adsorbed
on
a
cell. visible
seen
attaches
twined directly
on
the round
to
surface the
of surface
a projection
the
RBC.
of the of
an
Bar: RBC
infected
5 ƒÊm. (lower). HEp-2
The cell.Bar:
other 2
μm. Figs. Fig.
8-12. 8.
Electron Note
infected
micrographs HEp-2
of cells
ultrathin
adsorbing
sections. an
RBC
by
6 microvilli.
Bar:
1 ƒÊm.
that erythrocytes were bound to HeLa cells infected with mumps virus by either short cell processes or by several small protuberances. Uno et al (13) also showed that several microvilli of monkey cells infected with influenza virus twined round chicken RBCs, using a scanning electron microscope. The results are similar to Fig. 4 of this paper. In vaccinia-infected cells RBCs were most frequently adsorbed by two modes; cyto-HAD and microvillus-erthrocyte binding. If some external force is exerted on RBC adsorption represented by these two modes of binding, the stronger of the two
HEMADSORPTION
ON
VACCINIA-INFECTED
9
10
11
12
Fig.
9.
Two
HEp-2
cell,
Fig.
10. of
11. seen
but
The
brane Fig.
RBCs
the
(lower are
HEp-2 host
cell cell
Two
RBCs
(arrows).
Bar:
right)
do
by
many
bound has
2 RBCs
Bar:
and
attach
closely
to
Bar:
2 ƒÊm.
microvilli.
adsorbed
(arrows). (left
not
which
CELLS
the
attach
cytoplasmic
closely
to
599
membrane
the
of the
cytoplasmic
mem-
2 ƒÊm.
right)
are
moderately
distorted
and
tentacle-like
portions
are
2 ƒÊm.
Fig. 12. High magnification of an attachment zone between the cytoplasmic membranes of an HEp-2 cell and RBC. An electron-lucent gap of 10-40 nm in width is shown (arrows). While the contour of the RBC is smooth, that of the HEp-2 cell is irregular. Around the nucleus of the RBC a perinuclear cisterna is seen. Bar: 100 nm. HEp-2, HEp-2 cell; RBC, chicken red blood cell; N, nucleus. should
remain
intact.
erythrocyte polygonal between
the
mode
of
was
plays
We
had
binding
et
al
and
a
electron
and
in the
microscope
1,900 •~ is
a
useful
(13 •~ g
for tool
a
scanning
the
producing
binding
remains
105
was
10
min to
shown
from
the
disease fluid
found
(unpublished
microscope
that
move-
HEp-2 and
examine
which
Newcastle
force
mm),
forces
obscure
vaccinia-infected
Mannweiler electron
microvillus-
RBC,
with
shearing to
fields, the
it
infected
structures. used
it
connection,
cells
tube
three-dimensional authors
this
adsorbed
to
some
measure
cells,
considerable
centrifuge up
to
HEp-2 In
a
in
of
difficult
HeLa
RBCs
centrifugations
areas (13)
role.
withstand
in
is
that
deformation
infected
RBCs to
noted
in it
major
centrifuged
cultured
scanning surface
and
have
withstood A
RBCs the
we
resulted Because
enough
been
regarded
5).
adsorbed
strong
(11).
which
Uno
adsorbed
between
ment
large
(Fig.
binding
binding
this
binding
shapes
virus
In
membrane
cells that
the
data).
and
characterize
and
Rutter to
analyse
(10), the
600
Y. SABURI
ET AL
ultrastructure of HAD induced by mumps, influenza and vaccinia viruses, respectively, coming to a conclusion that the processes, protuberances or microvilli of the host cell played an important role in the adsorption of erythrocytes to the infected cell surface. Support for the assumption might be found in the following two reports. Kumon (9), studying HAD with scanning immunoelectron microscopy, demonstrated the presence of abundant virus-specific antigens along slender and long protrusions binding the erythrocytes. Ichihashi and Dale (7) also showed, using a ferritin-antibody technique, that the hemagglutinin appeared throughout the cell surface including microvilli of HeLa cells infected with vaccinia virus. With these techniques, it may be possible to reveal the correlations between the viral antigens at the cell surface and the erythrocytes adsorbed. The script. School Center
authors We also
are very wish to
grateful to Dr. P.I. Marcus for his advice and critical thank Dr. Y. Nagano, Department of Microbiology,
of Hygiene, for his interest, and Dr. K. Ishihara, Hospital, for his skillful technique on transmission
Department electron
reading of the manuKitasato University
of Dermatology, microscopy.
National
Cancer
REFERENCES
1) 2) 3)
4) 5) 6) 7) 8) 9) 10)
11) 12) 13)
14)
Baker, R.F., Gordon, I., and Stevenson, D. 1965. Electron microscope study of hemadsorption in measles virus infection. Virology 27: 441-445. Chu, C.M. 1948. Studies on vaccinia hemagglutinin. I. Some physicochemical properties. J. Hyg. 46: 42-48. Compans, R.W., Harter, D.H., and Choppin, P.W. 1967. Studies on pneumonia virus of mice (PVM) in cell culture. II. Structure and morphogenesis of the virus particle. J. Exp. Med. 126: 267-276. Compans, R.W., and Dimmock, N.J. 1969. An electron microscopic study of single-cycle infection of chick embryo fibroblasts by influenza virus. Virology 39: 499-515. Due-Nguyen, H. 1968. Hemadsorption of mumps virus examined by light and electron microscopy. J. Virol. 2: 494-506. Hotchin, J.E., Cohen, S.M., Ruska, H., and Ruska, C. 1958. Electron microscopical aspects of hemadsorption in tissue cultures infected with influenza virus. Virology 6: 689-701. Ichihashi, Y., and Dale, S. 1971. Biogenesis of poxviruses : Role of A-type inclusions and cell membranes in virus dissemination. Virology 46: 507-532. Ichihashi, Y., and Dale, S. 1971. Biogenesis of poxviruses : Interrelationship between hemagglutinin production and polykaryocytosis. Virology 46: 533-543. Kumon, H. 1976. Morphologically recognizable markers for scanning immunoelectron microscopy. II. An indirect method using T4 and TMV. Virology 74: 93-103. Mannweiler, K., and Rutter, K. 1975. High resolution investigations with the scanning and transmission electron microscope of hemadsorption binding sites of mumps virus-infected HeLa cells. J. Gen. Virol. 28: 99-109. Marcus, P.I. 1962. Dynamics of surface modification in myxovirus-infected cells. Cold Spring Harbor Symp. Quant. Biol. 27: 351-365. Stokes, G.V. 1976. High-voltage electron microscope study of the release of vaccinia virus from whole cells. J. Virol. 18: 636-643. Uno, F., Ueba, 0., Kumon, H., Tsutsui, K., Tawara, J., Kaneshige, T., and Kaneshige, H. 1975. Observations of the hemadsorption on influenza virus-infected cells. Igaku no Ayumi 95: A-577578 (in Japanese). Vogel, J., and Shelokov, A. 1957. Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture. Science 126: 358-359.
Requests for reprints should be addressed to Dr. Yasuo Sakuri, Department Okayama University Medical School, Shikatacho, Okayama 700, Japan.
of Virology,
