JOURNAL OF VIROLOGY, June 1976, p. 1040-1050Copyright ©) 1976 American Society for Microbiology

Vol. 18, No. 3Printed in U.S.A.

Strain Selection During Serial Passage of Trichoplusia niNuclear Polyhedrosis Virus

KATHLEEN N. POTTER, P. FAULKNER,* AND E. A. MAcKINNON

Department ofMicrobiology and Immunology* and Department ofAnatomy, Queen's University, Kingston,Ontario, Canada

Received for publication 30 December 1975

Two strains of a nuclear polyhedrosis virus (NPV) of Trichoplusia ni wereisolated on the basis of plaque morphology. They are designated as MP (havinggreater than 30 polyhedra per nucleus) and FP (having fewer than 10 polyhedraper nucleus). Serial, undiluted passage of plaque-purified MP nonoccluded virus(NOV) in tissue culture led to the production of the FP phenotype detectable atpassage 9. With continued serial, undiluted passage, FP became the predomi-nant strain. Comparative growth curves showed that FP NOV are releasedfaster than MP NOV. MP morphology was not observed after 14 serial, undi-luted passages of plaque-purified FP. By the plaque neutralization assay, NOVfrom both strains of virus was neutralized by the homologous and heterologousantisera. The FP phenotype was observed when FP virus was grown in cultureat 17, 22, and 27 C. Hence, the FP phenotype was not considered to be the resultof temperature-inhibited crystallization of polyhedrin under standard tissueculture conditions. The NOV ofboth strains killed insects when injected directlyinto the hemocoele of T. ni larvae. Only MP inclusion bodies were virulent peros. The FP inclusion bodies fed to cabbage looper larvae did not kill, and no in-fectious agent could be detected in the hemolymph. Electron micrographs ofMPpolyhedra showed bundles of nucleocapsids of normal length within the poly-hedra, whereas FP polyhedra contained heterogeneous, electron-dense material,which could account for their lack of pathogenicity.

The nuclear polyhedrosis viruses (NPV) ofinsects are members of the genus Baculovirus(22). Nuclei of infected cells contain polyhedral-shaped inclusion bodies (PIB), which consist ofvirions embedded in a noninfectious proteincalled polyhedrin (20). Under natural condi-tions of infection, the inclusion bodies dissolvein highly alkaline regions of the larval insectgut and release virions that initiate an infec-tion in the gut epithelium (9).

Several NPV have been propagated in con-tinuous insect cell lines. These -are initiallyinfected with virus by inoculating cultures withhemolymph (4, 8, 19) or with a cell extract frominsects previously fed NPV PIB (21). Infectedtissue cultures form PIB within their nucleiand also nonoccluded virions (NOV) at the cellmembrane (16). NOV may be used to propagatethe virus in tissue culture (4, 15).

Recently, a plaque titration technique wasapplied for assay of the NPV of Autographacalifornica in Trichoplusia ni cells (13). Twotypes of plaque morphology were observed:those with many PIB per cell nucleus (MP) andthose containing few PIB per nucleus (FP).

Electron microscope examination indicatedthat MP plaques contained inclusion bodies ofthe multiply embedded virus (MEV) type andFP plaques contained singly embedded virus(SEV) in inclusion bodies (17). The term MEVis used to describe PIB in which several nucleo-capsids are surrounded by a single, outer enve-lope to form bundles; the term SEV indicatesthat the nucleocapsids are singly dispersedthroughout the inclusion bodies.Working with the NPV of T. ni propagated in

a T. ni cell line, we have also observed twoplaque morphologies and in this report showthat they result from infection with separatestrains of NPV. Under conditions of serial pas-sage in vitro, the MP strain is replaced by anFP strain, probably due to mutation of the MPvirus, followed by selection pressure in the tis-sue culture system which favors growth of FPvirus.

MATERIALS AND METHODSTissue culture. An uncloned, continuous, insect

cell line, TN-368, derived from T. ni (12) was grownas monolayers in either 25-cm2 Falcon plastic flasks,

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150-cm2 Corning plastic flasks, or 35-mm diameterplastic petri dishes at 27 C. Tissue culture mediumBML/TC10 (7) was sterilized by positive-pressurefiltration through a series of membrane filters (Mil-lipore Corp.) with pore sizes of 1.2, 0.8, 0.45, and 0.22g.m and stored at 4 C. Before use, the medium was

supplemented with heat-inactivated fetal calfserum(10%, vol/vol) (Grand Island Biological Co.) andgentamicin sulfate (5 mg/100 ml of medium) (giftfrom Schering Corp., Montreal).

Virus. To provide virus suitable for infection oftissue culture, cabbage looper larvae were fed inclu-sion bodies of the MEV type (10). Hemolymph was

collected and after dilution and filtration was usedto infect the T. ni cell cultures (4). Progeny PIBfound in tissue culture were examined using an

electron microscope and were confirmed as being ofthe MEV type (16).

Plaque assay and plaque purification of viralstrains. NOV was assayed by plaque titration on T.ni cells by the method ofHink and Vail (13). After 72h at 27 C, plaques were observed and counted byusing an inverted light microscope.

Plaques were of two morphological types. Somefoci consisted of cells whose nuclei were packed withPIB (MP type) and others consisted of foci of cellsexhibiting hypertrophy and contained <10 PIB/nu-cleus (FP type). MP and FP strains of virus were

plaque purified three times on T. ni cells. Isolatedplaques on plates containing fewer than five plaqueswere picked, using 20-,l Microcaps (Drummond Sci-entific Company), and expelled into 1 ml of medium.This suspension was used as inoculum for a secondplaque assay. Isolated plaques were picked and re-

plaqued. Isolated plaques were again picked, andthis final plaque suspension was used to infect cellsin a 25-cm2 Falcon plastic flask. The resulting NOVsuspension was called passage 1 and was used as an

inoculum to grow larger virus stocks. All studieswere done with virus derived from a single plaquethat had not been passed more than three times intissue culture.

End-point dilution assay for NOV. The method ofBrown and Faulkner (1), in which cells were incu-bated in wells of a Falcon Microtest plate, was usedfor the end-point dilution assay of NOV. After incu-bation for 72 h at 27 C in a humid container toprevent evaporation, wells were observed under a

microscope and were scored for the presence or ab-sence of cytopathic effect. The 50% tissue cultureinfectious dose (TCID5,) was calculated by the sta-tistical method derived by Reed and Muench (18).The relationship 1 TCID5O, = 0.7 infectious unit wasapplied for calculations of multiplicity of infection(MOI) (15).Growth curves. Falcon plastic tissue culture

flasks (25 cm2) seeded with 10' cells were infected atan MOI of 4 with MP or FP NOV. At the end of thevirus adsorption period (1 h), unadsorbed virus wasremoved and the cells were washed twice with warmgrowth medium. The third wash was taken as timezero. Cultures were fed 5 ml of growth medium and,at regular time intervals thereafter, fluid from eachflask was removed and centrifuged at 1,000 x g for10 min to sediment floating cells (15). The superna-

tant was removed and stored at 4 C for assay ofNOV. Pelleted cells were resuspended in 5 ml offresh, warmed growth medium and returned to theculture flask. The flasks were incubated at 27 Cuntil the next sampling period.

Serial undiluted passage of NOV. Falcon plasticpetri dishes (35 mm) were seeded with 105 cellssuspended in 2 ml of growth medium. The cells wereallowed to settle for 1 h at 27 C. For the first pas-sage, cells were infected at an MOI of approximately100. The virus was left to adsorb for 1 h at 27 C, andthe inoculating fluids were removed. The cells werefed 3 ml of growth medium and returned to the 27 Cincubator for 48 h. At this time, 0.5 ml of superna-tant containing NOV was used to initiate the nextpassage. The undiluted inoculum was added to 105cells. After 1 h the inoculum was removed, replacedwith 3 ml of growth medium, and incubated at 27 Cfor 48 h. The process of undiluted serial passage wasrepeated up to the levels described in the Resultssection. Fluids for assay were stored at 4 C andanalyzed by plaque titration.

Quantitation of polyhedra per cell. Falcon plastictissue culture flasks (25 cm2) seeded with 106 cellswere maintained at 27 C for 1 h for cell attachment.Cells were infected at an MOI of 4 with plaque-purified NOV of the MP or FP strains, using virusfrom the second tissue culture passage. After 1 h theinoculum was removed and 5 ml of medium wasadded; 72 h later, cells were suspended in growthmedium by shaking the flask and pelleted by cen-trifugation at 3,000 x g for 30 min. PIB were re-leased by treatment of the pellet with 1 ml of 10%deoxycholate, 1 ml of Triton X-100, and 1 ml ofdeionized water for a minimum of 1 h at room tem-perature (4). The PIB were counted in a hemocytom-eter and, together with the cell number, the averagenumber of polyhedra per cell was determined.

Effect of temperature on polyhedra production.Falcon plastic petri dishes (35 mm) were seeded with5 x 105 cells per dish and infected at an MOI of 4with either MP or FP NOV. After virus adsorptionat 27 C, the inoculating fluids were removed andreplaced with 2 ml of medium. Duplicate disheswere then incubated at 17, 22, and 27 C. Five dayspostinfection, polyhedra were released from infectedcells, as described previously, and counted in a he-mocytometer.

Preparation of immune sera. Female rabbits (ap-proximately 2.5 kg) were injected intradermallywith plaque-purified MP or FP NOV from the thirdtissue culture passage. NOV was concentrated bycentrifuging virus-containing tissue culture fluidsat 1,500 x g for 30 min to remove cell debris and waspelleted at 80,000 x g for 30 min in a Spinco SW27rotor at 4 C. The virus was suspended overnight in 1ml of growth medium and centrifuged through 29 mlof20% sucrose at 73,000 x g for 1 h in a Spinco SW25rotor at 4 C (11). Virus pellets were suspended over-night in 0.5 ml of Freund complete adjuvant (DifcoLaboratories, Detroit, Mich.). An inoculum con-sisted of approximately 108 TCID5dO.5 ml of adju-vant. The rabbits were injected intradermally and 4weeks later were given three booster shots intrader-mally for two weeks at 3-day intervals. Ten days

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1042 POTTER, FAULKNER, AND MACKINNON

after the final booster, the rabbits were exsanguin-ated by cardiac puncture, using Vacutainers (Beck-ton, Dickinson and Co., Canada Ltd.), and the se-rum was prepared. Serum was heated at 56 C for 30min and stored in small aliquots at -20 C.

Plaque neutralization tests. Equal volumes (1ml) of serum dilutions and a constant amount ofvirus (100 PFU) were mixed and incubated for 90min at room temperature. Controls were (i) diluentand virus and (ii) normal rabbit serum and virus.The diluent was serum-free insect cell growth me-

dium. Mixtures were analyzed for unneutralized vi-rus by plaque assay.

Bioassays. PIB from cultured T. ni cells infectedwith second-passage MP and FP NOV were isolatedas described previously and purified by centrifuga-tion through 20 ml of a 40% sucrose solution at 6,700x g for 30 min. The purified suspension of PIB was

suspended in 1 ml of water, and the number of PIBwas determined with a hemocytometer. Aliquots (5,l) of dilutions were dispensed onto 0.83-cm2 disks ofcollard leaf (previously washed in 1::100 Tween 20),and the deposit was allowed to dry. Disks were fed to25 third-instar cabbage looper larvae, individuallyreared in shell vials (25 by 95 mm) plugged withcorks, per dilution. After 24 h, larvae that had eatenat least 75% of the disk were fed a piece of diet andreared for an additional 10 days at 25 C after whichtime mortality by NPV was recorded (14). Dilutionsof NOV strains were fed to larvae and scored in thesame manner.

Aliquots (1 ,ul) of NOV of MP and FP variantswere injected into 25 fourth-instar larvae per dilu-tion. One hour later, live larvae were fed syntheticdiet. After 24 h, larvae that had died because ofinjection trauma were removed from the assay. Theinsects were reared an additional 11 days at 25 C atwhich time mortality by NPV was recorded.Hemolymph was collected from infected larvae 5

days postinfection by puncturing the insect at thebase of the heart and withdrawing the hemolymphinto a Pasteur pipette. The hemolymph from fiveinsects was dispensed into 2 ml of growth mediumand filtered through a Millipore-Swinnex apparatuscontaining a 0.3-Am filter. The virus strain contentof the hemolymph suspensions was determined bythe plaque assay method.

Electron microscopy. Infected cells that had beenincubated for 72 h postinfection at 27 C were re-

moved from the surface of Falcon plastic petri dishesand prepared for electron microscope examinationas described previously (16). Purified PIB were pre-

pared for electron microscopy after release from cellswith Triton X-100 and deoxycholate (4).

RESULTSPlaque morphology and titration ofMP and

FP virus strains. Typical plaque morphologiesproduced by the MP and FP virus strains areshown in Fig. 1. MP plaques (Fig. 1B) are com-posed of cells containing many PIB (greaterthan 30 per cell), whereas FP plaques (Fig. 1A)are foci of rounded cells containing 0 to 10 poly-

hedra per cell. Both MP and FP plaques areapproximately 0.5 mm in diameter.The number of plaques on several plates was

determined on days 3, 4, and 5 postinfectionwith no change in plaque count. Thus, the over-lay (0.6% methylcellulose) allowed only local-ized transmission of progeny virus to adjacentcells. A linear dose response obtained by plaquetitration of the FP strain NOV is given in Fig.2.

Plates containing FP plaques were observedmicroscopically on days 3, 4, and 5 postinfec-tion. The FP plaques did not produce the quan-tity of PIB characteristic of MP plaques and,therefore, are not considered to be precursors ofMP plaques.Growth curves. Figure 3 shows the growth

cycles ofMP and FP NOV after infection of cellcultures at an MOI of 4. The titer of the re-leased virus was determined at intervals andthe accumulated titer was calculated. The la-tent period for both virus strains was 12 h fol-lowed by an exponential phase reaching themaximal titer in 2 days. The steeper logarith-mic-rise period indicates that virions of the FPstrain were released faster than those of theMP strain and, in two separate experiments,reached a maximal titer one-half log higherthan MP virus. The virus yield was 30 and 90infectious units per cell for MP and FP, respec-tively. If the DNA content of T. ni polyhedra istaken as 12.2 gg/mg (5), we calculated thatthere are approximately 330 nucleocapsids perPIB. Therefore, between 1 x 104 and 2 x 104nucleocapsids are produced per MP-infectedcell. In these experiments the intracellularNOV content was not measured. Titrations ofthe pooled, unadsorbed virus and two washingsindicated that both MP and FP NOV adsorb tocells with equal efficiency (76 and 80%, respec-tively).Plaque neutralization tests. Antisera made

against plaque-purified MP and FP NOV werereacted with both homologous and heterologousvirus. Figure 4 indicates that both MP and FPantisera neutralize both MP and FP NOV. Di-luent and normal rabbit sera had no effect onthe number of plaques observed for eitherstrain. These observations indicate that thesurface antigens of both MP and FP virions arevery similar in antigenic composition.Polyhedra production. PIB were released

from cells infected with NOV at an MOI of 4.Cells infected with MP NOV consistentlyyielded an average of greater than 30 PIB/cell,whereas cells infected with FP NOV yieldedless than 10.

Purified PIB from the third-passage level

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STRAIN SELECTION OF T. NI NPV 1043

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FIG. 1. Appearance ofFP (A) and MP (B) plaques on T. ni cells 72 h postinfection under an inverted lightmicroscope, x240. FP plaques exhibit rounded cells with hypertrophied nuclei with few PIB. MP plaquesexhibit rounded cells and hypertrophied nuclei filled with PIB, giving infected cells an overall blackappearance.

were examined in an electron microscope. PIBfrom the MP virus strain were of the MEV type(Fig. 5). The virus bundles consisted of two toseven nucleocapsids. Although many nucleo-capsids were occluded within the nucleus ofMP-infected cells, a few nucleocapsids buddedthrough the plasma membrane 72 h postinfec-tion.PIB from the FP strain did not contain rod-

shaped nucleocapsids (Fig. 6). Sectioned inclu-sion bodies often appeared empty, but some-times enveloped structures were seen. Thesecontained fragments of electron-dense mate-rial, which did not fall into a particular sizeclass and did not resemble viral nucleocapsids.Nucleocapsids were observed budding atplasma membranes ofcells infected with the FPstrain (Fig. 7). Virions were released in largenumbers and were more loosely enveloped thanthose released from MP-infected cells.NOV that budded from cells infected with FP

and MP strains contained nucleocapsids of sim-ilar lengths (335 nm).

Production of polyhedra at reduced incuba-tion temperatures. The possibility was consid-ered that the genome of the FP strain mayinstruct a temperature-sensitive inclusion bodyprotein that would not crystallize effectively at27 C and, hence, would give rise to the FPphenotype characterized by few inclusions percell.Table 1 shows that the number of PIB pro-

duced by the FP strain is not influenced bythese temperatures. PIB production by the MPstrain was the same at 27 and 22 C but wasreduced at 17 C. At 27 and 22 C, uninfected cellcontrols remained healthy, whereas at 17 Cthey began to deteriorate.

Infectivity of strains. The virulence of PIBand NOV was assessed in cabbage looper larvae(Table 2). Only MP strain PIB were pathogenicwith a 50% lethal dose (LD50) of 56 PIB perlarva. MP NOV was detected in the hemo-lymph after feeding MP PIB. FP strain PIBneither killed the larvae nor yielded an infec-tious agent in the hemocoels.

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1044 POTTER, FAULKNER, AND MAcKINNON

240 A second series of serial passages was initi-ated with plaque-purified MP virus (Fig. 9).MP virus remained homogeneous with regard'to phenotype for eight consecutive passages

200

but, beyond this level, FP plaques appearedand displaced production ofMP virus. FP virusremained homogeneous for 14 consecutive pas-sages with no reversion to the MP phenotype.

160 - DISCUSSION

nU /A plaque assay technique (13) was used toassay the NPV of T. ni and to provide foci ofinfected cells for plaque purification of the vi-rus. MP and FP foci (Fig. 1) were readily distin-

o / guished on plates inoculated with virus thatLi .had been grown for several passages in tissue

culture before plaque assay. Three cycles ofz / plaque purification were carried out to obtain

80 - virus strains that exclusively produced the MPand FP phenotypes.The plaque assay technique used here is

based on that of Dulbecco (2) for other animal

40-8

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01 02 03 04 0.5 06 M

RELATIVE VIRUS CONCENTRATION 7 mP

FIG. 2. Proportionality ofFP virus concentrationto plaque count. Twofold dilutions of virus were ana-lyzed by plaque assay in duplicate. Plaques were Eicounted, unstained, 72 h postinfection under an in- LOverted light microscope. l

NOV ofboth MP and FP strains were equally o0lethal for insects when injected directly into the °ihemocoel but did not kill after per os infection. o POLYHEDRVCELL YIELD

Plaque assays on the hemolymph from insects Lo MP 45injected with MP strain NOV indicated that a DFP3mixture ofMP and FP strains was present (97% EMP and 3% FP). Assays on hemolymph from 3insects injected with FP NOV remained homo- Ugeneous for the FP strain.Changes in phenotype after serial passage 4

of virus. MacKinnon et al. (16) reported thatthe average production ofPIB per cell decreasesas the NPV is serially passed in tissue culture.Subsequently, we investigated the distributionofMP and FP strains at up to 50 passages from E_E _. _. __the original isolation of the virus from hemo- 1 2 3 4lymph of insects fed PIB. The data in Fig. 8 TIME (DAYS)show that the predominant strain of virus at FIG 3. Growth curves of MP and FP NOV inpassage level 2 was MP (approximately 99%) tissue culture. Cultures were infected at an MOI of4,but that by passage level 5 the FP strain was and the extracellular infectivity was monitored atalready in excess and by passage level 25 had intervals. The accumulated log TCID5, per milliliterbecome the sole plaque-producing.strain in the was calculated at each interval. Symbols: 0, FPcultures. strain; *, MP strain.

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STRAIN SELECTION OF T. NI NPV 1045

viruses. The linear dose response obtained byplaque titration of the FP NOV strain shown inFig. 2 indicates that a plaque is caused by asingle infectious particle. If more than one vi-rus was required to initiate a plaque, the curvewould not be linear (3). By demonstrating thata single FP virion can initiate a plaque, it isconcluded that FP virions, although having theability to make only small amounts of polyhed-rin, are not defective particles and do not re-quire a "helper" virus for infection to proceed.Both plaque-purified strains were used to

raise antibodies, and each kind ofantibody neu-tralized both MP and FP viral strains (Fig. 4).It remains to be resolved whether host antigensare an integral part of the virion or could havecontaminated the antigen preparation. Ineither instance, cross-reaction between theviral strains would also be observed. However,the two strains of NOV appear to share manysimilar antigenic determinants, and it is possi-ble that the altered plaque morphology is due todifferences in the polyhedrin of inclusion bodiesrather than to major differences in virion nu-cleocapsids and envelopes.The FP strain shows a selective advantage in

growth in T. ni cells cultured in vitro. Virus isreleased more rapidly and rises to a higher titerthan is observed with the MP strain (Fig. 3).The production of inclusion bodies by the FPstrain relative to the MP strain is not consid-ered to be the result of incubation at a restric-tive temperature (Table 1). It is not knownwhether the FP genome codes for more polyhed-rin than is observed as crystallized PIB, or iflarge amounts of polyhedrin are synthesizedbut do not crystallize.When NPV derived from insect hemolymph

was serially passed in vitro there was a fall inthe average yield of PIB/cell corresponding to agradual selection of the FP strain (Fig. 8).Starting with the plaque-purified MP strain,there was a delay of eight passages before theFP phenotype appeared, but thereafter therewas rapid replacement of the MP strain withFP virus (Fig. 9). In over 14 serial passages noMP virus arose after the series was initiatedwith FP virus. These results bear out the hy-pothesis that FP strains grow more readily inthe T. ni cell line and, ifthey arise by mutation,have a selective advantage over MP strains.

Previous studies established that aberrantforms of virus are present in inclusion bodies inlate-passage virus but not in early-passage vi-rus (16). We examined the polyhedra and NOVin thin sections of cells infected with early-passage, plaque-purified MP and FP strains ofvirus. NOV consisting of enveloped nucleocap-sids from both strains had similar morphology.

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LOG ANTISERUM DILUTION

FIG. 4. Plaque neutralization of MP and FP

NOV. Equal volumes ofserum dilutions (anti-MP or

anti-FP) and approximately 100 PFU of virus were

mixed and incubated for 90 min at room tempera-ture. The mixtures were analyzed for unneutralizedvirus by plaque assay. The neutralization ofMP vi-rus (A) and ofFP virus (B) with MP and FP antiseraare shown. Symbols: 0, FP antiserum; 0, MP anti-serum.

Electron micrographs of NOV budding at thecell membrane reveal that occasionally morethan one nucleocapsid is enclosed within a sin-gle envelope, i.e., polyploidy. This is not consid-ered unusual since polyploidy is commonamong animal viruses that mature by buddingfrom cell membranes (6). Major differenceswere seen between sections ofMP and FP poly-hedra. Sections of MP inclusions appeared tohave typical MEV structure and were infec-tious per os (Fig. 5, Table 2). The polyhedracontained many bundles consisting of groups ofnucleocapsids enclosed by a common mem-brane. The morphology is similar to the MEVT. ni NPV described by Heimpel and Adams(10). It is the predominant structure observedwhen the T. ni NPV is passed in insects. Thefine structure of FP inclusion bodies was differ-ent in that the inclusions did not contain dis-tinct rod-shaped nucleocapsids. These inclu-sions were sparsely populated with envelopedstructures that contained some heavily stain-

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1046 POTTER, FAULKNER, AND MACKINNON

FPi..r e 0

FIG. 5. Thin section ofMP PIB 96 h postinfection. Third-passage PIB, x104,000.

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STRAIN SELECTION OF T. NI NPV 1047

4

FIG. 6. Thin section ofFP PIB 96 h postinfection. Third-passage PIB, x57,000.

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1048 POTTER, FAULKNER, AND MACKINNON J. VIROL.:;* $ri. ;

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FIG. 7. FP-infected cells. Infected cells were removed from the growth surface by shaking at 72 h postinfec-tion. The cells were pelleted and prepared for electron microscopy. Nucleocapsids are seen budding at the cellmembrane (arrows). x13,000.

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STRAIN SELECTION OF T. NI NPV 1049

TABLE 1. Effect of temperature on polyhedraproductiona

Incubation temp Polyhedra/cell(C) MP FP

27 70 522 67 617 30 5

a Effect of temperature on polyhedra production.Falcon plastic petri dishes (35 mm) were seeded with5 x 105 cells per dish and infected at an MOI of 4with either MP or FP NOV. After 1 h at 27 C,inoculating fluids were removed and replaced with 2ml of medium. Duplicate dishes were then distrib-uted to 17, 22, and 27 C incubators. Five days postin-fection, PIB were released from infected cells bytreatment with 1 ml of 10% deoxycholate, 1 ml ofTriton X-100, and 1 ml of deionized water for aminimum of 1 h at room temperature and were thencounted in a hemocytometer.

TABLE 2. Bioassays of cell culture productsa

Virus LD50 fed LDs0 injected(PIB/larva) (TCID5/insect)NOVMP 0 16 (97% MP, 3% FP)FP 0 29 (100% FP)

PolyhedraMP 56 (100% MP)FP 0 (no CPE in cell

culture)a Bioassays of viral tissue culture products. Cabbage

looper larvae were fed and injected with purified NOV andfed purified PIB. Control experiments showed that infec-tious NOV could be recovered from collard leaves on whichvirus had been deposited and dried as described in thetext. Numbers in parentheses refer to the proportion of MPand FP NOV detected in hemolymph by the plaque assay.

ing material but of no distinct form (Fig. 6).The FP inclusion bodies were not infectious peros (Table 2) and did not resemble the SEV of T.ni NPV. Thus, FP polyhedra do not appear toencapsulate FP nucleocapsids. If nucleocapsidmaterial is present in the FP inclusions, it mayhave been distorted by the crystallization proc-ess. An alternative is that cellular componentshave become trapped in viral material, andthese pseudovirions may have become oc-cluded.

Results given in Table 2 indicate that FPNOV arise in vivo. Although FP polyhedra arenot infectious per os, injected FP NOV werelethal and the virus was propagated as the FPstrain. When MP NOV were injected, the hem-olymph of diseased insects contained both MPand FP NOV (Table 2). This finding suggeststhat new strains with an FP phenotype ariseduring systemic replication of MP NOV.Should FP polyhedra be formed in vivo as a

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PASSAGE NUMBER

FIG. 8. Serial, undiluted passage of uncloned vi-rus. The virus was originally derived from infectioushemolymph as described previously (16). Sampleswere thawed and analyzed by plaque assay for theproportion of MP and FP plaques at the passagelevels indicated.

100

80C')

a 60 /CL/Xo40/

20

4 8 12 16 20PASSAGE NUMBER

FIG. 9. Serial, undiluted passage ofplaque-puri-fied MP NOV. Falcon plastic petri dishes (35 mm)were seeded with 105 cells per dish. After 1 h at 27 C,the cells were infected with MP virus at an MOI of100 and incubated for 48 h at 27 C. An aliquot (0.5ml) was used to initiate the subsequent passage. Suc-cessive passages were carried out in the same man-ner. Viral harvests were analyzed by plaque assay,and the proportion ofMP and FP plaques was deter-mined.

consequence of the strain change they wouldnot be replicated under natural conditions ofvirus transmission since FP polyhedra are notinfectious per os (Table 2).

ACKNOWLEDGMENTSWe gratefully acknowledge financial support of the Med-

ical Research Council, Ottawa, Canada.We thank R. P. Jaques, Canada Agriculture Research

Station, Harrow, Ontario, for assistance in the in vivobioassays and Sheila Leonard for technical assistance.

LITERATURE CITED1. Brown, M., and P. Faulkner. 1975. Factors affecting

the yield of virus in a cloned cell line of Trichoplusia

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6. Fenner, F., B. R. McAuslan, C. A. Mims, J. Sambrook,and D. 0. White. 1974. The biology of animal viruses,2nd ed. Academic Press Inc., New York.

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13. Hink, W. F., and P. V. Vail. 1973. A plaque assay fortitration of alfalfa looper nuclear polyhedrosis virusin a cabbage looper (TN-368) cell line. J. Invertebr.Pathol. 22:168-174.

14. Jaques, R. P. 1967. The persistance of a nuclear polyhe-drosis virus in the habitat of the host insect, Tricho-plusia ni. I. Polyhedra deposited on foliage. Can.Entomol. 99:785-794.

15. Knudson, D. L., and T. W. Tinsley. 1974. Replication ofa nuclear polyhedrosis virus in a continuous cell cul-ture of Spodoptera frugiperda: publication, assay ofinfectivity, and growth characteristics of the virus. J.Virol. 14:934-944.

16. MacKinnon, E. A., J. F. Henderson, D. B. Stoltz, andP. Faulkner. 1974. Morphogenesis of nuclear polyhe-drosis virus under conditions of prolonged passage invitro. J. Ultrastruct. Res. 49:419-435.

17. Ramoska, W. A., and W. F. Hink. 1974. Electron micro-scope examination of two plaque variants from a nu-clear polyhedrosis virus of the alfalfa looper, Auto-grapha californica. J. Invertebr. Pathol. 23:197-201.

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19. Sohi, S. S., and J. C. Cunningham. 1972. Replication ofa nuclear polyhedrosis virus in serially transferredinsect hemocyte cultures. J. Invertebr. Pathol. 19:51-61.

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