JOURNAL OF VIROLOGY, Sept. 1978, P. 713-7240022-538X/78/0027-0713$02.00/0Copyright X 1978 American Society for Microbiology

Vol. 27, No. 3

Printed in U.S.A.

Identification of a Herpesvirus Isolated from Cytomegalovirus-Transformed Human Cells

LASZLO GEDER, RICHARD W. HYMAN, MANUEL FIGUEROA, JOHN E. OAKES,t JEFFREY P.ILTIS, MARILYN S. DAWSON, AND FRED RAPP*

Department ofMicrobiology and Specialized Cancer Research Center, The Milton S. Hershey MedicalCenter, The Pennsylvania State University College ofMedicine, Hershey, Pennsylvania 17033

Received for publication 1 December 1977

Human cells transformed by cytomegalovirus and transplanted to athymicnude mice yielded a cytopathic virus, Hershey Medical Center virus, followingprolonged in vitro passage of the tumor cells. The virus is a double-envelopedherpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. Nosignificant antigenic relationship to herpes simplex virus was detected usingherpes simplex virus-immune sera in neutralization and immunofluorescencetests, but indirect immunofluorescence tests revealed cytomegalovirus-relatedantigenicity. Further immunological tests revealed that Hershey Medical Centervirus is antigenically indistinguishable from infectious bovine rhinotracheitisvirus. Thus, it appears that Hershey Medical Center virus is infectious bovinerhinotracheitis virus, which presumably appeared in the cell culture as a contam-inant from fetal calf serum.

We have reported that human embryo lung(HEL) cells infected in vitro with a genital iso-late of human cytomegalovirus (CMV) devel-oped a persistent infection resulting in oncogenictransformation. Infectious virus was not re-covered from the transformed cells, althoughimmunofluorescence techniques detected virus-specific antigens and microcytotoxicity tests es-tablished that the cells shared a membrane an-tigen(s) with hamster cells transformed by in-activated CMV (6, 7). The transformed humancells induced progressively growing tumors inweanling athymic nude mice, and cells culturedin vitro from the tumors demonstrated intracel-lular and membrane antigens related to CMV.One tumor cell line (CMV-Mj-HEL-2,T-1) dem-onstrated these antigens in a relatively highproportion ofthe cells and was therefore selectedfor further immunological studies. The cellswere maintained in vitro in serial passages understrict sterile conditions. After 8, 10, and 11months in culture, some of the cell culturesyielded an infectious herpesvirus, designatedHershey Medical Center virus (HMCV).Two sets ofexperiments were undertaken with

the newly isolated herpesvirus: (i) to testwhether or not the virus would transform humancells in culture, and (ii) to try to identify thevirus. The results of these two sets of experi-ments indicate that the virus isolate transforms

t Present address: Department of Microbiology and Im-munology, University of South Alabama College of Medicine,Mobile, AL 36688.

human cells in culture (L. Geder, R. Ladda, J.Kreider, M. Figueroa, and F. Rapp, manuscriptin preparation) and that, while we originallythought the new isolate might be an unknownherpesvirus, the isolate appears to be infectiousbovine rhinotracheitis virus (IBRV). We reporthere the latter set of experiments.

MATERIALS AND METHODSCells and viruses. CMV-Mj-HEL-2 and CMV-Mj-

HEL-2,T-1 human embryo lung cells were trans-formed with a genital strain of human CMV as de-scribed previously (6, 7). The tumor cell cultures wereprepared and maintained in passages as publishedelsewhere (6). HEL cells were obtained from HEMResearch, Inc. and maintained as described previously(23). Primary human embryo kidney (HEK), primaryhuman amnion (HA), RK-13 established rabbit kid-ney, and Flow 5000 human embryo cell cultures wereobtained from Flow Laboratories and maintained inHam medium with fetal calf serum (10%), sodiumbicarbonate (0.075%), penicillin (100 IU/ml), andstreptomycin (100 ,g/ml). The PS-1 human bladdercancer cells were established in our laboratory, main-tained in the same medium used for HEK cells, andwere in passages 70 to 80 at the time of the experi-ments. Human epithelioid kidney cancer (HKC) andhuman endometrium (HE) cell cultures were preparedin our laboratory and appeared to be in passages 1 to4 at the time of the experiments. Mouse embryofibroblast (MEF), hamster embryo fibroblast (HEF),and primary rabbit kidney (PRK) cell cultures wereprepared in our laboratory as primary cell culturesand, along with the Vero monkey cell line, were main-tained in routine passages using Dulbecco mediumwith 5% calf serum, 0.075% sodium bicarbonate, 100

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IU of penicillin per ml, and 100 Ag of streptomycin perml. Herpes simplex virus type 1 (HSV-1) (Patton),HSV-2 (333), CMV (AD169), CMV (Mj), and murineCMV (Smith) are available in this laboratory and areroutinely plaque purified before use. IBRV (LA) waspurchased from the American Type Culture Collectionand was grown in RK-13 cells. HMCV, as isolated inHEL and grown in PRK cells, was used in initialexperiments. Later, key experiments were repeatedusing HMCV that had been plaque purified threetimes in RK-13 cells and grown in RK-13 cells. Growth,titration, and neutralization of all viruses were carriedout as previously described (1).

Animals. Weanling athymic homozygous (nu/nu)nude mice (NIH Swiss 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, and CD-1 were obtainedfrom Charles River Farms; inbred LSH hamsters wereobtained from Lakeview Hamster Colony; andDla:(NZW) female rabbits were obtained from Dutch-land Lab Animals, Inc.Immune sera. Human antisera to CMV were ob-

tained from hospital patients. Some sera (no. 1 to 5)displayed indirect immunofluorescence antibody titersof 1/128 to 1/256 to CMV-infected HEL cells. TheHSV-2 immune rabbit serum had a greater than 50%neutralizing effect on 200 PFU of HSV-2 at a dilutionof 1/10 and reacted strongly with HSV-2-infected HELcells at a dilution of 1/4. Rabbit hyperimmune serumto CMV (AD169) was obtained from R. Lausch. Hu-man CMV-immune serum was adsorbed with CMV-infected HEL cells to remove CMV reactivity as de-scribed earlier (8). Mouse anti-murine CMV serum,obtained from mice 3 weeks after infection with mu-rine CMV, had a neutralizing antibody titer higherthan 1/10. Rabbit anti-HMCV serum was obtained byheart puncture 10 days after the 4th weekly injectionof 107 PFU of virus in an equal volume of Freundadjuvant and had a neutralizing antibody titer of 1/40.Bovine monospecific immune serum to IBRV (Colo-rado), obtained from P. Gupta, had a neutralizingantibody titer of 1/40. This serum was originally ob-tained commercially from Miles Laboratories, Inc.Immunological tests. Indirect immunofluores-

cence detection of virus-related antigens was as pre-viously described (6, 8). Plaque neutralization testswere carried out in PRK, RK-13, or Flow 5000 cellsfollowing standard procedures as previously described(1, 3). Microneutralization tests were carried out inmicrotiter plastic plates (MicroTest II, Falcon Plas-tics) in PRK cells as described elsewhere (4).

Ultracentrifugation. For band velocity sedimen-tation, [3H]thymidine ([3H]TdR)-labeled HMCVDNA was purified from the Hirt supernatant ofHMCV-infected cells by preparative sedimentationthrough a preformed glycerol gradient (11, 21). The3H-labeled HMCV DNA was concentrated by dialysisagainst Aquacide II (Calbiochem), and an aliquot wasmixed with '4C-labeled bacteriophage T4 DNA; halfthe mixture was sedimented through a neutral sucrosegradient and the other half was sedimented throughan alkaline sucrose gradient. The neutral and alkalinesucrose gradients were prepared, centrifuged, col-lected, processed, and counted as previously described(13, 14). For buoyant density centrifugation, ['4C]TdR-

labeled HMCV DNA was prepared as the Hirt super-natant of ['4C]TdR-labeled HMCV-infected RK-13cells (14, 21). [3H]TdR-labeled HSV-2 DNA was pre-pared as the Hirt supernatant of [3H]TdR-labeledHSV-2-infected Vero cells. Each Hirt supematant wasconcentrated by dialysis against Aquacide (Calbi-ochem) and contained small amounts of cellular DNA.The two supernatants were mixed, and solid CsCl wasadded to yield a density of 1.71 g/cm3. Centrifugationwas carried out, fractions were collected, and the filterswere processed as described previously (14). In someexperiments, the radiolabels were reversed, withoutchange in the results. In other experiments, the virusDNAs were isolated by lysing the infected cells withPronase and Sarkosyl, also without altering the re-sults.

Restriction enzyme cleavage and agarose gelelectrophoresis. 32P-labeled virus DNA was pre-pared by adding 25 uCi of 32PO4 (carrier free; Amer-sham/Searle) per ml to the medium of virus-infectedcells as previously published (19, 20). The virus DNAswere purified by sequential Hirt extraction, prepara-tive glycerol gradient sedimentation, and isopycnicbanding in CsCl (20, 21), followed by Pronase digestionand phenol extraction (20). Endonuclease EcoRI waspurchased from Miles Laboratories, Inc. and was usedas described previously (19, 20). The DNA fragmentswere separated by electrophoresis through a 0.5% aga-rose vertical slab gel as previously described (20, 25).Following electrophoresis, the gels were dried andplaced on Kodak Medical X-ray film RP/R2. Photo-scans of the autoradiographs were made on an Op-tronics P-100 photoscanner connected to a DigitalEquipment Corp. POP 11/40 computer as describedelsewhere (19).

RESULTSIsolation of HMCV from CMV-trans-

formed HEL cells. CMV-transformed HELcells (4 x 107 CMV-Mj-HEL-2 cells) in passage13 were injected subcutaneously into weanlingathymic nude mice. Twenty-seven days aftertransplantation of the cells, tumors (10 by 25mm) developed. Cell line CMV-Mj-HEL-2,T-1was isolated from one mouse tumor, and cellcultures were prepared as described (6, 7). Aftera confluent cell sheet was obtained, the tumorcells were maintained in continuous passages bydiluting the cells from one 75-cm2 plastic tissueflask at 2-day intervals with a twofold dilution.At 324 days after in vitro establishment (atpassage 90), some of the cell cultures developedfocal rounding of cells (Fig. la). CMV-like cyto-pathic effects (CPE) developed in HEL cellsinoculated with an extract of transformed cells(Fig. lb). Similar CPE developed at 1-monthintervals in later passages of the transformedcell cultures (passages 94 and 117), and cyto-pathic agents were again isolated from the trans-formed cell cultures in PRK cells. We designatedthe original isolate the Hershey Medical Centervirus (HMCV), deliberately giving the isolate asuperficial name, and, thus, specifically not pre-

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maturely identifying the virus isolate as a hu-man, mouse, or other animal virus, an HSV, orCMV. Preparations of HMCV-infected HELcells stained with hematoxylin and eosin re-vealed rounded cells with darkly stained nuclei.Some cells showed slight margination of chro-matin. A few had intranuclear bodies resemblingtype A inclusion bodies induced by herpesvi-ruses.

Herpesvirus-like particles were seen in thenuclei of infected HEL and PRK cells, and thedense virus nucleus was surrounded by doubleenvelopes (Fig. 2). That HMCV was an enve-loped DNA-containing virus was confirmed bythe facts that the isolate was inactivated byether treatment and iododeoxyuridine inhibitedvirus replication in PRK cells (data not shown).Characterization ofHMCV DNA. The size

ofHMCV DNA was determined by sucrose gra-dient sedimentation in neutral and alkaline gra-dients. Figure 3 shows the results of these meas-urements. With T4 DNA as a standard, HMCVDNA sedimented just behind T4 DNA in neu-tral sucrose gradients (Fig. 3a). By applying theBurgi-Hershey equation (2, 5), the molecularweight of HMCV DNA was calculated to be 96x 106. In alkaline sucrose gradients (Fig. 3b),HMCV DNA sedimented very heterogeneously;the largest size sedimented just behind T4 DNA.The buoyant density of HMCV DNA was mea-sured by equilibrium centrifugation in CsCl, andHSV-2 DNA was used as a standard. The resultsof the CsCl buoyant density centrifugation areshown in Fig. 4. HMCV DNA had a reproduciblyhigher buoyant density than HSV-2 DNA (1.729g/ml) and was far removed from the buoyantposition of human or mouse CMV DNA (1.717g/ml). With the radiolabels reversed, HMCVDNA still had a higher buoyant density thanHSV-2 DNA (data not shown). To measure thebuoyant density of HMCV DNA, 14C-labeledHMCV DNA was mixed with 3H-labeled HSV-2 DNA (1.729 g/cm3), Klebsiella pneumoniaeDNA (1.717 g/cm3), and cellular DNA (1.695g/cm3). The three DNAs of known buoyant den-sity were used to plot a line of density versusdistance (data not shown). From this standardcurve, the buoyant density of HMCV DNA is1.730 g/cm3, and, using the equation of Schild-kraut et al. (24), the guanine-plus-cytosine con-tent of HMCV DNA can be calculated to be71.5%.The endonuclease EcoRI cleavage pattern of

HMCV DNA was determined and directly com-pared to the patterns ofCMV, HSV-1, and HSV-2 DNAs. Figure 5 shows an example of an au-toradiograph of the EcoRI-cleaved DNAs: track1 is HMCV DNA, track 2 is CMV DNA, track3 is HSV-2 DNA, and track 4 is HSV-1 DNA.

The numbering/lettering nomenclature for thebands of HSV-1 and HSV-2 DNAs is used asdescribed by Hayward et al. (9, 10). The EcoRIcleavage pattern for HSV-1 (Patton) DNA(track 4) has a one-to-one correspondence withour previously published pattern (19) exceptthat band 2, a minor fragment, is not seen in ourpresent pattern. The pattern in track 4 (Fig. 5)is in excellent agreement with the EcoRI cleav-age patterns for HSV-1 DNAs previously pub-lished by Skare et al. (26) and by Hayward et al.(9). Similarly, the EcoRI cleavage pattern ofHSV-2 (333) DNA (track 3, Fig. 5) is in goodagreement with previously published patterns(9, 19, 26). The top band in track 3, band X, isan unexplained artifact. Band 6 of track 3 ap-pears as two closely migrating bands, whereasonly a single band is seen in our previouslypublished pattern (19). The important points forthe EcoRI digestion pattern of HSV-2 (333)DNA (track 3) are that the two largest frag-ments, bands 1 and 2, appear as submolar, fol-lowed sequentially by molar band A, minorbands 3 and 4, the dark doublet BC, and so forth,just as in the previously published patterns fromour own (19) and two other laboratories (9, 26).The EcoRI cleavage pattern of CMV (AD169)DNA published by Kilpatrick et al. (16) wasaccomplished on a 1% agarose gel and is there-fore not directly comparable to the pattern intrack 2 (Fig. 5). Nevertheless, certain compari-sons can be made. The largest band in bothpatterns has a molecular weight of about 15 x106. The pattern of Kilpatrick et al. (16) showsmore than 9 bands between the molecularweights 5 x 106 and 15 x 106; the pattern intrack 2 (Fig. 5) shows 11 bands. Therefore, asfar as can be determined, our pattern for EcoRI-digested CMV (AD169) DNA is in agreementwith the pattern published previously by Kil-patrick et al. (16). Thus, as clearly seen in Fig.5, the EcoRI cleavage pattern of HMCV DNAis totally distinct from the EcoRI cleavage pat-terns of the other three DNAs. The molecularweights of the HSV-1 DNA bands (26) havebeen graphed on semilog paper against the dis-tance migrated (data not shown). These pointsdetermine the standard curve and allow thedetermination of the molecular weights of theHMCV DNA bands. The molecular weights foreach of the specific EcoRI fragments of HMCVDNA are given in Table 1, as well as a calcula-tion of the relative molar amount of each band.The relative molar amounts of the largest (bandA) and smallest (band H) specific fragmentshave not been calculated because their molecu-lar weights are very uncertain. As seen in Table1, HMCV DNA bands B, C, E, and F appear tobe present in one molar amount. BandD appears

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FIG. 1. CPE caused by HMCV. (a) Development of CPE in CMV-Mj-HEL-2,T-1 tumor cell line due tospontaneous induction ofHMCV. Photograph of cell culture in tissue culture flask. x108. (b) CPE induced byHMCV in human embryo lung cells 48 h after inoculation. x108. (c) CPE induced by HMCV in rabbit kidneycells 24 h after inoculation. x108. (d) CPE induced by HMCV in adult human kidney cancer cells 72 h afterinoculation. x108.

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FIG. 2. Electron micrograph of HMCV propagated in rabbit kidney cells. Infected cells were fixed inKarnovsky fixative and postfixed in Dalton chrome-osmium, dehydrated in ethanol, and embedded in Sperrlow-viscosity plastic. Sections were cut with a Sorvall ultratome, stained with lead citrate and uranyl acetate,and examined with a Hitachi HU-12 electron microscope. x220,000.

to be present in two molar amounts and proba-bly represents two one-molar fragments thatcomigrated. Band G alone appears to be presentin a submolar amount.Biological properties of HMCV. The sen-

sitivities of HEL, HEK, HKC, HE, PS-1, HA,Vero monkey cell line, MEF, 3T3 establishedmouse cell line, HEF, PRK, RK-13, CMV-Mj-HEL-2, and CMV-Mj-HEL-2,T-1 cell lines tothe HMCV isolate were tested. The develop-ment and morphology of CPE and maximumvirus yield in these different cell cultures areshown in Table 2. Maximum virus yield wasobtained in PRK cells. Good yields were har-vested from RK-13, HEK, HA, Vero monkey,and HEF cells. More limited virus replicationwas observed in human embryo fibroblasts,adult HKC, HE, PS-1, MEF, and CMV-trans-

formed human epithelioid cells. No growth wasnoticed in 3T3 established mouse cells.The morphology of CPE induced by HMCV

depended largely on the cell type used forgrowth. In human and mouse embryo fibroblastcells, rounded and swollen cells developed intosmall foci. The spread followed the longitudinalaxis of the fibroblasts, thus resembling the CPEinduced by CMV. CPE spread slowly, like thatof CMV (Mj), and did not involve the whole cellsheet when the multiplicity of infection was low(Fig. lb). In PRK (Fig. lc), RK-13, HE, HA,Vero monkey, and HEF cells, virus-infected cellswere seen in foci as rounded cells. The spread ofCPE was fast, and the entire cell sheet wasinvolved within 2 to 4 days. In HEK and HKCcells (Fig. ld), rounded cells were nmixed withsyncytia having 10 to 50 nuclei. These charac-

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FIG. 3. Sucrose gradient sedimentation ofHMCVDNA. [3H]TdR_labeled HMCV DNA was isolatedfrom the Hirt supernatant and purified by glycerolgradient sedimentation. The HMCV DNA was di-alyzed and concentrated. An aliquot of 3H-labeledHMCVDNA (-) was mixed with '4C-labeled T4 DNA(0). Half the mixture was sedimented through aneutral sucrose gradient (a) and the other half wassedimented through an alkaline sucrose gradient (b).Sedimentation is from right to left.

teristics were similar to the CPE induced bycertain strains of HSV. The spread was fast andinvolved the full cell sheet in HEK cultures. Thespread was slow in HKC and HE cells, and thevirus-infected cell culture could be passed fourtimes before the whole cell sheet became in-volved. In CMV-transformed human cells, theCPE progressed slowly and consisted of small,well-defined foci of rounded cells. PS-1 humanbladder cancer cells appeared rounded butshrunken; foci spread slowly, and in most casesCPE regressed. Persistent infection was estab-lished by passing the virus-carrier HKC, HE,and PS-1 cells several times.Immunological studies with HMCV. In in-

direct immunofluorescence tests, high-titerCMV- and HSV-immune human sera reactedwith nuclear antigens ofboth 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 not react with theCMV and HMCV preparations tested, whereasthe reactivity of the serum with HSV-2-infectedcelLs remained unaltered (Table 3). HSV-2-hy-

perimmune rabbit and CMV-negative HSV-hy-perimmune human sera did not react withHMCV- and IBRV (LA)-infected HEL cells(Table 3 and Fig. 6). HMCV-immune rabbitserum reacted with IBRV-infected cells, but noreactivity was found with CMV (Mj)- and HSV-2-infected cells. Bovine IBRV-immune serumreacted strongly with HMCV- and IBRV-in-fected cells; faint nuclear reactions were ob-served with CMV (Mj)-infected cells, but noantigens were detected in cells infected withHSV-2 (Table 3). HMCV-immune rabbit andIBRV-immune monospecific bovine sera hadidentical neutralizing antibody titers to bothHMCV and IBRV (Table 4). No significant neu-tralization of CMV (Mj), HSV-2, and murineCMV was found with these sera at a dilution of1/10. CMV-immune human serum with a neu-tralizing antibody titer of 1/40 against CMV(Mj) did not neutralize HMCV, IBRV (LA),HSV-2, or murine CMV strains at a 1/10 dilu-tion. CMV-hyperimmune serum, prepared inrabbits with partially purified CMV (AD169)and having a CMV antibody titer of 1/1,000,neutralized 53% of the PFU of HMCV whenused in 1/10 dilution. Preimmune rabbit, HSV-2-immune rabbit, and murine CMV-immunemouse sera had no neutralizing effect against

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FRACTION NUMBERFIG. 4. Buoyant density centrifugation of HMCV

DNA. '4C_labeled HMCV DNA (0) and 3H-labeledHSV-2 DNA (0) were isolated from the Hirt super-natant. The Hirt supernatants also contained smallamounts of host cell DNA. The DNAs were mixedand sedimented isopycnically in a CsCl gradient. Atequilibrium, fractions were collected from the bottom.The meniscus is on the right. Density increases to theleft. For convenience, the bottom 20 fractions werenot graphed.

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keys, and rhesus monkeys did not neutralizeHMCV. Sera from six different strains of mice(C3H/He, DBA/2, BALB/c, C59B1/C, CD-1,and nude mice) had no neutralizing antibodiesto HMCV.Infection of experimental animals with

EHMCV. Subcutaneous, intramuscular, or intra-peritoneal inoculation of adult heterozygous andathymic nude mice, newborn mice, adult andnewborn hamsters, and rabbits with 106 PFU ofvirus suspensions failed to induce any clinicallymanifested disease or death in these animals.Intracerebral inoculation of adult hamsterscaused clinical symptoms, but all of the animalsrecovered. Meningitis and a mild encephalitiswere diagnosed based on histological prepara-tions. No reaction was induced by corneal scar-ification of the eyes of a rabbit with HMCV(Table 5).

DISCUSSIONWe reported previously that persistent infec-

tion of HEL cells with a strain of CMV isolatedfrom the prostate of a young boy (postmortem)

fb~G TABLE 1. Molecular weights and molar ratios ofb the EcoRI digestion products ofHMCVDNA

E H

HF

FIG. 5. EcoRI digestion products ofHMCV DNA.32P-labeled HMCV DNA, along with the standards,32P-labeled CMV (AD169), HSV-2 (333), and HSV-1(Patton) DNAs, were individually digested with re-

striction enzyme EcoRI. The digestion products ofeach DNA were separated by electrophoresis througha 0.5% agarose slab gel. The gel was dried andplacedon Kodak Medical X-ray film RP/P2. After an ap-propriate time, the film was developed. Track 1,HMCV DNA; track 2, CMV (AD169) DNA; track 3,HSV-2 (333) DNA; track 4, HSV-1 (Patton) DNA. Thespecific fragments ofHMCV DNA were given letterdesignations in order of increasing mobility (10). Thespecific fragments of HSV-1 and HSV-2 DNAs were

given letter or number designations as described pre-viously (11).

HMCV (Table 4). Pooled sera from normal rab-bits, hamsters, guinea pigs, African green mon-

Fragment Mol wt' (X10 Molar ratio'designation

A >25c NCdB 22 1.0C 18 1.3 ± 0.2D 11 1.8±0.6E 8.9 1.1 ± 0.1F 6.0 1.1 ± 0.1G 5.5 0.2 ± 0.05H 1.5c NCd

a Fragments are ordered on the basis of increasingelectrophoretic mobility and decreasing molecularweight.

b The mass of fragment was determined in arbitraryunits by graphically measuring the area under thepeak of a photoscan of an autoradiograph. The massis divided by the molecular weight to determine themolar amount in arbitrary units. The molar amountof fragment B was taken as equal to 1, and the molaramounts of the other fragments were calculated rela-tive to fragment B. The average relative molar amount(± one standard deviation) of each fragment was cal-culated from two autoradiographs from one EcoRIdigestion.

'The molecular weights of the largest (A) andsmallest (H) fragments are extremely approximatebecause both are far removed from the molecularweights of the standards. In particular, the molecularweight of fragment A can only be estimated as greaterthan 25 x 106.

d NC, Not calculated. Because of the large uncer-tainty in the molecular weights of the largest andsmallest fragments, their relative molar ratios havenot been calculated.

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TABLE 2. Development and morphology ofCPE and maximum virus yield in different cell cultures ofhuman and animal origin inoculated with HMCV

Progress of CPE (% of cell sheet)a Maximum virusCells

24 h 48 h 72 h 96 hType of CPE yield (PFU/ml)b

HEL (human) 0 30 50 50 CMV-like 5.0 x 104HEK (human) 50 95 100 100 HSV-like 7.0 x 105

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

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

roundedcells

PS-1 (human) 0 1 10 20 Atypical 1.1 x 104HA (human) 10 30 90 100 HSV-like 1.1 x 106

roundedcells

Vero (simian) 5 20 30 50 HSV-like 1.3 x 106roundedcells

MEF (mouse) 0 30 50 50 CMV-like 1.2 x 1043T3 (mouse) 0 0 0 0 None 1.0 x 100HEF (hamster) 10 90 98 100 HSV-Iike 3.0 x 106

roundedcells

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

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

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

CMV-Mj-HEL-2,T-1 0 10 90 95 Rounded 1.3 x 103(human) cellsa Inoculum: 0.3 PFU/cell.b Titrated in rabbit kidney cells.

TABLE 3. Indirect immunofluorescence tests in Flow 5000 cells for the identification ofHMCVSera (at 1/4 dilution)

Virus inoculum Mn Human Human Pre-HMCV-HMCV- Bovine HSV-2-im-ihm CMV-nega- CMV-ixII- CMV-Uiin Pnmue rbHCHMnCIRVi.HS-intive HSV-im- mune no. 1, 2, mune no. 1 it mune rabbit BRVim mune rabbit

mune 3, 4, 5 adsorbed it

None - - - - - - -

CMV (Mj) - +a-bHMCV - +-b +a,b +a,bIBRV (LA) - +b NDC - +a,b +a,bHSV-2 +a,b +a,b + +a,b

a Cytoplasmic fluorescence.b Nuclear fluorescence.c ND, Not determined.

resulted in transfornation of these cells. Trans-formed cells were transplanted to athymic nudemice. Of over 100 animnals inoculated, 62% de-veloped tumors after an average latent period of19 days. Cells reisolated from one of these tu-mors have been maintained for over 150 in vitropassages, and the line was designated CMV-Mj-HEL-2,T-1. These cells have shown a stable

expression of CMV-related membrane and intra-cellular antigens. The CMV-Mj-HEL-2,T-1 cellline, unlike the parental and other tumor lines,has undergone crisis several times. On threedifferent occasions (at passages 90, 94, and 117)CPE-like foci developed in the cell sheet, andthree virus strains were isolated in HEL or PRKcell cultures. One of the three virus isolates,

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FIG. 6. Photomicrograph of nuclear fluorescence. Fluorescence was detected in (a) HMCV-infected and (b)IBRV(LA)-infected cells. CMV-immune human immune serum was used with fluorescein isothiocyanate-conjugated anti-human goat serum in the indirect immunofluorescence test. (c) IBRV (LA)-infected cells showno fluorescence when treated with CMV-negative human serum. x450.

TABLE 4. Neutralization tests with HMCV in RK-13 and Flow 5000 cellsVirus

SeraHMCV IBRV (LA) CMV (Mj) HSV-2 Murine CMV

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

HMCV-immune 40 40 <10 <10 <10rabbit

IBRV-immune 40 40 <10 <10 NDbbovine

CMV (AD169)- 10 ND 1,000 ND <10immune rab-bit

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

HSV-2-immune <10 <10 <10 >10 NDrabbit

Murine CMV- <10 ND ND <10 >10immunemousea Reciprocal of dilution with >50% reduction in PFU as compared to the control.bND, Not done.

HMCV, isolated in HEL cells, has been studiedextensively. We have data, however, based onneutralization tests carried out with rabbit anti-HMCV immune serum, that all three virus iso-lates are identical.The immediate questions concerning HMCV

are: What is the virus and where did it comefrom? These questions are more easily answeredby first determining what HMCV is not. (i)HMCV is not a human CMV. The wide hostrange of HMCV is incompatible with all knownhuman CMV isolates, which only grow in humancells. In particular, the CMV (Mj) isolate used

to transform the original HEL cells grows onlyin human cells and then only poorly. HMCVgrows well in human, rabbit, hamster, and mon-key cells. HMCV does show some immunologicalcross-reaction with human CMV. The buoyantdensity of HMCV DNA and the EcoRI restric-tion pattern of HMCV DNA are additional evi-dence that HMCV is not a CMV. (ii) HMCV isnot a human HSV. HMCV does not react withantiserum prepared against HSV. Rodents in-oculated intracerebrally withHSV generally suf-fer a fatal encephalitis; rodents inoculated intra-cerebrally with HMCV survive. The buoyant

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TABLE 5. Infection of experimental animals with HMCVDose of injection Result after 6 weeks ofAnimal species No. injected Method of injectiona (PFU x 106Y observation

Adult mouse 5 SC 1 NegativeAdult mouse 6 IC 1 NegativeAthymic nude mouse 3 SC 1 NegativeNewborn mouse (<24 h) 7 SC 0.5 NegativeAdult hamster 2 IP 2 NegativeAdult hamster 7 IC 1 Meningitis and

encephalitiswithout mor-tality

Adult hamster 5 IM 2 NegativeNewborn hamster 8 SC 1 NegativeRabbit 1 IM 1 NegativeRabbit 1 SC 1 NegativeRabbit 1 Comeal 1 Negative

a SC, Subcutaneous; IC, intracerebral; IP, intraperitoneal; IM, intramuscular.

density ofHMCV DNA is close to but reproduc-ibly more dense than the buoyant density ofHSV-2 DNA. The restriction patterns ofHMCVDNA and HSV DNA are completely dissimilar.The sedimentation behavior of HMCV DNA onneutral and alkaline sucrose gradients is indistin-iguishable from several other herpesvirus DNAs(13-15, 27). (iii) HMCV is not murine CMV.HMCV grows well in human cells and not inmurine 3T3 cells. Mouse CMV grows well in 3T3cells but not in human cells (12), and antiserumprepared against mouse CMV does not reactwith HMCV. The buoyant density of HMCVDNA is far removed from the buoyant densityof mouse CMV DNA (18). (iv) Antibodies di-rected against HMCV could not be detected inthe normal sera from six strains of mice, includ-ing nude mice. These data suggest, but do notprove, that HMCV is not a virus common tomice. (v) Antibodies directed against HMCVcould not be detected in the normal sera fromrabbits, hamsters, guinea pigs, African greenmonkeys, and Rhesus monkeys. These data sug-gest that HMCV is not a virus common to anyof these animals. (vi) Bovine monospecificIBRV-immune serum neutralized HMCV and,conversely, high-titer anti-HMCV serum neu-tralized IBRV. High titer CMV-immune humansera reacted with nuclear antigens of bothHMCV- and IBRV-infected cells, but did notneutralize these viruses. Adsorption of CMV-immune serum to CMV-infected cell extract re-moved the reactivity against these antigenic for-mations.These immunological data suggest that

HMCV is IBRV. In addition, the host range andappearance of HMCV CPE are similar to thepublished host range and CPE of IBRV (17).The lack of pathogenicity of HMCV in testanimals has also been reported for IBRV (17).

The published buoyant density of IBRV DNA(22) is indistinguishable from the buoyant den-sity ofHMCV DNA. An EcoRI cleavage patternof IBRV DNA (26) is similar, but not identical,to the EcoRI cleavage pattern of HMCV DNA.These differences could be strain variation.The CMV-transformed cell cultures were

maintained in a laboratory where only humancells were being cultured. No viruses other thanhuman CMV and HSV were maintained in thesame room, and these were in other laminar flowhoods. The strictest biohazard regulations wereadopted when the CMV-transformed cells werepassed. A presterilized hood, separately pre-pared medium and trypsin, and previously uno-pened bottles of fetal calf serum and pipettecontainers were used. The isolation of HMCVwas made in HEL cells in their 11th in vitropassage. Uninoculated HEL cells, carried simul-taneously, remained negative. Despite these pre-cautions, it now appears probable that HMCVis a contaminant IBRV, presumably arising fromthe fetal calf serum. Thus, HMCV is similar toor identical with IBRV.The HMC isolate of IBRV transforms adult

human epithelioid cells in culture (Geder et al.,manuscript in preparation). We are currentlytesting the oncogenic potential of a standardreference strain (LA) of IBRV. In addition, fromimmunological evidence, it is clear that IBRVshares antigens with human CMV. This obser-vation must be confirmed and extended by DNAhomology studies currently in progress.

ACKNOVWLEDGMENTS

We thank J. Gruber, Office of Program Resources andLogistics, Virus Cancer Program, Division of Cancer Causeand Prevention, National Cancer Institute, Bethesda, Md., forsupplies of nude mice; R. Glaser for his valuable advice; R.Lausch for supplying us with the hyperimmune rabbit CMV-immune sera; and J. Gorodecki, A. Laychock, and L. Kudler

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for their technical assistance. We are especially grateful toDavid Porter, who brought to our attention the possibilitythat HMCV might be IBRV.

This work was supported by Public Health Service contractno. N01-CP-5-3516 within the Virus Cancer Program of theNational Cancer Institute and grants CA-18450, CA-16365,and CA-16498 awarded by the National Cancer Institute. J.P.I.is the recipient of Public Health Service fellowship CA-05677from the National Cancer Institute, and M.S.D. is a postdoc-toral fellow of the Damon Runyon-Walter Winchell CancerFund (DRG-161F). R.W.H. holds a Faculty Research Awardfrom the American Cancer Society (FRA-158).

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