Purification of plant viruses by zone electrophoresis

VIROLOGY

23, &h!io2

(1964)

Purification

Depnrtment

of

Plant

Viruses

of Microbiology, University Accepted

April

by Zone

of Steblenboxh,

Electrophoresis

South

Africa

16, 1964

The purification of plant viruses by a final step of zone electrophoresis is advn-cated as one of the most, versatile and eflkient methods of obtaining pure virus preparations suitable for immunological studies. The operation of a simple type of electropharesis apparatus is fully described; it allowed nine plant viruses propagat.ed in six different hosts to be separated from contaminating plant antigens. Detailed result,s are given of the purification achieved with cucumber mosaic virus. ISTRODUCTIOIL

It has been stressed previously (van Regenmortcl, 1964) that, for purii’ying the The techniyuc of conventional electroless stable plant viruses one must use gcntlc phoresis without support.ing medium has been used occasionally for purifying plant methods of sap clarification, which arc inviruses (Lauffer and Price, 1947; :Rahn et al., efEcient in the removal of the antigenic 1955), but its main application has been fraction 1 protein of green lcaves (Darner restricted to the testing of virus preparat,ions el al., 1958). As a result, it is impossible to avoid contaminat,ion of virus particles with for electrochemical hornogencit.y (Hartman and Lauffer, 1953). The complexity of the fraction I (F 1) protein during differentiai equipment for moving boundary electro- ultracentrifugation, and in order to obtain phoresis as well as sarnpling diflicultics are antigenically pure virus prcparations it is probably the main reasonsfor the reluctance essentialt,o include in the puriIicat.ion process procedures such as gradient, centzifugation of virologists to use it for purification (Hrakke, 19SO), adsorption chromatography The limitations of the moving purposes. (Tremaino, 1961)) agar gel filtration (Van boundary method have led to the devclopliegcnmortel, 1962a>, or zone clectrophorc:mcnt of zone clectrophoresis methods of sis (Brakke, 1955). xvhich the density gradients type has been The need for virus preparations free of the most successfulfor virus studies (Gamer 171 protein is especially great in st.udies 01 and Svensson, 1961). In this technique hy- serological relationships between pla.nt vidrodynamic stability is usually provided by ruses, for it has been shown that. all the a sucrose density gradient, which cffcct~ively plants gcncrally used for propagating viruses opposes both thermal convection and dis- cont,ain serologically related a.nt.igctns(van turbances during sampling. Ilegenniortef, 196%, 1963a). Zone eiectroA number of plant and animal viruses phoresis has been found previously (van have been purified by this method (Erakkc, Regenmort,el, 1964) to be an elEcient method 1955, 1956; Hrakke and St~aples, 1933; \Tan for separating two viruses from the I-(’f Itcgcnmortel 1960; 1961; see Cramer and protein of marrow plants. The prcscnt work Svensson, 1961; I’olson and Dceks, 1062) was undertaken to test the applicability of but in view of the advantages and simplicity zone clectrophoresis to the separation 01 a of this dectrophoretic technique, it is SLS- number of viruses from the normal antigms tonishing t,hat so few workers have used it. of various plants.

496

FIG. 1. Zone e1ect.rophoresis concent,ration gradient. is formed tical tube. For the significance text.

VAN

apparaks. in t,he long of symbols

R.ISGICNMORTEL

The versee

A ppwahs The zone electrophoresis apparatus1 used in this work is similar to the one used by I’olson and Cramer (1958) ; it is schematically represented in Fig. 1. It. consists of a Ii-tube of 23 mm internal diameter provided wit.11 a wide st,opcock A in one arm and cormccted to two clcctrode vessels of ri cm internal diameter, ending in two cupillaries RI and Kz of 2 mm internal diameter. The lower end of the straight arm of the U-tube is closed by a glass stopper t,hrough which pass two capillaries Cl and Cz of 1 mm internal diarneter. The apparatus is first filled with a borat,e buffer of pI1 8.6 with the following composition: 0.035 M ITaP,03; 0.0176 N SaOII; 0.0075 N HCl; 0.073 M XaCl. ‘i’hc advantages of this buffer have been discussed by Poison and Decks (1962). A 40 % SUCTOSCsolution in buffer is then introduced through Cz and fluid is allowed to

escape through B, so that the sugar solution attains the levels D1 and Dz. The sucrose solut,ion is made up in the borat,e buffer and because of the formation of a borate-sugar complex, the pH is readjusted to the original 8.6 by the addition of N NaOH. The stopcock is then closed. The sugar gradient is prepared by allowing 160 ml of 40 5% sucrose in buffer to pass i&o 150 ml of buffer contained in a closed erlenmcyer flask at the same rate as the mixture enters the column through Cl. The contents of tho closed flask are constantly mixed by a magnetic stirrer. The dilute solution which first enters the column just above the layer of 40% sucrose is displaced upward by the following more concentrated port,ion of t,he solution. The sucrose gradient, obtained in this way is approximately linear (cf. lcig. 2) and occupies about 30 cm in the column. Sat’uratcd SaCl solut,ion is next, introduced into the electrode vessels through B1 and H, urltil the Ag-AgC1 electrodes arc covered. In order to ensure that the 2-1111 virus sample (containing approximately lo-20 mg of the different viruses) ITill float on the 40 % sucrose layer, suflicicnt sucrose is added to obtain a 35 % concentration. Phenol red, which has a high electrophoretic mobility, is also added to the virus sample as a standard reference substance to indicate the progress of the electrophorcsis. With the stopcock open, the virus suspension is introduced through C1 with a syringe. The residual virus suspension in t,he Cl capillary is removed bs adding some buffer to the right electrode vessel (raising level 11,) and l&ting about. 10 ml of the 40 %~ su~rosc sol&on escape through Cl. All the experirnents reported in this work were carried out in an air-condit,ioned room kept at IVC, under standardized conditions of pH, ionic strength, and potential and concentzation gradients. If a lower temperature were required, tho apparatus, after it had been filled, could be immersed in an ice water bath so as to leave only a few cent,imeters above the wat.er surface. Tho potent,ial gradient after an cloctrophoretic run is recorded in Fig. 2 (see I’olson 1 Scicnt.ific Cape Town.

Glass

Blowers,

332 Victoria

Road,

19.31) were propagated as previou& described in vegetable marrow plants (Cue cwbita pepo 1,.). Infected leaves were homogenized for 30 seconds in a itiring blcndor with one part of 0.05 Ail p1-l: 6.7 &rate buffer (van ikgenmortei, bOO4),‘I’&: sap was clarified by chloroform cmulsiiication and concentrated by two eyclw of differential u!tracentrifugation. AI’ttcr kc first cycle (78,000 ~1 for 2 hours) pelleia were resuspended in 0.0005M citrate bufl’cr, and the final pellets (after lO5,N.N y for i hour) in 35 “/c SIKT~~S~ in pII 8.6 boraLc buEcr. i I6 Ii 26 2ki Tobacco veinal necrosis (PVT’n; a strait DISTANCE IN CM of potat,o virus Sr isolated from discasctl Frc:. 2. Sugar concentration gradient (O--e) potatoes) and potato virus X (1’7\%) KWC and voltage gradient (C- ---0) after an electropropagated in plants of Nicotiana lni~wwi phoresis run, plotted against distance in the zone L. var. White Kurley. Odu&oglossuiibriugclectrophori:sis coinmn. spot virms (ORY ; van Regenmortel (-1 al .f 1’364) was propagated in pIants o:’ Ch~zo-~ and Decks, 1962). So convect,ion disturb- podiu7r~ @noa Willd. and bean yellow ances occurred at 18” if the current was not mosaic virw (BYJR; a strain kolated raised abovc 25 ma. from diseased lupines) in plants of i’ic?,cc The lcvol 112occupied by the virus prcpvulgwih L. PIT Un. Jaba I,. ancX Phase&u aration at the beginning of the experiment I’VX, OILY, and RYAW were purified by is known as the origin, and the dist,ances the method used with \WSV, but 0. that the different components migrate are ascorbic acid and 0.01 34 xa,SOa were measured from this point. added to the buffer used in the lxmogcni-. Electrophoresis is allowed to proceed until eation. The prtwnce of &rate iona m~ty suufficienl: scparat:ion of the different com- found to prevent nlost of the partkic agponents of the sample has been obtained by gregation (Reichmann, 1959). upward movement in the gradient column. Cherry necrotic ringspot virus (S RV) With a curreut of 23 ma and a pot,ential was propagated in plants of CULW~~S saiicu~ difference of 200 volts, the time nccdcd L. and purified by the calcium phosphate usually varies from 18 to 30 hours. adsorption method of I!‘ulton (1950) as By illumination with a vertOicallight beam, described previously (van Itegcwnor~~~ hands of opakscence are generally detectable and Engelbrccht, l962). Tobacco ~;crosis in tlrr co!umn after elcctrophoresis, and their virus (‘L’SV, a strain isolated from the roots position is recorded by photography. The of Il’ragaiia wsca 1,. plants and kindl:y prostopcock is then closed, and the contents of vided by I>. J. Engclbreeht.) was propagated the column are run out through C1 and in pla.nts of Phawolus zmlgaris I,. ard puri-collected in diff went fract,ions according to fied by the method used for SfLY. Turnip the fractionation desired. To prevent, any yellow mosaic virus (TUr\lrV) was propapossiblt contamiriation, e.g., by slight, adgated in plants of ~~7~~ssictx chir~ensis 1,. sorptio11 t,o the walls of the sampling capiland purified by the mclhod used for C.\Li:. lary, zones in the upper part of the colunui All the methods used in 1:hc preiiminarg tnay bc rc~movcd from the t,op by meansof a purificat,ion yielded virus susp~~rwionscow Pasteur pip&to with a bent t,ip. talninated with a high conecntration of I- j Viruses and I’wli~~~~inury Pwi;lication protein easily dctccted by Ouchterloq ge! precipitin tests. ‘I’hese tests were pcrformctd Watcrrwdon mosaic virus (T;liMV; van using the same ltegenmort,cl c!f al., 1962) and cucumber as tlcscribod previolAy, mosaic virus (CAIV; van Itogenmortel, antiaorlmi to the F I protciu of C~~l~$itfd

498

VAN

RBGEN,MORTEL

1963a, 1964). PePo- (van Rcgenmortel This antiserum reacted with the normal antigens of all the plants used in the present study. After fractionation in the zone electrophorcsis column the positions of the different viruses and plant antigens were located by serological and infectivity tests and correlated with the various bands of opalescence. The 2530% sugar concentration appeared to have no influence on the infectivity titre and the samples could be titrated without prior dialysis.

between sedimentation and electrophoretic migration, and after 42 hours the WMV opalescent band was still located 4 cm from the origin. Xuch of this aggregation was prevented by homogenizing the leaves and resuspending the pellets in citrate buffer, and in this case WMV moved about 5 cm in 24 hours. When aggregation was not prevented and the virus material, purified by electrophoresis, wm titrated in C. pepo, a ten- to hundredfold reduction in infective titre compared to that before electrophoresis was obtained. The original titre, however, could be restored by t,he action of 20 kc ultrasonic waves for 40 seconds; prolonging the ultrasonic treatment, for 80 or 160 seconds rapidly destroyed all the virus activity. Electron micrographs showed that this eff cct was due to the dispersion of aggregates leading to more particles becoming free to act as individual infective units. After prolonged treatment these units themselves

RESULTS

Watermelon Mosaic Virus In initial experiments phosphate buffer was used for homogenizing the leaves, and under these conditions WMV became increasingly aggregat,ed during electrophoresis. An equilibrium was eventually achieved TABLE RESULTS

OF

ZOSE

ELECTROPHOI~ESTS

1

EXPEIIIMENTS

ON

SISE

PLAST

!

I

VIRUSES

Position

(in cm) of

I

Virus

Host

/

.-__

plant

Titrated

in

,

! --

-..

Watermelon mosaic virus Oclontoglossum rinyspot virus Bean yellow mosaic virus Bean yellow mosaic virus Potato virus Y Potat. virus X jGIecrotic ringspot virus Tobacco necrosis virus Turnip yellow mosaic virus Cucumber mosaic virus

I

.~

Virus 0palescence”i

pep0

--I -~Cucurbita -~

/ Chenopodium

Chenopodium

quinoa

ranticolor

faba

Phaseolus ’ ATicotiana Avicotiana I Cuczcmis Phaseolus

vulgaris

Brassica

chine&s

Cucurbita

pepo

35

vulgaris

i

/ Phaseolus

vulga?is

/ /

Nicotiana tabacum Gomphrena globosa Cucumis sativuse

--

1 15-16

’ 18.-20

vulgaris

/

Hrassica

chinensiv

~

/ Chenopodium ranticolor”

ama-

4.54

:

5.57.5

15-17

5-8

15-17

/ 6.5-8

3.5 6 0.5-1.5 11.5-12.5

/ Phaseolus

I

--

Phenol red

escence” -____

9-11.5

ama-

Phaseolus

tabacum tabacum sativus vulgaris

pepo

I

Opal-

~

Cucurbita

Vicia

Plant antigens

’ 12-14

7-10.5 4-8 6-8.5

19-22 16-18 1.8-20

1.5-2.5

;

s-10

17.20

IO-14

~

7-11

21-24

5-7

’

12-13

14-16

a Maximum infectivity in all cases coincided with this opalescent band. b Dctorminod by gel-precipitin serological test.s, using an antiserum to fraction c The position of r\TRV, TYMV, and CMV was also established by gel-precipitin using antisera to these viruses.

1 protein of C. pepo. serological tests,

PVRIFICATION

OF PLANT

break up and the titre falls (Newton, 1951). These results emphasize the necessity of being careful in interpret~ing inff33tivit.y data on virus yields when the extent of particle aggregation is unknown. WMV produced a strong opalescent band in the electrophoresis column, and its position as well as that of the 1”‘1 protein is presented in Table. 1. Since WMV and F 1 protein of C. pepo are cha,raetcrized by very low and high eleatrophoretic mobilities, respectively, an ideal separation of the two can be effected.

VIRUSES

499

conditions to have a very low mobility and it was rapidly separated from tobacco antigens (Table 1).

The results of preliminary experiments made it seem unlikely that this virus could be purified by zone electrophoresis (SW van Rcgenmortel, 1962a), for it was fourd that after 16 hours of electrophoresis the virus zone coincided with a green band of plant mat,erial. Subsequc:nt tests, however, showed that this green band was nonantigenic and that it, could be separated from C.khatoglossurn Ringspot Virus the infective opalescent zone by prolonging This virus mult,iplies systemically in C. the experiment for 24 hours. In this case quirloa. and reaches a high conccnt,raDion in the distance separat.ing NRV from the P 5 this plant. (van Regcnmortel el al., 1964). protein of C. satiulcs (Table I) was greater After elcctrophoresis two strongly opalescent than that obtained by density gradient, bands were visible in the column; the faster centrifugation (van Regenmortel and I+:ngeimigrating component was found to be ORV, brecht, 1963), and it appeared that zone and the slower one the F 1 protein of C’. elect,rophoresis is a superior method for y’uinoa, seroIogicall~y related to that of C. obtaining antigenically pure NRV preppepo (‘Table 1). The high mobility of ORV arations suitable for antiserum production is in agreement with the known dectro(van Regenmortel and Meyer, 1963), phoretic behavior of the serologically related tobacco mosaSiovirus. (Hawdcn and Kleczkowski, 19ji; Townsley, 19.39). This virus has a low mobility at, phP 8.6, and it was rapidly freed of the contaminating Bean Yellow Mosuic Virus antigens of I’. vulgaris. In initial cxperimcnt,s r/. f&u plants were used to propagate BYMV? but the purification procedure used yielded a concentraEven aft.er 30 hours of electrophoresis, tion of virus too low to be detected after completa separation of TYMV and h”. elcctrophoresis either visually or by infec- chir~nsis antigens was not achieved. Only tivit.y tests. However, when 1’. o?clga& was one opalescent band appeared 7-l-4 cm from used, the result shown in Table 1 was ob- the origin; the lolver part consisted of plant tained. BYMV moved as a weakly opales- material, the remainder being virus (Table cent zone 4.5-6 cm from the origin and was I>* separated from the normal bean antigens after 14 hours of electrophoresis. The purification of CMV by zone eleetrophoresis has been described previously Tl1-is virus was separated from the F 1 (van Regenmortel, 1961). Ah&ough distilled protein in 26 hours of elect.rophorcsis, two watm was found at the time to be a suitable bands of opalcscencc being visible in the medium for homogenizing the infected plant column (Table 1). tissue, later work showed that it was advantageous to homogenize the leaves in 0.05 M citrate buffer, pH 6.7 (van RcgcnAs anticipated from’provious work mortcl, 1964). Following the report of Scott (Rawden and Kleczkowski, 1957; Town&y, (1963) t.hat showed CMVinfected tissuo 1959) this virus was found under present, to be homogenized in 03 31 citrate huffcr,

500

VAN

REGENMORTEL

FIG. 3. C’ltracentrifugc pattern of CXV purified by zone clectrophorcsis at a concentration of about I mg/ml suspended in 0.006 M cit,rate IsuRer pH 6.7. The photograph was taken 64 minutes after a speed of 17,250 rpm had been reached in the Spinco model li: ultracentrifuge. Schliercn angle 45 degrees. Sedimentation is from left to right.

pI1 6.5, a comparison was made hetcvoen distilled water, 0.05 Ad and 0.~3 M citrate buffer? pH 6.7, for their efficiency in extracting CMV from C. pep0 leaves. Aqueous phases obtained after emulsification with chloroform svere titrated in C. amaranticolo~ and t,he concentration of 1: I protein was determined by quantitative gel precipitin tests (SW Poison and van ~Regenrnortcl, 1961). Extracts made with 0.05 X and 0.5 M citrate buffer cont.aincd, respectively, twice and four times more 1” 1 protein than tho water extract, and at a dilution of 1: 100, showed lesion counts (average of ten leaves) of 110 and 85 against a count of 70 for the water extract. In the present work the tissue was homogenized in 0.05 M citrate buffer and the pel1et.s were resuspended in 0.005 M citrat.e buffer. After 16 hours of electrophoresis, CMV was located 5 7 cm from the origin (Table 1). After it had been

sampled, this mat,erial was concentrated at 105,000 9 for 60 minut.es; spect.rophotometric determinations (van Itegenmortel, 1964) showed that the virus yield was approximately 280 mg of virus per kilogram of fresh tissue. Amounts of up to 60 mg of virus could be purified in a single experiment, but the virus band present in the column was t,hen 5-4 cm wide. When examined at. a concentrat,ion of about 1 mg’ml in the Spinco model E analytical ult,racemrif ugc, tho purified virus showed a single component (l’ig. 3). When CMV was examined in t)he analytical ultracentrifuge prior to zone electrophoresis, a second small peak corresponding to the P 1 protein (Table 1) with a sedimentalion constant of about. 20 S was observed. All the viruses used in the present study produced visible opalescent bands in the

column; their migration elcctrophoresis could therefore easily be followed and sarnpliug was greatly facilitated. This is a dccisivc advantage over other types of clectrophoresis. In order to compare moro easily the mobilities of the E’ 1 protein from different host plants, only results of cxperimoms at pH 8.6 have been reported. However, other buffers could also be used successfully; CAW, WMV, and TYMV, for example, have been purified using 0.03 M phosp1rat.e buffer, pl I 7.5. Considerations of virus stability should determine the choice of buffer and pII. The concentration of F 1 protein in plant. sap may bo greatly reduced by using less gen-tic methods of clarification than those used in this work. I’or example in the case of ‘l’YJl\~, precipit,ation of the green sap with ethanol would remove most of the F 1 protein. Milder methods of clarificat,ion were used in this work, however, because t,hey are more widely applicable to the less stable viruses, and it is in this field that zone dectrophoresis may be of greatest help in removing host cont,aniinants from virus preparations. In the present work the main emphasis was laid on separating the differont~ viruses from contaminating plant antigens. Gel diffusion serological tests arc, known to be one of the most sensitive means for cvaluating the purity of virus preparations (Le I3ouvier et al., 1957 ; van Regenmortel and i\Ieycr, 19G3). Thti presence of contaminating host antigona not dctect,ablc by physicochemical methods may be revealed in the virus preparation &her by testing it with an antiserum to host material or by preparing an antiserum against. the supposedly pure virus. Very low conccntratioris of plant antigens which do not form visible procipit,ation lines may still elicit t,hc production of ant,ibodios and t-hus be detected indirectly in the resultiug antiserum (van Ilegenmortcl and YIeyer, 1963). This test of purity was also applied t,o some of t,he virus preparations (WXIV, I’VYn, SRV, C&IV) obtained in this work and showed that the F 1 protein fraction had been reduced to a level below det,cction by the methods cmployccl. The rclevancc of these

findings to current work on serological relationships between plant viruses is obvious (van ltegenmortel, 1963a,b), and it is worth emphasizing again the impor-tancc of including adequate controls with host an& gens when testing for these relationships. The technique of zone clectrophoresis seems to be able in most cases to separate plant ant,igeus from virus particles (‘l’ablc I), and it is thcreforo a useful final step in purifying viruses for immunological studies. Since the plant antigens from different hosts vary in their &ctrophoretie mobilities, it will oft~en bc possible t.0 s&et a hostvirus combination where the mohilitie~ of the component,s to be scparatcd differ significantly. Because of the hi&r mobility of the vegct~able marrow antigens, for cxample, it is more con\-eriien’i to purify t.:Liar from C. pc;po than from ,V. tfzbnCI~117plants (Table 1) . In the case of viruses with widely different mohilities, zone clectrophorcsis is also an ideal method for separating viruses from a mixt,ure as, for example, in the case of C.\IJ and W-\lV (van I~egcrimortel and l.‘owivict, 1962). Alfalfa mosaic virus and potato virus X, which often occur together in infected potato plants (potato calico d%Ydse), ill&~ also be sclparatctd in this way (unpublishctl results) ~ lleceritly Scott (1063) reported that a purified preparation of C 11 V showed two peaks in the analytical uitracent,rifuge. lu the prescut, work on CMV two components were also observed, but only when zone electrophoresis was not included in the purification proccidurc. ‘I%(: second small component was identified serologically as I: I protein and was rcmovcd by clectrophoresis (Table 1, I’ig. 3). Although Scot,t (ll163) states that his slow component is not present in extracts fr01u healthy tobacco lcavcs, this does not, preclude it front being a polymerized normal plant, component prescnt, in higher concentration in diseased plan&. Commoner fi al. (1052), for exampier reported an increase in t,hc: concenbraiion of plant proteins during the period of rapid tobacco mosaic virus biosymhesis in tobacco plants, and Bawden and Kieczkowski (!9X) found no consistent correlation bctw~~ctn the

concentration of normal proteins and virus production. Unpublished experiments perforined in this laborat.ory with TYMV, PVYn, CMV, and WMV also showed no consistent trend, t.he concentration of F 1 protein in infected sap sometimes being higher, sometimes lower than in the healthy plant controls. The main advantage of zone electrophoresis for purification purposes probably lies in its independence of size and shape parameters, thereby making it an ideal complement to diff ercntial ultracentrifugation. Plant components likely to contaminate virus preparations often aggregate during the initial stages of the purification, which may prevent. their removal by procedures such as gradient centrifugation or agar gel filtrahon.

(1958). Proteins of green leaves. VIII. The distribution of fraction 1 protein in the plant, kingdom as detected by precipitin and ultracentrifugal analysis. Biochim. Hiophys. Acta 29, 24@245. ALTOS, R. W. (1959). Purification of sour cherry necrotic ringspot and prune dwarf viruses. Virology 9, 522-535. ?&RTUN, R. I%, and LAUE'FBR, M. A. (1953). An application of electrophoresis to the identification of biological activity with characteristic particles. J. Am. Chem. Sot. 75, 62056209.

KAHN, R. P., I)ES.JARDTNS, P. R., and SEKSBXEY, C. A. (1955). Biophysical characteristics of the tomat. ringspot virus. Phytopathology 45, 334337. LAIXFER, -M. A., and PRICE, W. C. (1947). Electrophoretic purification of sout,hern bean mosaic virus. Arch. Biochem. 15, 115-124. LE BOCVIER, G. L., S~IIWERDT, C.E., and SCHIZFFIR, F. L. (1957). Specific precipitates in agar ACKNOWLEDGMENT with purified poliovirus. Virology 4, 590-593. The author is grateful to Dr. A. Polson for NEWI'OS, N. (1951). Some effects of high-intensity ultrasound on tobacco mosaic virus. Science introducing him to the electrophoretic field, as well as for performing the analytical ultracen114, 185-186. trifuge determinations. These det,erminations POLSOS, A., and CIXVIER, R. (1958). Zone elecwerecarried out in the Virus Research Unit, of the trophoresis of type I poliomyelitis virus. HioUniversity of Cape Town, which is supported by a chim. Riophys. Acta 29, 187-192. Public Health Services Research Grant AlPOLSOS, A., and ~)EEKS, n. (1962). Zone electro04044-02 from the Nalional Institutes of He&h, phoresis of enteroviruses. J. Hyg. 60, 217-234. Bethesda, Maryland. POI,SOI\‘, A., and YAS REGEXMORTEL, M. H. V. (1961). A new m&hod for determination of sedimentation constants of viruses. Virology 15, 397403. BAWDSX, F. C., and KLECZKOWSKI, A. (1957). An Rsrcn~-ash-, M. E. (1959). Potato X virus. II. electrophoretic study of sap from uninfected and Preparation and properties of purified, nonvirus-infected tobacco plants. ‘virology 4, 26-40. aggregated virus from tobacco. Can. J. Chem. BURKE, M. K. (1955). Zone electrophoresis of 37,4-10. dyes, proteins and viruses in densit.y-gradient SCOTT, H. (1963). Purification of cucumber mosaic columns of sucrose solution. Arch. Biochem. virus. Virology 20, 103-106. Biophys. 55, 175-190. TOWXSLEY, 1’. M. (1959). The electrophoretic BURKE, M. K. (1956). Stability of potato yellowanalysis of plant virus preparations. &an. dwarf virus. Virology 2, 463 476. J. Biochem. l’hysiol. 37, 119-.126. BRAKKE, M. K. (1960). Density gradient centrifTREMAIS%:, J. H. (1961). Removal of host antigens ugation and its application to plant viruses. from plant virus preparations by ion-exchange Advan. virus Ties. 7, 193-224. chromatography. Can. .J. Botany 3Y, 1705-1709. BRAKKE, M. K., and SWPLES, R. (1958). CorrelaVAN REQEN~MORTEL, M. 1%. V. (19Cfi). Zone election of rod length with infectivity of wheat trophoresis and electron microscopy of a waterstreak mosaic virus. Virology 6, 14-26. melon mosaic virus from South Africa. Virology COMMOXER, B., NEWMARK, P., and RODMSBE:HG, 12, 127-130. 8. I). (1952). An electrophoretic analysis of VAN REGIINMORTEL, M. H. V. (1961). Zone electobacco mosaic virus biosynt,hesis. Arch,. Riotrophoresis and particle size of cucumber mosaic them. Biophys. 37, 1536. virus. ‘Virology 15, 2X-223. CRaMEn, I’., and Svnsssos, H. (1961). Density VAN REGESVORTEL, M. H. V. (1962a). Purification gradient electrophorcsis as a now tool in virology. Experientia 17, 49-57. of a plant virus by filtration through granulated DORSER, R. W., KAHS, A., and WILDMAN, S. G. agar. Virology 17, 601-602.

VAS I~X~ENYORTRT., X. H. V. (1962b). Fraction 1 protein--a common contaminant in plant virus preparations. S. dj~ican J. Lab. Clin. Ned. II, 1GI. v.hv REGE.~~~CIORTE~, 31. 11. i7. (19G3a). Serologitally related plant contaminants in preparations of partially purified plant viruses. Virology 21, G&G;js VAS ~~EGENYOR~L, M. H. V. (19631s). Comment on the reported serological relat.ionship between turnip yellow mosaic virus and wild cucumber mosaic virus. Phytopathol. Z. 48, 197-199. vnx lisc;~;.suo~tTwr~, M. II. V. (1904). Separation of an antigenic plant protein from preparations of plant viruses. Ph:ytopathology 54, 282-289. VAS KEGESMOR~‘EI., i%. II. v., and Ih-osrmmx'~', I). J. (1962). The rapid diagnosis of necrotic ringspot virus infection of stone fruit.s by scrological means. 8. African J. Agr. Sci. 5,607-G13.

VAX lt~assmmmr,, >I. H. \‘~, and R~G~.BRE~I~T, 1). J. (1963). Purification and biophysical prayserties of necrotic ringspot virus of stone Eruits. S. African J. dgr. Sci. 6, 505-516. VAS ItsaEsMorw:r., M. H. V., and hWl.E, !a. f:, (1962). Separation of two plant viruses by zone elcctrophoresis. 8. Aj’ka?L J. ilg~. ~5%. 5, 51$ VAN

~tEGESMORl'EI.,

?d.

ii.

xi.,

Uct

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