Download An Electron Microscope Study of Tissue Cultures Infected with Visna

VIROLOGY
14, 463475
An
(1961)
Electron
Microscope
Study
Infected
with Visna
HALLDOR
Institute
for
Expetimental
of Tissue
.Virus’
Cultures
THORMAR
University
Pathology,
Accepted
April
of Iceland,
Keldur,
Reykjavik
26, 1961
Sections of tissue culture cells infected with visna virus were studied in the electron
microscope. The ultrastructure
of the nuclei and the cytoplasm of infected cells was
found to look much like that of uninfected control cells. In the former, however, a
number of spherical particles was seen on the external cell surface. The particles averaged 85 rnp in diameter, were bounded by a single membrane, and contained a
centrally located electron-dense core. They were found in three types of tissue cultures infected with various strains of the virus. The particles appeared 2-3 days after
inoculation, concurrently with the typical cytopathic changes and the rise in virus
titer of the fluid. They seemed to be formed by a budding process in the cell membrane and to be released as double-walled
bodies which later developed to form the
characteristic particles. It is suggested that the particles represent visna virus, and
their apparent similarity to certain cancer viruses is pointed out.
INTRODUCTION
Visna
nervous
is a virus disease of the central
system
of
sheep,
causing
demy-
elination and destruction of white matter in
the brain, the cerebellum, and the spinal
cord. The disease, which has been described
by Sigurdsson and associates (1957) and
Sigurdsson and Palsson (1958)) develops
extremely slowly, clinical signs often appearing months or even years after infection,
Visna virus has been propagated in tissue
culture, where the characteristic cytopathic
changes are found to be formation of multinuclear giant cells, often with long processes
stretching from the surface (Sigurdsson et
al., 1960). If tissue cultures are inoculated
with large amounts of virus, almost every
cell in the cultures will be typically affected
‘Work
supported
by the Icelandic
Science
Foundation
and by a grant (No. B-2425) of the
Department
of Health, Education, and Welfare,
Public Health Service, National
Institutes
of
Health, Bethesda, Maryland.
’ Present address: Statens Seruminstitut, Amager
Boulevard 80, Copenhagen S., Denmark.
in 4-5 days. Not until a couple of days later
do the cells begin to detach from the glass
wall.
Since a high percentage of the cells in a
visna-infected tissue culture shows typical
cytopathic changes before the cell sheet begins to degenerate and come off the glass, it
was considered that such a culture might be
suited for an electron microscope study of
the ultrastructure
of infected cells. The
study did not, however, reveal any characteristic structural changes in the nucleus
or in the cytoplasm of the cells. On the
other hand, characteristic “viruslike” particles were seen to accumulate on the external surface of the cells in an infected
culture. In the present communication,
these particles will be described and data
will be presented which suggest that they
represent infectious visna virus.
MATERIALS
AND
METHODS
Tissue culture. Monolayer cultures of serially propagated cells derived from the
choroid plexuses of sheep brain were used
for most of the present work. The method
463
464
THORMAR
of cultivation has been described previously
in detail (Sigurdsson et aZ., 1960). These
cultures will hereinafter be referred to AS
plexus cultures.
Cultures of kidney epithelial cells and of
liver cells from sheep were also employed.
They were prepared by trypsinization
of
small pieces from the cortex of the kidney
and from the liver. All cultures were grown
in roller tubes using medium 199 with 20%
sheep serum as growth medium. The maintenance medium in the inoculated cultures
consisted of medium 199 with 2% sheep
serum. All media contained 100 units of
penicillin and 100 pg of strept,omycin per
milliliter.
Virus. Strain K485 of visna virus adapted
to growth in tissue culture (TC) through
more than 30 passages (Sigurdsson et al.,
1960) was used in the greater part of the
present work. Strains K643, 644, 682, and
796 in their eleventh or twelfth TC passage
were used in a few cases.
The virus was titrated by inoculating tenfold dilutions of infectious TC fluid into
roller tube cultures (Thormar, 1961) ; the
50% infectivity end point was calculated by
the method of Reed and Muench. Infectious
TC fluid was employed for inoculation of
cell cultures used for electron microscopy.
The fluid had an infectivity titer of IO6 to
lo7 TCID60 per 0.1 ml, and the inocula were
chosen so that they would contain more
than one infectious unit per cell.
Preparation of specimens for electron microscopy.
Routine methods were used for
preparation of tissue cultures for electron
microscopy. The cells were rinsed once in
Hanks’ salt solution and fixed at 4” for 1520 minutes, using 1% solution of osmium
tetroxide, buffered at pH 7.3-7.4, according
to the method of Palade. After fixation, the
cells were rinsed quickly with distilled water and covered with 70% ethanol, The cell
layer was then detached from the glass wall
with a metal spatula. It came off as small
flakes that were transferred to a centrifuge
tube and collected by low speed centrifugation, The cells were dehydrated in graded
dilutions of ethanol and embedded in a mixture of 75% butyl and 25% methyl methacrylate, containing 2% benzoyl peroxide.
The polymerization
was carried out ovcr-
night at’ 45”. Sections were cut on a Cambridge Micro Section Rocking microtome,
using glass knives. They were floated onto
the surfacr of a lOc/;~mixture of acetone in
water, spread with xylene vapor (Satir and
Peachy, 1958) and picked up on Formvarcoated copper grids. Before examination in
the electron microscope, which \vas a Sicmens Elmiscope I, the sections were usually
stained with a saturated solution of lead
acetate, according to the method of Watson
(1958).
RESULTS
UT&infected Plexus Cultures
Plexus cultures incubated with maintenance medium at 37” for 6-7 days served as
control cultures to be compared with cultures infected with visna virus. At the time
of fixation for electron microscopy, the uninfected cultures appeared normal, as observed in the light microscope.
In the electron microscope, the cytoplasm
of uninfected cells varied considerably in
appearance from one culture to another, but
usually looked rather poor in structural
constituents. Only a few mitochondria were
seen. Ergastoplasm was more often found
well preserved, and the cytoplasm often
contained a number of small vacuoles. The
nuclei showed a finely dispersed matrix, and
nucleoli were sometimes observed.
Plexus Cultures Infected
with Visna Virus
Plexus cultures inoculated with infectious
TC fluid showed widespread cytopathic
changes, by light microscopy, after incubation at 37” for 6-7 days (Fig. 1). They were
then fixed and prepared for electron microscopy.
In the electron microscope the cytoplasm
of cells from infected cultures looked similar to that of uninfected cells, appearing
rather lacking in electron-dense structure.
Mitochondria were sometimes seen but were
usually not well preserved, showing swelling and vacuolization. Ergastoplasm was observed in most of the cells. The internal
structure of the nuclei was similar to the
structure of nuclei of uninfected cells.
The most striking difference between uninfected and infected cultures, as revealed
VISNA VIRUS INFECTION
465
FIG. 1. Plexus culture 6 days after inoculation with visna virus. The characteristic cytopathic changes consist in formation of multinucleated
cell syncytia, the nuclei being arranged in a rosette-like pattern. Most of the cells in this culture show pathologic effects.
Giemsa-stained. Magnification : X 146.
by electron microscopy, was the appearance
in the latter of small round particles on the
external surface of the cells. Often the particles lay side by side along the cell membranes and sometimes they formed large
clusters, containing dozens of particles. Figure 2 shows a section through cells from an
infected plexus culture. A few round particles, each with a dense central body, can be
seen scattered along the surface of the cells,
and at the top of the figure there is a cluster
of particles. A swollen and disrupted mitochondrion is seen at the lower right corner,
and remnants of ergastoplasm and mitochondria are found scattered through the
vacuolized cytoplasm.
Occurrence of Extracellular Particles in Infected Kidney and Liver Cell Cultures
Confluent monolayers of kidney cells were
inoculated with visna virus and incubated
at 37”. After 6-7 days, cytopathic changes
with the formation of multinuclear giant
cells were seen by light microscopy of fixed
and Giemsa-stained infected cultures. The
cytopathic changes increased during the following days and the infectivity titer of the
culture fluids rose to lo6 to 10’ TCIDsO per
0.1 ml. On the seventh to tenth day of infection, kidney cell cultures were prepared
for electron microscopy together with normal-looking uninfected cultures of the same
age. Electron microscopic examination of
THORMAR
FIG. 2. Section
from a plexus culture fixed 7 days after inoculntion
Visna particles can be seen on the external cell surface. Magnific.ation:
sections from the infected cultures showed
extracellular particles having the same appearance as those observed in infected
plexus cultures. No particles of this type
were seen in sections of uninfected kidney
cell cultures, which resembled sections from
infected cultures with respect to the ultrastructure of the cytoplasm and the nuclei.
Monolayer cultures of liver cells inoculated with visna virus showed cytopathic
X
with \-isna virus.
22,000.
changes similar to those seen by light microscopy of infected plexus and kidney cell
cultures. Electron microscopic examination
of the infected liver cell cultures revealed
the presence of round extracellular particles,
containing a dark central body.
Altogether, more than twenty plexus, kidney, and liver cell cultures infected with
visna virus have been examined in the electron microscope. In all these cultures the
VISNA
VIRUS
characteristic particles were seen, usually in
great number. They have never been observed in uninfected cultures, although these
have been thoroughly searched. These particles will, in the following sections, be referred to as visna particles.
The Size and the Structure
Particles
of the Visna
Figure 3 shows a cluster of visna particles
tt a magnification
of 120,000 times. The
Iarticles seem to have a slightly ovoid
shape, but as the longest axes of most of
;hem are oriented in the same direction, it is
relieved that the apparent ovoid shape is
lue to compression of the section, caused by
(he microtome knife, and that the particles
Ire in fact spherical. The irregular shape of
:ome of the particles might be caused by
meven polymerization
or shrinkage of the
nethacrylate.
The size of the visna particles was estinated by measuring their diameters in mirographs. About 200 particles were meas(red and in each one the average between
he longest and the shortest axis was conidered to be the true diameter. The average
liameter of all particles measured was 85
np. About 90% were within the range of
O-100 rnp and the remaining 10% were
ither slightly smaller or bigger. Particles
onsiderably bigger than 100 rnp in diameter
Jere seen occasionally.
The visna particles are bounded by an
pparently single membrane and contain a
entrally located osmiophilic core, separated
rom the outer membrane by a zone of low
lectron density. The central cores seem also
3 be surrounded by a membrane. Their
verage diameter was found to be 35 rnp
nd about 80% of those measured ranged in
ize from 30 to 40 rnp.
No difference was found in the appearrice of particles seen in the various types
f tissue culture, either in size or structure.
‘ormation of Spherical Bodies at the Surface of the Cells
In sections of tissue cultures infected with
isna virus, cell membranes are often seen
1 be covered with a great number of
ouble-walled buds (Fig. 41. Also, in in:cted cultures, small extracellular
bodies
INFECTION
467
enveloped by double membranes are sometimes found intermingled with visna particles near the surface of the cells (Fig. 5).
Figures 6 and 7 show a number of protrusions of the cell membrane, varying in size.
Some appear as a slight thickening and
bulging of t’he membrane, others have protruded farther, having the shape of hemispherical buds. Still others are assuming a
spherical shape or seem to be breaking away
from the membrane as double-walled spherical bodies. The various protrusions apparently represent steps in t,he formation of the
free extracellular
bodies, which are seen
near the surface of the cells together with
visna particles.
The phenomena seen in Figs. 6 and 7 are
illustrated
at a higher magnification
in
Figs. 8 and 9. In Fig. 8 a small bulge is
seen at the right border of the field, two
buds are seen near the center, and at the
left a spherical body is seemingly detaching
from the membrane. A dark dot is visible
in the center of this body and in the one
shown in the inset. Figure 9 shows a couple
of buds of almost hemispherical shape and
a bud assuming a spherical shape and separating from the cell surface. Above it two
visna particles are found. Free spherical
bodies are visible near the right border of
the field and at the lower left corner. Figures 8 and 9 show that the inner membrane
of the double-walled
buds and bodies is
more electron dense than the outer.
The extracellular bodies have the same
shape as visna particles but appear on the
whole slightly bigger. Some of them are seen
to have a structure similar to that of visna
particles, containing a dense central core,
which is, however, much smaller and less
distinct than the central core of visna particles (cf. Fig. 8). The bodies appear in
sections of infected cultures more rarely
than do visna particles and always accompanied by the latter.
The buds and the extracellular bodies are
believed to represent precursors of the visna
particles.
1
Relationship
between Xumber
of Visna
Particles and Infectious Virus Titer
In order to study if there were a relationship between the number of visna particles
THORMAR
468
FIG. 3. Cluster
of visna
Magnification
: X 120,000.
particles
from
a plexus
culture
fixed
7 days
after
inoculation
VISNA
FIG. 4. Cell
double-walled
FIG. 5. The
walled
bodies
which
apparently
VIRUS
469
INFECTION
from a plexus
culture
7 days after inoculation.
The membrane
buds. Magnification
: X 22,000.
same culture
as in Fig. 4. Visna particles
are seen intermingled
near the external
cell surface.
A few buds are also seen on the
is disintegrating.
Magnification:
X 22,000.
is covered
cell
with
with dou Iblemembr ‘ane ,
470
THORMAR
FIG. 6. Cell from a plexus culture 7 days after inoculation, showing protrusions of the,
cell membrane which are believed to represent stages in the formation of free extracelluh11
bodies. A few visna particles are also seen near the cell surface. Magnification:
x 22,000.
FIG. 7. Plexus cultures 7 days after inoculation, showing the same phenomenon as in Fig.
6. Buds of various lengths are protruding from the cell membrane, some of them pinching
off to form spherical bodies. A great number of visna particles are seen covering the cell
surface. The section seems to be a little compressed. Magnification:
X 22,000.
VISNA
VIRUS
INFECTION
FIG. 8. Section of a plexus culture 7 days after inoculation,
showing a piece of cell membrane. Protrusions in various stages of their development into free bodies are seen along the
membrane, and in the inset an apparently free spherical body from the same section is
shown. Note the dense centers of the bodies. Magnification:
X 120,000.
seen in an infected tissue culture and the
virus titer of the surrounding fluid the following experiment was made: A number of
young monolayer plexus cultures were inoculated, each with 0.1 ml of infectious TC
fluid, and incubated in a roller drum at 37”
for 6 hours, a period which has been found
sufficient to ensure maximum adsorption of
virus (unpublished data). One culture was
then fixed for electron microscopy. The remaining cultures were washed with six
changes of medium 199 and the fluid of the
sixth washing was saved for titration of
virus. The cultures were then incubated further and at daily intervals for 7 days one
culture was harvested for fixation. The fluid
from each tube was saved, deep-frozen, and
later assayed for infectious virus. About
half of the cell layer in each culture was detached from the glass and prepared for
electron microscopy, while the remaining
cells were Giemsa-stained for examination
by light microscopy.
Sections of about 100 different cells from
each culture were examined in the electron
microscope, and all visna particles seen in
3-4 sections of each of the 100 cells were
counted. The results are summarized in
Table 1 and Fig. 10. Table 1 shows that
visna particles appear in a detectable number on the second to the third day of infection concurrently with the development of
cytopathic changes in the culture and at the
same time as the main rise in virus titer occurs in the fluid, i.e., from 1000 to l,OOO,OOO
TCID5,, per 0.1 ml. As illustrated in Fig. 10,
the number of particles increases from the
second to the fifth day of infection, almost
in parallel with the infectious virus titer.
After this time, the titer did not rise sig-
THORMAR
FIG. 9. Another section from the same culture as in Fig. 8, showing cell membrane with
buds, free bodies, and two visna particles. The buds and the bodies arc bounded by double
membranes, the inner membrane being more distinct than the outer. Magnification:
:a’
120.000.
nificantly but the number of visna particles
and the cytopathic changes increased until
the end of the experiment.
Four days after inoculation, visna particles were seen on the surface of almost
every cell examined in the electron microscope, but only one or very few in each section. On the fifth to the seventh day the
number of particles surrounding a cell was
greatly increased, reaching a maximum on
the seventh day, when clusters of particles
were often seen. On the sixth and the seventh day, cell membranes were often found
to be covered with the small buds that are
believed to represent precursors of visna
particles being released from the cells. Visna
particles were not observed within the cells
at any time, either in the cytoplasm or in
the nucleus.
This experiment has been repeated, both
with plexus cultures and with cultures of
kidney cells, always with similar results.
DISCUSSION
The present study has shown that infection of tissue cultures with visna virus causes
the appearance of characteristic particles
which accumulate on the surface of the cells.
The particles have been observed by electron
microscopy of plexus cultures infected with
five strains of visna virus which have been
propagated in TC, and they have been seen
in cultures of sheep kidney and liver cells
inoculated with visna virus. These three
types of cell cultures support the growth of
the virus and undergo similar cytopathic
changes. The size and the structure of the
particles is the same regardless of the strain
of visna virus used for infection and of the
t,ype of tissue culture employed.
VISNA
CYTOPATHIC
VIRUS
473
INFECTION
TABLE 1
OF VISNA PARTICLEG, AND VIRUS TITER IN PLEXUS CULTURES
HARVESTED AT VARIOUS TIMES AFTER INOCULATION WITH VISNA VIRUS=
CHANG
Time of harvest
s, RELATIVE
NUMBER
Relative number of
visna particles
Cytopathic (CP) changes
Virus titer
of fluid
6
Hours
None
No particles
observed
30
Hours
None
No particles
observed
103JJ
Slight CP changes; 5-10% of the
cells with two or more nuclei
(2)
104.6
2 Days
1O’.Ob
3
Days
Increased CP changes;
the cells multinuclear
late form
4O-5Oyo of
or of stel-
68
106 .o
4
Days
50-60~0 of the cells with CP changes
408
10°J
5
Days
70-8Oojo of the cells with CP changes
1668
106 .r
6
Days
Cells beginning to degenerate
detach from the glass wall
increase
106 .’
7 Days
and
Increasing disintegration,
but multinuclear
and giant cells still
present in great number
Further
Innumerable
10’ .o
(1Cytopathic changes were studied by light microscopy of Giemsa-stained cultures. Relative number
of visna particles in the various cultures was obtained by counting all the particles seen in sections from
about 100 cells from each culture, using 34 sections of each cell. The infectious virus titer is expressed
as TCIDw per 0.1 ml of culture fluid. b Fluid of the sixth washing.
The visna particles
are viruslike,
being
spherical
and containing
a dense internal
body. Their size is within the size range of
viruses,
the average
diameter
being about
85 rnp. There is a considerable variation
between individual particles but definite
size groups have not been found. The relative size of the internal bodies varies somewhat, but as a rule their diameter is less than
one half of the diameter of the particles.
In the present study, visna particles were
seen in all tissue cultures infected with
visna virus and showing widespread cytopathic changes. They have, on the other
hand, not been found in uninfected control
cultures. This fact, together with their viruslike structure, suggests that the particles
represent visna virus. The observations (1)
that visna particles do not appear in a detectable number in infected tissue cultures
until the cells have begun to release virus,
and (2) that the number of visna particles
increases during the infection in a similar
manner as the infectious virus titer render
a further indication in favor of the viral nature of the particles.
The apparent similarity between visna
particles and the double-walled spherical
bodies formed by budding of the cell membrane, and their appearance together on the
surface of the cells, suggests a relationship
between the bodies and visna particles, possibly that the former are precursors of the
latter. This is in agreement with the fact
that the particles have never been observed
intracellularly,
although found in a great
number on the external cell surface. Structures likely to be precursors of visna particles have not been observed within the
cells either. However, on the basis of the
present work no conclusion can be drawn
concerning the effect of visna infection on
the ultrastructure of tissue culture cells. The
cytoplasm of infected and control cells had
474
THORMAR
FIG. 10. Increase in virus titer (left ordinate) and in relative number of visna particles
(right
., ordinate) in tissue cultures incubated at 37” for various time periods after inoculation
with visna virus (abscissa).
a similar appearance, as observed by electron microscopy, looking rather poor in
electron dense structure. Whether this lack
of inner structure is due to an unspecific
degeneration of the tissue culture cells, to a
disruption of the cell components during the
polymerization of methacrylate, or to some
other cause is not known at the present
time.
Considering further the suggestion that
visna particles are formed by a budding
process in the cell membrane and are released as double-walled spherical bodies, it
should be noted that intermittent stages between these and mature visna particles have
not been observed in the electron microscope. However, double-walled bodies with
a dense center surrounded by more diffuse
material have been seen (cf. Fig. 8). Since
the bodies often appear somewhat bigger
than most of the visna particles, a contraction of their double membrane might be the
last step in the process of particle formation, occurring
so rapidly that there would
be little probability of fixing a body during
this step. If the inner membrane contracts
more than the outer, a dense central core
will be formed, leaving a zone of lesser
density between the core and the outer
membrane. Assuming that the visna particles represent visna virus, the present electron microscope study thus suggests that
visna virus is formed at the surface of the
host cells at the time of extrusion.
Franklin (1958) has put forward the hypothesis that animal viruses which contain
lipids as an essential component are completed as they pass to the external surface
of the host cell by obtaining a lipid coat
from the cell membrane. Such viruseswhich include, for example, myxoviruses,
Western and Eastern equine encephalomyelitis viruses, and certain cancer virusesare released continuously from their host
cells over a long period of time unlike those
viruses which are released by a burst
(Franklin, 1958). Visna virus seems to fit
into this pattern, since it has been found to
be ether sensitive (Thormar, 1961), thus
indicating that it is lipid containing, and
the viral particles are apparently formed at
the cell surface, as suggested by the present
work. Furthermore, the observation that tissue culture cells show cytopathic changes,
VISNA
VIRUS
INFECTION
475
electron microscope. I arn grateful to Dr. Herdis
by light microscopy, shortly after inoculavon
Magnus and civil engineer Aksel Birch-Andertion with visna virus and remain intact on
sen,
Statens Seruminstitut, Copenhagen, Denmark,
the glass wall for at least 5-6 days of incu- for valuable
criticism of the manuscript, and to
bation while the virus titer in the fluid grad- Miss Anne-Grete Overgaard for preparation of the
ually rises to maximum (cf. Table 1) is photographic prints.
compatible with the conception of continuREFERENCES
ous virus release over a long period of time.
The protrusions observed on the surface DE HARVEN, E., and FRIEND, C. (1960). Further
of cells infected with visna virus show a
electron microscope studies of a mouse leukemia
striking resemblance to those seen on the
induced by cell-free filtrates. J. Biophys. Biothem. Cytol.
7,747-752.
surface of mouse mammary carcinoma cells
(Lasfargues et al., 1959; Moore et al., 1959; FRANKLIN, R. M. (1958). An hypothesis to explain
the relation between the synthesis and release of
Goldfeder et al., 1960)) and mouse leukemic
animal viruses from infected cells and the lipid
cells (de Harven and Friend, 1960). In the
content of the viruses. Experientia 14, 346-348.
case of these tumor cells, the protrusions
FURTH, J., and METCALF, D. (1958). An appraisal
also develop into double-walled
bodies
of tumor-virus
problems. J. Chronic
Diseases,
which are released from the cell membrane.
8,88-112.
In some instances, these bodies-which
are GOLDFEDER, A., GELBER, D., and MOORE, D. H.
believed to represent viral particles-later
(1960). An electron microscope study of sponseem to be rearranged to form a sac, contaneous mammary carcinomas in a subline of
strain DBB mice. J. Natl. Cancer Inst. 25, 827taining a central nucleoid (Lasfargues et
846.
al., 1959; Goldfeder et al., 1960).
In this connection it is of interest to note LASFARGUES,E. Y., MOORE, D. H., MURRAY, M. R.,
HAAGENSEN, C. D., and POLLARD, E. C. (1959).
that visna infection in sheep seems to have
Production of the milk agent in cultures of mamcertain characteristics
in common with
mary carcinoma. J. Biophys. Biochem.
Cytol.
5,
mouse mammary carcinoma and mouse leu93-96.
kemia, namely a long initial period of la- MOORE, D. H., LASFARGUES, E. Y., MURRAY, M. R.,
tency and-once
clinical signs have apHAAGENSEN,
C. D., and POLLARD, E. C. (1959).
peared-a
slow progressive course which
Correlation of physical and biological properties
leads to death. These are the characteristics
of mouse mammary tumor agent. J. Biophys.
of “slow infections,” as defined by SigurdsBiochem.
Cytol.
5,85-92.
SATIR, P. G., and PEACHEY,
D. D. (1958). Thin
son (1954). Furthermore, it is noteworthy
sections. II. A simple method for reducing comfor visna infections that they have in many
pression artifacts. J. Biophys.
Biochem.
Cytol.
instances been found not to cause forma4,345348.
tion of circulating neutralizing antibodies
B. (1954). Rida, a chronic encephalitis
(Sigurdsson et aZ., 1960), apparently also a SIGURDSSON,
of sheep. With general remarks on infections
characteristic of the above-mentioned neowhich develop slowly and some of their special
plastic diseases (Furth and Metcalf, 1958).
characteristics. Brit. Vet. J. 110,341-354.
Finally, as suggested by the present work, SIGURDSSON, B., and P~LSSON, P. A. (1958). Visna
visna virus grown in tissue culture seems to
of sheep. A slow, demyelinating
infection. Btit.
J. Exptl.
Pathol.
39,519-528.
be formed by a budding process similar to
that reported in the case of the mouse mam- SIGURDSSON,B., P~~LSSON,P. A., and GR~MSSON,H.
(1957). Visna, a demyelinating
transmissible dismary tumor agent and of Friend leukemia
ease of sheep. J. Neuropathol.
Exptl.
Neurol.
virus, the viral membrane being derived
16,389403.
from the membrane of the host cell. It would
B., THORMAR, H., and P&,soN, P. A.
be interesting if this peculiar mechanism of SIGURDSSON,
(1960). Cultivation
of visna virus in tissue culvirus formation and a low antigenicity of
ture. Arch. ges. Virusforsch.
10,368--381.
the virus turned out to be characteristic
THORMAR,
H. (1961). Stability of visna virus in
traits of slow virus infections, in addition to
infectious tissue culture fluid. Arch. ges. Virusthe protracted course of the disease.
forsch.
10,501~509.
ACKNOWLEDGMENTS
The author wants to thank Dr. Halldor Grimsson
for much help during the work with the
M. L. (1958). Staining of tissue sections
for electron microscopy with heavy metals. II.
Application
of solutions containing
lead and
barium. J. Biophys.
Biochem. Cytol. 4, 727-730.
WATSON,