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Identification of a herpesvirus isolated from cytomegalovirus-transformed human cells.

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0022-538X/78/0027-0713$02.00/0

CopyrightX 1978 AmericanSocietyforMicrobiology Printed in U.S.A.

Identification of

a

Herpesvirus Isolated from

Cytomegalovirus-Transformed Human Cells

LASZLOGEDER, RICHARD W.HYMAN, MANUELFIGUEROA, JOHN E. OAKES,tJEFFREY P. ILTIS, MARILYN S.DAWSON, AND FRED RAPP*

DepartmentofMicrobiologyandSpecializedCancer ResearchCenter, The Milton S.Hershey Medical

Center,ThePennsylvaniaState UniversityCollege ofMedicine, Hershey,Pennsylvania 17033

Receivedforpublication1December 1977

Human cells transformed by cytomegalovirus and transplanted to athymic nude mice

yielded

acytopathic virus, Hershey Medical Center virus, following prolonged in vitropassage of the tumorcells. The virus is adouble-enveloped herpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. No significant antigenic relationship to herpes simplex virus was detected using herpes simplex virus-immune sera in neutralization and immunofluorescence

tests, but indirect immunofluorescence tests revealed cytomegalovirus-related

antigenicity.Further immunologicaltestsrevealed thatHersheyMedicalCenter virus is antigenically indistinguishable from infectious bovine rhinotracheitis virus. Thus, it appears thatHershey Medical Center virus is infectious bovine rhinotracheitisvirus, whichpresumablyappeared in the cell cultureas a

contam-inant fromfetal calfserum.

We have reported that human embryo lung

(HEL) cells infected in vitro witha genital iso-late of human cytomegalovirus (CMV) devel-opedapersistent infectionresultinginoncogenic transformation. Infectious virus was not

re-covered from the transformed cells, although

immunofluorescencetechniquesdetected virus-specificantigensandmicrocytotoxicitytests

es-tablished that the cells sharedamembrane an-tigen(s) with hamster cells transformed by in-activated CMV (6, 7). Thetransformed human cells induced progressively growing tumors in

weanlingathymic nudemice,and cellscultured in vitro from thetumorsdemonstrated intracel-lular and membrane antigens related toCMV. Onetumorcellline(CMV-Mj-HEL-2,T-1) dem-onstrated these antigens in a relatively high

proportion of the cells andwasthereforeselected for further immunological studies. The cells

weremaintained in vitro in serialpassagesunder strict sterile conditions. After 8, 10, and 11

months in culture, some of the cell cultures

yielded an infectious herpesvirus, designated HersheyMedical Center virus (HMCV).

Twosetsofexperimentswereundertakenwith the newly isolated herpesvirus: (i) to test

whetheror notthevirus wouldtransformhuman

cells in culture, and (ii) to try to identify the

virus. The results of these two sets of

experi-mentsindicate that the virusisolate transforms

tPresent address: Department ofMicrobiology and Im-munology,UniversityofSouth AlabamaCollegeofMedicine, Mobile,AL 36688.

human cellsin culture (L. Geder, R. Ladda, J.

Kreider, M. Figueroa,and F. Rapp, manuscript

in preparation) and that, while we originally

thought the new isolatemight be an unknown

herpesvirus, the isolateappearstobe infectious bovine rhinotracheitis virus (IBRV). We report

here the lattersetofexperiments.

MATERIALS AND METHODS

Cellsandviruses. CMV-Mj-HEL-2and

CMV-Mj-HEL-2,T-1 human embryo lung cells were trans-formed with a genitalstrain of human CMV as

de-scribed previously (6, 7).Thetumorcellcultureswere

prepared and maintained in passages as published

elsewhere (6). HEL cells were obtained from HEM Research, Inc. and maintained as described previously (23). Primary human embryokidney(HEK), primary human amnion (HA), RK-13 established rabbit kid-ney, and Flow5000humanembryocellcultures were obtained from FlowLaboratories and maintained in Ham medium with fetal calf serum (10%), sodium bicarbonate (0.075%), penicillin (100 IU/ml), and

streptomycin (100,g/ml).ThePS-1 human bladder

cancercellswereestablished inourlaboratory, main-tained in thesamemedium used for HEK cells, and were in passages70to80 atthe time of the experi-ments.Humanepithelioidkidney cancer (HKC) and human endometrium (HE) cellcultures were prepared

inourlaboratoryandappearedtobe in passages 1 to

4 at the time of the experiments. Mouse embryo fibroblast(MEF), hamsterembryo fibroblast (HEF), andprimary rabbit kidney (PRK) cell cultures were preparedin our laboratory as primary cell cultures

and,alongwith theVeromonkeycellline,were

main-tained in routine passages using Dulbecco medium with 5% calfserum, 0.075% sodiumbicarbonate, 100 713

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714 GEDER ET AL.

IU ofpenicillin per ml, and 100

Ag

of streptomycin per ml. Herpes simplex virus type 1 (HSV-1) (Patton), HSV-2 (333), CMV (AD169), CMV(Mj),and murine CMV(Smith)areavailable in this laboratory and are routinely plaquepurified beforeuse.IBRV (LA) was purchased from the AmericanTypeCulture Collection andwasgrownin RK-13 cells.HMCV,asisolated in HEL and grown in PRK cells, was used in initial experiments. Later, key experiments were repeated using HMCV that had been plaque purified three times in RK-13cellsand grown in RK-13cells.Growth, titration, and neutralization of all viruseswerecarried out aspreviously described (1).

Animals. Weanlingathymic homozygous (nu/nu) nude mice(NIHSwiss Webster, 6th backcross gener-ation) and NIH Swiss heterozygous mice were ob-tained from Life Sciences. Mouse strains C3H/He, DBA/2, BALB/c,C58B1/C, andCD-1 were obtained fromCharles River Farms; inbredLSH hamsters were obtained from Lakeview Hamster Colony; and Dla:(NZW) female rabbitswereobtainedfrom Dutch-land LabAnimals,Inc.

Immunesera.Human antiseratoCMV were ob-tained from hospital patients.Some sera (no. 1to 5) displayed indirect immunofluorescence antibody titers of 1/128 to 1/256 to CMV-infected HEL cells. The HSV-2 immune rabbitserumhadagreater than 50% neutralizing effecton 200PFU ofHSV-2atadilution of 1/10 and reactedstrongly withHSV-2-infected HEL cellsat adilution of 1/4. Rabbithyperimmuneserum toCMV (AD169)wasobtained from R. Lausch. Hu-manCMV-immune serum wasadsorbed with CMV-infected HELcellsto remove CMV reactivityas de-scribedearlier (8). Mouse anti-murine CMV serum, obtained from mice3weeks after infection with mu-rine CMV, had aneutralizing antibody titer higher than 1/10. Rabbitanti-HMCVserum wasobtained by heart puncture10daysafter the 4thweeklyinjection of 107 PFU of virus in an equal volume ofFreund

adjuvantand hadaneutralizing antibodytiter of 1/40.

Bovinemonospecificimmune serum toIBRV (Colo-rado), obtained from P. Gupta, had a neutralizing antibody titer of 1/40. Thisserum wasoriginally ob-tainedcommerciallyfrom MilesLaboratories, Inc.

Immunological tests. Indirect

immunofluores-cence detection of virus-related antigenswas as pre-viously described (6, 8). Plaque neutralization tests werecarriedout inPRK, RK-13,or Flow5000 cells

followingstandardproceduresaspreviouslydescribed

(1, 3). Microneutralization tests were carried out in microtiter plastic plates (MicroTest II, Falcon Plas-tics) in PRKcellsasdescribed elsewhere(4).

Ultracentrifugation. For bandvelocity

sedimen-tation, [3H]thymidine ([3H]TdR)-labeled HMCV DNA was purified from the Hirt supernatant of HMCV-infected cells by preparative sedimentation through apreformed glycerol gradient (11, 21). The

3H-labeledHMCVDNA wasconcentrated bydialysis

againstAquacide II (Calbiochem), andanaliquotwas mixedwith '4C-labeledbacteriophage T4 DNA; half the mixturewassedimentedthroughaneutral sucrose gradient and the other halfwassedimented through

analkalinesucrosegradient.Theneutral and alkaline sucrose gradients were prepared, centrifuged, col-lected,processed,and countedaspreviouslydescribed (13,14). Forbuoyantdensitycentrifugation,

['4C]TdR-labeled HMCVDNA wasprepared as the Hirt super-natant of ['4C]TdR-labeled HMCV-infected RK-13 cells (14, 21). [3H]TdR-labeledHSV-2 DNAwas pre-pared as the Hirt supernatant of [3H]TdR-labeled HSV-2-infected Vero cells. Each Hirt supematantwas concentrated by dialysis against Aquacide (Calbi-ochem)and containedsmallamountsofcellularDNA. Thetwosupernatantsweremixed, and solid CsCl was addedtoyieldadensityof1.71g/cm3. Centrifugation wascarried out,fractionswerecollected, and the filters wereprocessedasdescribedpreviously (14). Insome experiments, the radiolabels were reversed, without change in theresults. In other experiments, the virus DNAswereisolatedbylysingtheinfectedcells with Pronase and Sarkosyl, also without altering the re-sults.

Restriction enzymecleavage and agarose gel

electrophoresis. 32P-labeled virus DNA was

pre-pared by adding 25 uCi of32PO4 (carrier free;

Amer-sham/Searle) per mltothemedium of virus-infected

cellsaspreviouslypublished(19, 20). The virus DNAs werepurified bysequentialHirt extraction, prepara-tive glycerol gradient sedimentation, and isopycnic banding in CsCl (20, 21), followed by Pronase digestion andphenol extraction (20). Endonuclease EcoRI was purchased from MilesLaboratories, Inc. and was used asdescribedpreviously(19, 20). The DNAfragments wereseparated by electrophoresis through a 0.5% aga-rosevertical slabgelaspreviously described (20, 25).

Following electrophoresis, the gels were dried and

placed onKodakMedical X-rayfilmRP/R2.

Photo-scans of the autoradiographs weremade on an Op-tronics P-100 photoscanner connected to a Digital EquipmentCorp. POP 11/40 computer asdescribed elsewhere (19).

RESULTS

Isolation of HMCV from

CMV-trans-formed HEL cells. CMV-transformed HEL cells(4 x 107CMV-Mj-HEL-2 cells)inpassage 13were injected subcutaneously intoweanling athymic nude mice. Twenty-seven days after transplantation of the cells, tumors (10 by 25 mm) developed. Cell line CMV-Mj-HEL-2,T-1

was isolated from one mouse tumor, and cell

cultureswerepreparedasdescribed(6, 7). After aconfluent cell sheet wasobtained, the tumor

cellsweremaintained incontinuouspassagesby

dilutingthecellsfromone 75-cm2plastic tissue

flask at2-day intervals with atwofolddilution.

At 324 days after in vitro establishment (at

passage90),someofthecellculturesdeveloped

focalroundingofcells(Fig. la).CMV-like

cyto-pathic effects (CPE) developed in HEL cells

inoculated with an extract of transformed cells

(Fig. lb). Similar CPE developed at 1-month

intervals in later passages of the transformed

cell cultures (passages 94 and 117), and

cyto-pathicagentswereagainisolated from the trans-formed cellculturesin PRKcells.Wedesignated

theoriginal isolatetheHershey MedicalCenter

virus (HMCV), deliberately givingtheisolatea

superficialname,and, thus,specificallynot pre-J. VIROL.

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maturely identifyingthe virus isolate as a

hu-man,mouse, orotheranimal virus, anHSV, or

CMV. Preparations of HMCV-infected HEL cells stained with hematoxylin and eosin

re-vealed rounded cells withdarklystained nuclei. Some cells showed slight margination of chro-matin. Afew had intranuclear bodiesresembling

type A inclusion bodies induced by herpesvi-ruses.

Herpesvirus-like particles were seen in the nuclei of infected HEL and PRK cells, and the dense virus nucleuswas surrounded by double

envelopes (Fig. 2). That HMCV was an

enve-loped DNA-containingvirus wasconfirmed by

the facts that the isolate was inactivated by

ethertreatmentandiododeoxyuridineinhibited virusreplicationin PRKcells(datanotshown). Characterization ofHMCV DNA. The size ofHMCV DNAwasdeterminedbysucrose

gra-dient sedimentation in neutral andalkaline

gra-dients.Figure3showsthe results ofthese

meas-urements. With T4 DNAas astandard,HMCV

DNAsedimentedjust behind T4 DNA in

neu-tralsucrosegradients (Fig.3a).Byapplying the

Burgi-Hershey equation (2, 5), the molecular

weight of HMCV DNA wascalculatedto be 96

x 106. In alkaline sucrose gradients (Fig. 3b),

HMCV DNA sedimentedveryheterogeneously; thelargest size sedimentedjust behind T4 DNA.

The buoyantdensityof HMCV DNAwas

mea-suredby equilibrium centrifugation in CsCl,and

HSV-2 DNAwasusedas a standard. The results

of the CsCl buoyant density centrifugation are

shown inFig.4.HMCV DNA hadareproducibly higherbuoyant density than HSV-2DNA (1.729

g/ml) and was far removed from the buoyant position of humanor mouse CMV DNA (1.717 g/ml). With the radiolabels reversed, HMCV

DNA still had a higher buoyant density than

HSV-2 DNA (datanotshown).Tomeasurethe

buoyant density of HMCV DNA,

14C-labeled

HMCV DNAwasmixed with

3H-labeled

HSV-2 DNA (1.729

g/cm3),

Klebsiella pneumoniae DNA (1.717

g/cm3),

and cellular DNA (1.695

g/cm3).

The threeDNAs of known buoyant

den-sity were used to plot a line of densityversus

distance (data notshown). From this standard

curve, the buoyant density of HMCV DNA is

1.730

g/cm3,

and, using the equation of

Schild-krautet al. (24), theguanine-plus-cytosine

con-tent of HMCV DNA can be calculated to be 71.5%.

The endonuclease EcoRIcleavage pattern of

HMCVDNA wasdetermined anddirectly

com-paredtothepatterns ofCMV,HSV-1,and

HSV-2 DNAs. Figure 5 shows an example ofan

au-toradiographof theEcoRI-cleavedDNAs: track

1 is HMCVDNA,track 2is CMVDNA, track

3 is HSV-2 DNA, and track4is HSV-1 DNA.

The numbering/lettering nomenclature for the bands of HSV-1 and HSV-2 DNAs is used as

describedby Haywardetal. (9, 10). TheEcoRI

cleavage pattern for HSV-1 (Patton) DNA

(track 4) has a one-to-onecorrespondence with our previously published pattern (19) except

that band2,aminorfragment, isnotseeninour

present pattern. Thepatternintrack4(Fig. 5) is inexcellent agreementwiththe EcoRI

cleav-agepatterns for HSV-1 DNAspreviously

pub-lishedbySkare et al. (26) and byHaywardetal.

(9). Similarly, the EcoRI cleavage pattern of

HSV-2 (333) DNA (track 3, Fig. 5) is in good agreement with previously published patterns (9, 19,26). The topband in track3, band X, is

an unexplained artifact. Band 6 oftrack 3

ap-pears as two closely migrating bands, whereas

only a single band is seen in our previously publishedpattern (19).Theimportant pointsfor the EcoRI digestion pattern of HSV-2 (333)

DNA (track 3) are that the two largest

frag-ments, bands 1 and2, appear as submolar,

fol-lowed sequentially by molar band A, minor

bands3and4,the darkdoubletBC, andsoforth, justasinthepreviously publishedpatternsfrom

our own(19) andtwootherlaboratories (9, 26).

The EcoRIcleavage pattern of CMV (AD169)

DNA published by Kilpatrick et al. (16) was

accomplishedon a 1%agarosegel and is there-fore not directly comparable to the pattern in track2 (Fig. 5). Nevertheless, certain

compari-sons can be made. The largest band in both

patterns hasa molecular weightofabout 15 x

106. The pattern ofKilpatricket al. (16) shows

more than 9 bands between the molecular

weights 5 x 106 and 15 x 106; the pattern in track 2 (Fig. 5) shows 11 bands. Therefore, as

faras canbedetermined,ourpatternfor

EcoRI-digested CMV (AD169) DNA is in agreement

with the pattern published previously by Kil-patrick et al. (16).Thus, asclearlyseen inFig.

5, theEcoRIcleavage pattern of HMCV DNA istotally distinct from the EcoRIcleavage

pat-terns of the otherthree DNAs. The molecular weights of the HSV-1 DNA bands (26) have beengraphedonsemilogpaperagainst the

dis-tance migrated(data notshown).These points

determine the standard curve and allow the determination of the molecular weights of the

HMCV DNAbands.The molecularweights for

each of thespecific EcoRIfragmentsof HMCV

DNA aregiveninTable 1, aswellas a

calcula-tion ofthe relative molaramountof eachband.

Therelativemolar amounts ofthelargest(band

A) and smallest (band H) specific fragments

have not beencalculated because their

molecu-lar weightsareveryuncertain.As seen inTable 1,HMCVDNAbandsB, C, E, andFappear to

bepresent in one molar amount. Band Dappears

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716 GEDER ET AL.

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4.-*4-FIG. 1. CPE caused byHMCV. (a) DevelopmentofCPE in CMV-Mj-HEL-2,T-1 tumor cell line dueto spontaneousinductionofHMCV.Photographofcell culture in tissuecultureflask.x108.(b)CPE inducedby

HMCV in humanembryolungcells 48 hafterinoculation. x108.(c)CPE inducedbyHMCV in rabbitkidney

cells24hafterinoculation. x108.(d)CPEinducedbyHMCV in adulthumankidneycancercells72hafter

inoculation. x108.

J. VIROL.

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FIG. 1.c-d. VOL. 27,1978

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718 GEDER ET AL.

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FIG. 2. Electron micrograph ofHMCVpropagated in rabbitkidney cells. Infected cells werefixed in

Karnovsky fixativeandpostfixedin Daltonchrome-osmium, dehydratedinethanol,and embeddedinSperr low-viscosity plastic.SectionswerecutwithaSorvallultratome,stained with lead citrateanduranylacetate, and examined withaHitachiHU-12 electronmicroscope. x220,000.

tobe present intwomolaramountsand

proba-bly represents two one-molar fragments that

comigrated. Band G aloneappearstobepresent in asubmolaramount.

Biologicalproperties of HMCV. The

sen-sitivities ofHEL, HEK, HKC, HE, PS-1, HA,

Vero monkey cell line, MEF, 3T3 established

mouse cell line, HEF, PRK, RK-13,

CMV-Mj-HEL-2, and CMV-Mj-HEL-2,T-1 cell lines to the HMCV isolate were tested. The develop-ment and morphology of CPE and maximum

virus yield in these different cell cultures are

shown in Table 2. Maximum virus yield was

obtained in PRK cells. Good yields were

har-vested from RK-13, HEK, HA, Vero monkey,

and HEF cells. More limited virus replication

was observed in human embryo fibroblasts,

adultHKC, HE, PS-1, MEF, and

CMV-trans-formed humanepithelioidcells. Nogrowthwas

noticed in 3T3 establishedmousecells.

The morphology of CPE inducedby HMCV

depended largely on the cell type used for

growth. In human and mouseembryofibroblast

cells,roundedandswollen cells developed into small foci.Thespreadfollowed thelongitudinal

axis of thefibroblasts, thusresemblingtheCPE

inducedby CMV. CPEspreadslowly, likethat

of CMV(Mj),and didnotinvolvethe wholecell

sheet when themultiplicityof infectionwaslow

(Fig. lb). In PRK (Fig. lc), RK-13, HE, HA, Veromonkey,andHEFcells,virus-infectedcells

were seenin fociasrounded cells.Thespreadof

CPE was fast, and the entire cell sheet was

involved within 2 to 4 days.InHEK and HKC

cells (Fig. ld), rounded cells were nmixed with

syncytia having 10 to 50nuclei. These

charac-J. VIROL.

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HERPESVIRUS IBRV 719

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FRACTION NUMBER

FIG. 3. Sucrose gradient sedimentation of HMCV

DNA. [3H]TdR_labeled HMCV DNA was isolated

from the Hirt supernatant andpurifiedby glycerol gradient sedimentation. The HMCV DNA was di-alyzed and concentrated. An aliquot of3H-labeled HMCVDNA (-)wasmixedwith'4C-labeledT4 DNA

(0). Half the mixture was sedimented through a

neutralsucrosegradient (a) and the other half was sedimentedthroughanalkalinesucrosegradient (b). Sedimentation isfromrighttoleft.

teristics were similar to the CPE induced by certainstrains of HSV. Thespreadwasfast and involved the full cell sheet in HEK cultures. The spreadwasslow in HKC and HEcells,andthe virus-infected cell culture could be passed four times before the whole cell sheet became in-volved. In CMV-transformed human cells, the CPEprogressed slowly and consisted ofsmall, well-defined foci of rounded cells. PS-1 human bladder cancer cells appeared rounded but shrunken; foci spreadslowly,and inmostcases

CPEregressed. Persistent infection was estab-lished by passing the virus-carrier HKC, HE, andPS-1 cells several times.

Immunologicalstudies withHMCV.In

in-direct immunofluorescence tests, high-titer

CMV- and HSV-immune human sera reacted withnuclearantigensofboth HMCV-and IBRV

(LA)-infected HEL cells (Table 3 and Fig. 6).

CMV-immune serum adsorbed to a

CMV-in-fected HEL cell extract did notreact with the

CMV and HMCV preparations tested, whereas

thereactivity of the serum with HSV-2-infected

celLs remained unaltered (Table 3).

HSV-2-hy-perimmune rabbitand CMV-negative HSV-hy-perimmune human sera did not react with HMCV- and IBRV (LA)-infected HEL cells

(Table 3 and Fig. 6). HMCV-immune rabbit

serumreacted with IBRV-infectedcells, butno

reactivitywasfound withCMV(Mj)-and HSV-2-infected cells. Bovine IBRV-immune serum

reacted strongly with HMCV- and IBRV-in-fected cells; faint nuclear reactions were ob-served with CMV (Mj)-infected cells, but no

antigens were detected in cells infected with HSV-2 (Table 3). HMCV-immune rabbit and IBRV-immune monospecific bovine sera had identical neutralizing antibody titers to both HMCV and IBRV(Table4).Nosignificant

neu-tralization of CMV (Mj), HSV-2, and murine CMVwasfound with thesesera at adilution of 1/10. CMV-immune human serum witha

neu-tralizing antibody titer of 1/40 against CMV

(Mj) did not neutralize HMCV, IBRV (LA), HSV-2, ormurine CMV strainsat a 1/10 dilu-tion. CMV-hyperimmune serum, prepared in rabbits with partially purified CMV (AD169)

and having a CMV antibody titer of 1/1,000,

neutralized 53% of the PFU of HMCV when used in 1/10 dilution. Preimmunerabbit, HSV-2-immune rabbit, and murine CMV-immune

mouse sera had no neutralizing effect against

CV)

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FIG. 4. Buoyant density centrifugation of HMCV DNA. '4C_labeled HMCV DNA (0) and3H-labeled HSV-2 DNA (0) wereisolatedfrom the Hirt super-natant.TheHirt supernatants also contained small amounts of host cell DNA. The DNAs were mixed andsedimentedisopycnicallyin aCsCl gradient. At equilibrium, fractionswere collectedfrom thebottom. The meniscus is ontheright. Density increases to the left. Forconvenience, the bottom 20fractions were notgraphed.

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720 GEDER ET AL.

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keys, and rhesus monkeys did not neutralize

HMCV. Sera from six different strains of mice (C3H/He, DBA/2, BALB/c, C59B1/C, CD-1, and nude mice) had no neutralizing antibodies

toHMCV.

Infection of experimental animals with

EHMCV. Subcutaneous, intramuscular,or

intra-peritoneal inoculationof adult heterozygous and

athymic nude mice, newborn mice, adult and newbornhamsters, and rabbits with106PFU of

virussuspensionsfailedtoinduce any clinically

manifested disease or death in these animals.

Intracerebral inoculation of adult hamsters

causedclinicalsymptoms, but all of the animals

recovered. Meningitis and a mild encephalitis

were diagnosed based on histological

prepara-tions. No reactionwas induced bycorneal

scar-ification of the eyes of a rabbit with HMCV

(Table5).

DISCUSSION

Wereported previously that persistent

infec-tion ofHELcellswith a strain of CMV isolated

from theprostate of a young boy (postmortem)

fb~

G TABLE 1. Molecularweightsand molar ratiosof b theEcoRIdigestion products ofHMCV DNA

E H

H

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F

FIG. 5. EcoRI digestion products ofHMCVDNA. 32P-labeled HMCVDNA, alongwith thestandards, 32P-labeled CMV(AD169),HSV-2 (333), and HSV-1 (Patton) DNAs, wereindividuallydigested with

re-striction enzymeEcoRI. The digestion products of each DNAwereseparated byelectrophoresis through a0.5%agaroseslabgel.Thegelwasdried andplaced

onKodakMedicalX-ray filmRP/P2. Afteran

ap-propriate time, the film was developed. Track 1, HMCVDNA; track2, CMV(AD169) DNA;track3, HSV-2(333) DNA;track4,HSV-1(Patton)DNA. The specific fragments ofHMCV DNAweregivenletter designationsin orderofincreasing mobility(10).The specific fragments ofHSV-1 and HSV-2 DNAswere

givenletterornumberdesignationsasdescribed

pre-viously (11).

HMCV(Table 4).Pooledserafromnormal

rab-bits, hamsters, guinea pigs, Africangreen

mon-Fragment Molwt'(X10 Molar ratio' designation

A >25c NCd

B 22 1.0

C 18 1.3±0.2

D 11 1.8±0.6

E 8.9 1.1±0.1

F 6.0 1.1±0.1

G 5.5 0.2±0.05

H 1.5c NCd

aFragmentsareorderedonthe basisofincreasing

electrophoretic mobility and decreasing molecular

weight.

bThemassoffragmentwasdetermined inarbitrary units by graphically measuring the area under the peak ofaphotoscanofanautoradiograph. Themass is dividedby the molecular weight todetermine the molaramount inarbitraryunits. The molaramount offragmentBwastakenasequalto1, and themolar amountsof the otherfragmentswerecalculated rela-tivetofragmentB. The averagerelative molaramount (±onestandarddeviation) of eachfragmentwas cal-culated from two autoradiographs from one EcoRI digestion.

'The molecular weights of the largest (A) and

smallest (H) fragments are extremely approximate

because both are far removed from the molecular weightsof thestandards.Inparticular,the molecular weight offragmentA canonly be estimatedasgreater than 25x 106.

dNC,Notcalculated. Because of thelarge uncer-tainty in the molecular weights ofthe largest and

smallest fragments, their relative molar ratios have

notbeencalculated.

1

A

B

C

D

E

F

G

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TABLE 2. Developmentandmorphologyof CPEand maximum virusyieldindifferent cellculturesof human and animal origin inoculated with HMCV

ProgressofCPE (% ofcellsheet)a Maximum virus

Cells 24 h 48 h 72 h 96 h Typeof CPE yield

(PFU/ml)b

HEL (human) 0 30 50 50 CMV-like 5.0x 104

HEK(human) 50 95 100 100 HSV-like 7.0 x105

syncytial

HCK (human) 0 10 30 50 HSV-like 5.0x 104

syncytial

HE(human) 0 10 30 50 HSV-like 5.0x 104

rounded cells

PS-1 (human) 0 1 10 20 Atypical 1.1 x104

HA(human) 10 30 90 100 HSV-like 1.1 x 106

rounded cells

Vero(simian) 5 20 30 50 HSV-like 1.3x106

rounded cells

MEF(mouse) 0 30 50 50 CMV-like 1.2x 104

3T3(mouse) 0 0 0 0 None 1.0x 100

HEF(hamster) 10 90 98 100 HSV-Iike 3.0x 106

rounded cells

PRK(rabbit) 10 95 100 100 HSV-like 2.7x 107

rounded cells

RK-13(rabbit) 10 95 100 100 HSV-like 5.0x 106

rounded cells

CMV-Mj-HEL-2 (hu- 0 10 50 50 Rounded 6.0x 103

man) cells

CMV-Mj-HEL-2,T-1 0 10 90 95 Rounded 1.3x 103

(human) cells

aInoculum:0.3PFU/cell. bTitrated in rabbitkidneycells.

TABLE 3. Indirectimmunofluorescencetests in Flow5000 cellsfortheidentificationof HMCV Sera(at1/4dilution)

Virusinoculum Mn Human Human Pre-HMCV-HMCV- Bovine

HSV-2-im-ihm CMV-nega-

CMV-ixII-

CMV-Uiin

Pnmue

HCHMnCIRVi.HS-in

rb

tiveHSV-im- mune no. 1, 2, mune no.1 it munerabbit BRVim munerabbit mune 3, 4, 5 adsorbed it

None - - -

-CMV(Mj) - +a-b

HMCV - +-b +a,b +a,b

IBRV(LA) - +b NDC - +a,b +a,b

HSV-2 +a,b +a,b + +a,b

aCytoplasmicfluorescence.

bNuclear fluorescence. cND, Notdetermined.

resultedintransfornationofthesecells.

Trans-formedcells weretransplantedtoathymic nude

mice. Ofover 100 animnals inoculated, 62%

de-velopedtumorsafteranaveragelatentperiod of

19days. Cells reisolated from one ofthese

tu-morshavebeenmaintainedforover150in vitro

passages,andtheline wasdesignated

CMV-Mj-HEL-2,T-1. These cells have shown a stable

expressionof CMV-related membrane and

intra-cellular antigens.TheCMV-Mj-HEL-2,T-1 cell

line, unlike theparentaland other tumor

lines,

has undergone crisis several times. On three

different occasions (atpassages90, 94,and

117)

CPE-like foci developed in the cell sheet, and

three virusstrainswereisolatedin HELorPRK

cell cultures. One of the three virus

isolates,

HERPESVIRUS IBRV 721

VOL. 27,1978

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722 GEDER ET AL.

FIG. 6. Photomicrographof nuclear fluorescence. Fluorescencewasdetected in(a) HMCV-infectedand(b)

IBRV(LA)-infected cells. CMV-immune human immune serum was used with fluorescein

isothiocyanate-conjugatedanti-human goatserumin the indirectimmunofluorescencetest.(c)IBRV(LA)-infectedcellsshow

nofluorescence when treated withCMV-negativehumanserum.x450.

TABLE 4. Neutralizationtestswith HMCV in RK-13 andFlow5000cells Virus

Sera

HMCV IBRV(LA) CMV (Mj) HSV-2 MurineCMV

Pre-HMCV-im- <1Oa <10 <10 <10 <10

munerabbit

HMCV-immune 40 40 <10 <10 <10

rabbit

IBRV-immune 40 40 <10 <10

NDb

bovine

CMV (AD169)- 10 ND 1,000 ND <10

immune rab-bit

CMV-immune <10 <10 40 <10 <10

human

HSV-2-immune <10 <10 <10 >10 ND

rabbit

Murine CMV- <10 ND ND <10 >10

immune mouse

aReciprocal of dilution with >50% reduction in PFUascomparedtothecontrol.

bND,Notdone.

HMCV,isolated in HEL cells, has been studied

extensively. We have data, however, based on

neutralizationtests carried out withrabbit

anti-HMCVimmune serum, that all three virus

iso-lates areidentical.

Theimmediate questionsconcerning HMCV are: What is the virus and where did it come

from?These questions are more easily answered

by first determining what HMCV is not. (i)

HMCV is not a human CMV. The wide host

range ofHMCV is incompatible with all known

humanCMV isolates,which only grow in human

cells. Inparticular, the CMV (Mj) isolate used

totransform theoriginalHELcellsgrowsonly

in human cells and then only poorly. HMCV

growswellinhuman, rabbit, hamster,and

mon-keycells.HMCVdoes show someimmunological

cross-reaction with humanCMV. Thebuoyant

density of HMCVDNAand theEcoRI

restric-tion pattern ofHMCVDNAareadditional

evi-dence thatHMCV isnot aCMV. (ii) HMCV is

not ahumanHSV. HMCVdoesnot reactwith

antiserum prepared against HSV. Rodents

in-oculatedintracerebrallywithHSVgenerally

suf-ferafatalencephalitis;rodentsinoculated

intra-cerebrally with HMCV survive. The buoyant

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TABLE 5. Infectionof experimental animals with HMCV

Dose of injection Resultafter 6 weeks of Animalspecies No.injected Method ofinjectiona (PFU x 106Y observation

Adult mouse 5 SC 1 Negative

Adult mouse 6 IC 1 Negative

Athymicnude mouse 3 SC 1 Negative

Newbornmouse(<24 h) 7 SC 0.5 Negative

Adult hamster 2 IP 2 Negative

Adult hamster 7 IC 1 Meningitis and

encephalitis without mor-tality

Adult hamster 5 IM 2 Negative

Newbornhamster 8 SC 1 Negative

Rabbit 1 IM 1 Negative

Rabbit 1 SC 1 Negative

Rabbit 1 Comeal 1 Negative

aSC, Subcutaneous; IC,intracerebral; IP, intraperitoneal;IM,intramuscular.

density of HMCV DNA is closetobut reproduc-ibly more dense than the buoyant density of

HSV-2DNA. The restriction patterns of HMCV DNAand HSVDNAarecompletely dissimilar.

The sedimentation behavior of HMCV DNAon

neutral andalkaline sucrose gradients is

indistin-iguishable

fromseveral otherherpesvirusDNAs

(13-15, 27). (iii) HMCV is not murine CMV. HMCV grows well in human cells and not in murine 3T3 cells. Mouse CMV grows well in 3T3 cells butnotin human cells (12), and antiserum prepared against mouse CMV does not react

with HMCV. The buoyant density of HMCV DNA isfar removed from the buoyant density

of mouse CMV DNA (18). (iv) Antibodies

di-rectedagainst HMCV could not be detected in thenormalserafrom six strains ofmice,

includ-ing nude mice. These datasuggest, but do not prove, that HMCV is not a virus common to

mice. (v) Antibodies directed against HMCV could notbe detected in the normal sera from rabbits, hamsters, guinea pigs, African green

monkeys,and Rhesusmonkeys.These data sug-gest thatHMCV is not aviruscommon to any of these animals. (vi) Bovine monospecific IBRV-immune serum neutralized HMCV and,

conversely, high-titer anti-HMCV serum

neu-tralized IBRV.HightiterCMV-immune human

sera reacted with nuclear antigens of both HMCV- and IBRV-infected cells, but did not neutralize these viruses. Adsorption of CMV-immuneserum toCMV-infectedcell extract

re-moved thereactivity againsttheseantigenic

for-mations.

These immunological data suggest that

HMCVisIBRV.Inaddition,the host range and appearance of HMCV CPE are similar to the

published host range and CPE of IBRV (17). The lack of pathogenicity of HMCV in test

animalshasalso been reported for IBRV (17).

The published buoyant density of IBRV DNA

(22) isindistinguishable from the buoyant den-sity ofHMCV DNA. An EcoRI cleavage pattern ofIBRV DNA (26) is similar, but not identical,

tothe EcoRIcleavagepattern of HMCV DNA. Thesedifferences could be strain variation.

The CMV-transformed cell cultures were

maintained in a laboratory where only human cellswerebeing cultured. No viruses other than human CMV and HSV were maintained in the

sameroom, and thesewerein otherlaminar flow hoods. The strictest biohazardregulationswere

adopted when the CMV-transformed cellswere

passed. A presterilized hood, separately

pre-pared medium andtrypsin, andpreviously

uno-pened bottles of fetal calf serum and pipette

containers were used. The isolation ofHMCV

was made in HEL cells in their 11th in vitro passage.UninoculatedHELcells,carried simul-taneously,remained

negative.

Despite

these

pre-cautions, itnow appears probable that HMCV isacontaminantIBRV,presumably arisingfrom the fetal calfserum. Thus,HMCV is similarto oridentical with IBRV.

The HMC isolate of IBRV transforms adult

humanepithelioid cells in culture (Geder et al.,

manuscript in preparation). We are currently testing the oncogenic potential of a standard reference strain (LA) of IBRV. In addition, from

immunological evidence, it is clear that IBRV sharesantigenswith human CMV. This

obser-vation must be

confirmed

andextended by DNA

homologystudiescurrently in progress.

ACKNOVWLEDGMENTS

We thank J. Gruber, Office of Program Resources and Logistics,VirusCancerProgram, Division of CancerCause

andPrevention,NationalCancerInstitute, Bethesda, Md.,for

suppliesofnudemice;R.Glaserforhis valuableadvice;R. Lauschforsupplyinguswith thehyperimmunerabbit CMV-immunesera; and J.Gorodecki,A.Laychock,andL.Kudler

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724 GEDER ET AL.

for theirtechnical assistance. Weare especially gratefulto

DavidPorter, who broughtto ourattention thepossibility that HMCVmight be IBRV.

Thiswork wassupportedbyPublic HealthServicecontract

no.N01-CP-5-3516 within the Virus CancerProgramof the National Cancer Institute and grants CA-18450, CA-16365, and CA-16498 awardedbythe National Cancer Institute. J.P.I. istherecipientof Public HealthServicefellowshipCA-05677 from the National CancerInstitute,and M.S.D. isa postdoc-toralfellow of the DamonRunyon-Walter Winchell Cancer Fund(DRG-161F). R.W.H. holdsaFacultyResearch Award from the American CancerSociety(FRA-158).

LITERATURE CITED

1. Albrecht, T.,and F.Rapp. 1973. Malignant transfor-mationof hamsterembryofibroblastsfollowing expo-sure toultraviolet-irradiated humancytomegalovirus. Virology 65:53-61.

2. Burgi, E.,andA. D.Hershey.1963.Sedimentationrate asa measureof molecularweightsof DNA.Biophys.J. 3:309-321.

3.Doller, E.,R.Duff,and F. Rapp.1973. Resistance of hamster cellstransformedbyherpessimplexvirustype 2 tosuperinfectionby herpessimplexviruses. Intervi-rology1:154-167.

4. Figueroa,M.,and A. Zambrance. 1976.Elherpes gen-ital in Honduras ysurelacionenel carcinoma delcervix uterine. Rev. Latinoam. Microbiol. 18:111-116. 5. Freifelder,D. 1970. Molecularweightsofcoliphagesand

coliphageDNA. IV. Molecularweightof DNA from bacteriophages T4,T5 and T7 and thegeneral problem ofdeterminationof M. J. Mol.Biol. 54:567-577. 6.Geder,L.,J.Kreider,and F.Rapp.1977. Human cells

transformed in vitro byhuman cytomegalovirus: tu-morigenicityinathymicnude mice. J. Natl. Cancer Inst. 58:1003-1007.

7. Geder, L.,R. N.Lausch,F. J.O'Neill,and F.Rapp. 1976.Oncogenic transformation of human embryo lung cells by human cytomegalovirus. Science 192: 1134-1137.

8. Geder, L, and F. Rapp. 1977. Evidence for nuclear antigens incytomegalovirus-transformed humancells. Nature(London) 265:184-186.

9. Hayward, G.S.,N.Frenkel,and B.Roizman. 1975. Anatomy ofherpes simplexvirus DNA: strain differ-encesandheterogeneityinthelocations of restriction endonuclease cleavage sites. Proc. Natl. Acad. Sci. U.S.A.72:1768-1772.

10. Hayward,G.S.,R.J.Jacob, S.C.Wadsworth,and B.Roizman. 1975.Anatomyofherpes simplexvirus DNA:evidence for fourpopulationsofmolecules that differ inthe relative orientation of theirlongand short components. Proc. Natl. Acad. Sci. U.S.A. 72:42434247.

11. Hirt, B. 1976.SelectiveextractionofpolyomaDNA from infected mousecellcultures. J.Mol. Biol.26:365-369. 12. Hudson, J. B., V. Misra, and T. R. Mosmann. 1976.

Properties of the multicapsid virions ofmurine cyto-megalovirus. Virology 72:224-234.

13.Hyman, R. W., J. E. Oakes, and L. Kudler. 1976. In vitrorepairof thepre-existingnicksand gaps in herpes simplex virus DNA. Virology 76:286-294.

14.iltis, J.P., J. E.Oakes,R. W.Hyman, and F. Rapp. 1977. Comparison of the DNAs ofvaricella-zoster vi-rusesisolated fromclinical cases of varicella and herpes zoster. Virology 82:345-352.

15. Kieff, E. D., S. L Bachenheimer, and B. Roizman. 1971. Size,composition, and structure of the deoxyri-bonucleicacid ofherpes simplex virus subtypes 1 and 2. J.Virol. 8:125-132.

16. Kilpatrick, B. A., E.-S. Huang, and J. S. Pagano. 1976. Analysis of cytomegalovirus genomes with restriction endonucleases HinD III and EcoR-1. J. Virol. 18:1095-1105.

17.McKercher, D. G. 1973. Viruses of other vertebrates, p. 428-441.In A. S.Kaplan (ed.), The herpesviruses. Ac-ademicPress, New York.

18.Mosmann, T. R.,and J. B. Hudson. 1973. Some prop-erties of the genome of murine cytomegalovirus (MCV). Virology 54:135-149.

19.Oakes, J. E., R. W. Hyman, and F. Rapp. 1976. Genome location of polyadenylated transcripts of herpes simplex virus type1 and type 2DNA. Virology 75:145-154. 20. Oakes, J. E., J. P.Iltis, R. W. Hyman, and F. Rapp.

1977.Analysisby restriction enzyme cleavage of human varicella-zostervirusDNAs. Virology82:353-361. 21. Pater, M. M., R. W. Hyman, and F. Rapp. 1976.

Isola-tionofherpes simplex virus DNA from the "Hirt su-pernatant."Virology 75:481483.

22.Plummer,E., C. R.Goodheart,D.Henson, and C. P. Bowling. 1969.Acomparative study of the DNA den-sityand behavior in tissue culture of fourteen different herpesviruses. Virology39:134-137.

23. Rapp, F.,LGeder,D.Murasko, R. Lausch, R. Ladda, E.Huang,and M.Webber.1975.Long-term persist-ence ofcytomegalovirus genome in cultured human cells ofprostaticorigin.J. Virol. 16:982-990.

24. Schildkraut, C. L., J. Marmur, and P. Doty. 1962. Determination of the basecomposition of deoxyribo-nucleic acid from its buoyant densityin CsCl. J. Mol. Biol. 4:430443.

25. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection oftworestrictionendonucleaseactivities in Haemophilusparainfluenzaeusing analytical agarose-ethidium bromide electrophoresis. Biochemistry 12:3055-3063.

26. Skare,J., W. P.Summers,and W. C. Summers. 1975. Structureand function of herpesvirus genomes. I. Com-parison offive HSV-1 and two HSV-2 strains by cleav-age of their DNAwith Eco R Irestriction endonuclease. J. Virol.15:726-732.

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strand interruptions in HSV-1 DNA. J. Gen. Virol. 21:453467.

J. VIROL.

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Figure

FIG.1.spontaneouscellsHMCVinoculation. CPE caused by HMCV. (a) Development of CPE in CMV-Mj-HEL-2,T-1 tumor cell line due to induction ofHMCV
FIG. 1. c-d.
FIG. 2.low-viscosityandKarnovsky Electron micrograph of HMCV propagated in rabbit kidney cells
FIG. 3.alyzedHMCVDNA(0).gradientDNA.sedimentedneutralSedimentationfrom Sucrose gradient sedimentation ofHMCV [3H]TdR_labeled HMCV DNA was isolated the Hirt supernatant and purified by glycerol sedimentation
+5

References

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