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In Vitro Morphology and Maturation of Lymphocystis Virus

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MIDLIGE AND MALSBERGER

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FIG. 1-4.Electronimicrographsofthinisections of

inifected

BF-2cells. Thebarin eachfigure represents I,u.

FIG. 1.Sectioni of cells harvested4days P1showing clumps of extracellularvirus (ECV). Cellsotherwise

re-semble normalcellsexcept that theniumerous cytoplasmicprocesses havewithdrawn. X

9,000.

FIG. 2.Sectionofcellsharvested 8daysP1 showingthe

periniuclear

iniclusion

body

(1),niucleus

(N),

anidnumerous

mitochondria (M). X 18,000.

FIG. 3-4. Section ofcells harvested 15days P1showing inzclusionz (1),

fenestrationis (F),

ancd

niumerousvirionts

(V) adjacenlt to theamorphous iniclusiontsubstanice and withini the

fenestrationis.

Note

conitinuiiity

of feniestrationis

with thecytoplasmicmatrix.FiG.3, X 16,000.FIG. 4, X 11,000.

832 J. VIROL.

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INVITRO STUDIES OF LYMPHOCYSTIS VIRUS 833

I4

f

446

...~ ~ ~ ~

4

-,..f

FIG. 5-7. The bar in eachfigure represents I A. Sectionsofcells harvested28days P1 showing thecomplete

disintegration ofcellularstructures,the persistence ofthe inclusion substance (1), and the massive accumulation

ofvirusparticles (V) withtin thecells. No release wasnoted inanysections examined. FIG.5, X 6,000. FIG. 6,

X15,000. FIG. 7, X 80,000.

VOL. 2, 1968

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834 MIDLIGE AND MALSBERGER J. VIROL.

5'

/

S

11

FIG. 8-1 1.Electron2

microgralphs

of

ntegatively

stainied lymphocystis virlusparticles.

FIG.8-9. Thtebarineach

figuire

represents Ij1.Negatively

stain2ed

LDVparticles fromBF-2cells,28daysP1,

anidassociatedcell debris. No capsomerestructure was

evidelnt.

Close

inispectioni in7dicates

a

membranious

capsid

collapsed abolut the core. FIG. 8, X 31,000. FIG. 9, X 60,000.

FIG. 10-I1. The bar ineach

figutre

represenlts 0.1

.I.

Negativelystailed LD VparticlesfromBF-2 cells, 28days

P1,showinigpresuimptive spikes (S) orfilamen7tsprojectinigin7tothesuirrouniidinigphosphotuniigstic acidstainl. FiG.

10, X 220,000.FIG. 11, X 240,000.

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[image:3.490.59.451.76.576.2]
(4)

IN VITRO STUDIESOF LYMPHOCYSTIS VIRUS

inclusions

that contain DNA as determined by Feulgen staining and

acridine-orange

ultraviolet

fluorescence.

The inclusions associated with the amphibian viruses appear to be ofamorediffuse type than those of LDV and the insect viruses. Both these amphibian viruses and the insect iridescent viruses replicate in a more rapid temporal sequence than doesLDV, andallexcept

Tipula iridescent

virus

acquire

anaccessory mem-brane by a

budding

process at the cytoplasmic membrane during

liberation

from the cell. LDV differs from these viruses

by

virtue of its larger diameter and lack of an accessory membrane.

LDVmay be related to the

amphibian

and insect viruses mentionedabove, although defectivewith

regard toacquisition of an accessory membrane.

It is obvious that LDV does not fitinto any of the establishedgroups ofthepresent classification scheme.

ACKNOWLEDGMENTS

This investigation was supported

by

grant E-402 from the American Cancer Society and grant GB-6228from the NationalScienceFoundation.

LTERATURE CITED

1. Bird,F. T.1962. On the

development

of the

Tipula

iridescent virus particle. Can. J. Microbiol.

8:533-534.

2. Bellett, A. J. D., and E. H. Mercer. 1964. The

multiplication of Sericesthis iridescent virus in cell cultures from Antheraea eucalypti

(Scott).

I. Qualitative experiments. Virology 24:645-653.

3. Darlington,R.W.,A.Granoff,and D.C. Breeze. 1966. Viruses and renal carcinoma of Rana

pipiens. II. Ultrastructural studies and sequen-tialdevelopment of virus isolated from normal andtumor tissue.Virology 29:149-156. 4. Dunbar,C.E., and K. Wolf. 1966. The cytological

course of experimental lymphocystis in the bluegill. J. Infect. Diseases 116:466-472. 5. Leutenegger, R. 1964. Development of an

icosa-hedral virus in hemocytes of Galleriamellonella (L).Virology 24:200-204.

6. Lunger, P. D., and P. E. Came. 1966. Cytoplasmic viruses associated with Lucke tumor cells. Virology30:116-126.

7. Lunger, P. D. 1966. Amphibia related viruses. Advan. Virus Res. 12:1-33.

8. Parsons, D. F. 1963. Mitochondrial structure: Two types of subunits on negatively stained mitochondrial membranes. Science 140:985. 9. Walker, R. 1962. Fine structure of lymphocystis

virus offish.Virology 18:503-505.

10. Walker, R. 1965.Conformity of light and electron

microscopicstudies of virusparticle distribution inlymphocystistumor cells of fish. Ann. N.Y. Acad.Sci. 126:375-385.

11. Walker, R., and K. Wolf. 1962. Virus array in

lymphocystis cells of sunfish. Am. Zoologist 2:566.

12. Wolf, K. 1962.Experimental propagation of

lym-phocystis disease of fish. Virology 18:249-256. 13. Wolf, K. 1966. The fish viruses. Advan. Virus.

Res. 12:35-101.

14. Wolf, K., and C. P. Carlson. 1965.Multiplication oflymphocystis virus in thebluegill (Lepomis macrochirus). Ann. N.Y. Acad. Sci. 126:414-419.

15. Wolf, K., M. Gravell, and R. G.Malsberger. 1966. Lymphocystis virus; isolation and propagation in centrarchid fish cell lines. Science 151:1004 1005.

VOL. 2, 1968 835

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JOURNAL OFVIROLOGY, Aug. 1968, p. 836-849

Copyright ©1968 AmericanSociety for Microbiology

Vol.2, No.8

Printedin U.S.A.

Purification

of

Rables Virus

Grown

in

Tissue Culture

FRANTISEK SOKOL, ERNEST KUWERT, TADEUSZJ. WIKTOR, KLAUS HUMMELER, AND

HILARY KOPROWSKI

The WistarlhtstituteofAnatomyandBiology,the WorldHealthOrganiization International Reference Centerfor

Rabiesatthe WistarInistitute,and TheChildren'sHospital of Philadelphia, Phliladelphia, Penniisylvania19104

Receivedforpublication4 May 1968

Extracellular rabies

virus,

grown in monolayer cultures of BHK21 cells in the

presenceof mediumsupplemented with bovineserumalbumin,was purified by the

following procedure. Virus was precipitated from infectious tissue culture fluid by

zincacetateandwasresuspended inasolution ofethylenediaminetetraacetate. The

suspension was filtered through a Sephadex column and was treated with

ribo-nuclease anddeoxyribonuclease. The virionswerethenpelleted by centrifugationat

high speed andwere resuspended in buffer solution. Banding ofthevirusby

cen-trifugation in a sucrose density gradient wasthe final step in the purification

pro-cedure. Purified preparations contained bullet-shaped virus particles of variable length and little (up to 5%) contaminatinghost-cell material. Most ofthe virions

were "complete", i.e., 180 nm long, but some virus particles were shorter. The

length distribution of the virions was nonrandom. Shorter virions seemed to be noninfectiousand showedmarkedlydecreasedhemagglutinating activity. The

com-plement-fixing activity andtheribonucleic acid toproteinratio of thevirions were

not relatedto the length of the virus particles. Although the properties of

extra-cellular and intracellular viruses were similar, the procedure was not suitable for

purification ofintracellular rabies virus.

Large

quantities of

highly purified

virus are

necessary for the

study

of the

antigenic

structure

and the chemical

composition

of the virion.

Modified

methods for

cultivating

rabies virus in

tissue

culture,

described in the present report, have made it

possible

to obtain increased

yields

of virus. Similar

improvement

in methods for

quantitative

assay of the virus

(24)

had

previously

opened

the way to

purification

of rabies virus

grown in

tissue

culture. This report summarizes the

results of

experiments

in which an efficient recovery rate

of

highly purified

virus was

achieved.

Some

properties

of

purified

rabies virons are also described.

MATERIALS AND METHODS

Virus straini. The Pitman-Moore (PM) and

Flury

high egg passage(HEP)strains of rabiesvirus,adapted

togrowthinhuman

diploid

cell strainWI-38and prop-agated in these cells for 52and 16passages, respec-tivelv(30),werethreetimes

clone-purified by plaquing

inagarose-suspendedBHK/13Scells (24).Seedpools

of viruswerepreparedinBHK21cellcultures.

Tissue cultures. Two hamster cell

lines,

BHK21

clone 13 (17) and Nil-2 cells (4), were

propagated

in 1-liter Blake bottlesas described

previously

(14).

Buiffer

solution2s

used in virus

purification.

NT

buffer was composed of 0.13 M NaCl and 0.05 M

tris(hydroxymethyl)aminomethane

(Tris)

- chloride

(pH 7.8); NTEbuffer was composed of 0.13 MNaCl,

0.001 M ethylenediaminetetraacetate (EDTA), and 0.05 M Tris-chloride (pH 7.8).

Inifectionz

ofcells and virus

propagationi.

After re-moval of the growth medium, cell cultures were in-fected with 2 ml of seed virus containing 50 ,g of

diethylaminoethyl(DEAE) dextran per ml (Pharmacia Fine Chemicals, Piscataway, N.J.). DEAE dextran

wasaddedtoincrease the efficiency of virus adsorption andpenetration (14). After 1 hr of adsorption at 35

C,

theinoculumwasremoved; the cell monolayers were washedoncewithwarmphosphate-buffered saline (6), andwererefedwith growth medium containing either 10%heat-inactivated calf serum or

0.4%'o

bovine serum albumin (BSA; fraction V; Nutritional Biochemicals Corp., Cleveland, Ohio) and a doubled amount

(0.44%) of sodium bicarbonate. Cultures were then incubated for 60 to 72 hr at 32 to 33 C. The input multiplicity of infectionwasbetween0.5and2

plaque-forming units (PFU) of virus per cell. No specific changes could be detected in the infected cells when cultureswereexamined with the light microscope at the time ofharvest.

Assay ofmycoplasma. The infectedandnoninfected cell culturesused inthe present studyweretested for the possible presence of mycoplasma by a method describedpreviously (31). No cultures were contami-nated.

Titrationl

of

viruts

infectivity. Viruswasassayedbya

plaque technique in agarose-suspended BHK/13S cells. The original method (24) was modified as

836

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PURIFICATION OF RABIES VIRUS

follows. Petri dishescontaining onelayerof nutrient agarose and a superimposed layer of agarose-cell mixture (31) were incubated for 24 hr and then infected with 0.1 ml of serial virus dilutions. The virus inoculum was placed on the surface of the cell-con-taining agarose layer and was allowed to penetrate. Infected plates were incubated for 7 daysat 35 C in a humidified CO2 incubator before the addition of neutral red.Plaques were counted 4 hr later. In some

experiments, the PM virus was also titrated by in-tracerebral inoculation of 4- to 5-week-old white

Swiss mice. One MLD5o was found to

correspond

to

approximately 10 PFU of this virus strain.

Assay of viral hemagglutinin (HA). The method devised recently by Halonen et al. (8a) was used. Serial twofold dilutions of virus

preparation

were

made inasolution of 0.12 M NaCl and 0.05 M borate (pH 9.0), containing 0.4%, BSA, and the dilutions were kept at 4 C for 30 min. An equal volume of

0.25% gooseerythrocytes suspendedin 0.15 M NaCl-0.2Mphosphate solution (pH6.4) wasthen added to

each wellofaplastic plate.The endpointof the

reac-tionwasreadafter incubationfor 45 minat4C. The

reciprocalof thehighestfinaldilution

showing

partial hemagglutination was considered to represent the number of hemagglutination units (HAU) per

milli-liter. When titers were so expressed, the results of titrations carried out in plastic plates (0.1 ml final

volume) and in test tubes (1 ml final volume) were

identical.

Complemenitfixation (CF) test.The

microtechnique

describedbySever (25)wasused.The volumeof each

ingredient used was 0.05 ml. The amount of anti-rabiesimmune serum used fortitrationof theantigen correspondedto 10

antibody

units,

as determinedon

the basis of50% hemolysisintubetests.Thisamount

ofserumdidnotshowaprozonephenomenoninblock

titration. Thereciprocalof thehighest initial dilution

ofantigen active in the test was considered as the number of complement-fixing units (CFU) per ml. This wayofexpressingthetitersgaveidentical results for titrations carriedout with plastic plates (0.25ml finalvolume) and for titrations carried outwith test

tubes (1 mlfinal

volume).

Immuiize sera. Antirabies immune sera, obtained from rabbits immunized with concentrated and

par-tially purifiedviralantigen (strain PM),was depleted

of host cell antibodiesbyabsorptionwith Nil-2 cells.

A serum dilution of 1:60,000 (0.05 ml) reduced by

50% the number ofplaques formed by 40 PFU of rabies virus. When diluted as much as 1:1,200, the serumreacted in the CFtestwith 10antigenunits of rabies virus. It did not reactwithhamster cellantigens

orBSA.

Anti-BHK-cell serum, obtained from rabbits im-munized with disrupted cells, reacted in the CF test with 10units ofhomologous antigen when diluted as

much as1:320.

The anti-BSA serum was purchased from Nutri-tional BiochemicalsCorp.

Agargel precipitation. The precipitin test was per-formed in

%lo

agarose gel. A 0.2-ml amount of

im-mune serum was placed in the center well, and

0.1-ml amounts ofantigen in the peripheral wells. The

distancebetween the central and peripheral wellswas

1 cm. Theplates werereadafter incubation in a wet chamber for 7 daysat37 C.

Protein determination. Protein was determined

according to the method of Lowry et al. (16), with BSAasthestandard.

Determination ofradioactivity.Acid-insoluble radio-activitywasdetermined inaBeckman liquid

scintilla-tion counterby the usual techniques.

Electronzmicroscopy. The preparations were exam-ined by the negative-contrast technique. Suspensions ofvirus were transferred to carbon-coated Formvar grids by means ofaplatinum loop. A 4%solution of phosphotungstic acid (pH 6.8) was added, and the

excess fluid was removed from the grid with filter

paper.Thespecimenswerephotographed at a magnifi-cation of 10,5000 X or 53,000 X in a Siemens Elmis-kop I electron microscope.

Preparation ofradioactive virus. To prepare rabies virions containing labeled ribonucleic acid

(RNA),

the medium added tovirus-infected cellcultures after virus adsorption was supplemented with 1.5 uc of 3H-uridine per ml (16 c/mmole: Nuclear-Chicago Corp., Des Plaines, Ill.). The labeled precursor re-mained inthe cultureduring the whole period of virus propagation.

Precipitationof thevirusbyzinc acetate.The

proce-dure of D.Spicer and B. H. Sweet(personal communi-cation; see also 20) wasusedwithslight modification. Fifty parts ofinfectious tissue culture fluid, freed of cell debrisbycentrifugation at 1,000X gfor20min, was mixed withonepart of 1 Mzinc acetate solution (pH 5.0). Aftermixing, the pH of the virus suspension dropped from 7.2 to 7.4 to 6.7 to 6.9. Themixture was allowed to standat4 C for 20min andthen was cen-trifugedat1,000 X g for 40 min; the supernatant fluid

wasdiscarded. Thepelletwassuspended in a saturated solution of disodium EDTA (about 11.7 g/100 ml of water), adjusted to pH 7.8 by the addition of solid Tris. The volume of the EDTAsolution used for sus-pending the virus was 1.25%of that oftheinfectious tissue culture fluid. Theresulting solution was clari-fiedbycentrifugationat1,000 X g for 20 min.

Filtration throughi Sephadex column. Columns (33 X 3cm) of Sephadex G-75 (particle size 40to120,u;

Pharmacia FineChemicals)wereequilibratedwith NT buffer. Crude virus suspension (10 ml) concentrated

byprecipitationwith zinc acetate was then put on the topof the

column,

and theviruswaselutedbywashing with NT bufferat aflowrateof 1 ml per min.

High-speed ceintrifugation. The virus was pelleted bycentrifugationat49,000 X g at 4 C for 60 min. The supernatant fluid was discarded and the pellet was suspended in 0.5 to 0.7 ml of NTEbuffer. With virus grown in the presence ofserum, sonic treatment for

30 to 60 sec at 10 kc (Raytheon, model DF 101

sonicoscillator) wasnecessary to disper.se the pellet. Theresultingsuspensions were then clarified by

cen-trifugationat 1,000 X gfor 20min.

Sucrosedensity gradient centrifugations. About0.5

ml ofsample was layered on 28 ml of a linear gradient of sucrosein NTE buffer and was centifuged at 4 C in the SW 25.1 rotor of a Spinco centrifuge. When 10 to 60% (w/v) sucrose gradients were used, the

837

VOL. 2, 1968

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Figure

FIG. 8-1anidcollapsedP1,10, 1. Electron2 microgralphs ofntegatively stainied lymphocystis virlus particles.FIG

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