Micromorphological Aspects of the Development of Rubella Virus in BHK-21 Cells

JOURNALOFVIROLOGY, Apr. 1969,p.439-444

Copyright@ 1969 AmericanSocietyforMicrobiology

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

Micromorphological

Aspects

of

the

Development

of

Rubella

Virus

in

BHK-21

Cells1

MERCEDES R. EDWARDS, SOPHIA M. COHEN, MINERVA BRUNO, AND RUDOLF DEIBEL

DivisionofLaboratories andResearch,New York StateDepartmentofHealthl,Albany,NewYork12201

Received for publication 29 November 1968

Sequentialeffects ofrubellavirusinfectioninBHK-21cells were studiedby

elec-tronmicroscopy of thinsections ofcontroland infectedcells,2to 7daysafter

infec-tion. Vacuolization of cytoplasm in Golgi areas apparently preceded budding of

virions from vacuole membranes and involvement ofthe endoplasmic reticulum.

Newly formed endoplasmic reticulum cisternae encircled and segregated

virion-forming vacuoles together with other cellularelements. Large vacuolarcomplexes

withnumerousvirusparticlesdeveloped,and virus release from theseareasoccurred with disruption atthe cellperiphery. The viral particles, with a meandiameterof

about 56nm,consistedofanelectron-dense coresurroundedbyaless densecapsid,

enveloped by a typical unit membrane derived from the vacuole membrane.

The rubellavirusparticle hasbeendescribedin

recent studies of strains propagated in cell

cul-tures of BHK-21 (3, 5, 6),RK(6; T. E. Hobbins

and K. 0. Smith, Bacteriol. Proc., p. 181, 1968),

and SIRC(8).Thefindings,ingeneralagreement,

haveindicated that rubella virus is not a

myxo-virus as suggested by an earlier report (13) but

mayfall intoataxonomicgroup yet tobedefined

(8, 9, 11). Limited data on the development of

rubella virions inSIRC cells have beenreported (8). The progressive events, however, in the development of the rubella virions in infected

cells have not been clarified. The present study

describes theultrastructuralchanges of BHK-21

cellsinfected with rubella virus and thesuccessive

stages in the formation and maturation of the

virions.

MATERIALS AND METHODS

Virus strain. M33 (N.Y.S.no.65-0287),isolatedby

Parkmanetal.(12),wasobtainedfrom The American Type Culture Collection asthe 20th passage in

pri-marycultures of African greenmonkeykidney cells,

and was adapted in this laboratory to the BHK-21

clone13cellline and used in the 9th and 11th passages for electronmicroscopy.

Cell cultures. The BHK-21 cells were grown in monolayers withEagle's modified mediumcontaining

10% fetal bovineserumand10%tryptosephosphate broth. Twoto4daysafter seeding, thecultureswere

inoculated withvirusat amultiplicityof about 0.2to

1.0TCID15 per cell. Maintenance mediumwasEagle's

IPresentedin partattheFirstInternational Congress for Virol-ogy,Helsinki, Finland, July15,1968.

with 3% fetal bovine serum. Control and infected cells wereexaminedfrom 2to7days post-infection.

Preparation for electron microscopy. Cell mono-layers were fixed withglutaraldehyde-osmium tetrox-ide (15) or chrome-osmium tetroxide (A. J. Dalton,

Anat. Rec., p. 281,1955), washed withbuffer,scraped into a fresh change ofbuffer, andsedimentedby

cen-trifugation. After dehydration in a graded series of

ethyl alcohol solutions followed by propylene oxide,

the material was infiltrated and embedded in an

Eponmixture (7). Thin sections were obtained with

Reichert or LKB ultratomes, stained with uranyl acetateandleadcitrate (17), andphotographedwith SiemensElmiskopmodels I and IAatmagnifications

of20,000 to80,000 X.

RESULTS

Inthe infected cell cultures, cytopathic effects

wereevident in 48 hr. Infectivity titers of

super-natant fluids in repeated tests were 1058to 106 5

TCIDso per ml at48 hr and 1015 to 107-5 at 72

hr.Thepresenceof rubellaviral antigenwas

con-firmed by immunofluorescence with rabbit homologous antiserum or human convalescent-phaseserum (4) before examination ofduplicate

cell cultures for electron microscopy.

Control cellsexhibitednormalGolgistructures

(1), generally abundantinactively growing cells,

withcloselypacked stacksoflamellaeand a

pro-fusion ofsmallvesiclesusually buddingoff from

the lamellae. Large vesicles or vacuoles were

rarely found. Profiles of the endoplasmic reticu-lum varied widely in different cell sections.

Typicalmitochondria,free orattachedribosomes,

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activity with little further change from normal cells (Fig. 1). Involvement of the endoplasmic reticulum was evident after the appearance of virions in vacuoles. Profiles of the endoplasmic reticulum were readily identified around these vacuoles containing virions (Fig. 2), sometimes encircling them completely (Fig. 7). Virions budding from the vacuole membrane were fre-quently found (Fig. 2, 4). The triple-layered

structure ofatypicalunit membrane (14) could

clearly be seen inthe vacuole membrane and in the viralenvelope (arrow Fig. 2, 8, 9). Budding

fromplasma membraneswasnotobserved inour

preparations.

Cellsat moreadvancedstagesofviralinfection,

4and7days,werein variousstagesof

disintegra-tion. Infectivity titers of fluids from the 4-day

culture were 105-5 TCID50 per ml. Later effects of infectionwerecharacterizedby segregation of cortical cytoplasmicareasthat contained numer-ous membranous structures and large vacuoles with increased numbers of viral particles. The newly formed smooth endomembranes appeared

to originate from the rough endoplasmic reticu-lum which branched in various directions. They encircled theareas containing virus particles and

separated themfrom other portions ofthe

cyto-plasm. The segregation process, generally

exten-sive, also enclosed normal

organeiles,

mitochondria and lysosomes, with resulting

structuresthatmaycorrespondto thephagocytic

vacuoles described by Holmes et al. (6). The vacuolar complexes apparently became

perme-able, dilated, and burst atthe cell surface,

dis-charging masses of virions and cell debris (Fig. 3, 10).

Mature spherical virions ranged in diameter

from about 45 to65 nm (Fig. 8to 10). The

ru-bella virion with a diameter of 57.5 nm was

enveloped by a triple-layered single membrane

approximately 8 nm in width (Fig. 8). The

interior of the particle consisted of a highly electron-dense core, ca. 18.5 nm in width,

sur-roundedbya capsidofmedium tolightelectron density, ca. 11.5 nm wide. Variations in density

and size ofcores (Fig. 8,9) may be due to

differ-entplanesofsectioningorpreparatory methods.

Our observations on sequential effects of rubella virus infection in BHK-21 cells revealed preliminary signs of infectionintheGolgiregions

followed by budding of virions from vacuole

membranes and involvementofthe endoplasmic reticulum cisternae. Frequently, the endoplasmic reticulum cisternae developed around initially small vacuoles containing virions. They

appar-ently continuedtoproliferate, segregating

exten-sive affected areasand enclosing large, complex, vacuolar structures and also islets of cytoplasm

with organelles such as mitochondria and

lyso-somes. Formation of such structures by

con-fluence of the small vacuoles seems to be an

autophagic process rather than phagocytic as

suggested byHolmesetal. (6). Increased

perme-ability and dilation of the vacuoles couldatfinal

stages cause theirdisruptionatthecellperiphery

with virus release. The virus particles were

clearly seen to be enclosed by a typical unit membrane derived from thevacuole membrane.

Ourfindings arein general agreementwiththose of others onthesize andmorphology of rubella virions of different strains isolated and

propa-gated in various cell lines.

A paper by Murphy et al. (10) on electron

microscopy of the development of rubella virus

in BHK-21 cells appearedjust asthispaper was

being completed. Their observations differ from

ours in several important respects: no

morpho-logical changeswere seen ininfected cellsbefore budding of virusfrom cell membranes; budding

was more prominent from the cell membrane

than from endomembranes and was seen in

cisternae of the endoplasmic reticulum; the

envelope of the virion was not indicated as a

typical unit membrane; latent hamster virus seemed to be more numerous. Certain ofthese differences may have resulted from the high

passage level of their virus strain in BHK-21

cells (38ascomparedwith 9or 11 inourstudy)

or from the preparatory methods.

ACKNOWLEDGMENTS

Theauthorsacknowledge the excellent technical assistanceof RobertJ. Bendon.

This investigation was supported by Public Health Service

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FIG. 1.Portions oftwoadjaceintcellsslhowingnucleuis (n), mitochonidria (m), dilated Golgi cisternae (g), scarce

elements of the endoplasmic reticulum (er), cytoplasmicfilaments, and free ribosomes. Note, at intercellular

space,virions(arrows)and pinocytoticvesicles inthisregion. Avirion (v) isseenin a small vesicle.

Glutaraldehyde-osmiumfixation. X 30,000.

FIG. 2. Portion of infectedcellrevealing vacuoles presumablyderivedjfrom Golgicisternae (g) and virions (v).

At the lowerleft(arrow), notethecontinuationbetween vacuolemembraneand envelopeof virion. Profilesofthe

endoplasmic reticuluim (er) in the viciniity of vacuolesare in,dicated. Glutaraldehyde-osmium fixation. X 60,000. 441

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.44

FIG. 3. Virionis (arrows) inipartiallydlisruipted vacuiole,orvacutoleswhichalsocoIItainl cdgentercatedmembranous

organellesanld amorphousmaterial.Gluitaraldeytyde-osmilum

.xationl.

FIG. 4. Budcldinigparticle exhibitilng enlvelope still conttinuous wit/i the vacuiolemembrane (lobug arrow). Short

arrowpoinlts to acloutbleparticle. Glittaraldehyde-osmium fixation. X 120,000.

FIG.5.Douible virio,iwit/hcommontbaseatpoinltqIoriginiltvacucolemembranle(arrow).Gllutaralclehydle-osnmiluI

fixatioui. X 120,000.

FIG. 6.FCJrtherexampleojdoublevirionls. Glutaraldehyde-osmiumfixationu. X 120,000.

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FIG. 7. Cytoplasmicportionis of adjacent cells, one of which exhibitsinumerouts virions withiln a vacuole

com-pletelyencircledbyacisternalelementof theendoplasmic reticulum (er). Mitochondria and attachedorfree

ribo-somesareseeninbothcellportions. Chrome-osmiumfixation. X30,000.

FIG.8. Maturevirioni exhibiting electron-dense core, capsid, and enveloping membrane (unit membrane).

Clhrome-osmiumfixation. X 240,000.

FIG.9. Virionswithslightlylessdensecoresthan that ofFig.8,butalsoenvelopedbyunitmembranes. Glutaral-dehyde-osmiumfixationt. X 200,000.

FIG. 10. Portioniofdegenerating cell,atadvanicedstageofinfection,displayingmassesofvirionsassociated with

brokent membranesanidamorphous material. Note virions with coresofvarying density (v, vi).

Glutaraldehyde-osmiumfixation. X 60,000.

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Lancet2:237-239.

4. Deibel, R., S. M. Cohen,andC.P.Ducharme.1968. Serology of r-ubella. Virus neutralization, immunofluorescence in BHK21 cells, and hemagglutination inhibition. N.Y. State J. Med.68:1355-1362.

5. Holmes,I.H.,and M.F. Wairburton.1967. Is rubellaan arbo-virus? Lancet 2:1233-1236.

6.Holmes, 1. H.,M. C.Wark, I.Jack, and J. Grutzner. 1968.

Identification oftwo possible types ofvirus particle in rubella-infected cells. J. Gen. Virol. 2:37-42.

7. Luft, J. H. 1961. Improvements in epoxy resin embedding methods.J. Biophys.Biochem.Cytol. 9:409-414. 8. McCombs, R. M., J. P. Brunschwig, and W. E. Rawls. 1968.

Morphology of rubella virus. Exptl. Mol. Pathol. 9:27-33.

615-618.

14. Robertson,J. D. 1959. Theultrastructureofcell membranes and their derivatives. Biochem. Soc. Symup. (Cambiidge, England) 16:3-43.

15. Sabatini, D. D., K. Bensch, and R. J. Barrnett. 1963. The preservation of cellular ultrastructure and enzymatic

ac-tivity of aldehyde fixation. J. Cell Biol. 17:19-58.

16. Thomas, A. J.,E. Delain, and E. Hollande. 1967. Morpho-genese d'un virus du hamster associe a la souche BHK-21

oua destumeurs.Compt. Rend. 264:785-788.

17. Venable, J. H., and R. Coggeshall. 1965. Asimplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25:407-408.

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