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JOURNAL OF VIROLOGY, Sept. 1969, p. 271-282Copyright C) 1969 American Society for Microbiology

Vol. 4, No. 3Prilited in U.S.A.

Morphogenesis of Aura VirusEDUARDO 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 ofgroup A arboviruses, was studied by electron microscopy in suckling mouse brainand chick embryo cultured cells. Virus precursors, budding particles, and completevirus particles were first detected 10 hr after infection in chick embryo cells and24 hr after inoculation in mouse brain. Virus precursors were generally seen alignedalong cytomembranes, and were less frequently seen closely associated with viro-plasm-like foci, tubular aggregates, or scattered in the cytoplasmic matrix withoutan apparent connection to any other structure. The assembly of mature virus wasobserved to take place by a budding process of the virus precursor from the plasmamembrane into the extracellular space, and from the cytoplasmic membranes intothe lumina of vacuoles and cisternae. It was demonstrated that the endoplasmicreticulum participates in the assembly of intracellular virions. Indirect evidence was

found to indicate that the Golgi complex may also form mature virus. Aura virionshad a size, shape, and structure similar to those of the previously described group Aarboviruses.

Aura virus was first isolated by Causey et al.(5) in Belem, Brazil. It has been demonstrated tobe a group A arbovirus which is closely related toWestern equine, Sindbis, and Whataroa viruses(5, 33). So far, Aura virus has been found infect-ing mosquitoes of only South American forests(2, 5). It is pathogenic for the newborn mouse,growing well in tissue culture cells; apparently,however, it is not pathogenic for man.

Previous electron-microscopy studies have pro-vided important information on the develop-mental characteristics of group A arboviruses.Among other findings, they have shown thepresence of virus precursors in cells infected withseveral members of the group (1, 11, 16, 20), andthe budding of virus particles at the cell surface(1, 6, 16). Electron-microscopy studies have alsodemonstrated the final assembly process of maturevirus from portions of the plasma membrane (1,12) modified by the virus infection (12). Allstudies of thin sections of group A arboviruseshave also shown intracellular virions within thelumina of vacuoles (1, 6, 16, 20, 24). However, aclear demonstration of the assembly process ofintracellular virus is still lacking. Some studies,have suggested that precursor particles acquireenvelopes and form mature virus as they crossvacuolar membranes (7, 10, 12, 16), but it hasalso been theorized that the assembly may takeplace within the lumina of cisternae (14) and

vacuoles (7). Different hypotheses have postulatedthe sites of formation of virus precursors (1, 10,12, 16) and the origin of membranes involved intheir final development into mature virus (6, 10,16).

In this electron-microscopy study, the assemblyof Aura virions from the plasma membrane andintracellular membranes is demonstrated. In-direct morphologic evidence sugegsting that theGolgi complex may also form mature virus ispresented.

MATERIALS AND METHODSThe A. se-rraltis strain of Aura virus (2) was used

throughout this study. The eighth mouse brain pas-sage since isolation was used for experiments in mice,and the 10th chick embryo cell passage (18th passagesince isolation), for experiments in tissue culture cells.

Litters of 1- to 2-day-old albino mice were infectedby intracerebral inoculation with 0.02 ml of brainsuspension containing 10:3 LD,o of Aura virus. Three tofive mice were sacrificed at different intervals there-after, and brains were collected for electron micros-copy and control of infectivity. Samples used forpreparing the micrographs shown in this study hadvirus titers of 109"8to 1 _lLD5o per g of brain tissue.

Chick embryo fibroblasts were grown in a mediumcomposed of Hank's Balanced Salt Solution (BSS)with 0.5'c lactalbumin hydrolysate, 10'- tryptosephosphate, 10%' fetal bovine serum, 100 units penicil-lin per ml, and 100 tg of streptomycin per ml. Mono-layers of about 107 cells per bottle were obtained.After three washings with Hank's BSS, each bottle

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LASCANO, BERRIA, AND BARRERA ORO

was incoulated with 1 ml of undiluted chick embryotissue culture supernatant fluid that contained 107TCD,o of Aura virus. The inoculum was left to adsorbat room temperature for 2 hr and was then removed.The monolayers were washed three times with Hank'sBSS and fed with maintenance medium in whichHank's BSS was substituted by Earle's BSS, and thefetal bovine serum was reduced to 2.5%',; the othercomponents remained the same as those of the grow-ing medium. Bottles were incubated at 36 to 37 C;at certain intervals, some of them were removed forstudy by electron microscopy and control of infec-tivity of cell-associated virus. Samples for the micro-graphs of this study showed virus titers of 10 to 107TCD;Io per ml of cell-associated virus.

Brains of mice inoculated with brain suspensionsfrom healthy mice, and chick embryo monolayersinoculated with tissue culture supernatant fluids fromuninfected chick embryo cells were used as controls.

For ultrathin-section electron microscopy, chick-embryo cell pellets were fixed in Millonig's buffered(15) 1% paraformaldehyde for 20 min; minced mousebrain was fixed in Millonig's buffered 4%,/; paraformal-dehyde for 1 hr. Thereafter, both materials wereequally treated as follows. They were washed twicewith buffer, refixed in I % osmium tetroxide for 1 hr,washed four times with veronal buffer, immersed in50',, ethyl alcohol-saturated uranyl acetate for 2 hr,dehydrated in graded alcohols, immersed in propyleneoxide, and embedded in Epon 812 (13) or Vestopal(23). Sections were cut with glass knives, stained withuranyl acetate (32), and then stained with lead citrate(22). Specimens were examined in a Siemens ElmiskopI electron microscope.

RESULTSThe three developmental stages of group A

arboviruses (virus precursors, budding particles,and mature virus particles) were found in thinsections of Aura virus-infected suckling-mousebrain and chick embryo cultured cells. They werefirst detected 10 hr after infection in chick embryocells and 24 hr after inoculation in mouse brain,but were more frequently found at times whenmaximal virus titers were obtained, i.e., 16 to24 hr after infection in chick embryo cells and 48to 72 hr after inoculation in mouse brain (Fig. 1,2).

Virus precursors. Dense particles of 28 to 31nm in diameter and having clearly defined con-tours were seen in the cytoplasmic matrix, fre-quently connected to membranes. A thin sectionof mouse cerebellum is shown in Fig. 3. Numerousprecursors are aligned along the cytoplasmic sideof the triple-layered membranes which surroundflat saccular spaces having the appearance ofcisternae (thick arrow). Membranes showing virusprecursors are distinctly thicker (thick arrow)than those of endoplasmic reticulum (thin arrow).In suckling-mouse brain, virus precursors were

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FIG. 2. Growth curve of cell-associated Autra virusin chick embryo cultured cells. Titrationis were carriedoutt in chick embryo tuibe culltutres.

also found associated with the Golgi complex.Sometimes the virus precursors were directly ap-plied to the cytoplasmic side of cisternae of thiscomplex (Fig. 4).

Three types of aggregates were found in thecytoplasm near the nucleus: foci of electron-dense,finely textured material, tubular aggregates, andmixed aggregates in which tubules seem to haveformed from the dense background. Sometimes,virus precursors were seen associated with these

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MORPHOGENESIS OF AURA VIRUS

aggregates (Fig. 5, 6). More frequently, however,the aggregates showed no virus precursors (Fig.7). These foci were always found in close proxim-ity to the endoplasmic reticulum (Fig. 5, 6, 7).Virus precursors were occasionally scattered in

the cytoplasmic matrix without apparent connec-tion to any other structure.

Virus assembly. Virus precursors were de-veloped into mature virus across several cellularmembranes. Virus particles in the process of bud-

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FIG. 3. Mouse cerebellum 48 hr after inoculation. Virus precursors are aligned along the cytoplasmic side oftriple-layered membranes (thtick arrow). Mature virus particles are seen within the lumina of cisternae (brokenarrow). Membranes withl precursors (thick arrow) are distiiictly thicker than those of endoplasmic reticulum (thinarrow). X 80,000.

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FIG. 4. Virus precursors aligned alonig cisternae of the Golgi complex (arrow). Mouse cerebellum 53 hr afterinioculation. X 40,000.

ding from the cell surface into the extracellularspace are shown in Fig. 8. Virus precursors, lyingone behind the other beneath the plasma mem-brane (arrows), look as if they all were going tobud from the same place on the membrane.

Intracellular formation of virus. Figure 9 showsdifferent stages of five particles budding into theperinuclear cisterna from a protrusion of thebordering, rough, endoplasmic reticulum. In thelower right of the figure, a cluster of virus particlesis being released into the perinuclear cisterna.Figure 10 shows a big cytoplasmic vacuolesituated near the nucleus of an Aura virus-infected chick embryo cell. Several invaginationshave virus precursors lining their cytoplasmicsides; one particle is budding from the membranethat encircles an invagination (see inset of Fig.10). Several virus particles are seen within thelumen of the vacuole. In Fig. 11 (an enlargement

of Fig. 3) virus precursors partially enveloped bycytoplasmic membranes appear to be initiatingbudding processes (arrows). The triple-layeringof membranes is clearly seen in this figure.

Virus morphology and localization. Aura virusshowed a morphology similar to that of the pre-viously described group A arboviruses (1, 6, 12,16, 19, 20, 21, 24, 26, 27). In thin sections, it dis-played a roughly spherical and sometimes polyg-onal profile (Fig. 8, 9, 10, 12-14). The virusparticle was composed of an inner core (nucleo-capsid) closely surrounded by a triple-layeredmembrane (Fig. 12). Only the middle (electron-clear) and the outer (electron-dense) leaflets wereclearly visible. The electron-dense, inner leafletwas generally masked by the similar density of thecore to which it was applied (Fig. 12). This wouldexplain why core diameters (32 to 35 nm) wereapparently larger than those of precursors (28 to

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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-tions were seen extending from the viral membrane(Fig. 12, 13). The diameter of the virus particleminus length of the projections was 50 to 54 nm.The most frequent localization of mature virus

was the extracellular space. Intracellular virus wasfound less frequently and in less quantity. Oneobvious location of Aura virus (Fig. 14, a micro-graph taken from suckling-mouse brain) is withinthe lumen of a vesicle of the Golgi complex(arrow). Bunyamwera virus has recently beenshown within Golgi vacuoles of mouse brain(18) .

DISCUSSIONSite of assembly of virus precursors (nucleo-

capsids). The virus precursors observed in thecytoplasm of Aura virus-infected cells are veryprobably the morphologic equivalent of the ribo-

nucleoprotein 140S particles, or nucleocapsids,found in other group A arboviruses (9, 29, 30).In this discussion, "virus precursor" and "nucleo-capsid" will be synonymous.

"Viroplasmic" foci of electron-dense materialshave been described in arboviruses (17, 25, 28,31) as well as in other ribonucleic acid (RNA)and deoxyribonucleic acid viruses. Recent elec-tron autoradiographic studies of Chikungunyavirus have shown viral RNA in these foci (N.Higashi, First International Congress for Virol-ogy, Helsinki, Finland, 1968). In this study, virusprecursors were found within the viroplasmicfoci (Fig. 5). Although these observations havebeen obtained from different viruses, their sup-port for each other tentatively postulates that theelectron-dense masses observed in this study areassembly sites for virus nucleocapsids. The closeproximity of the foci to the endoplasmic reticulum

VOL. 4, 1969 275

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276 LASCANO, BERRiA, AND BARRERA ORO J. VIROL.

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FIG. 6. Aggregate of tuibules (arrow) and virus precursors (broken arrow) in the cytoplasm of a chick embryocell 24 hr after infection. Endoplasmic reticiulum (ER). Mitochiondria (M). X 100,000.


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VOL. 4,1969 MORPHOGENESIS OF AURA VIRUS 277

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FIG. 8. Virus particles budding,from the plasma membrane into the extracellular space (arrows) where severalvirions 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 ofparticles is being released in the cisterna (righit lower corner). X 120,000.

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FIG. 10. Huge membrane-bound vacuole ofa chick embryo cell sampled 16 hr after inifectionz. Virions and severalinvaginations 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 buddinigfrom the membrane which borders an invagination (brokenz arrow). X 40,000. The inset shows the budding particleat 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 eniveloped by triple-layered cytomembranes seem to beinitiating 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. Somefine projections can be seen extending from the viral membranes. X 300,000.

279VOL. 4, 1969

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(Figs. 5-7) would explain frequent nucleocapsidsattachment to cytoplasmic membranes; further,this explanation would fit in with earlier (16) andmore recent (10) suggestions concerning the role

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FIG. 1 3. Virus particle budding at tlhe cell surface.Fine projections are seeui at the bottom and on tlle leftside of the particle. Moutse cerebelluim 72 hr after in1ocIu-lation. X>200,000.

of cytomembranes in the assembly of virus pre-cursors. The tubular structures that were seenassociated with viroplasmic foci (Fig. 7) andvirus precursors (Fig. 6) would participate in theassembly of the latter in a way which at presentis obscure. No convincing evidence was foundwhich suggested that nucleocapsids originatedfrom the tubular structures. Cross sections oftubules roughly coincided with diameters of pre-cursors (Fig. 6), but in other preparations notshown in this study they were larger. In Keme-rovo virus, "viral matrix" and tube-like struc-tures very similar to those of the present study wereseen in close association with virions (25, 31).Biochemical studies have provided data indicat-ing that the process of nucleocapsid formation isvery rapid in group A arboviruses (3, 8). Thus,any morphologic expression of this process, suchas those suggested by this study (Fig. 5-7), shouldbe a relatively infrequent finding.

Virus assembly. The findings presented in this

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FIG. 14. One particle of Auira viruts is seen withint the hlmen of a vesicle (arrow) of the Golgi complex. Mouisecerebelluim 72 hr after inoclulcation. X 80,000.

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paper support previous suggestions of a singlemechanism of viral assembly for several membersof group A arboviruses (6, 10, 12, 16). In Auravirus, a similar process of budding was seen to oc-cur across different cellular membranes. Pre-formed, intracytoplasmic nucleocapsids acquiredenvelopes from the cellular membranes, thusforming mature virus particles that were finallyreleased into extra- or intracellular spaces. Thesingle mechanism for the development of thevirus precursor into mature virus was observedboth in the suckling-mouse brain and in culturedchick embryo cells. No indication of other waysof virus formation (7, 14) could be found forAura virus.The discovery of virus particles budding into

the perinuclear cisterna from the bordering,rough, endoplasmic reticulum (Fig. 9) was for-tunate, for it clearly confirmed previous hypoth-eses on the participation of the membrane in theassembly of intracellular virions (6, 10; E. F.Lascano et al., in press). Although no actual bud-ding particle from the Golgi complex could beseen, the presence of virus precursors (Fig. 4)and virus (Fig. 14) closely associated to it sug-gests that membranes of this complex may alsoparticipate in the process of virus assembly. Thepresence of the virus precursors and virions makesit very difficult to speculate on the origin of somecytomembranes showing budding particles (Fig.3, 11). They could be heavily altered endoplasmicreticulum, but they could also be modified mem-branes of the Golgi complex, or both.

ACKNOWLEDGMENT

We thank Kendall 0. Smith of the Laboratory of Biophysicsand Biochemistry, Division of Biologics Standards, NationalInstitutes of Health, Bethesda, Md. for reviewing this manuscript.

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