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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
iniclusionbody
(1),niucleus(N),
anidnumerousmitochondria (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.
Noteconitinuiiity
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
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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.
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11
FIG. 8-1 1.Electron2
microgralphs
ofntegatively
stainied lymphocystis virlusparticles.FIG.8-9. Thtebarineach
figuire
represents Ij1.Negativelystain2ed
LDVparticles fromBF-2cells,28daysP1,anidassociatedcell debris. No capsomerestructure was
evidelnt.
Closeinispectioni in7dicates
amembranious
capsidcollapsed 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, 28daysP1,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]IN VITRO STUDIESOF LYMPHOCYSTIS VIRUS
inclusions
that contain DNA as determined by Feulgen staining andacridine-orange
ultravioletfluorescence.
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, andallexceptTipula iridescent
virusacquire
anaccessory mem-brane by abudding
process at the cytoplasmic membrane duringliberation
from the cell. LDV differs from these virusesby
virtue of its larger diameter and lack of an accessory membrane.LDVmay be related to the
amphibian
and insect viruses mentionedabove, although defectivewithregard 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 theTipula
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 thepresenceof 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 arenecessary for the
study
of theantigenic
structureand the chemical
composition
of the virion.Modified
methods forcultivating
rabies virus intissue
culture,
described in the present report, have made itpossible
to obtain increasedyields
of virus. Similar
improvement
in methods forquantitative
assay of the virus(24)
hadpreviously
opened
the way topurification
of rabies virusgrown in
tissue
culture. This report summarizes theresults of
experiments
in which an efficient recovery rateof
highly purified
virus wasachieved.
Someproperties
ofpurified
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),werethreetimesclone-purified by plaquing
inagarose-suspendedBHK/13Scells (24).Seedpools
of viruswerepreparedinBHK21cellcultures.
Tissue cultures. Two hamster cell
lines,
BHK21clone 13 (17) and Nil-2 cells (4), were
propagated
in 1-liter Blake bottlesas describedpreviously
(14).
Buiffer
solution2s
used in viruspurification.
NTbuffer 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 viruspropagationi.
After re-moval of the growth medium, cell cultures were in-fected with 2 ml of seed virus containing 50 ,g ofdiethylaminoethyl(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
ofviruts
infectivity. Viruswasassayedbyaplaque 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
toapproximately 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
weremade 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) permilli-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 determinedonthe 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 ofim-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