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 immunofluorescencetests, 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 (1.729g/cm3),
Klebsiella pneumoniae DNA (1.717g/cm3),
and cellular DNA (1.695g/cm3).
The threeDNAs of known buoyantden-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 ofSchild-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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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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[image:6.501.125.394.85.437.2]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)
9
-.5
9
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5t
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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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[image:7.501.253.447.386.587.2]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 DNAE H
H
[image:8.501.262.454.371.471.2]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
rbtiveHSV-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 threedifferent 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
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[image:9.501.59.452.451.558.2]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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[image:10.501.49.453.76.259.2] [image:10.501.58.456.325.508.2]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
thesepre-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 DNAhomologystudiescurrently 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
on November 10, 2019 by guest
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[image:11.501.53.449.91.248.2]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).
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