Characterization of Bluetongue Virus Ribonucleic Acid

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Characterization of Bluetongue

Virus

Ribonucleic Acid

D. W.VERWOERD, H. LOUW,AND R. A. OELLERMANN

MolecularBiology Section, Veterinary Research Institute, Onderstepoort, South Africa

Receivedfor publication 2 September 1969

Animproved purificationprocedureyieldedbluetongue virus free from any

single-stranded ribonucleic acid (RNA) component. Double-stranded RNA obtained

frompurified virus or isolatedfrom infected cells was fractionated into 5 compo-nents by means ofsucrose gradient sedimentation analysis, and into 10 compo-nentsbyelectrophoresisonpolyacrylamidegels. The size of these components vary from 0.5 X 106to 2.8 x 106daltons,with atotal molecular weight estimate of about 1.5 x 107 forthe viralnucleic acid. Thedenaturationof thegenome and separation of theresulting fragments are also discussed.

A striking similarity has been found between

the nucleic acid moieties of bluetongue virus (BTV) and reovirus, although the virions differ

insize andmorphology. Bothgenomesconsist of double-stranded ribonucleic acid (RNA) which

has been found, after isolation, to consist of a

number of segments ofreproducible size. In the case of reovirus, the presence of 10 double-strandedsegments was demonstrated in fraction-ation experiments utilizing polyacrylamide gel electrophoresis (12, 20),whereaselectron

micros-copy studies using the Kleinschmidt technique indicated 9 to 11 fragments (15). In addition, a

single-stranded,

adenine-rich component has

beenfound (1,

11).

Previous studies on BTV-RNA indicated the presenceofatleast threesizegroupsofsegments,

by theuseof sucrosegradient sedimentationfor

fractionation (16).

Inthe

experiments reported here,

the presence

or absenceofa

single-stranded

RNAcomponent inBTVwasinvestigated,and thedouble-stranded viral RNA was

subjected

tofurther

purification

and characterization.

MATERIALS AND METHODS

Buffers. The STE buffer consisted of 0.1 M NaCl,

0.005Mtris(hydroxymethyl)aminomethane (Tris),and 0.001 Methylenediaminetetraacetic acid (EDTA), pH

7.4; the SSC buffer consisted of 0.15 M NaCl and

0.015 M sodium citrate, pH 7.2.

Virus.BTVserotype 10has been usedthroughout

this work. Methods for the production of virus in

BHK-21 cellcultures and forplaquetitrations in

L-cells have been described (4).

Reovirus type 1 wasobtained fromN. F. Stanley,

Melbourne, Australia; grown in BHK-21 cells, and

purified according to the method of Bellamy et al.

(1).

Purification of BTV. The following modifications

have been introduced into the procedure described

previously (16). After extractionwith Tween 80 and

ether, sucrose was added to the aqueous phase

con-tainingthevirus toafinal concentrationofabout4%,

and the salt concentration was adjusted to 0.1 M. It

wasthen layered over 2ml ofa40% sucrosesolution

in STE, and the viruswaspelletedthrough thislayer

for2hrat102,000X g.Thepelletsweredissolved in

asmall volume of 0.002 MTris-hydrochloride atpH

8.0 and finally purified by centrifugation through a

10 to 30% sucrose gradient in the same buffer at

78,000 X g for 70 min. Thesemodifications arebased

ontheobservation thatpurifiedBTVformaggregates

in0.1Mbuffer,withdisaggregationat asalt

concentra-tion of 0.002 M. Gradients were fractionated in a

model D fractionator (Instrumentation Specialties

Co., Inc., Lincoln, Neb.) and assayed for virus

ac-tivity by meansof plaque titrations;thefractions

con-tainingviruswerepooled for phenol extractionorfor pelleting ofthe virus ifrequired.

Phenol extraction. The procedure described by

Scherrer andDarnell (10) wasfollowed forthe

isola-tion of RNA from infected cells. In the case of

purified virus, the pooled fractions were dialyzed

against STEbuffer, and sodium dodecyl sulfate was

added to a concentration of 1%. Deproteinization

withphenolwascarried out atpH 7.4 atroom

tem-perature.

Isolation of double-stranded BTv-RNA from

in-fected cells. Cells were harvested about 20 hr after

infection andwerecollected bylow-speed

centrifuga-tion. A cytoplasmic extract was prepared according

to the methodofBellamy et al. (1), and theextract

wasdeproteinizedwithphenol.Afterprecipitationof

thetotal RNA withtwovolumes of coldethylalcohol,

transfer and double-stranded RNA were selectively

dissolved in 1 M NaCl. After dialysis against STE,

the double-stranded RNA was finally purified by

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meansofchromatography on a methylated albumin-kieselguhr (MAK) column (18). The double-stranded

RNA peak, eluted at 0.7M in the NaCl gradient, was

pooled, precipitated overnight in two volumes of cold ethyl alcohol, and dissolved in 0.1 X SSC.

MAKcolumn chromatography. MAKcolumns were

preparedaccording to thesimplified method described

by Osawa and Sibatani (9). Elution wascarried out

withalineargradient of 0.2 to 1.2 M NaClbufferedto

pH 6.7 with Tris-hydrochloride.

Density gradient sedimentation analysis. Purified

viruswasdisruptedby theadditionof0.5 M ureaand

0.1 Msodium acetate, and its RNA wasanalyzed in

sucrose density gradients containing 0.1% sodium

dodecyl sulfate (SDS) by the method of Bellamy et al. (1).

Analysis of isolated RNA was carried out in

gradients prepared from ribonuclease-free sucrosein

STEbuffer containing0.025 M EDTA.Centrifugation wasroutinely carriedoutfor5 hrat200,000 X gin

aSpinco SW50rotor. Gradientswerefractionated in

anIsco model D gradient fractionator.

Polyacrylamide gelelectrophoresis. Electrophoresis

onpolyacrylamidegelswascarriedoutbythemethod ofLoening(7), with themodificationthat0.1%SDS

wasadded to thebufferandethylenediacrylate(0.2%),

wasused ascross-linkingagent. Gels(3%), prepared

in SDS-containingLoening's buffer, were used

rou-tinely, andseparationwasnormallyfor 4hr, at100v

in 10-cm gelsat roomtemperature. Thesamebuffer

wasused in the electrode chambers. To resolve the

high-molecular-weight components, the gel

concen-tration was increased to 3.6% and the time was

extended to 7hr. Gelswere cutinto 1-or2-mmslices

asrequired, dissolvedin 0.2 ml of1Mpiperidine, and

counted inKinard's (6) scintillator solution.

Thermal denaturation. Melting curves were

deter-mined by heating the samples in theheating

attach-ment of a DK-2A spectrophotometer (Beckman

Instruments Co., Inc., Fullerton, Calif.) in

glass-stopperedcuvettes.

Radioactive precursors.The

'4C-uridine,

3H-uridine

and 32P, asorthophosphate, were obtainedfromthe

Radiochemical Centre, Amersham, England.

Radioactivity assays. All determinations of radio-activity were made by scintillation counting in a

Packard Tri-Carb liquid scintillation spectrometer

(Packard InstrumentCo., Inc.,DownersGrove, Ill.).

Aqueoussamples were counted inBray's scintillator

(2) and gel slices in Kinard's solution (6). The 32p_

containing sampleswerecountedwithoutscintillator, utilizing Cerenkov radiation.

RESULTS

Purity of the isolated virus. Earlier studies

pro-vided evidence for a

single-stranded

RNA

com-ponent present invaryingamountsinthe

double-stranded RNA preparations isolated from puri-fiedBTV(16).Thesimilarity ofthe BTVgenome to that ofreovirus naturally led to the specula-tionthat this componentmight be equivalent to the single-stranded,

adenine-rich,

2S material reported in reovirus (1, 11). The

irregularity

of

the amount present in various preparations,

however, indicated the possibility of this compo-nent being a contaminant. Subsequent modifica-tion of the purification procedure (see Materials and Methods) resulted in many preparations containing no single-stranded RNA, thus strengthening this view. The possibility still existed, however, that such a low-molecular-weight fragment could have been lost during the

isolationofviralRNA. The fact that 3H-uridine was normally used for labeling viral RNA might have been a contributory factor in view of its low concentration in the adenine-rich compo-nent. To resolve this question, it was decided to label BTV with 32p and to liberate the RNA from purified virus directly onto sucrose gradients.

Although the modified purification procedure resulted in better yields of virus with a higher degree ofpurity, two virus bands were still com-monly encountered inthe sucrosegradients

con-stituting the final purification step. After 32p_ labeling and purification, the two bands were collected separately, the viruswas solubilized by

adding urea and sodium acetate, and the RNA was fractionated by centrifugation in 15to 30%

sucrose gradients containing 0.1% SDS (1).

Results are shown inFig. 1. Band B, the

faster-sedimenting band always present, contained only high-molecular-weight material equivalent

to the 8 to 165, double-stranded components

previously demonstrated. Band A, which was

absent in some experiments and presentin

vari-10.

C.~

5

.

o10 15 20

Fractionnumber

FIG. 1. Sucrose density gradient sedimentation

analysisoffractionsAandBobtainedduring

purifica-tion of 32P-labeled BTV. Virus was solubilized with

ureaandacetateandlayered directly onto 15to30% ribonuclease-free sucrose gradients in STE buffer.

Gradients were centrifuged for 5 hr at 200,000 X g

and

fractioilated

as described; the total number of

countsin eachfractionwasdetermined.Sedimentation

isfrom left to right. The positions of

14C-uridine-labeled ribosomal RNAfrom BHK-21 cells, addedas

markers, are indicated by arrows. Symbols: 0,

fraction A; *,fractionB.

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able amounts in others, contained, in addition,

some ribonuclease-sensitive material in the 2 to

45 region. This material was not available in

sufficientquantities to allow a base analysis. Various tests were performed onthe two frac-tions to exclude the possibility that the second

band represented contamination with another related virus or serotype of BTV and to see whether any characteristic difference could be found which would explain the presence of two bands.

Negativelystained preparationsexamined in an electronmicroscopedid not reveal any

morpho-logical difference. Both bands contained similar numbers of empty and fullparticles.

Antiserawere produced againstboth A and B

virusin rabbits, and complement fixation as well as cross-neutralization tests were performed as

previously described (3). The same single BTV type 10-specific antibody was found in the

anti-serum, and no neutralization of other serotypes could be demonstrated (P. G. Howell, personal communication).

RNA isolated from both fractions was

frac-tionated on a MAK column and by

polyacryla-mide gel electrophoresis. No difference between A and B could be detected by either method. Both preparations eluted from the MAK column at asaltconcentrationof 0.68 to 0.7M, and both yielded the standard electrophoretic pattern for

double-stranded RNA shown in Fig. 3. Other data collected during this investigation are

sum-marized inTable 1.

Itisobvious thatthemaindifferences between

the twobands arethevariation in buoyant den-sity,theconsiderably higher specific infectivity of

the faster-sedimenting B band, and a large

TABLE 1. Comparative data obtained for bands A

andBfound during purification ofBTVby

zonalcentrifugation in sucrosegradients

Characteristic Band A Band B

Approximate

sedimen-tation constanta... 520 S 750 S Buoyant

densityb...

1.39 1.38

Specific radioactivityc... 7,620 8, 500 Specific

infectivityd

... 2.1 X 109 4.0 X 10'°

aCalculated according to the method of

Mc-Ewen (8).

bDeterminedbymeansofequilibrium

centrifu-gationin CsCl density gradients.

cExpressed as counts per minute per absorbancy

unit at260nmn.

dExpressed as plaque-forming units per milli-liter perabsorbancy unit at 260 nm, determined by

theplaque assaypreviouslydescribed (16).

difference in sedimentation velocity. The dif-ferences indensity and specific infectivity are com-patiblewith the evidence that the A band contains some additional low-molecular-weight RNA, as

revealed by density gradient sedimentation anal-ysis. It is more difficult to explain the difference in sedimentation value. One can only speculatethat the RNA contaminant might be "tailing" from the spherical virus particles, causing a consider-able increase of the frictional coefficient.

Intheabsence of any evidence to the contrary, it is concluded that band B represents purified virusfree from the single-stranded, 2 to4S RNA componentfound inprevious preparations,which represents acontamination and forms no essential partof the BTV genome.

Fractionation of double-stranded RNA by sedi-mentation in sucrose gradients. The fractionation of the BTVgenomeinto fragments of three size groups by means of sucrose gradient sedimenta-tion analysishas been described (16). The

resolu-tion obtained, however, was never asgood as in the case of the reovirus genome (12, 20). In an attempt to improve the resolution by extended

centrifugation and by collection ofsmaller

frac-tions, itwasfound thatthe lossofresolutionwas

causedby thepresenceofmore than threepeaks. These peaks could be partly resolved by using the

improved technique mentioned.Thefractionation pattern obtained isillustratedin Fig. 2. The pat-tern obtained with reovirus RNA isincludedfor

E

(.1

Fraction number

FIG. 2. Fractionation pattern of BTV-RNA on

sucrosegradientscomparedtothatobtainedfor reovirus RNA. Both viruses were labeled with 14C-uridine

(specificactivity, 510mc/mr)at afinalconcentration

of0.01juc/ml. BTVwaspurifiedasdescribed. Reovirus

was purified by the method of Bellamy et al. (1).

Both viruspreparations weresolubilized withureaand

acetate, and the liberated RNA wasfractionated in

sucrose gradientsasdescribedin thelegendtoFig. 1.

Sedimentation valueswerecalculatedfrom the relative

positionsofcellular RNA markers indicatedinFig. 1.

Symbols: 0, BTV; *, reovirus.

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comparison. Five peaks are resolved with ap-proximate sedimentationconstantsof 8.5, 10, 12, 14, and 15.5S. Sedimentation values are based on

thesedimentation rates of cellular transfer RNA and18Sribosomal RNA, preparedfrom BHK-21

cells and added as markers. Calculated on the

basisof therelationshipS = 0.0882 X molecular

weight'-306,

deduced by Studier for

double-stranded DNA (14), these sedimentation con-stants correspondto values of 0.5 X 106, 0.8 X

106, 1.5 X 106, 2.3 X 106,and 3.0 X 106daltons, respectively, for the molecular weights of the

fragments.

Polyacrylamide gel electrophoresis of the BTV genome. Tritium-labeled, double-stranded RNA isolated from purified BTV was resolved into 10 componentsby means ofelectrophoresis in3.6%

polyacrylamide gels. These components,

num-bered 1to 10,areshowninFig. 3.The fractiona-tion pattern is similar, but not identical, to that obtained forreovirus (12). The 10 peaks are not as clearly groupedinto threesizecategories as in

the case ofreovirus, mainly because the largest and smallest components, possessing the lowest and highest rates ofmigration, respectively, are

well separated from their neighbors. The general impressiongiven by thepattern isagroupinginto fivecategories rather thanthree.This would cor-relatewellwith theseparation of BTV-RNA into fivecomponentsinsucrosegradients.

To obtain a more detailed

comparison

of the migrationrates ofthe components derived from reovirus andBTV, tritium-labeledRNA derived from bothwere fractionated under

strictly

iden-Fraction number

FIG. 3. Fractionation of double-stranded RNA

derivedfromtritium-labeledBTVbymeansof

electro-phoresisin3.6%polyacrylamidegelscontaining0.1%

SDS. Virus was labeled with 3H-uridine (specific

activity, 1.5 c/mM) at a final concentration of 1.0

c/ml.

Migration

is

from left

to

right. Electrophoresis

wascarriedoutfor7hrat25 Cat a voltage gradient of

IOv/cm. Gelswere cut into 1-mm slices.

tical conditions. Electrophoresis was carried out in3 % gels,10 cm long, for 4 hr at 10 v/cm.Under these conditions, reovirus RNA is separatedinto five components only, the first two peaks

cor-responding to the unresolved large and medium

size groups, the small group being resolved into three components. The BTV-RNA pattern, in contrast, is resolved into nine componentswhich

areclearly discerniblein Fig. 4.

The double-stranded RNA from reovirus has

been reasonably wellcharacterized, and the mo-lecular weights of the main components have been determined by several authors. Therefore, the

average molecular weights (1, 12, 19) of the five

peaks resolved in Fig. 4A (2.5 X 106, 1.4 X 106, 0.92 X 106,0.76 X 106, and 0.63 X 106 daltons,

respectively) were plotted against the distances they migrated to obtain a reference curve. A

linearrelationship was obtained (Fig. 5). Themolecular weights of the BTV-RNA com-ponents in Fig. 4B were then determined from

Fractionnumber

FIG. 4. Comparison of the electrophoretic patterns of (A) reovirus RNA and (B) BTV-RNA. Purified

tritium-labeled virus was solubilized with urea and

acetate and layered directly onto 3% gels containing

SDS. Timefor separation was5 hr; otherwise,

condi-tions werethesame asfor Fig. 3.

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this plot on the basis of their relative

electro-phoreticmobilities. The values thus obtained for peaks 1to10areshown in Table 2. Asafurther test for the validity of these figures, an analysis was made of the distribution of radioactivity

among thepeaks in Fig. 3. The total countsper

minute for all peaks was divided by the total

estimated molecular weight of 1.5 x 107,and the

resultantfigure was multiplied by the estimated

molecular weight of each peak to obtain the estimatedcountsperminute for that peak. These

valuesarecompared with the observedcountsper

minute in Table 2. The good agreement between estimated and observed countssuggests that the molecular weight figuresarereasonablyaccurate.

Melting behavior. Identical thermal denatura-tion curves were obtained with the

double-stranded RNAisolated from highlypurified BTV and with that isolated from infected cells. The firstdenaturationstepobserved in previous

prepa-.l

3:

to

75 ixio1

u

0

5X105

2 3 4 5 6 7 8

Distance migrated (cm)

Fio. 5. Relationship between molecular weight of

reovirus type I components and their electrophoretic

mobility in 3% gels.

TABLE2. Molecular weight of, anddistribution of

radioactivity in, the electrophoretic components

of BTV-RNA inFig.4B

Distance Estimated Estimated Observed Peak mit(mm) molecular weight counts/mina counts/min

1 27.0 2.8 X 10* 3,320 3,760

2

332.

2.2

X 106 5,460 5,490

3 32.0 2.4 X 106

4 42.0 1.7 X 106 2,020 1,920

5 46.0 1.5 X 106 1,780 1,700

6 48.0 1.4 X 106 1,660 1,510

7 63.5 0.90 X 106 1,060 1,040

8 67.0 0.80 X 106 950 910

9 69.5 0.74 X 106 880 860

10 83.0 0.49 X 106 580 620

aForcalculation, seetext.

rations (16) was no longer present. As expected,

Tm values were dependent on theionic strength andpH of thesolution (Table 3).Aninteresting observation wasthe effectof Trisonthe melting point of the RNA. At all concentrations, the sample in the Tris buffer had a Tm value about

4Chigher thanthat of an equivalent sample in SSC. As all buffers were preparedwith deionized

water, this phenomenon cannot be explained in terms of divalentions forming complexeswith the citrate-containing SSC. The absolute Tm values

varied withina rangeof about 2 C in different

ex-periments. Toensurecomparibilityof the data in Table 3, one RNA preparation was used, after

dilution

in thevarious buffers, for all

determina-tions. The Tmvaluesin SSC arefairly similar to thosereportedforreovirusRNA atdifferentionic strengths (1). The hyperchromic increase after melting also showed some variation reflecting the degreeof purity ofthe preparation, but was

usually about 30to36%.

Rapid coolingin anice bath afterdenaturation invariably led to about 50% renaturation, as

judged by the decrease in absorption (Table 3).

Asecond cycle ofthermal denaturation resulted inreversible melting at a much lowertemperature ofabout 40to60C,

indicating

thatthis decrease inabsorptiononcoolingrepresents thefoldingof

the single strands and is not true renaturation (Fig. 6).

Denaturationwithchemicalagents. Attempts to

fractionate the single-stranded components, ob-tained by thermal denaturation, by means of sucrose

density

gradient sedimentation or

poly-TABLE 3. Thermaldenaturation dataforBTVt-RNA

in variousbuffersof different ionicstrength

andpH values

Per cent hyperchromicity Buffer Concn pH Tm

After After

melting cotling

inice

SSC... 0.165 7.4 95.0 33.0 17.0

Tris/hydro-chloride... 0.2 7.4 99.0 34.0 18.0

0.1 X SSC... 0.0165 7.4 80.0 30.0 16.0

0.01 X SSC.... 0.00165 7.4 74.0 34.0 18.0

0.01 X SSCa... 7.4 58.0 46.0 45.0

Tris/hydro-chloride.... 0.002 9.0 75.0 38.0 23.0

aPlus8%formaldehyde.

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acrylamide gel electrophoresis invariably failed due to partial degradation of the single strands.

Various chemical agents have been employed by other workers to reduce the temperature of

de-naturation, such as formamide, formaldehyde, and dimethyl sulfoxide (DMSO). DMSO has

been used extensively for the denaturation of reovirus RNA (1, 5, 19). Under identical

condi-tions, however, denaturation of both BTV-RNA andreovirusRNA wasusually incomplete inour

hands,and partialrenaturationwasencountered

afterprecipitationof thedenaturedmaterialfrom theDMSO. Therefore, no satisfactory fractional

tion pattern could be obtained after DMSO

treatment.

Formaldehyde at a concentration of 8%

in-duced completedenaturationat atemperatureof

60Cand anionic strength of 0.002 M. No

degra-dation and no folding ofthe single strandswere

encounteredafterformaldehydetreatment.

Frac-tionation of the denatured material by sucrose

gradient sedimentation yieldedthepattern shown in Fig. 7, in which it is compared with that of

normal double-stranded RNA. Approximate S

values for the single-stranded RNA fragments

are11.0, 13.0, 16.5, 19.5, and 22.5,

corresponding

to molecular weights of0.24 x 106, 0.34 x 106, 0.56 X 106,0.8 X 106,and 1.1 X 106,respectively, according to the empirical relation molecular

weight=1,150X S2 lderivedbySpirin (13).These

valuesareallslightlyless thanhalf ofthe

equiva-lent molecular weights derived for the

double-strandedfragments. This

discrepancy

isprobably caused by the absence ofsecondary structure in the denatured strands due to the formaldehyde treatment, which would tend to lower the

sedi-mentationrates. Noseparation of

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formaldehyde-0c

FIG. 6. Thermaldenaturationprofile of BTV-RNA.

The RNAsample, in0.02m Tris-hydrochloride buffer

at pH 7.2, was heated to 98 C, cooledquickly by

immersion inasalt-icebath,heated againto60 C, and

allowedtocooldown, recording theabsorptionat260

nmat regular intervals.

Dv _v iv . ,,

Fraction4r.

FIG. 7. Sucrose gradient sedimentation analysis of

nativeandformaldehyde denatured BTV-RNA.Samples

werecentrifugedat200,000 X g for S hr in 15to30%

gradients of ribonuclease-free sucrose. Symbols: 0,

untreateddouble-strandedRNA; @,samepreparation

of RNA after heating for 10 min at 60 C in 0.01 X SSC buffer containing 8%formaldehyde.

denatured fragments could be obtained by gel

electrophoresis under our standard conditions.

DISCUSSION

The basic principle of construction of the

double-stranded RNA genomes of BTV and

reo-virus isobviously very similar, but there are some finer points of distinction. The single-stranded

component found in partly purified BTV, and thought to be equivalent to the adenine-rich com-ponentof reovirus, was shown to be absent from

highly purified BTV. Further work isneeded to ascertain whether the presence or absence of a

single-strinded componentconstitutesa real dif-ferencebetween thetwo viruses.

Fractionationof the double-stranded RNAby

means ofpolyacrylamide gel electrophoresis re-veals the presenceoftensegments in the genomes of both viruses. Differences in theelectrophoretic patterns, reflecting differences in electrophoretic

mobilities of thecomponentsconcerned, indicate slight variations in the molecular sizes of the segments. Because of thereproducible variations

in the electrophoretic patterns obtained for dif-ferentserotypes of reovirus(12),itisperhaps not

surprising that the serologically unrelated BT

virusandreovirusarestillmoredissimilarin this respect.

Thedifferent sizedistributionof the segments is

also reflected in the patterns of separation

ob-tainedinsucrosegradientsedimentation analysis.

Incontrasttothedistinctdistribution of

compo-nents into three size categories in the case of

reovirus, five poorly resolvedpeaksarefoundfor

BTV. This

reproducible

poorresolution of

BTV-RNAonsucrosegradientsthrows some doubt on

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the accuracy of the molecular-weight estimations

derived by comparison with ribosomal RNA markers. However, molecularweights determined from gels by comparison of the electrophoretic mohilities of BTV components with those of reovirus segments agree closely with the values obtained by sedimentation analysis. The values are also consistent with thedistribution of radio-activity among the peaks. The molecular weight estimates for components 1 to 10given in Table 2 can therefore be accepted with reasonable con-fidence.

A comparison of the molecular-weight esti-mates for BTV-RNA components obtained by the 2 methods moreover clearlyreveals a

correla-tion between the 5 peaks resolved in sedimenta-tion analysis and the 10 components resolved in gel electrophoresis. The 15.5S peak obviously

corresponds to component 1, the 14S peak to

components 2 and 3, 12S to 4, 5, and 6, 10S to

7, 8and 9,and 8.5S to component 10.

The significance of the segmented structure of the genomes of the double-stranded RNA viruses

remains to be elucidated. It does seem to be a

common characteristic of this viral group, how-ever, although it is not yet clear whether the

segmentationis a result of theisolationprocedure. African horsesickness virus also has recently been shown to possess a similar double-stranded ge-nome, separable into at least six components by gel electrophoresis (R. A. Oellermann et al., in

press). The fact that other viruses of animal,

plant, andinsecthosts arenowknown to possess asimilarstructureand to belong to alarge group

of related viruses (17) adds new interest to the

investigation of the biological functions of the

various partsofsuch acomplex genome.

ACKNOWLEDGMENTS

We thank J.H. Broekmnan and P. A. M. Wegeforexcellent

technicalassistance, P.G. Howell forassistance with the

sero-logicaltests,and theDepartment ofAgricultural Technical

Serv-icesfor thefacilities provided.

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