JOURN-AL OFBICTEFRI OLOGYVol. 87, No. 2, p. 454-460 February, 1964('opyright ©1964 by the Amiierican Soeietx forlMicrobiology

Pr'inted ilt U.SNA.

ELECTRON- \I CROS('OPIC STRUCTURE OF A I YXOi\A VIRUTS-NI1) SOAIE REACTIVABLE DEIVATIVES

IBILLIIE L. PAI)(1lEJTT, MARGARIT' J. WRIGHT, ANNE1 JAYNF, ANI) DWARDI) L. WALKEIRIcpartmoents of illJorlhl .lhi robiolorly (i0(1 Bioch(10li.stir!, Unircisitly of 1 'isconsin, .lladison, WIis(onsin

Iteceive(l for pulblictation 14 September 1963

A B STRA CTI

PADGET'T, Bi.r,iiS, L. (UniversIity ()f Wiscolsin,M\Ladison), Mi.ARGARE.T J. WVIIGHT, ANNE JAYNE,ANI) DUARI) IL. WALKER. ElectroI1 imicroscop)icStructure of myxonia virus and some react ivalblede1rivatives. J. BIacteriol. 87:454-4(iO. 1964.-TheplhosphotUingstate negative staining techniq(ullewas used to studv the fine st ructutire of mlvxomiitvirus particles an(i Of react ivablle derivIatives in alnelectron mnicroscope. 'I'wo general types of parti-cles were observed in prel)arations of mvxoiuA.virus grown in rablbit kidney cells in tissuie cutltutre.1hl mIiost common formiii is iroutnded ori rectangulllar,with ain average lenigth atnd( width of 296 by 254mllt. Thie sturface of this typ)e of particle is (OIn-voluted, aiid is colnmpose(l of tubular elementsaratll' ed in pa coillex fashion; thet particle isfre:uently sturrounde(l by membrane. The secondfor-mll, which was seen less frequently, is miioorerectangular and slightly larger. It halts a closely fit-tnInielllbrante, a finely granular surface, and thieoii1v internial st ructtir'es discernible are present ina zo%ne about 150 A wvide, jtist beneat h the iniemll-brane. Reactivablle imvxoma virus plarticles werepIreplared by treatinig mvIlXomIna virtis with heat,uCreal, sodtiiiiii dodecyl sulfate, and etlIivl ether. T'lheapp)earance of the lItrticles after inactivation byhieat, urea, anId so(liuini dlodecyl sulfate is de-scribed.

M\lvxoma and fibroma viruses are in a subgroupof the pox viruses, xwhich is sep)arate(l fIonII otbherlpox viruses (Fenner an(i Burnet, 1957) by a lackof antwienic relatiOshils whenll convale(sCelnt seIrale u(seld, anId by th1ir stisceplti)iIitv tO inacttivat-tion by ethlv1 ether. Berrv and I)Dedrick (193(i)obIserved tle reactivation (If lheat-killed imvxomavirl1tis by active fiblromna virus. Althoughi this plue-11(m1e1tnon \\waS Widlelv stu(ied, the structtire Of ther(eactivaile virus p(articles was n1ot iInvestigate(d,nId(1 the structure (If nivxoma vCCireive(l littleattention since the w-ok (If Farrant and Fenner(1953). It an electron micrOscople, the pIhOspho-tutngstate nlegative staining te(hlii(1tie Of lBrennerand( Horime (1 959) proed very effective in reveal-

ing, the surface struictutre of virtises, aIi(l the struc-ttiral relatimishilpb5Ietweetl variouts v-iral com1-

Iboiients. 'l'his relport (lescrilbes the structure ofmvxoma viruts andl of some reactivable dleriva-tives, as revealed Ily the negative staining tech-

MATERIALS, AN) MIETHODS

Ilyxoma virus. 'T'he i\loses strain of mvxomaVirus was o(ltainel f'rom the Amierican lyl)e Cul-tute CollectiOn. It was mainitained in this labora-tory by serial passage in lprimtary rabbit kidney(1Rh) cells. Infected Rh1 cells in medium 199 and3,, ( calf sertum wvere in(cubate(l at 35 C until exten-sixvc cytolpathic effects we-re evident (generally 5days). The cell,s were collected in a smiall volumeof the miie(litIllu ano(l xere (lisrupted bya5 mimi ofOIlnic ibrati with a i\Iullard ultrasoinic disinte-grator (20 kc/sec). Cell (lebris was removed bylow-splced centriftugation, aii(l the supernatantfluid(l was used as the sourI(ce of virus f'or wo-oirk onthe structure of myxoIma virus. Virus x hich hadbeen lpartiallv plurifie(l by three cycles of differen-tial centrii fugation ill a Spin(o I(reparative centri-fuge was use(l in the reactiv ation studies.

Fibromna virus. The Ptatuxent strain of fibromaxvirus was(obltaiInedl fromi the American Type Cul-tture Collection, anol was maintaine(d bv serial lias-sage in RK cells. For use in re(activation attemnlts,fibroma virus was (ob)tained(l from inf'ected RNcells as des(ribe(d for mvxoma virus.

Incactivatiooi of mlyqxcwa virvis. Alyxoma virus in

me(lium 199 aind 10'(' c,alf semiim was seale(l ingl,jass tamlpiles alhel} (ated iIn a x ater llath at (65 Cfor 12 min. lellets of mvxoina virus oIbtainedl bvh1i1-speed cenltriftig,ttioin \ ere resuspeiided( in S\i uIrea (C)I1ta.iIni 0.001 'm efthlIenediaminetetra-iatetic aci(l (ED1)TA) at 1pH 10.2, an(I were heldat 37 C for 2 hr. Pellets (If myxoma xiruts werere-stuspended ini 1 sodliuiml dodecyl suilfate (S1)S)cmiltatining 0.001 mLIE)TA (pH 10.7), sealed inglass almptiles, and( sublmerged in a wv-ater bath at(iO C foI 2 Iltin. AIJvxoma viruis in ile(litillmu 199 alnd3 ',( calf ser-tuim was shaken with ani equitl voltime

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STRUCTUR El OF iI YX( )IAVIRS455

of ethyl ether for 30 min at room temperature.The mixture was centrifuged, the aqueous layerwas carefully recovered, and residual ether wasremoved hy nitrogen gas.

Test for inactivation of mnyxoma virus. Treatedmyxoma virus (1 ml) was inoeulate(l subcutane-ously into a New Zealand white rabbit, and therabbit was observed for 2 weeks. D)ilutions oftreated virus w-ere also inoculated onto plaquebottles of R 1 cells (lPadgett, :Moore, and AW'alker,1962). The virus was considered noninfectious ifthe rabbit failed to (levelop symp)toms of my-xo-matosis, and if no miyxoma plaques developed inthe RK cells.

LElectron microscopy. Saml)les of mvxoma virus,before or after ina(tivation, were centrifuged at41,000 X g for 30 min. The lellets were resus-pended in I ( ammonium a(ctate or in distilledwater, and were mixed with an equal volume of2(' phosphotungstic acid (lPTA) which had beenadjusted to neutrality. The lprel)arations w-eresprayed onto carbon-coate(d collodion films on(ol)l)er grids, and ecre examined in a SiemensElmiskol) electron microscolpe with double-con-densor illumination. All electron l)hotomicro-gral)hs were taken at a magnification of 40,000diameters.

Reactitation attemnpts. I nactivated mivxomnavirus, and an equal volume of fibroma virus, were

added to tubes of RK cells or centrifuged ontoRK cells in vials (Padgett and Walker, 1962).MAIyxoma virus wvas used at a multiplicity of10 to 100lplaque-forming units (PIdT) per cell,based on plaque titer before inactivation; fibromavirus was us,ed at a multiplicity of 1 to 10 I'llITper cell. After incubation at 37 C for 2 to 4 hr, theinoculum w-as replaced by maintenance mediumn.The fluid was collected and replaced at 2 days,and, at 5 days, the cells and fluid N-ere harvestedand sonically treated. T-he presence of infectiousm!vxoma v-irus in the 2- and 5-day specimens wasdetected by inoculating 1 ml of each into a NewZealand white rabbit. In all (ases of successfulreactivation, the rabbits (develolped generalizedmyxomatosisw;-ithin 7 days. In somne casVSe,myxoma virus was detected by inoculating dilu-tions of the specimens onto plaque bottles of RNcells; hoN-ever, if no typical mvxoma plaques wereobserved, the specimens w-ere also tested in r1ab-bits before being declared negative.

RESULTS

Structuere of the iyxoroma tirus particle. T-he ma-jority of the miyxoma virus )articles had a con-voluted surface which was comlposedl of tubularelements seen along their length. 'fhe IparticlesNvere either rounded (thimble-shaped), or rectan-gular with convex ends (Fig. 1). M\Ieasurement of

FIG. 1. !yxomIa virulls particles with convolutedl surfaces eumbedded in phosphotuinqlstate. (A) A. rectangull-lar particle with convex enris. So077e of the convolutions comn prisiny the surface of the particle extendcl acrosSits enti-e widlth. (B) A rouindledi or thiu1ible-shaped( particle with indications of a central *cense at-ea. Mlagnifi-cation: X 192,000.

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PAI)GETT ET AL.

FIG. 2. Myxoma viruts particles enclosed in mtiembranes. (A) This particle has a membrane separatedfrom the tubules by a narr owt, space which is fille(d wvith PTA. Tlhe c(Ie/e of the parti cle is serrated, and someof the tuibules seeti. to cross each other, resuilting in a herringjbone pattern. (B) This particle has a closelyfitting membrane which tends to obscture the tuibuiles. The hollow nature of the tuibules is apparent (arrows).M1agnification: X 248,000.

47 particles gave ani average leIngth1 of 2%6 "n' for uraniul-shadowed v-irus. Their particles(range, 250 to 330 tii) and( an average Nwidth of measured 286 ± 15 mug by 230 ± 20 mi. Occa-254 mru (range, 218 to 293 ruip). These figures comn- sionall p)articles were seen with a central opaquepare well with those of Farrant and Feimer (19.%,3) area, as described 1y \Nagington and Horne

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STRUCTURE ()F IN'XO.\IA VIRUSRLT7

(1962) for vaccinia virus. In some instances, thetubules Nere seen to cross a pJarticle from side toside (Fig. IA), but a pattern of arrangemeintcould not be discerned clearly. This may havebeen due in part to the presence of other materialbetween and around the tubules. WVhere the edgeof a particle was distinct, it had a serrated apl)ear-ance (Fig. 2A), and there appeared to he from 10to 12 tubules down the length of the particle. Thetubules on a given l)article were of uniform thick-ness, with a diameter between 120 and 140 A. Insomlie instances, the tubular elements had a less-dense central region which appeared to be filledwith PTA (Fig. 213, arrow). A closely fitting mem-brane could be seen around some of the particles(Fig. 2B), but the tubules were still discerniblebeneath this membrane} Other particles were sur-rounded by a slightly looser membrane, but onewhich was still closely associated with the lparticle(Fig. 2A).A second form of mvxoma virus particles w-as

observed. These particles were more rectangularand slightly larger than the first, ith an averagelength of 325 mu (range, 285 to 355 m,u) and anaverage width of 278 m,u (range, 245 to 328 m,u).

Their margin was wavy or scalloped, and theyhad a clearly demarked outer zone about 150 Aw-ide within w-hich, in some instances, a regularlyspaced pattern of parallel projections, each about40 A wide, could be seen (Fig. 3). The surface ofthese particles was composed of a uniform finestructure, and, aside from the projections, no in-ternal structures w-ere apparent. This type of par-ticle comprised about 10'>" of the population inone preparation of m)Yxoma virus. These l)articlesappeared to be similar to the type 2 vaccinia par-ticle of Nagington and Horne (1962).

Structure of the reactivable virus particles. Tensamples of mvxoma virus inactivated by heatwere all reactivable. The heated virus l)articleshad a slightly ragged appearance, and the tubuleswere less distinct in outline, but there were nogross alterations in the appearance of the parti-cles. Six samples of myxoma virus treated withurea were all reactivable. Figure 4 show s a typicalgroup) of myxoma virus lparticles after inactiva-tion with urea. 'The margins of the particles had araveled appearance, and tubules could not berecognized. Many of the larticles were more per-mneable to PTA, and they hadl a tendency to ad-

FIG. 3. Second type of mtyxoma virus particle. This particle is rouighly rectangutlar and has a wavyedge. An outer zone, about 150 A wide, contains parallel projections, each about 40 A in diameter. No otherinternal structures are apparent. Mlagnification: X 288,000.

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PAI)GETT JYTF AL.

Fi(.. 4. ifyjxoma cirius particles after treatmtient wvith urea. T'hese particles rire typical of all those observed.Y'heir edries are raveled, the tuibuilar structures are not evident on a miiajority of the particles, and the1y adherein maelsses. Magnification: X 100,000.

AFIG. 5. .M1yxoma ciruis particles after treatipent wivth SI)S. (A ) T'he m)1ost commonly observedt type of par-

ticlecfowulnr after tre(atmcent wtith SDS. T'his particle has no memmibrane, is very irreguilar in outline, and is7muchh more petricable to PTA.. The tubu7lar structures have been lreatly altererl. Maginification: X 232,000.(B) This type of particle was seen infrequently. The tabldar stituctures are irlentiJiable, blut the PTA haspenetrated more dleeply arotun(d them; the structure of the particle seemt,s loosser than in uintreated prepara-tions. A very wavy memc))lbrane is pr esentt. Magnification: X 202,000.

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VTI8,1TTRUCTURE OF AlYXO()A V'IRUt'S

here in masses. A few Iparticles w-hich x-ere Inotaltered as drastically were observed, but no infec-tious virus was detected in these prel)arations.The majority of the l)articles observed after treat-ment of myxoma -irus wvith S1)S reseembled thatshown in Fig. 5A. 'T'he particles wtere mnuch morelermeable to PT'A, their margins were very irreg-ular, and there w-ere no mnembranes around themii.The appearance of the tubules was greatly al-tered, and they did not show any regular arrange-iment. A few particles like that in Fig. 3B wereseen. These were not altered as extensively. Amembrane wvas l)resent, and tubules were stillidentifiable; how-ever, the structure seemecd looserthan l)reviously, and the P'T'A had l)enetratedlmore deeply between the tubules. Both SDS andurea al)l)eared to extract some material from thelparticles. Two samples of mv-xoma viruxs ere in-activated with SDS at 60 C, and both were reac-tivable. live other samples were treated withSDS at 65 C for 2 min. No structures identifiableas viral w-ere observed in the electron microscol)e,and none of these p)rel)arations was reactivable.W1'e (onfirme(l the report of Kilham et al. (1958)that ethyl ether regularly converts my;xoma intoa reactivable state. len samples of myxoma v-irusinactivated with ether \-ere all reactivable, butwe were unable to obtain electron photomi-crogral)hs of ether-treated virus. The difficultyal)l)arently resulted fromn a resi(lue of ether in thelprel)arations. 'T'he sx)raved drol)s dicl not slprea(lon the film, but drie(l in thick l)atches.

I)iscussIoN

Of the other pox xviruses which were examinedby the negative staining technique, the most de-tailed studies were ma(le with orf (Nagington andHorne, 1962) and vaccinia viruses (Herzberg etal., 1961; Nagington and Horne, 1962; Noves1962a, b). Structurally, mnyxoma virus l)articlesreseimble those of vaccinia more (loselv than thoseof orf virus. 'T'he dimensions of the mvxoma andvac(inia virus l)articles are comi)arable; p)articlessimilar to the two major forms of mrvxomia viruswere observ-ed by Nagington and Horne (1962) inprel)arations of vaccinia xvirus. Howev-er, the cen-tral opaque area which is coommonly seen in vac-cinia virus l)articles was not a consl)icuous featureof the myxoma virus l)article. Also, myxooma -irusparticles were, not observe(l enclosed in a looseenvelope. Su(h an envelope might have been dis-rulted by the sonic vibration used during thel)rel)aration of specimnens for ele(tron rnicroscop)y;

how(>ev-er, observations of sampI)les pirepared (di-rectl- from infected c(ells disrupted by freezingand thawing revealed only the forms d(escribed.Wl-e have no data bearing on the relative mnatuirityof the forms described, and at l)resent we (annotsay which formn of the mv-xonma virus lparticle isinfectious.The tubules of mnvxoma virus have a lar-geri

diameter than has been relported for any of theother l)ox viruses, but whether this difference issignificant is uncertain. Although the tubules ofmnixoma virus did not exhibit as highly orderedan arrangement as do those of orf xvirus, occasionall)articles were observedelvith indications of anorderly l)attern (fig. 2A). Noyes (1962a, b) sug-gested that the cords of vaccinia xirus, structurescorresx)onding to the mvxoma virus tubules, maybe formed from sl)herical units by denaturationduring l)urifi(ation. BIecause our observationswere made with viius which had been .subjectcdonly to sonic vibration an(l ccntrifugation, an(lbecause particles having the same surface struc-ture wtere observed in debris of infecte(d (ells (lis-rupte(l by freezing an(l thawing, we feel that thetubules are natural stiuctures. Tlhey may (er-tainlv be constructedl of subunits, ho-ever.The procedures that convert mvxoma virtus

particles into a noninfectious, reactivable foimap)l)arently affect the l)rotein of the virus, whlilenot (lenaturing the nucleic aci(l. Joklik (19(62),using rabbit-pox virus ra(lioactively labeled in thel)rotein, foun(d that conversion to the reactivablestate by heat or 6. M urea was accoml)anicd by aloss of some l)rotein fromii the viius. S1)S anid urea

both (ause a separation of the l)rotein and nucleicacid of somie l)lant viruses (Bawden and Pirine1940a, b). In the l)resent work, heat l)roduce(lvery little alteration in the gross appearance of tlhemyxomna virus p)articles, whereas S1)S and urea

al)l)eare(l to extract some material fromii the parti-cles, resulting in a greater l)ermeability to PTA.React ivability, howvever, was associatedl withmaterial sedi mentable by (entrifugation at41,000 X g for 30 min andl still recognizable as

p)articles in the electroni microscol)e.

ADDENDUM IN I)ROOF

Since this m11anllscript XXas submitted, ouri at-tentioni has been (Irawin to the paper on theelectron miicros(opy of mnyxoma -virus by 1'. J.Chapple and ,J. C. N. Westwood (Nature 199:199,1963). These authors also used1 the p)hosp1hotung-

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4PAD)GElTT' E' AL.

stie aci(l negative stainingT, techri(lUe, aI(l theyob)served1 twco types of myxonia virus particles.

ACKNOWLEDGMEATS

'I'h;is work wvas aid(d ly research g,ran1ts fromthle INatioial C(ancer Insititute 1andl the Nationalinstitute of Allergy and Infectious Diseases, U.S.public Health Service.

LITERATURP,E CITED

BAWVDEN, F. C., AND N. XW'. PIR{IE. 1940o(. Th}e inac-tivationi of some phuit viruses by urea. Bio-clemII. J. 34:1258-1277.

BANN-DEN, F. C., AND) N. XV. PInIE. 1940b. Thfieeffects of alkali an(d SoD1e siimjple organie sub-stances oni thr('e planit vi 'utses. Biochlen. J.34:1278-1292.

BERRY, G. 1'., AND H. L. I)DERICK. 193(6. A miiethodfor changing the vi iUs of rabbit fibroma(Sliope) into that of infectious imvxomnatosis(Sanarelli). J. Bacteriol. 31:50-51.

BRENNER, S., AND) It. XV. HORNE. 1959. A. negativestaining mnethodl for higih resolutioni electionmicroscopy of viruses. Biochiim. Biophys.Acta 34:103-110.

FARRANT, J. L., ANI) F. IFENNER. 1953. A coImpari-soII of the morphology of vaccinlia and mvx-onia viruses. Australian .J. Exptl. Biol. Aled.Sci. 31:121-12G.

FENNER, F., ANI) F. \I. BURNET. 1957. A shortdescriptionl of thCe poxvirins group) (vacCiniaandirelated viIIuses). Virology 4:305-314.

HERZBERG, K., A. KILEIMNSCIMII)T, t). LANG, ANDK. RiEUSS. 1961. VaICCinevirus Und KanarieII-PockenvirUs elekt ronenni kroskopisch bei Ne-

gativkont rastierung. Nat urwissenschaften 48:725-726.

JOKIAK, XX'. K. 19C,2. 'T'lhe p)reparation and charalc-teristics of highly putrifiecd radioactivelylabelled poxvirins. liochiinli. Biophvs. Acta61:2910-301.

KIILHAM, L., I']. LERNER, C. HIATT, ANT) J. SHACK.1958. Properties of nvxonlltt vi ius t raiisfoinii-ing atgenit. lroc. Soc. ELxptl. Bliol. AMed. 98:689-692.

NA(;IN(OToN, J., ANT) It. XV. HORNE. 1962. AMor-p)hological s-tudies of orf a(l vacciniia viiruses.Virology 16:248-2G0.

NoYES, Wr. 1F. 1962a. The surface finie strIuctuire ofv,taccinlia viirius. Virology 17:282-287.

NOYEs, XV. F. 1962b. Fvurther studies oIn tthe struc-turc of vacciinia viIrus. Virology 18:511-516.

PADGiETT, 1B. L., M. S. MO00RE, AND) 1). L. WALKER.1962. Plaque assays forI nyxoma and fibroiniavirutses and differenitiatiomi of the viruses byplaque formii. Virology 17:462-469.

PAI)ETT, B1. L., ANT) D). L. XVAlIKER. 1962. t'se ofcenitr ifiugal force to promote adsorption ofmvnxoria virtus to cell monlolavers. Proc. Soc.Exptl. Biol. M\ed. 111:364-367.

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