Purification and properties of tomato spotted wilt virus

VIROLOGY

67,

11-19

Purification

(1974)

and

Properties

of Tomato

Spotted

Wilt

J. J. ,JOUBERT,lm 4 J. S. HAHN,Z M. B. VOX WECH;\IAR,”

M. H. V. Tobacco

Resenrch Stellen,hosc:h,2

4?;n

REGENR’IORTJ~X3

VAN

Institute, Rustenburg,’ ad Departmetkt of Microlriology and Department of Microbiology, UwiversitU of Cape Accepted

Virus

September

n/~tl Virologlg, 7’011Jrk,

Sollth

[‘/ritsersit!/ .4 fricn”

of

12, lY?S

Tomato spotted wilt virus was purified by chromatography on colttmns of calcittnr phosphate, precipitation with polyethylene glycol, density gradient centrifugat.ion, and ascending zone electrophoresis in a sucrose density gradient. Examination of the virus by electron microscopy revealed particles of different, shapes in different Sus. pending media. Particles fixed with glutaraldehydr were spherical and sonl~~what flat,tened and had a corrected diameter of 85 nm. Antisera with a titer of l/l% against the virus were prepared which did not react wit,h plant antigens. The virtts had a sedimentation coefficient c3SU.~of 530 S and, when fixed in O..i(; glutaraldehyde, it, had an electrophoretic mobility at pH 7 of -1:l.g x lo-6 ctnz V- I

INTRODUCTION

Studies of the physicochemical properties of tomato spotted virus (TSWV) have been impaired by the lack of a suitable purification method yielding sufficient quantities of virus (Ie, 1970). Although numerous purification methods have been described no single procedure has gained general acceptance. Best and Balk (I 964)) Best (1966)) and Tsakiridis (1971) were unable to purify TSWV by the method of Black et al. (1963) while Tsakiridis (1971) also found the methods of Best and Palk (1964) and Best (1966) unsatisfactory. Black et al. (1963) homogenized infected tobacco leaves with an equal weight of 0.1 na phosphate buffer containing 0.01 M Na2S03 , and found that the virus was precipitatcd by low speed ccntrifugation. The virus could bc dispersed again by resuspcnsion in 0.01 M sodium sulfite and was further purified by differential and density gradient centrifugation. But this method does not always give good results (see Ic, 1970); the 4 Present address : Department of Microbiology and Virology, University of Stellenbosch, South Africa. 11 Copyright All rights

0 1974 lw Acadamir Press, of reprodurtirm in any form

Inc. reserved.

purified virus is not sufhcicntly free of cellular contaminants and the yield of virus is low. Best’ (1966) homogenized diseasedtobacco leaves in five times their weight of 0.01 M phosphate buffer (pH 7) containing 0.07 M Na2S04 , 0.01 hf Na,SO,, and 10d4 M sodium cthylenediaminetetraacet~at~r and clarified the homogenate by low speedcentrifugat,ion. li’ollowing diff erontial and density gradient centrifugation, he obtained a virus yield of 1 mg/lOO g infected tissue (Best 1968). Additional procedures have been described by Martin (1964) who clarified infected root extracts with calcium glyccrophosphate and by Tsakiridis (1971) who used I’rcon 114 for clarificat)ion. r3ur attcmptjs to purify TSWV by any of the above methods failed. Differential centrifugation led to a considerable lossof virus and the final virus preparations were still severely contaminated with plant material. We have developed a IWW purificat8ion method which yielded 6 mg purified virus per 100 g infected tobacco Icavcs. The purit.y of the virus material was dcmonst,ratcd by

12

JOUBERT

failure to elicit antibodies in rabbits. MATERIALS

AND

to plant antigens

METHODS

A South African strain of TSWV isolated from infected Oobacco was passaged through several single thrips-induced local lesions, and was propagated in Nicotiana glutinosa L plants. Forty-eight hours after the expression of systemic symptoms, leaves were harvested and stored in liquid nitrogen until required. Purification. All work was done in a cold room at 4 C. Infected leaves were homogenized in pH 7.2, 0.02 M sodium phosphate containing 0.01 M sodium sulfite. The homogenate was centrifuged for 10 min at 10,OOOg and the supernatant was further clarified by chromatography on calcium phosphate columns (Tavcrnc et aE., 1958). The brushite form of calcium phosphate was prepared a’s follows; equal volumes of 0.5 M CaClz and 0.5 M Na2HP04. 12H20 were pumped into a flask on a magnetic stirrer at a flowratc of 5 ml/min. The precipitate was washed five times by decantation with distilled water. Glass columns (4.5 x 45 cm) were packed with 600 ml adsorbent. The plant sap was allowed to flow through the column and clution was carried out with the pH 7.2 phosphate sulfite buff cr. The virus was concentrated by ccntrifuging the eluate in the Spinco No. 30 rotor at 25,000 rpm for 40 min and by precipitation with 1.5% polyethylene glycol (PEG, MW 6000) and 0.5% sodium chloride. Further purification was achieved by density gradient centrifugation and elcctrophorcsis. Sucrose gradient columns for the Spinco SW 25.2 rot.or were set up according to Brakke (1967). Solutions of 10, 20, 30, and 40 % ribonuclease-free sucrose (Milcs-Seravat, Cape Town, South Africa) in phosphate sulfite buffer were layered above each other in volumes of 6, 12, 12, and 10 ml, respectively. After 16 hr equilibration at 4 C, 2 ml of the conccntratcd virus extract from the PEG precipitation were layered onto each gradient and the tubes were centrifuged at 20,000 rpm for 60 min. The gradients were pumped through a Beckman fraction recovery system and a LKB Uvicord dcnsitometer at a flowrate of 0.5 ml/min. Zone

ET

AL.

electrophoresis (van Regenmortcl, 1972) was performed in a sucrose density gradient prepared in pH 7.2 phosphate sulfite buffer. Analytical ultracentr~~uyation. Sedimentation coefficients were determined at 4 C in a Spinco Model E analytical ultracentrifuge using schlieren optics. Experimental values were converted to s20,uvalues in the usual way (Chervenka, 1969). Electrophoretic mobilities were determined at 2.5 C in a Tiselius cell of the Spinco Model H clectrophoresis diffusion apparatus as described previously (van Regcnmortel, 1972). Electron, micrcscopy. Carbon collodion coated grids were examined in a Siemens Elmiskop 1 microscope. Grids were floated with the filmside downward on virus suspensions for lo-60 min and were washed afterward with a few drops of phosphotungstate solution. Grids were also prepared by the dip method in distilled water (Brandes, 1964) or in glutaraldehyde solution (Milne, 1970). Carbon collodion coated platinum grids were used for determining the size of TSWV. The grids were shadowcasted in a Siemens UBG 500 shadowcaster with tungstic oxide at a 30” angle. Tobacco mosaic virus was used as an internal standard (Brandcs, 1964). Serology. Antisera were prepared in rabbits by intramuscular injections of virus preparations emulsified in Freund’s incomplete adjuvant. Rabbits were given three weekly injcct,ions of 2 mg of purified virus and wcrc bled at weekly intervals. Immunodiffusion tests were performed in 0.75% agar in buffered saline and were used t,o determine the tit,carsof antisera. RESULTS

The virus isolate used in this study was identified as TSWV by the following properties (see Best, 1968): 1. The isolate was derived from a single, thrips-induced lesion; 2. Symptoms produced on tomato and tobacco were typical of TSWV; 3. Loss of infectivity contained in extracted plant sap occurred after 45 min; 4. Retention of infectivity in plant sap extended to -50 hr upon addition of sodium sulfite; 5. Destruction of infcctivit,y in plant sap occurred after 10 min at 46 C.

PURIFIED

TOMATO

SPOTTED

Purijicution Batches of 100 g of frozen infected leaves in ptast,ic bottles wcrc pulverized with an iron rod. Small quantities of the frozen material w-erc mixed wit,h phosphate sulfite buffer preheated to 37 C, and wcrc homogcnizcd in a Waring Blendor. n-0 success was obt,ained whrn t’ho purification methods of Black et al. (1963), Martin (1964), and Best (1966) were used with our strain of TSWV. The amount of infectivity rtltaincd afkr each step was dct,c:rrninpd by local lesion counts and was cxprclss(>das a pctrcentage of the lesions produced by thr homogcnatc. With the method (Jf l3laclc et al. (1963) (NY: Introduction) onlp I 5 ‘;G of t#hctotal inf&ivity of the preparation was rctaincld in the solution which was to 1~: submitted t,o density gradient ccntrifugation. This did not produce a disccrnable zone in tho &bnsity gradient’, and the cantircl tube was c~ontaminatcd wit,11 d(lnse grconish plant material. Whc:n thcx mothod of Best (1966) was uscld only 5 ‘? of t,hr infectivity was rctaincsd in the prtparation to be submitted to gradient ccntrifugation. With thtl method of Martin (1964) only 55 of th(l infcctivitjy was Ictaincd afkr clarification with calcium glyccrophosphate and two cyc*l~sof diff(~rc~ntialcelkifugation. Pollowing t hclseinitial atkmpts, different altc~rnntivc~ clarification agentjs wcrc tested. No S~~CPRS was obtained with bcntonitc fkc~ulat~ion (Ihnn and Hitjchborn, 1965) and :hsorptic)n t’o hydrated calcium phosphatcl (I~‘ulton, l%!l), or charcoal (Gnlvc~z, 1964). (111t,h(ao&r hand, considcrabk SIC COBS was achicbvcd with chromatography on c~alciumphosphak (brushit caolumns. Aftc~r holnc,gonizatioIl and ccntrifugation at 10,OOOqfor 10 min, t,h(i parUg clarified sap (X0 ml) was poured ont80 a hrushitc cwlumn :ind \vas cllutcd wit’h 0.02 il/, pH 7.2 phosphatc~huff~r containing 0.01 nr sodium suliitc,. Most’ of t,hctgrc’c’riplant mat,cGl \vas rctnirrcld on th(a top half of the columnsand SO’2 of thcb virus activity was rccovcrcid in an &ate volume of about 400 ml. The cJu:ttc was cclritrifugcld at 25,000 rpm for 40 min in a Spinco Xo. 30 rotor and the pcllcts wcrc rctsuspcndcd in 6 ml of t,hc phosphate sulfite buffer .

WILT

VIRUS

13

Attempts were made to further purify the resuspended pellets by density gradient centrifugation in lo-40 70 sucrose. After centrifuging at 20,000 rpm for 60 ruin in a Spinco SW 25.2 rotor: about 50”;’ of the initial infectivity was present in a zone situated I~~-24 mm from the meniscus. After conrctntrating this material by ultraccnt,rifugat,ion, ckctron microscopy showed that considerahl(1 impurities remained and that the virus particles varied in shap and genvral appc~aranc~c~. After n second strxp of zonal ccnt,rifugatiuu only 107;~of that i&ial virus activity was rcbcbovcxrt>d hut thcl virus preparations still contained plant mntt~rial. Attcbmpts to remove tlic rcmnining impuriticIs 1)~.ZOIWcl(>rtrophoresis failed. l’h~ light, scattt>ring zone of virus matcCJ that was introducc,d in t h(b ck%rophor& c%olumn disappc>arcd aftor 3 hr of elrctrophorcsis. This indicakd that, th(l virus JV:\Zunstable in 40’6. sucrose and that it \I-ould f)c IWWSsary to wtahilizt: thcl virus pnrtic~l(~shclf’ore submitting th(lni to zon(~ c~i(‘ctrc)plic,rc,sis. Stabilization 1~:~sachicvctd t)y :&ling :m equal volumc~ of 1 :‘; glut:\r:Ll(l~,l~~d(, in 0.01 II/ phosphatc~l)ufYc~rto thci virrls SWprwic tn.

Ttic, virions in thcl top band i’rolll thcb c:lc,~trol,horc,sis ~olunm TWW cv)uwntratt~d by ultrn~t,rit,rifilg:ttioil and \vor~’ uwd to inmiiinizcl rabbits. Xntis;cLraprotluccd in this mann(~r still rcactod strongly n-ith t’raction 1 plant prot c>in(van Rqqnmort(~l. 1966). ( ;(:I diffusion t&s sho\vcd that thv virus I)rc’p:iration ot~t:~incd aftc,r grudknt cbc,lltrifugation (tk sl (lp prior to (,lc~c:troplic,rt‘sis)aIs0 contninod a considcrabl(, amormt of l’rac:t,ion I protckl. That in&irkncy of su(‘ros;(’tl(lnsit,> gradient cc>ntrifugation for ronloving plant antigcans is will documc~ntc~d(wn Rcgcnmortcl, 1966). On the other hand, it is possiblc that tlicl fnilurc of zone c~lcctro~)llorc~sis

100 g infected

leaves

+ 350 ml 0.02 M, pH

7.2 phosphate-sulfite

buffer

Homogenize

for

2 min

Supernatant

Eluate

and

squeeze

Precipitate

through

(discard)

Layer onto a calcium the phosphate-sulfite

phosphate buffer

Centrifuge rotor

at 25,000

column

and

for 40 min

rpm

with

in the Spinco

No. 30

I Supernatant Resuspend

(discard)

in 6 ml phosphate-sulfite

Add 0.36 ml of a 25% solution. Mix thoroughly Centrifuge

for

5 min

PEG

buffer

and

0.12 ml of a 25%

Resuspend

in 6 ml distilled

(discard) water

Layer onto lO-4Oya sucrose density with the phosphate-sulfite buffer

gradient

Centrifuge 25.2 rotor

in the

Withdraw

for

60 min

virus

zone

Centrifuge rotor

for 35 min

at 20,006

rpm

at 35,000

rpm

Resuspend

in 0.2 ml phosphate-sulfite

Add 0.2 ml fite buffer

1% glutaraldehyde

Withdraw

virus

Centrifuge rotor

in the Spinco

7.2 phosphate-sulfite

at 35,000

rpm

in the Spinco

I

FIG.

1. Purification

wt)

Procedure 14

No. 40

zone

Supernatant (ea. 6 mg dry

SW

in 0.01 M phosphate-sul-

I

preparation

Spinco

buffer

in 0.02 J! pH

for 35 min

I Pellet

prepared

I (discard)

Supernatant

Zone electrophoresis buffer (16 hr)

NaCl

at 10,OOOg

Supernatant

virus

elute

(460 ml)

I Pellet

Final

cheesecloth

of TSWV.

(discard)

No. 40

PURIFIED

TOMATO

SPOTTED

to produce antigenically pure virus preparations is due to t’he binding of plant proteins ont,o the virions by glut’araldehyde. It seemed essential therefore to remove the plant antigens during the initial stages of purification. To do this the pellets obtained aftor centrifuging the clarified sap were rcsuspendcd in 6 ml phosphate sulfite buffer. To this virus suspension were added 0.36 ml of a 25 % polyethylene glycol solution and 0.12 ml of a 25 % XaCl solution to give final concentrations of 1.5 % PEG and 0.r) % NaCl, respectively. The virions were precipitated by centrifugation at 10,OOOgfor 5 min and were resuspendedin 6 ml distilled water. This suspension was used for gradiclnt ccntrifugation and yielded a virus zone situated 18-24 mm from the meniscus. The virions wore concentrated by ultracentrifugation and appeared fret of plant antigens in immunodiffusion tests. Aft’er glutaraldehydr fixation and zone electrophoresis in pH 7.2 phosphate sulfite buffer, only one band with a R+ value of 1.00 (van Regenmortel? 1972) was visible after 16 hr, while aggregates of denatured material were obsclrvcd in the lower part of the column. The purification method which was finally adopted is reprcsent)ed as a flow diagram in Pig. 1.

WTLT

VIRUS

15

When PEG precipitation was omitted from the purification scheme virus part’icles with a sedimentation coefficient of ~20,~~ = 614 S we’re obtained (average of two d&rminations of 610 and 618 S), indicating that some extraneous material might have become attached to the virions during glutaraldehydc fixation. Unfixed TSWV part’icles obtained after PEG precipitation and gradient centrifugation and suspended in the phosphate sulfite buffer had an s?O,~,; = 530 f 7. After fixation and zon(~ olectrophoresis the particles had an S valur of 583 f

s.

It seemsreasonable to accept the S value’ of 530 as the best,value since it was o1~tainc.d from purified preparat)ions unaltcbrchd 1)~ glutaraldehydr fixation. This values is in close agreement with the S valucb of .iX reportrd by Best (1966). Electrophoresis

The electrophokc mobilitv of fixed TSWV particles was determined in a Tiscblius cell at 2.6 C. In the 002 iii phosphate 0.01 iI4 sulfite buffer at pH 7.0 a mobility of -18.7 X 10-j cm2 V-’ SN -I n-as obtained. In the pH 7.0, 0.1 ionic strrng;th buffer of

Purified TSWV obtained as outlined in Fig. 1 was used for injecting t,hree rabbits. Antisera with a titer of 352 and >i2s were obtained which did not react with plant antigens (Fig. 2). When PEG precipitation was omitted from the purification procedurc, and the final virus preparation was used to immunize animals, antisera reacting with TSWV (up to f{zs dilution) and with plant antigens (up t.o >{e dilution) were obtainrd. A~~al~tica~b Ultracentrifugation

Virus preparations obtained at different stages of the purification procedure were examined in a Model E analytical ultracentrifuge. Partially purified TSWV preparations in 0.01 M sodium sulfite obtained sftrxr gradient centrifugation had a sedimentat’ion coefficient of s20,w = $1 f 13 S (avaragScof 11 expcxriments).

FIG. 2. Precipitin patterns with TSWV preparation in pH 7 phosphatesulfite salineagar. The central well was filled with an antiserum dilut,ed >$ prepared by immunizing rabbits with purified TSWV. Wells l--7: twofold dilutions of a virus preparation obtained as outlined in Fig. 1. Well 8 = healthy plant, juice.

16

JOUBERT

Miller and Golder (1950) a mobility of - 19.8 X 1W5 cm2 V-l see-’ was determined for TSWV. Electron Microscopy Systemically infected leaf tissue was crushed and spread in a drop of distilled

ET AL. water or 1% glutaraldehyde solution in pH 7.0 phosphate buffer on carbon coated collodion films (Milne, 1970). Grids were examined at a magnification of approximately x 40,000. Glutaraldehyde-fixed virus particles always appeared round (Fig. 3b) whereas unfixed pa,rticles showed consider-

1s of TSWV particles negatively stained with 20/, FIG. 3. Electron micrographs of dip preparation water. In C a cluster of virus particles is neutral PTA. A and C: dip preparations spread ont o distilled in 0.1 M neutral phosphate enclosed in a membrane. B: dip preparation spread onto 1% glutaraldehyde in a purified preparation. The bar represents 50 nm. buffer. The bars represent 250 nm. D: virus particles

PURIFIED

TOMATO

able variation in shape (Pig. 3a). Clusters of virus particles enclosed in a membrane were occasionally observed with both methads (Fig. 3~). The morphology of TSWV particles was also studied at different stages of the purification proccdurc. xo changes in general

SPOTTED

WILT

VIRUS

17

appearance of the part’icles could be demonstrated when preparations obtained after ultraccntrifugation, after gradient centrifugation, and aftw zonr’ elwtrophoresis were compared. Howvw, fixation \vith glutaraldehydc and the type> of rcsuspwding buffer greatly uffcctchd th(l Inorphology of the,

FIG. 4. Micrographs of partially purified TSWV preparations PTA. A: resuspended in Best,‘s (1966) neutral phosphate buffer, I)tlfYer (pN 7.2), C: resuspended in distilled water, I): resuspended with gllltaraldehydc. The lmrs represent X0 nm.

negatively stained with 25.; ne~~tral B: resllspended in phosphate stdfite in phosphate slllfite hlltfer x11(1 fixed

18

JOUBERT

vii-ions at all stages of purification (Fig. 4). When the final pellet was resuspended in distilled water, TSWV appeared as pleomorphic particles with characteristic taillike or bud-like structures (Fig. 4~). When resuspendedin Best’s (1966) phosphate buffer, the particles tended to be dumbbellshaped (Fig. 4a), whereas in the phosphate sulfite buffer they were only slightly distorted. Before zone electrophoresis, fixed particles always had the same regular appearance, but after eleetrophoresis about 30 % of the particles appeared less dense and partly filled with the phosphotungstate stain. The effect of pH on particle shape was also determined with unfixed preparations obtained after gradient centrifugation. Virus suspensions in distilled water were mixed with equal volumes of 2 % solutions of phosphotungstic acid adjusted to pH values between 3 and 9. At pH 3 the particles were roughly spherical, at pH 5 they were all partly filled with the stain, at pH 6 they were highly distorted with numerous tails, and at pH 8 t,hey were pleomorphic with numerous dumbbell shapes. All attempts made to disrupt TSWV particles (with peroxide free ether, sodium deoxycholate or Tween 20), in order to visualize the internal nucleoprotein, failed to reveal any recognizable filamentous structures such as those found with influenza viruses (Fenner, 1968). The periphery of particles showed protuberances of 5 nm diam (Fig. 3d). The diameter of the virus particle was determined with fixed purified preparations shadowed with tungsten trioxide, using tobacco mosaic virus particles as a standard. The apparent average diameter of the TSWV particles was determined as 112 nm. From measurements of the shadow of individual TSWV particles (Fig. 5) the height of the particles above the supporting film of the grid was calculated. The average ratio of the height to diameter of the particles was 0.48, indicating a considerable flattening of the particles. A corrected diameter for spherical particles was calculated by transforming the apparent diameter of the elliptical particles into that of the corresponding spherical particles using the relationship between diameter, height, and circumference of an

ET AL.

FIG. 5. Composite electron micrograph of TSWV particles shadowed with tungsten oxide at an angle of 30“. The bar represents 200 nm. The particles had an apparent diameter of 112 nm. The height of 350 individual particles was calculated from measurements of the shadow and was used to calculate a corrected diameter of 85 nm.

ellipse described by Marks (1946). From the apparent diameter of 112 nm in shadowed preparat,ions a normal corrected diameter of 85 nm was calculated for the TSWV particle. DISCUSSION

The extreme in oitro instability of TSW is responsible for the difficulties encountered in attempting to purify this virus. We found it necessary to stabilize the virus by fixation with 1% glutaraldehyde during the later stages of the purification procedure. By comparing areas under the schlieren peaks of TSWV preparations at successive stages of purification it was obvious that a considerable loss of virus occurred during the purification procedure. About 50% of the virus was lost during the zone electrophoresis step. In spite of this, virus yields were 6-8 mg/lOO g of leaf tissue. This compares favorably with the yield of 1 mg/lOO g tissue obtained with another strain by Best (1968). Differences in the diameter of TSWV particles reported by various authors (Martin, 1964; Best, 1968) may be attributed to the flattening of virus particles on the microscope grid. By transforming the diameter of flattened ellipsoid virus particles to

PURIFIED

TOMATO

that of spherical particles with the same circumference, a particle diameter of 85 nm was calculated for TSWV. The dumbbell-shaped particles reported by Best (1966) appear to be artifacts of the preparation procedure. When different solvents were used for resuspending the virus, a considerable variation in particle shape was observed (see Fig. 4). Variations in particle appearance which depended on the pH of staining solutions bear some resemblance to those reported by Wolanski and Francki (1969) for lettuce necrotic yellows virus and may be caused by osmotic and imbibition phenomena as suggested by t,hese authors. REFERENCES BEST, 11. J. (1966). Preparation and properties of tomato spotted wilt virus (strain E). Enzymologia 31, 333-346. BF;ST, R. J. (1968). Tomato spotted wilt virus. Advan. Virus Res. 13, 65-146. BEST, R. J., and POLK, B. A. (1964). Electron microscopy of strain E of tomato spotted wilt virus and comments on its probable biosynthesis. Virology 23, 445-460. BL~cK,L. M., BRAKKE;, M. K., and VATEK, A. E. (1963). Purification and electron microscopy of tomato spotted wilt virus. Virology 20, 120-130. BRAKKE, M. K. (1967). Density gradient centrifugation. la “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 2, pp. 93-118. Academic Press, New York and London. BRANDES, J. (1964). Mitt. Biol. Bundesanstalt Land Forstwirtschaft. Berlin-Dahlem 110,1-130. CHERVENKA, C. H. (1969). “A Manual of Methods for the Analytical Ultracentrifuge.” Spinco Division of Beckman Instruments, Inc., Palo Alto, California. DUNN, I). B. and HITCHBORN, J. H. (1965). The use of bentonite in the purification of plant viruses. Virology 25, 171-192.

SPOTTED

WILT

VIRUS

19

“The Biology of Animal FENNER, F. (1968). Viruses.” Vol. 1, 474 pp. Academic Press, New York. FULTON, R. W. (1959). Purification of sour cherry necrotic ringspot and prune dwarf viruses. Virology 9, 522-535. GALVEZ, G. E. (1964). LOSS of virus by filtration through charcoal. T’iroZogg 23, 307312. IE, T. S. (1970). Tomato spotted wilt virus. Descriptions of Plant Viruses No. 39, Commonwealth Mycological Institute, Kew. MARKS, L. S. (1946). “A Mechanical Engineers Handbook,” p. 107 McGraw-Hill, New York. MARTIN, M. M. (1964). Purification and electron microscope studies of tomato spotted wilt virus (TSWV) from tomato roots. Virology 22, 645649. MILLER, G. L., and GOLDEJK, It. H. (1950). Buffers of pH 2 to 12 for use in electrophoresis. Arch. Biochem. 29, 420-423. MILNE, R. G. (1970). An electron microscope study of tomato spotted wilt virus in sections of infected cells and in negative stain preparations. J. Gen. Viral. 6, 267-276. TAVERNE, J., MARSHALL, J. H., and FULTON, F(1958). The purification and concentration of viruses and soluble antigens on calcium phosphate. J. Gen. Microbial. 19, 451-461. TSAKIRIDIS, J. P. (1971). Biological and serologica characterization of tomato spotted wilt virus in Greece. M.Sc. 1)issertation Nort,h Carolina State University. v.4~ R.WXNMORTEL, M. H. V. (1966). Plant virus serology. Advan. Virus Res. 12, 207-271. V.4N RICGENMORTJSI~, M. H. V. (1972). JiXertrophoresis. In “ Principles and Techniqlles in Plant Virology” (C. Kado and H. 0. Agrawal, eds.), pp. 390-412. Van Nost,rand Reinhold, New York. WOI,.~XSKI, B. S., and FRANCKI, 1:. I. B. (1969). Structure of lettuce necrotic yellows virus. II. Electron microscopic studies of the effect, of pH of phosphotungst’ic acid stain on the morphology of the virus. Virolog?l 37, 437-447.