Isolation and Characterization of a Herpesvirus from Leukemic Guinea Pigs

(1)

Isolation

and Characterization

of

a

Herpesvirus

from Leukemic

Guinea Pigs

D. P. NAYAK

DepartmentofMedicalMicrobiologyandImmunology,Schoolof Medicine, Universityof California, LosAngeles, California 90024

Received for publication 7 June 1971

A guinea pigherpesvirus (GPHV) hasconsistently been isolated fromleukemic

lymphoblasts of strain-2 guinea pigs. GPHV is serologically related to the guinea

pigherpes-like virus isolatedby HsiungandKaplow. The virions of GPHVconsist

of an icosahedralcapsidcontaining a dense nucleoprotein core enclosed in a

double-layered membrane.The averagediametersofGPHVvirionandcapsidwere 166 and

101 nm,

respectively.

Studies onthemorphogenesis of GPHVrevealed that, as in

otherherpesvirusinfections, onlythenakedcapsidswith or withoutthenucleoprotein

corewerefound in the infectedcellnuclei;itwasalso learned that thevirionacquired

its

envelope by budding

from thenuclear membrane oftheinfectedcells. However,

GPHV-infected cell nuclei also contained dense fibrous rods, resembling nucleo-protein core outside the capsids, and tubules resembling viral core protein. The

capsids were often embedded in dense granular antigen. GPHVdeoxyribonucleic

acid (DNA) has a density of 1.716 g/ml in cesium chloride compared to herpes simplexvirus DNA (p = 1.728g/ml) and cellularDNA (p = 1.700g/ml).

Opler's

observation

(18)

of

C-type particles

in lymphoblasts of leukemic

guinea pigs suggested

a

viral

etiology

for

guinea

pig

leukemia. Nadel et

al. (14), and more recently Feldman and Gross

(6),

alsoobserved such

particles

in

leukemic,

but

notinnormal, guinea

pigs.

However,no onehas

successfully

demonstrated that these

C-type

particles

are the

leukemogenic

agent in

guinea

pigs. We also studied the role of viral agentsin guinea

pig

leukemia and

consistently

found C-type particles budding from the

endoplasmic

reticulum of leukemic

lymphoblasts.

However,

we were unable to

produce

this agent in tissue

culture in sufficient

quantity

to characterize its

biological

and

biophysical properties.

During

this

investigation,

we

consistently

noted the

activation

ofasecond virusinleukemic

lympho-blasts culturedinvitro. Thisagent hasthe

prop-erties

of

herpesvirus.

A similar virus bas been

isolated from normal and leukenic

strain-2

guinea

pigs by Hsiung

and

Kaplow

(9)

and more

recently

fromother strains of

guinea pigs

aswell

by Bhatt et al.

(2).

This report describes some

morphological

and biochemical characteristics

of this

guinea

pig

herpesvirus (GPHV)

which

wasisolatedfromleukemicstrain-2

guinea pigs.

MATERIALS AND METHODS

Guineapigs. All the strain-2guinea pigs (both in-fected anduninfected) used in theseexperimentswere

kindly supplied by S. Opler of Stanford University.

Leukemia in these animals is maintained by

inoculat-ingtissues ofinfected animals (spleen, blood, tumors, etc.)into healthy animals.Noinfectious leukemogenic

agenthas yet beenisolated from theseleukemicguinea pigs. Infectedanimals develop leukemia within 2 to 3 weeks afterinoculation. Theanimalsin terminal phase

ofleukemiawereexsanguinated bycardiacpuncture.

Gross andhistopathology ofthediseasehave been

de-scribedbyOpler (19). HartleyandEnglish short-hair

strainsofguineapigswerepurchasedlocally.

Cell culture. Usually, circulating lymphoblasts or

lymphoblastsfromalocal tumor,mesentericlymphoid

tumors, and spleen of leukemic guinea pigs were

placedintissueculture. ModifiedEagle medium

con-taining inactivated fetal calf serum (10 to 20%),

tryptosephosphate broth

(10%,)

penicillin (200IU/

ml), and streptomycin (200,Ag/ml) was used. The

medium was normally changed twice a week or as

needed. Guineapig embryocultures (GE) were pre-pared from 1-to 1.5-month-oldembryos byusingthe

usual procedureofcell culture.

Labeling ofinfected cells. Monolayers of cultures

were infected with GPHV. When the initial

cyto-pathic changes became evident (usually 6 to 8 days

after infection), the old medium was replaced with

medium containing 3H-thymidine (specificactivity, 25 Ci/mM; 20 uCi/ml) and dialyzed fetal calf serum (10%).Thefresh mediumwasincubated for48to 72

hruntilall of thecells rounded up,atwhich time the

medium and thecellswere harvested and processed

forvirus purification as well as for the isolation of intracellularDNA.

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NAYAK

Viruspurification. The culture medium was freed from cells and subcellular debris by two cycles of

centrifugationin an RC-2B Sorvallcentrifuge(10,000

rev/min for 10 minin an SS 34 rotor).Viruswas con-centrated twice at the interface of 70 and 20% sucrose

andfinally purified by density-gradient centrifugation

in apreformedlinear70 to20%sucrosegradient (15).

Aftercentrifugation,thedensity, radioactivity, and

in-fectivity ofeachfractionwereanalyzed,andpeak

frac-tionswereexamined bynegative stainingandthin

sec-tioningfor the presence of virusparticles.

Isolation and analysis of viral and cell DNA.

Deoxy-ribonucleic acid (DNA) frominfected cellsand

puri-fiedviruswasisolated andanaylzedin a cesium

chlo-ride gradient asdescribed by others (21, 25). Thecells

orfractionscontaining viruswerelysed with sodium

dodecyl sulfate (3%), dialyzed, and treated with

self-digested Pronase (200,ug/ml). DNA soisolated

wasmixedwith concentratedcesium chloridetomake

asolution withadensityof 1.700g/mlandcentrifuged

at35,000rev/min for72hr at 20 C inanSW 50 rotor.

Fractionswerecollectedandanalyzed for densityand

radioactivity.

Electronmicroscopy.Smallpiecesofselectedtissues,

culturedcells, or pelleted virus materialswere

imme-diately fixedin2%glutaraldehydeinphosphate buffer

(pH 7.2 to 7.4) for 30 to 60 min, washed in three changes of phosphate buffer for 1 min each, and treatedwith

1%

osmium tetroxideinphosphate buffer

(pH 7.2 to 7.4) for 30 min. Thetissuewasthenrinsed

inRinger's solutionandblock-stained for 30min in

1% aqueousuranylacetatebroughttopH4.9to5.0

withNaOH.All of theforegoing solutionsweremade

slightlyhypertonic withsucrose.Dehydrationwas

ac-complished with the usual ethanol series. Thetissue

blockswereinfiltratedfirstwitha1:1 mixture of

ab-solute ethanol and embedding media and then

em-beddedinUNOXepoxy (Spurr).Fornegative

stain-ing, virus particleswerefixed on carbon-coatedgrids

andstained with1%phosphotungstic acid.

RESULTS

Isolation

of the GPHV. Spleencells, lymphoid

tumors, local tumors, and

circulating

leukemic

lymphoblasts

wereplaced in culture in modified

Eagle medium containing inactivated fetal calf

serum

(10

to

20%)

and 10%tryptosephosphate

broth. The medium was changed

twice

aweek.

In such cultures, most of the cells remained in

suspension, andover 80% weredead in72hras

determined by dye exclusion. Some fibroblasts

adhered tothe bottom ofthe dishes in about a

week. Bythesecond week, a few foci ofclumped

cells

appearedinculture.

Finally,

all ofthe

cells

would round up and die around the fourth or

fifth week ofculture. It is at this time that the

herpes-like virus (GPHV) can be first seen in

the electron microscope. The results were the sameifafecderlayerof GEculturewasusedfor

these leukemic

lymphoblasts.

Morphology. The morphology of GPHV was

studied both bynegative staining of intact virions isolated from sucrose-density gradientsaswellas

by positive staining ofa thin section of

virions

and infected cells. It was clear that the tissue

culture-activatedGPHV wascompletelydifferent

from mature and

immature

C-type

particles

ob-served in vivo in leukemic lymphoblasts by

Opler

(18), Nadel et al. (14), Feldman and Gross (6), andbyus (Fig. 1). TheseC-type viruseswith an

average diameterof 101 nm bud fromthe

endo-plasmic reticulum into the cytoendo-plasmiccysternae.

GPHVon the otherhandhas themorphological

characteristics ofa herpesvirus (Fig. 4-9; Table

1).

Mature GPHV consists of an icosahedral

capsid containing

a dense

nucleoprotein

core

surrounded bya double-layered membrane. The

outermembraneoften extends with the

formation

ofasmall tail. Mature GPHVis spheroidal

and

has an averagediameter of 166 nm. The

icosa-hedral capsid is about 101 rum. There is a dense material between the capsid and the outer

en-velope (Fig.5and7). Negative staining of purified virions andcapsids also revealed thesame

struc-tural components. Large virion-like structures

containing

several

(as

many as

eight) capsids

(Fig. 12) and naked capsidswere oftenfound in

some

preparations

of purified virion isolated

fromsucrosegradients(Fig. 9).

Biological and serological properties. Once the

virus is activated it can be passed serially in

normal guineapig embryonic culture. Itusually

takes 7 to 14days to

produce visible

cytopathic effect (CPE; Fig. 2 and 3). CPE consists of in-fected cells which become round, refractile, and loose and which often clump together. In GE

cells,

themaximum titeris around 104infectious

unitsasdetermined by endpoint dilution in tissue culture. This titer is50- to 100-foldovertheinput

virus.

Freezing and thawing once reduced the

infectivity at least 10-fold. The infectivity of GPHV is also sensitive to ether treatment. CPE

in GE cultures after infection was enhanced by incubation in reduced temperature (33C), in acidic culture

medium,

and in medium

deficient

in

arginine.

All of

these

conditions

have

been

found to activate the EB virus inBurkitt's

lym-phoma

cells

(7,

9).

GPHV also causes CPE in

primary rabbit kidney

cells

and Vero monkey kidney cells and is

serologically

relatedto GPHLV

(9)

as determined by neutralization test and by immunofluorescence technique. Virus neutraliza-tion test was carried out on

guinea pig embryo

cultures as described by Bhatt et al. (2);

virus

titerwas reduced 1,000-fold

by

thehomologous antisera and the antisera

against

GPHLV

pro-vided

by

G. D.

Hsiung.

Morphogenesis.Morphological developmentof

subviralcomponents and

assembly

ofthese

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VOL.8,1971 HERPESVIRUS FROM LEUKEMIC GUINEA PIGS 581

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FIG. 1. ImmatureC-type

particles

inleukemic

lymphoblasts budding from

endoplasmic

reticulum. X

84,000.

FIG. 2. Uninfected guinea pig

embryo

culture. X125.

FIG. 3. Guineapig

embryo

culture

infected

with GPHV.

Cytopathic

effectwasevident 7

days

postinoculation.

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FIG. 4. ThinsectionofGPHV presentoutsideoftheinfectedcells. X 63,000. FIG. 5. Guinteapigherpesvirus. Notethemembranous tail. X 113,000. FIG. 6. Guineapigherpesvirus.Notethe segmentation of nucleoid. X 113,000.

FIG. 7. Guinea pigherpesvirus. Notethe eccentricpositionof thecapsidandpresenceofdensematerialbetween

thecapsid andenvelope. X84,000.

FIG. 8. Guinea pig herpesvirus enclosed in apinocyticvacuoleon the cellmembrane. X113,000.

FIG. 9. Negativelystainednaked capsids without any envelope isolatedfromsucrosegradient. X84,000.

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HERPESVIRUS FROM LEUKEMIC GUINEA PIGS

TABLE 1. Morphological characteristics ofguinea pig herpesvirus (GPHV) and its components

Determination Avg diameter(nm)

GPHV virion... 166 i 15

Capsid... 101 i 5

Electron-dense nucleoid inside

thecapsid ... 50-70 Electron-lucent core inside the

capsid... 71.5i 5

Intranuclear tubules... 71i 5

Intranuclear fibrous rods

re-sembling viral nucleoprotein. 12 or24

ponentsintomorphologicallymaturevirionswere

studied in GE cell monolayers grown over a

plastic sheet and infected with GPHV. When

infected foci became evident, the cellswere fixed

andembedded; specificfoci of cellswere selected

under the light microscope, sectioned either in

parallel or vertical plane to themonolayer, and

examined with the electron microscope. In this

way, cells inthe same foci atdifferent stagesof

infection could be observed.

Ingeneral, the morphological development of

GPHV followed closely that of other

herpes-viruses. The nuclear phase of GPHIV consisted

ofnaked icosahedral capsids,mostofwhichwere

empty (Fig. 10). The electron-dense material,

when found inside thecapsids, appearedin

differ-ent stages of condensation: ring form or dense

nucleoid (Fig. 10 and 13). Some capsids possibly

containedonlycoreproteinasapparent fromthe

lack of electrondensity (Fig. 13).Thesestructures

representing different stages in the development

of GPHVcapsidswere similar to those found in

other herpesvirus infection (3, 4). Also found

inside the nucleuswere dense,rod-like structures

as reported in some herpesvirus-infected cells

(4). The rods resembled DNA protein core and

wereoften found in close association withcapsids

(Fig. 13). Inside the nucleus, capsids wereoften

found embedded in a spherical electron-dense

material (Fig. 11) which was similar to

intra-nuclear granular antigens inherpesvirus-infected

cells (16, 23). Inlater stages ofinfection, these

intranuclear structurespossibly disintegrate into

smaller pieces containing one or more capsids and are released into the medium after lysis of

infected cells. This could explain the origin of

somelarge virus-like particles containing multiple

capsids found in the preparation of purified

virions(Fig. 12).This denseintranucleargranular

antigen is present also around single capsids, is

often eccentric in arrangement, and resembles

closely the dense material often found between

the capsid and the envelope (Fig. 4, 5, 7). A

similar dense material between the envelope and

the capsids in the herpesvirus associated with

frog renal adenocarcinoma has been reported

(24). Tubular structures which have the same

density and diameter as the empty coreproteins

found inside the capsids can also be seen in the

nucleus (Fig. 14). Such tubules have beenfound

in guinea pig cytomegalovirus infection (13).

Complete virionswere not found in the

nucleus;

however, cytoplasmic invagination containing

matured virions was observed inside thenucleus

(Fig. 15). The virions acquire their envelope

during budding from the nuclear membrane (Fig. 16) as has been reported by others (4).

Matured virions were found in closeassociation

with the plasma membrane and outside the cell

membrane(Fig. 8), but very few could be located

inthe cytoplasmic vacuoles.

Putification of viiions and characterization of

viral genome. The morphology and the

matura-tion of the virions suggest that this agent is a

herpes-type virus. Next. experiments were de-signed to purify the virus and characterize the

viral genome. Cells were infected and labeled with 3H-thymidine (3H-TdR) when foci

became

visible.After labeling for 72 hr, themediumwas harvested. The virus was concentrated on 2 M

sucroseandpurifiedonasucrose-densitygradient

(20 to 70%) as described above. A sample of eachfractionwascounted, and the fractions

con-taining

the peak ofradioactivity were examined

by negative staining for virus particles. The in-fectivity in thepeakfractions of the gradientwas

around 105-5 units/ml and coincided with the peak ofradioactivity. The recovery ofthe total

infectious virus in the sucrose gradient was only

10 to 15% of the totalinfectious units used in the

purification procedure. DNA was isolated from purified

GPi-V

andwasfoundtobeheavierthan GE cell DNA (p = 1.700 g/ml).

GPkIV

DNA had a density of 1.716 g/ml in cesium chloride

(Fig. 17).

When DNA was isolated from infected cells

after

labeling with 8H-TdR,

as

above,

the newly synthesized viralDNAalso had apeak of 1.716

g/ml

(Fig. 18). 14C-TdR-labeled DNA ofHeLa

cells

infected with

herpes

simplex

viruswasused

as a marker in the same

gradient.

HeLa cell DNAand HSV DNA hadadensity of 1.700 and

1.728 g/ml, respectively, incesium chloride (20).

3H-labeled DNA isolated from purified GPHV and 14C-labeled DNA isolated from

GPHV-infected cells had the samedensity if run inthe

same cesium chloride gradient. Further proof

thatthe nucleicacidin GPHVvirionisDNA was

demonstratedby its deoxyribonucleasesensitivity, resistance to ribonuclease, and resistance to

alkali.

VOL.

81

1971 583

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541

FIG|.1.y. Mutil capsids...embdde '"ingrnua anie in...th nuli .Als not Sinl casd suroude by.t,.

FI.1!Ngtvl stie viu-lk patces cotainingFmutil casd islae fro sucrose grains

X84.000. GH asd

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FIG. 13. Sectionofaninfected nuclei. X84,000. (a) Fibrousrodresemblingviralnucleoprotein. (b)Ring form

of nucleoprotein inside the capsid. (c) Capsids containing protein core only. (d) Section of a densenucleoprotein

core. (e)Sectionsof tubules.

FIG. 14. Sectionofmassesof tubulesfound in infectednuclei. X84,000.

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586

NAYAK J.VIROL.

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FIG. 15. Invagination cytoplasmic vacuole containing matured GPHV inside the nucleus. Nucleus (N), Cy

(cytoplasm), INM (inner nuclearmembrane), ONM(outer nuclearmembrane). X84,000.

FIG. 16. BuddingGPHVfromnuclearmembrane. X84,000.

300

DROP NUMBER

FIG. 17. Cesium chlorideequilibrium centrifugation of3H-labeledguinea pig herpesvirusDNA (a) along with I4C-labeled guinea pig embryocell DNA(b).

DISCUSSION

Characteristics and morphogenesis of GPHV.

GPHVreportedinthispaperisclosely related, if

notidentical,toGPHLVisolatedby Hsiung and

Kaplow (9) and Bhattetal. (2) and is, therefore,

different from guinea pig cytomegalovirus. The

morphological and biochemical data indicate

that GPHV has the characteristics of a

herpes-virus. It isanenvelopedviruswithaicosahedral

capsid which contains a nucleoprotein core. Its

=5.0

i- 1:-;E

T

o00

*200

*100

3 b c

814

100 200 300 0R0p NU M B E R

400

180

170

_

160

FIG. 18. Cesium chloride equilibriumcentrifugation

of3H-labeled guinea pig cell DNAinfectedwith guinea

pigherpesvirusandU4C-labeledHeLacellDNAinfected

with herpes simplex virus. (a) Intracellular herpes simplex DNA. (b) Intracellular guinea pig herpesvirus DNA. (c)HeLacell DNA.

nucleic acid is double-stranded DNA. The

as-sembly

of

capsids

and

nucleoprotein

takes

place

in the cell nucleus, and the outer envelope is acquired

during budding

from the nuclear mem-brane. The virion is released in the medium

by

reverse

pinocytosis

or

by

lysis

ofcell. The

large

numbers of empty capsids in the nucleus, the large number of fibrous structures

resembling

DNA-containing protein core, and the

paucity

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HERPESVIRUS FROM LEUKEMIC GUINEA PIGS

of

complete

virions either

in the cytoplasm or

outside the cell indicate a

possible

defect in the assemblyprocess.

Relationship ofGPHVwith guinea pig leukemia.

Twodifferentviruses have, therefore, been found

in lymphoblasts of strain-2 leukemic guinea

pigs. (i) A C-type

virus particle

hasbeen observed in vivo, but usually not in vitro (Fig. 1).

(ii)

GPHV, a herpes-type DNA

virus,

is

isolated

fromguinea pig cells only in vitro culture andnot

invivo.

Itis

possible

that

guinea

pig leukemia is caused

by oneofthesetwovirusesor

by

the

interaction

of both viruses or

by

none. Wepsic et al.

(27)

and Sarma etal.

(22)

havesuggested that certain forms of

guinea pig

leukemia may not

possibly

be

caused

by

aninfectiousvirus. Studies

by Opler

(18), Nadel et al.

(14),

and Feldman and Gross

(6),on theotherhand,suggestthataC-type virus

is the cause of the

guinea pig

leukemia even though

physicochemical

characterization of this

virionand its definitive role inguinea pigleukemia

remains to be done. Their strongest argument,

thatsuch C

particles

arepresent

only

in leukemic guinea

pigs,

has been weakened

by

the recent

finding of C-type

particles

resembling

GPLV-Opler in germinal centers of lymph nodes of

Hartley guinea pigs, a

strain

not

susceptible

to

leukemia

(11).

However, the presence of

C-type

particles

in nonleukemic

guinea pigs

does not

ruleoutits

etiological

rolein

guinea

pig

leukemia.

GPHV, ontheother

hand,

hasnowbeen

isolated

from leukemic and

nonleukemic

strain-2

guinea

pigs (2,

9).

Preliminary

datasuggestthat

GPHV,

when inoculated

directly,

fail toinduce

leukemia

in strain-2 or Hartley

guinea

pigs (unpublished

data).

Inability

to induce

immunity against

a

similar

herpesvirus isolated by

Bhatt et al.

(2)

indicated the tolerance of

guinea pigs

to GPHV

antigen,

suggesting

either

congenital

or

hereditary

transmission of GPHV. The characteristics of

GPHV are very similar to those of EB virus

(EBV) found inBurkitt'slymphoma.EBVis also activated in

lymphoblasts only

in tissue culture andnotfound in

lymphoblastic

cells invivo

(5).

Theconditions ofactivation forEBV and GPHV

are very similar

(7,

8).

The

density

of GPHV

DNA

is similar

tothatofEBVDNA

(26)

which

is also

similar

to

cytomegalovirus

DNA. The

infection of EBV is also widespread and not

limited to persons with Burkitt's lymphoma.

Herpes-typeviruses havebeenfoundasthecausal

agent in

lymphoma

inmonkeys

(12)

and Marek's

diseaseinchickens (17). Possible role ofGPHVin inducing guinea pig leukemia has recently been

suggested (10).

Whether GPHV aloneorin

asso-ciation withaC-typevirus has any

etiological

role inguinea pigleukemia remains to be established.

ACKNOWLEDGMENTS

The author gratefully acknowledges the assistance of Zane Price for electromicroscopy and acknowledges his indebtedness to G. D.Hsiung of Yale University School of Medicine and Veterans Admimstration Hospital, West Haven, Conn., for her suggestions andalso for supplying the immune sera to GPHLV (9).

This work was supported by a SeniorDerhnam Fellowship

(Dl52),American Cancer Society,California Division, and also by California Institute of Cancer Research, and Cancer Research Coordinating Committee, University of California.

LITERATURE CITED

1. Abraham, A., and P. Tegtmeyer.1970. Morphological changes in productive andabortive infection by feline herpesvirus. J.Virol. 5:617-623.

2. Bhatt, P. N., D.H.Percy, J. L.Craft, and A. M. Jonas. 1971. Isolation and characterization of a herpes like (Hsiung-Kaplow) virus from guinea pigs. J. Infec. Dis. 123:178-189. 3. Cook, M. K., and J. F. Sears, 1970. Preparation of infectious

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5. Epstein,M. A.,and B. G. Achong. 1970. The EB virus, p. 231-248. In D. P.Burkitt andD.H.Wright (ed.), Burkitt's

lymphoma.E & S Livingstone,London.

6. Feldman, D. G., and L. Gross. 1970. Electron microscopic study of the guinea pig leukemia virus. CancerRes. 30:2702-2711.

7. Henle,W., and G.Henle. 1968. Effect of arginine-deficient media on the herpes-type virus associated with cultured

Burkitttumorcells.J. Virol.2:182-191.

8. Hinuma,Y.,M.Konn, J.Yamaguchi, D.J.Wudarski, J. R. Blakeslee, Jr.,and J. T.Grace, Jr. 1967.Immunofluorescence

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9. Hsiung, G. D., andL.S.Kaplow. 1969 Herpeslike virus iso-lated fromspontaneouslydegenerated tissue culture derived fromleukemia-susceptible guinea pigs.J.Virol. 3:355-357. 10. Hsiung, G.D.,and L. S.Kaplow. 1970.Theassociation of

herpes-likevirus andguinea pigleukemia.Bibl. Haematol. 36:578-583.

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