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JOURNAL OF VIROLOGY, Sept. 1969, p. 271-282 Vol. 4, No. 3
Copyright C) 1969 American Society for Microbiology Prilited in U.S.A.
Morphogenesis of Aura Virus
EDUARDO F. LASCANO, MARIA I. BERiA, AND JULIO G. BARRERA ORO
Instituto Nacional ce Microbiologica "Dr. Carlos G. Malbri4n," A t'nicla Velez Sarsfield 563, Blieiios Aires,
Argentinia
Received for publication 24 April 1969
Aura virus, a member of the Western equine-encephalitis-Whataroa subgroup of
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group A arboviruses, was studied by electron microscopy in suckling mouse brain
and chick embryo cultured cells. Virus precursors, budding particles, and complete
virus particles were first detected 10 hr after infection in chick embryo cells and
24 hr after inoculation in mouse brain. Virus precursors were generally seen aligned
along cytomembranes, and were less frequently seen closely associated with viro-
plasm-like foci, tubular aggregates, or scattered in the cytoplasmic matrix without
an apparent connection to any other structure. The assembly of mature virus was
observed to take place by a budding process of the virus precursor from the plasma
membrane into the extracellular space, and from the cytoplasmic membranes into
the lumina of vacuoles and cisternae. It was demonstrated that the endoplasmic
reticulum participates in the assembly of intracellular virions. Indirect evidence was
found to indicate that the Golgi complex may also form mature virus. Aura virions
had a size, shape, and structure similar to those of the previously described group A
arboviruses.
Aura virus was first isolated by Causey et al. vacuoles (7). Different hypotheses have postulated
(5) in Belem, Brazil. It has been demonstrated to the sites of formation of virus precursors (1, 10,
be a group A arbovirus which is closely related to 12, 16) and the origin of membranes involved in
Western equine, Sindbis, and Whataroa viruses their final development into mature virus (6, 10,
(5, 33). So far, Aura virus has been found infect- 16).
ing mosquitoes of only South American forests In this electron-microscopy study, the assembly
(2, 5). It is pathogenic for the newborn mouse, of Aura virions from the plasma membrane and
growing well in tissue culture cells; apparently, intracellular membranes is demonstrated. In-
however, it is not pathogenic for man. direct morphologic evidence sugegsting that the
Previous electron-microscopy studies have pro- Golgi complex may also form mature virus is
vided important information on the develop- presented.
mental characteristics of group A arboviruses.
Among other findings, they have shown the MATERIALS AND METHODS
presence of virus precursors in cells infected with The A. se-rraltis strain of Aura virus (2) was used
several members of the group (1, 11, 16, 20), and throughout this study. The eighth mouse brain pas-
the budding of virus particles at the cell surface sage since isolation was used for experiments in mice,
and the 10th chick embryo cell passage (18th passage
(1, 6, 16). Electron-microscopy studies have also since isolation), for experiments in tissue culture cells.
demonstrated the final assembly process of mature Litters of 1- to 2-day-old albino mice were infected
virus from portions of the plasma membrane (1, by intracerebral inoculation with 0.02 ml of brain
12) modified by the virus infection (12). All suspension containing 10:3 LD,o of Aura virus. Three to
studies of thin sections of group A arboviruses five mice were sacrificed at different intervals there-
have also shown intracellular virions within the after, and brains were collected for electron micros-
lumina of vacuoles (1, 6, 16, 20, 24). However, a copy and control of infectivity. Samples used for
clear demonstration of the assembly process of preparing the micrographs shown in this study had
virus titers of 109"8to 1 _lLD5o per g of brain tissue.
intracellular virus is still lacking. Some studies, Chick embryo fibroblasts were grown in a medium
have suggested that precursor particles acquire composed of Hank's Balanced Salt Solution (BSS)
envelopes and form mature virus as they cross with 0.5'c lactalbumin hydrolysate, 10'- tryptose
vacuolar membranes (7, 10, 12, 16), but it has phosphate, 10%' fetal bovine serum, 100 units penicil-
lin per ml, and 100 tg of streptomycin per ml. Mono-
also been theorized that the assembly may take layers of about 107 cells per bottle were obtained.
place within the lumina of cisternae (14) and After three washings with Hank's BSS, each bottle
271272 LASCANO, BERRIA, AND BARRERA ORO J. VIROL.
was incoulated with 1 ml of undiluted chick embryo
tissue culture supernatant fluid that contained 107
TCD,o of Aura virus. The inoculum was left to adsorb
at room temperature for 2 hr and was then removed.
The monolayers were washed three times with Hank's
BSS and fed with maintenance medium in which
Hank's BSS was substituted by Earle's BSS, and the 6
fetal bovine serum was reduced to 2.5%',; the other
components remained the same as those of the grow- a5
ing medium. Bottles were incubated at 36 to 37 C;
4
at certain intervals, some of them were removed for -
study by electron microscopy and control of infec-
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tivity of cell-associated virus. Samples for the micro- I
graphs of this study showed virus titers of 10 to 107
TCD;Io per ml of cell-associated virus. oF 2
Brains of mice inoculated with brain suspensions -
from healthy mice, and chick embryo monolayers r- BEGINNING OF EM
V POSITIVE FINDINGS
inoculated with tissue culture supernatant fluids from I l
uninfected chick embryo cells were used as controls. 10 24 48
For ultrathin-section electron microscopy, chick- HOURS
embryo cell pellets were fixed in Millonig's buffered rw FIG. 1. Growth curve of Aura virus in suckling-mouse
(15) 1% paraformaldehyde for 20 min; minced mouse brain. Eachl point slhows the virus titer qf 3 to 5 pooled
brain was fixed in Millonig's buffered 4%,/; paraformal- brainis. Titrations were determined in ntewbornt mice by
dehyde for 1 hr. Thereafter, both materials were intracerebral inioculationt.
equally treated as follows. They were washed twice
with buffer, refixed in I % osmium tetroxide for 1 hr,
washed four times with veronal buffer, immersed in
50',, ethyl alcohol-saturated uranyl acetate for 2 hr,
dehydrated in graded alcohols, immersed in propylene
oxide, and embedded in Epon 812 (13) or Vestopal
(23). Sections were cut with glass knives, stained with
uranyl acetate (32), and then stained with lead citrate
(22). Specimens were examined in a Siemens Elmiskop
I electron microscope.
RESULTS
The three developmental stages of group A
arboviruses (virus precursors, budding particles,
and mature virus particles) were found in thin
sections of Aura virus-infected suckling-mouse
brain and chick embryo cultured cells. They were
first detected 10 hr after infection in chick embryo
cells and 24 hr after inoculation in mouse brain, o3
V
BEGINNING OF EM
POSITIVE FINDINGS
but were more frequently found at times when
maximal virus titers were obtained, i.e., 16 to 24
1O URS
48 72
24 hr after infection in chick embryo cells and 48
to 72 hr after inoculation in mouse brain (Fig. 1, FIG. 2. Growth curve of cell-associated Autra virus
in chick embryo cultured cells. Titrationis were carried
2). outt in chick embryo tuibe culltutres.
Virus precursors. Dense particles of 28 to 31
nm in diameter and having clearly defined con-
tours were seen in the cytoplasmic matrix, fre- also found associated with the Golgi complex.
quently connected to membranes. A thin section Sometimes the virus precursors were directly ap-
of mouse cerebellum is shown in Fig. 3. Numerous plied to the cytoplasmic side of cisternae of this
precursors are aligned along the cytoplasmic side complex (Fig. 4).
of the triple-layered membranes which surround Three types of aggregates were found in the
flat saccular spaces having the appearance of cytoplasm near the nucleus: foci of electron-dense,
cisternae (thick arrow). Membranes showing virus finely textured material, tubular aggregates, and
precursors are distinctly thicker (thick arrow) mixed aggregates in which tubules seem to have
than those of endoplasmic reticulum (thin arrow). formed from the dense background. Sometimes,
In suckling-mouse brain, virus precursors were virus precursors were seen associated with theseVOL. 4. 1969 MORPHOGENESIS OF AURA VIRUS 273
aggregates (Fig. 5, 6). More frequently, however, the cytoplasmic matrix without apparent connec-
the aggregates showed no virus precursors (Fig. tion to any other structure.
7). These foci were always found in close proxim- Virus assembly. Virus precursors were de-
ity to the endoplasmic reticulum (Fig. 5, 6, 7). veloped into mature virus across several cellular
Virus precursors were occasionally scattered in membranes. Virus particles in the process of bud-
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- ,
. {/ t
/
~... . }
t (* @S* '4
FIG. 3. Mouse cerebellum 48 hr after inoculation. Virus precursors are aligned along the cytoplasmic side of
triple-layered membranes (thtick arrow). Mature virus particles are seen within the lumina of cisternae (broken
arrow). Membranes withl precursors (thick arrow) are distiiictly thicker than those of endoplasmic reticulum (thin
arrow). X 80,000.,~.N
274 LASCANO, BERRIA, AND BARRERA ORO J. VIROL.
4w~~~~
IL
iX~
Z * ~ ~ $ < #Bs-~VOL. 4, 1969 MORPHOGENESIS OF AURA VIRUS 275
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'441. )IS .,i;w.
ot v LX
'k
- Al. 4*¢s *, s 'lb Si At
.7-k
.
0
I..
.
.
.. A; w
'%Ot. *
.,
FIG. 5. Clhick embryo cell 16 hr after infrctioni. A viroplasnm-like aggregate qf denise, fintely textuired miiateri(al,
anid virus precuirsors are seez iin the cytoplasm, izear the iucleuis (N), closely associated to the enidoplasnmic reticillilm
(ER). X 100,000.
31 nm). In favorable thin sections, fine projec- nucleoprotein 140S particles, or nucleocapsids,
tions were seen extending from the viral membrane found in other group A arboviruses (9, 29, 30).
(Fig. 12, 13). The diameter of the virus particle In this discussion, "virus precursor" and "nucleo-
minus length of the projections was 50 to 54 nm. capsid" will be synonymous.
The most frequent localization of mature virus "Viroplasmic" foci of electron-dense materials
was the extracellular space. Intracellular virus was have been described in arboviruses (17, 25, 28,
found less frequently and in less quantity. One 31) as well as in other ribonucleic acid (RNA)
obvious location of Aura virus (Fig. 14, a micro- and deoxyribonucleic acid viruses. Recent elec-
graph taken from suckling-mouse brain) is within tron autoradiographic studies of Chikungunya
the lumen of a vesicle of the Golgi complex virus have shown viral RNA in these foci (N.
(arrow). Bunyamwera virus has recently been Higashi, First International Congress for Virol-
shown within Golgi vacuoles of mouse brain ogy, Helsinki, Finland, 1968). In this study, virus
(18). precursors were found within the viroplasmic
foci (Fig. 5). Although these observations have
DISCUSSION been obtained from different viruses, their sup-
Site of assembly of virus precursors (nucleo- port for each other tentatively postulates that the
capsids). The virus precursors observed in the electron-dense masses observed in this study are
cytoplasm of Aura virus-infected cells are very assembly sites for virus nucleocapsids. The close
probably the morphologic equivalent of the ribo- proximity of the foci to the endoplasmic reticulum276 LASCANO, BERRiA, AND BARRERA ORO J. VIROL.
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4.
*I.:: 4'.¶
4
V .'. j
* ,
FIG. 6. Aggregate of tuibules (arrow) and virus precursors (broken arrow) in the cytoplasm of a chick embryo
cell 24 hr after infection. Endoplasmic reticiulum (ER). Mitochiondria (M). X 100,000.VOL. 4,1969 MORPHOGENESIS OF AURA VIRUS 277
~
4.
1. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ M
ttt*~~
-
W-
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Fr.6R-
'~~~~~~~~~~~'I-
--#i
- *7W.
$ S
A@
k i - .z.
p
.4 -
to
~~~4%~~ ~~~~~1%
whic
Mouse
7.
FIG. 72 hr afte inoculation wit a rounded mass o electron-dense mterial from
cerbellum
structures see tob4 rgntn.Tewoemasi
tubular lotcmltl nirldb nolsi eiuu
100,000.~~~~~~~~~~~
(ER).
X
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*''-F.
FIG. 8. Virus particles budding,from the plasma membrane into the extracellular space (arrows) where several
virions can be observed. Virus precursors are lying, one behind the other, beneath the budding particles (arrows).
Membranes and cytoplasmic invaginations lined with dense particles are seen within the cell. Clhick embryo fibro-
blast 16 hr after infection. X 80,000.
FIG. 9. Portion of nucleus (N), n,ucleolus (Nu), and cytoplasm (C) of chick embryo cell 16 hr after infection.
Five particles in different stages are shown budding into the perinuclear cisterna from a protrusion of the rough endo-
plasmic reticulum. A cluster of particles is being released in the cisterna (righit lower corner). X 120,000.
278VOL. 4, 1969 MORPHOGENESIS OF AURA VIRUS 279
.1
f,
..,? -4.1-, qv_ AP
ip %1.11: ..Ol
*-. .,.
.1 -4
I
lw '' *
v ......
_ 9
-~~~~~~~~~~-
6 .~~~~~~~~~~..I&
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a II
t -
k-
& 4* ,U. ...I
I
T
I
ik
.t
FIG. 10. Huge membrane-bound vacuole of a chick embryo cell sampled 16 hr after inifectionz. Virions and several
invaginations with virus precursors lining their cytoplasmic sides are seeni within the vacuole lumeni. Virus precursors,
apparently lying free (arrow), probably belong to a tangentially cut cytoplasmic protrusion. One particle is buddinig
from the membrane which borders an invagination (brokenz arrow). X 40,000. The inset shows the budding particle
at higher magnification2. X 120,000. Nucleus (N), endoplasmic reticulum (ER), mitochondriont (M), plasma mem-
brane (PM).
FIG. 11. Enlargement of Fig. 3. Virus precursors partially eni veloped by triple-layered cytomembranes seem to be
initiating budding processes (arrows). X 200,000.
FIG. 12. Aura virions in a thin section of chick embryo cells. The densely stained core is surrounided by a triple-
layered membrane, clearly showing only the middle (electron-clear) and the outer (electron-dense) leaflets. Some
fine projections can be seen extending from the viral membranes. X 300,000.280 LASCANO, BERRIA, AND BARRERA ORO J. VIROL.
(Figs. 5-7) would explain frequent nucleocapsids of cytomembranes in the assembly of virus pre-
attachment to cytoplasmic membranes; further, cursors. The tubular structures that were seen
this explanation would fit in with earlier (16) and associated with viroplasmic foci (Fig. 7) and
more recent (10) suggestions concerning the role virus precursors (Fig. 6) would participate in the
assembly of the latter in a way which at present
is obscure. No convincing evidence was found
which suggested that nucleocapsids originated
*ost
:
from the tubular structures. Cross sections of
$s, b
ws
w_ tubules roughly coincided with diameters of pre-
.? X ., Z cursors (Fig. 6), but in other preparations not
shown in this study they were larger. In Keme-
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'< J
,,rqt 't w-s
tO w.#}
; rovo virus, "viral matrix" and tube-like struc-
_ @E Si -
.4. tures very similar to those of the present study were
s!, ,,S seen in close association with virions (25, 31).
4: J. Biochemical studies have provided data indicat-
ing that the process of nucleocapsid formation is
very rapid in group A arboviruses (3, 8). Thus,
any morphologic expression of this process, such
FIG. 1 3. Virus particle budding at tlhe cell surface. as those suggested by this study (Fig. 5-7), should
Fine projections are seeui at the bottom and on tlle left
side of the particle. Moutse cerebelluim 72 hr after in1ocIu- be a relatively infrequent finding.
lation. X>200,000. Virus assembly. The findings presented in this
d , .06
~'...- *.L M 41a
FIG. 14. One particle of Auira viruts is seen withint the hlmen of a vesicle (arrow) of the Golgi complex. Mouise
cerebelluim 72 hr after inoclulcation. X 80,000.VOL. 4, 1969 MORPHOGENESIS OF AURA VIRUS 281
paper support previous suggestions of a single 9. Friedman, R. M., and I. K. Berezesky. 1967. Cytoplasmic
fractions associated with Semliki Forest virus ribonucleic
mechanism of viral assembly for several members acid replication. J. Virol. 1:374-383.
of group A arboviruses (6, 10, 12, 16). In Aura 10. Grimley, P. M., I. K. Berezesky, and R. M. Friedman. 1968.
virus, a similar process of budding was seen to oc- Cytoplasmic structures associated with an arbovirus infec-
cur across different cellular membranes. Pre- tion: loci of viral ribonucleic acid synthesis. J. Virol. 2:1326-
1338.
formed, intracytoplasmic nucleocapsids acquired 11. Higashi, N. 1966. Electron microscopy of the multiplication
envelopes from the cellular membranes, thus of Chikungunya virus in cell cultures. Japan. J. S. Asian
forming mature virus particles that were finally Studies 4:88-94.
released into extra- or intracellular spaces. The 12. Higashi, N., A. Matsumoto, K. Tabata, and Y. Nagatomo.
1967. Electron microscope study of development of Chikun-
single mechanism for the development of the gunya virus in green monkey kidney stable (VERO) cells.
virus precursor into mature virus was observed
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Virology 33:55-69.
both in the suckling-mouse brain and in cultured 13. Luft, J. H. 1961. Improvements in epoxy resin embedding
chick embryo cells. No indication of other ways methods. J. Biophys. Biochem. Cytol. 9:409-414.
14. McGee-Russell, S. M., and G. Gosztonyi. 1967. Assembly of
of virus formation (7, 14) could be found for Semliki Forest virus in brain. Nature 214:1204-1206.
Aura virus. 15. Millonig, G. 1961. Advantages of a phosphate buffer for OSO4
The discovery of virus particles budding into solutions in fixation. J. Appl. Physiol. 32:1632.
the perinuclear cisterna from the bordering, 16. Morgan, C., C. Howe, and H. M. Rose. 1961. Structure and
development of viruses as observed in the electron micro-
rough, endoplasmic reticulum (Fig. 9) was for- scope. V. Western equine encephalomyelitis virus. J. Exp.
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eses on the participation of the membrane in the 17. Murphy, F. A., P. H. Coleman, A. K. Harrison, and G. W.
assembly of intracellular virions (6, 10; E. F. Gary, Jr. 1968. Colorado tick fever virus: an electron micro-
scopic study. Virology 35:28-40.
Lascano et al., in press). Although no actual bud- 18. Murphy, F. A., A. K. Harrison, and T. Tzianabos. 1968. Elec-
ding particle from the Golgi complex could be tron microscopic observations of mouse brain infected with
seen, the presence of virus precursors (Fig. 4) Bunyamwera group arboviruses. J. Virol. 2:1315-1325.
and virus (Fig. 14) closely associated to it sug- 19. Mussgay, M., and R. Rott. 1964. Studies on the structure of
a hemagglutinating component of a group A arbovi-
gests that membranes of this complex may also rus (Sindbis). Virology 23:573-581.
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22. Reynolds, E. 1963. The use of lead citrate at high pH as an
electron-opaque stain in electron microscopy. J. Cell Biol.
ACKNOWLEDGMENT 17:208-212.
23. Ryter, A., and E. Kellenberger. 1958. L'inclusion au polyester
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and Biochemistry, Division of Biologics Standards, National 24. Saturno, A. 1963. The morphology of Mayaro virus. Virology
Institutes of Health, Bethesda, Md. for reviewing this manuscript. 21:131-133.
25. Shestopalova, N. M., V. N. Reingold, L. G. Karpovich, and
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