[CANCER RESEARCH 28, 1311-1318,July 1968]

Virologie Studies in Human Leukemia and Lymphoma: TheHerpes-type Virus

Frank J. Rauscher, Jr.

National Cancer Institute, Bethesda, Maryland

SUMMARY

Selected background information is presented and discussedin the light of newer facts and leads from studies constitutinga broad effort aimed at determining whether viruses similar(or different) to those known to be associated with the induction of leukemia and lymphoma in laboratory and domesticanimals are also involved in the etiology of human leukemiaand lymphoma. Emphasis was placed on studies concerningthe establishment and experimental use of hematopoietic celllines and of a herpes-type virus frequently associated with thecultures. Based on information published or contributed to theSpecial Virus-Leukemia Program of the National Cancer Institute, as of July 1967, over 125 cell strains have been established from patients with cancer or other diseases and fromnondiseased controls. At least 61 of the "cancer" and 5 of the"noncancer" derived lines show herpes-type virus by electron

microscopy. The virus (es) has been detected in cultures frompatients and controls in laboratories in England, United States,Africa, Australia, New Guinea, Japan, and Sweden, and in theUnited States from several chimpanzees and 1 rhesus monkey—the latter with myeloid leukemia.

That the significance of these and other findings to canceretiology remains unknown is discussed in relation to keystudies that rapidly advancing technology and production ofindustrial quantities of virus will make possible.

INTRODUCTION

The primary purpose of this paper is to present a briefreview of recent background and current facts and leadsemerging from studies on the detection and characterizationof viruses and virus-like particles in human leukemia andlymphoma materials. Of the biologic agents, antigens, or antibodies most frequently detected or isolated from human leukemia and lymphoma patients to 1967, the unidentified herpes-type virus (HTV) and type "C" particles are those which have

received the attention of most investigators who seek to determine whether viruses similar (or different) to those knownto be associated with the induction of leukemia and lymphomain laboratory and domestic animals are also involved in theetiology of human leukemia and lymphoma. In considerationof other papers presented at this symposium, and in view ofthe intense current interest in recently established humanhematopoietic cell lines and an associated virus, particularemphasis is given to the "state of understanding" of the herpes-

type virus. Information and technical procedures developedin studies of animal leukemia and candidate human "C" type

particles which appear to have particular relevance to the detection of HTV are included.

DETECTION AND DISTRIBUTION OF HTV

Epstein et al. (13) in 1964 reported successful tissue culture propagation of cells from several Burkitt lymphomabiopsies. This was followed nearly concurrently by Iwakataand Grace (42) who were able to culture cells derived fromthe peripheral blood of a patient with chronic myeloid leukemia. Both patient sources yielded cell lines in which weredetected particles having morphologic and other characteristicsof herpes-type viruses. Over 125 cell lines from cancer andnoncancer patients have been established by investigators inlaboratories in England, Australia, New Guinea, Japan, Sweden, Africa, and the United States (e.g., 11, 13-19, 25, 39, 42,

43, 45, 50, 53, 54, 59, 62, 68, 72, 74; B. Clarkson, personalcommunication). Table 1 presents data which approximatethe number of lines which had been established as of July1967 according to disease and nondisease categories and according to the presence or absence of a herpes-type virus.Viruses other than HTV have not been reported in these lines.It is important to note that the numbers and percentages ofthis table are approximate. The data were kindly provided bymany investigators throughout the world known to be workingin this area and in particular working within or related to theSpecial Virus-Leukemia Program of the National Cancer Institute. The cells of nearly all of these cultures are large,mononucleated, and resemble lymphoblasts. They usually donot attach to the surface of the tissue culture vessel and appearto grow best in static suspension cultures. However, Dr. GeorgeMoore at Roswell Park Memorial Institute has had considerable success in growing large quantities of some cell lines inspinner cultures (53).

Comparative immunofluorescent and electron microscopystudies have shown that only 1-5% of the cells of most ofthese lines are positive at any one time for the antigen andthe virus particle, respectively (22, 24, 35, 36, 47). This apparently low incidence of infected cells has made it extremelydifficult to extract and purify adequate quantities of virus forfurther characterization work. Faced with these and otherproblems imposed by relatively small' numbers of apparently

infected cells, investigators at Roswell Park attempted to increase virus production by selecting clones of more heavily

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Table l

Patients cultured/Patienteattempted"CategoryLymphomaLymphoid

leukemiaMyeloidleukemiaSarcoma-carcinomaTotal

(cancer)Other

diseasesNormalTotal

(noncancer)No.31/6115/11134/13825/54105/3645/5414/8119/135Percent511425462991714Patients

HTV + »/PatientsculturedNo.19/314/1514/347/2544/1051/53/144/19Percent6127412842202121Lines

HTV + VLinesculturedNo.23/414/1723/10011/4661/2041/54/205/25Percent5624232430202020

Summary: Herpes-type virus (HTV), prototype Epstein's EBI, and human cell "lines."«Approximate. Successful attempts understandably are nearly always reported; nonsuc-

cesses, usually not.*These numerators are not identical because several positive lines have been started from

the same patient and up to 6 lines have been started from the same patient of which only1 or 2 are positive.

infected cells (39). Using a cloning technic in semisolid agar,49 sublines were derived from the P3J Burkitt lymphoma line.Following 4 months of propagation, one of these sublines, designated P3HR-1, showed 15-40% of the cells infected. Whenthe cells were incubated at 35°Cor 32°Cfor 9-15 days with

out refeeding, up to 75% of the cells became immunofluorescentpositive and showed particles by electron microscopy.

A number of investigators have shown that both the directand indirect immunofluorescence technics detect cells whichproduce herpes-type virus particles. Earlier work by Finket al. (22, 24) and Henle and Henle (35) revealed a good correlation between electron microscopic determination of virusproducing cells and immunofluorescence. This firm but independent correlation was surprising since the Fink-Mahngrendirect reagent was made in rabbits to centrifugal concentratesof human leukemic plasma containing type "C" particles while

Henle and Henle used an indirect test which employed selectedpools of human gamma globulin. In addition, it was thoughtthat since approximately IO6 virus particles were necessary for

an electron microscopic search to be positive, this particlevisualization technic would be far less sensitive in the detectionof small quantities of virus than either immunofluorescentmethod.

The original studies of Fink et al. (21, 23) are importantnot only because they appeared to provide the first supplementary detection technic to electron microscopy, but alsobecause the reagent and the procedure held promise of bridgingan actual and/or intellectual gap regarding the possibility that2 widely different groups of "viruses" (C and HTV) might be

associated with human leukemia and lymphoma.Fink et al. (23) employed antigen derived from leukemic

plasma for the induction of antibodies in heterologous hosts(rabbits and monkeys). The antibody was then conjugatedwith fluorescein isothiocyanate and used as a specific stain forthe detection of antigen in cells of the buffy coat, bone marrow and biopsies, or tissue culture expiants thereof, derivedfrom human patients with leukemia and lymphoma.

The plasma antigen used for inducing the antibody reagentwas a pooled concentrate separated by differential ultracen-

trifugation from 5 cases of human leukemia in which "virus-like" particles had been observed by A. J. Dalton, but in which

mycoplasma-like particles were not seen on electron microscopic examination. Absorption of the serum reagent wascarried out with various constituents of normal blood—eryth-

rocytes representing the known ABO blood groups and majorRh factors, platelets, and plasma derived from 10-14 humans—untilno reaction with normal blood factors could be demonstrated by hemagglutination or micro double-diffusion studiesand the presence of a slight excess of human plasma proteinscould be demonstrated. In addition to controls on the technicalprocedures, serum controls included: (a) rabbit antiserumprepared against the concentrated plasmas of 5 normal humansubjects in which "virus-like"' particles had not been observed;

(6) antibody prepared against the plasma of one leukemicpatient, in which "virus-like" particles were not demonstrated,but in which many "mycoplasma-like" forms were easily iden

tified by electron microscopy; and (c) antibody preparedagainst a mouse leukemia virus (Rauscher strain). In addition,reagent antibody was prepared in adult rhesus monkeysagainst a second pool of human plasmas containing "virus-like" particles.

Control studies on antigens included testing of the variousantisera against: (a) 11 different human strains of myco-plasma; (6) cells infected in tissue culture with pure culturesof the Yaba, herpes simplex, herpes zoster, and influenzaviruses, and the Eaton PPLO agent; and (c) 25 bone marrowspecimens from either normal human volunteers or patientsill with other diseases.

Of 72 leukemic patients tested (23), 49 (08%) showed reaction with the antihuman leukemic fluorescent antibody.Eight of them also showed positive reactions to the anti-Rauscher virus fluorescent antibody, as did 8 other patientswho failed to react positively with the antihuman leukemicreagent. None of the specimens tested showed positive reactions against other control sera, nor did any of the antigen-control specimens react positively with the ant ¡human leukemia fluorescent antibody. These studies were confirmed

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Herpes-type Virus

substantially by Dabieh et al. (8), Levine et al. (46), and in

part by Yohn and Grace (73).One of the most recent and most comprehensive studies to

assess the significance of electron microscopy and immuno-fluorescent findings in relationship to the stage of disease wasreported by investigators of Roswell Park Memorial Institute,Buffalo, New York. Levine et al. (46) examined 128 bloodsamples from 73 patients with leukemia and from 11 nonleu-kemia patients. The samples were studied by electron microscopy using thin sections of plasma pellets and by immuno-fluorescence in peripheral blood cells. Their results showedthat 29% of acute lymphocytic leukemia, 45% of acute myelo-

cytic leukemia, and 17% of chronic myelocytic leukemiapatients had at least 1 buffy coat specimen interpreted asimmunofluorescent positive. The difference between acute myelocytic leukemia and chronic myelocytic leukemia is statistically significant. This could mean that there was less chronicmyelocytic leukemia specific antigen in the plasma pellet usedto prepare the antisera for the fluorescent antibody test, orthat chronic myelocytic leukemia cells contain less detectableantigen. Another interesting comparison is that 27% of thetotal specimens collected from all 3 types of leukemia werefluorescent antibody positive, 52% of the type "C" particle

positive (by electron microscopy) specimens were fluorescentantibody positive, and only 21% of the electron microscopynegative specimens were fluorescent positive. This indicatesa significant correlation between the presence of type "C"

particles in plasma and fluorescent antigen in buffy coat cellsof leukemia patients. Within this same group of patients, 7%of those in remission and 19% of those in relapse were positive for virus-like particles. Comparatively, 20% of the bonemarrow cells of patients in remission and 35% of patientsin relapse showed positive immunofluorescent reactions.

Of considerable additional interest is the recent report byYohn and Grace (73) that pooled normal marrow is capableof completely absorbing the Fink type immunofluorescent antibody prepared against human "C" type virus-like particles.

This finding suggested the following alternatives for consideration: (a) the immunofluorescent antibody prior to absorptionwith bone marrow is capable of detecting an antigen of immature leukocytes which under ordinary conditions rarelyappears in the peripheral blood, or (6) a virus or virus antigenrelated to virus-like particles recovered from human leukemicplasma was present in the pool of adult human bone marrow.The latter consideration would be consistent with the reasonable assumption that while less than 1% of the populationcan be expected to develop leukemia, the vast majority ofpeople are infected with a virus(es) which may be capable ofinducing leukemia. This assumption would also be in line withthe known relationship between infection and disease in polioand other "necrotizing" viruses. If this relationship between

infection and disease with necrotizing viruses were true ofleukemia viruses, then it would be reasonable to expect thatthe bone marrow may be the site for latent retention of virusand/or of virus antigen. This latter hypothesis, while attractive, does not appear to be supported by Fink et al. (23),who failed to detect immunofluorescent antigens in the bonemarrows of normal individuals or of patients with diseases

other than leukemia or lymphoma. One would expect that ifsufficient antigen were present in bone marrow to allow complete adsorption of immunofluorescent antibody, there shouldalso be enough antigen to be detectable with the unadsorbedimmunofluorescent antibody. Fink (20), however, suggests that"the bone marrow of normal individuals might be expected

to contain latent leukemia virus, present in an amount sosmall as to be undetectable when only a few cells were examined, but in an amount sufficient to reduce the titer ofspecific antibody when measured in dry weight in grams."

Further studies by Fink et al. (21) with the antihuman "C"

type fluorescent antibody showed that cells in tissue culturederived from biopsies of 9 separate cases of Burkitt lymphomaand leukocyte cell lines derived from 6 cases of human leukemia all contained positive fluorescent reactions and "herpes-like" viral particles when examined by electron microscopy.

Fink et al. (22) interpret their findings to support the thesisthat the antigen detected by immunofluorescence with theantihuman leukemia antibody "is concerned with the leukemic process." Although these data are concerned with the

hypothesis of a viral etiology of human leukemia, it remainsto be determined whether the antigen being detected is ofviral origin.

Further similar studies by Henle et al. (38) and Mayyasiet al. (49) using an indirect immunofluorescent method showedthat human sera which were immunofluorescent positive contained antibodies directed against the virion as evident fromelectron microscopic determination of antibody coating andagglutination of virus particles extracted and concentrated fromBurkitt cell lines. Additional studies by Henle and Henle (34)revealed that on pulse exposure of cells containing HTV tothymidine-3H both viral and cellular DNA became labeled.

Later, fluorescent cells showed the label to be present in thecytoplasm, whereas in nonfluorescent cells it remained restrictedto the nucleus. The labeling of cellular DNA was substantiallylower in cells lethally X-irradiated 4—7days prior to exposureto the isotope. This treatment, however, did not prevent thelabeling of immunofluorescent cells. Unequivocal proof thatimmunofluorescent cells are those that contain HTV was reported by zur Hausen et al. (75), who were able to pick immunofluorescent cells for electron microscopic examination. Theresults showed clearly that immunofluorescent negative cells revealed the presence of virus particles.

Several groups of investigators have employed virus-containing Burkitt tumor cells to detect antibodies in human sera byimmunofluorescence (35), complement-fixation (1, 26), im-munodiffusion (55), and antibody coating technics (38, 49).Using appropriate immunofluorescence tests with Burkitt lymphoma cell lines, Henle and Henle (34) have shown that theage distribution of antibodies in noncancer American andAfrican children revealed a pattern similar to that for othercommon viruses. The incidence of positive sera was high (70%at age 0-3 months, declined between age 4-24 months, and thenat age 4 years rose to 50% where it remained until adolescence).Greater than 80% of sera from people at age 40 showed antibodies to HTV. Various investigators using different serologiedetection technics have shown that nearly all sera from Burkittpatients contain HTV antibodies and in most cases at levels

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higher than sera from nondiseased controls. There is some indication that Burkitt patients in remission contain generallyhigher levels of antibody, whereas on reoccurrence of the tumor,the antibody level declined. Very intriguing information wasreported by Old et al. (55), who showed an unusually highincidence and level of HTV antibodies in the sera of patientswith postnasal carcinoma. Gerber and Birch (26) using asensitive complement-fixation test with partially purified HTVprepared from the Burkitt P3 line by investigators of the PfizerCompany, Maywood, New Jersey, showed that 90% of serafrom healthy adults and patients with malignant diseases hadantibodies to HTV. They also tested 5 species of nonhumanprimates and found that all except baboons had a high incidence of complement-fixing antibodies to the P3 antigen andthat none of the sera from 9 species of laboratory or domesticanimals reacted with this antigen. Their finding that 10 of 10chimpanzees had relatively high levels of HTV antibody isparticularly interesting in view of the report by Landon et al.(45), who have established hematopoietic cell lines from 3 un-inoculated chimpanzees, all of which contain a herpes-typevirus. The virus in the chimpanzee cell lines is antigenicallysimilar, if not identical, to HTV contained in the human lines(J. L. Landon, personal communication). Studies by variousinvestigators have shown clearly that HTV is not related antigenically to any of the known human or animal herpes viruses,nor is it related to other nonherpetic human and animal virusesthat have been tested. Although additional work needs to bedone, there is good evidence to suggest that the herpes-typeviruses in at least some of the Burkitt lymphoma lines frompatients in Africa and the United States, as well as cell linesstarted from American leukemic patients, are related (49). Itis obviously important to determine whether more than onemajor antigenic type of the virus exists in the more than 60virus-containing human cell lines already established.

HTV has been detected in the peripheral leukocytes or tissues of several leukemia patients and in peripheral leukocytesfrom 1 normal person prior to propagation of cells in tissueculture and in biopsy materials from cases of Burkitt lymphoma (30). In one study, investigators of the Pfizer Company,Maywood, New Jersey, E. Jensen et al., personal communication) detected by electron microscopy HTV in peripheral cellsfrom a child with congenital leukemia. The virus persisted inthe cells following propagation in tissue culture. HTV wasalso detected in the peripheral blood of the mother but notof the father of this child. In this regard, Moore et al. (personal communication) and Gerber and Birch (26) have established cell "lines" from buffy coat cells of normal, nondiseaseddonors. Several of these "lines" were shown to contain HTV.

In view of this, it may be necessary to reevaluate the previousassumption that the cells of lines started from human leukemiaand lymphoma patients were neoplastic rather than normalor non-neoplastic. Definitive karyotypic analyses have, however, been done with several of these lines, the results of whichsuggest that some of the cells of at least some of these linesare neoplastic. As an example, the chronic myeloid leukemiapatient from which the M-2 (11) line was established was shown

to be positive for the Philadelphia chromosome (48). Periodic

chromosome analyses on the cultured cells showed retention ofthe Philadelphia marker.

Perhaps related to these observations is the report by Griffinet al. (28, 29), who detected by electron microscopy the presence of virus particles morphologically of the herpes type inthe pulp of a tooth removed from a Burkitt lymphoma patient with an active maxillary tumor. Similarly, they detectedherpes particles in the tooth of an apparently normal child,who 5 years previously had been successfully treated for aBurkitt lymphoma of the maxilla. Unfortunately, virus isolationand identification tests were not performed, and therefore it isnot known whether these particles represented herpes simplexvirus, other known herpes viruses, or HTV. Herpes simplexvirus has, in fact, been isolated from biopsy specimens ofBurkitt jaw tumors (3, 65, 71) as well as from the throats ofnormal children resident in the same area of Africa (65).

BIOLOGIC ACTIVITY OF HTV

Until very recently, infectivity or other kinds of biologicactivity had not been shown with any of the HTV isolates. Thevirus appeared to replicate (persist) only in the tissue culturecells in which it was originally detected and numerous attemptsto extract HTV infective for other cell cultures and for animalswere unsuccessful. More recently, however, several investigators have succeeded in showing infectivity of HTV and inseveral cases apparent disease induction in animals followingtheir inoculation with cell and cell-free materials containing thevirus. Stewart et al. (67) showed that hamsters developed aprogressive encephalitic syndrome leading to death following in-tracerebral inoculation with human cells or cell extracts knownto contain HTV. Investigators of the Pfizer Company, May-wood, New Jersey (F. Dürr,personal communication) confirmed this observation and showed in addition that variousstrains of mice developed a similar disease following intra-cerebral inoculation. Grace et al. (personal communication) atRoswell Park Memorial Institute showed that newborn kittensalso developed this disease following intracerebral inoculationwith HTV. In none of these studies, however, were virus particles detected by electron microscopy in thin sections of neuraltissue.

Henle et al. (37) have detected a reasonably frequent chromosome aberration in tissue culture lines begun from human leukemia and lymphoma patients and known to contain HTV. Thismodification of chromosome No. 10 and replicating virus particles also appeared in some normal human cell lines followingexposure to HTV.

At this point it appears important to reiterate that whileparticles of the HTV generally are indistinguishable in morphology and size to other known members of the herpes virus group,there are several quantitative and qualitative differences. First,Dalton et al. (personal communication) reported apparent morphologic differences between the unidentified herpes-type virusand known members of the herpes group. Particles associatedwith tissue cultures of human leukemia and lymphoma cellsacquired a finely granular coat as distinct from an envelopeformed from the nuclear or plasma membrane. No such coatwas found to develop in relation to the known herpes viruses.

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Increased electron density of peripherally condensed chromatinand beading of mitochondria were also characteristic of cells ofthe human leukemia and lymphoma tissue cultures which contain virus particles. Such features are not seen in cells infectedwith known herpes viruses. These investigators cautioned, however, that the latter phenomena could be nonspecific host-cellreactions to viral infection. Secondly, the usual findings in attempts to replicate more common herpes viruses, especiallyherpes simplex virus, is that the particles nearly consistentlycontain nucleoids (J. T. Grace et al., personal communication).Conversely, a persistent finding in the human leukemia andlymphoma cell lines is that many to most of the particles ofHTV are devoid of nucleoids. Thirdly, most attempts to showHTV infectivity have been initiated with partially purifiedvirus following various procedures, including density gradientcentrifugation, sonication, freeze-thaw cycles, and Chromatographie column separation. Based upon the reasonable assumption that these factors and procedures may have been deleterious to infectivity, if not antigenicity, it appears, in retrospect,not unusual that attempts to show infectivity have beenalmost invariably unsuccessful. A fourth and perhaps equallyimportant factor is that even when apparently intact particleswere separated from cells, the amount of virus (complete and"incomplete") available per unit inoculum was low and, in

fact, may have been too low to initiate infection. In this regard,it is important to recall the original observation of Bryan et al.(5) who showed with the Rous sarcoma virus that ability toinfect, latent period, severity of disease, and recoverable viruswere dependent on the amount of virus contained in the infecting dose. These observations were confirmed by others (31, 63)and most recently were shown to apply to at least one murineleukemia virus (58).

Zeve et al. (In vitro Cell Free Transfer of Burkitt AssociatedVirus to Human Leukocytes. Submitted for publication toScience, September 1967) successfully transferred HTV froma Burkitt cell line (MOB-8) to another human buffy coatculture (RPMI-2367) the latter of which has been negative forvirus and virus antigen when examined electron microscopicallyand immunofluorescently, respectively. Using a Belco dual spinner flask the MOB-8 cells were placed on one side of a milliporefilter (220 m^, GS) and the RPMI-2367 cells on the oppositeside. Following cultivation for 164 hours, the RPMI-2367 cellswere shown by electron microscopy to contain particles morphologically identical to HTV. Appropriate tests to determinothe integrity of the millipore filter strongly suggested cell-freeinfection by HTV of the recipient cell line.

Grace et al. (personal communication) at Roswell ParkMemorial Institute very recently showed that a virus-free lineof cells started from buffy coat of a myeloid leukemia patient(6410) rapidly declined in viability with death 5 days laterafter exposure to high multiplicities of HTV (derived from theHR1-K clone of the P3J Burkitt lymphoma cell line) of theorder of 500-1,000 virus particles per cell. Electron microscopyand direct immunofluorescent staining indicated that almostall cells were infected and that large amounts of new virus particles were produced. A virus carrier state of 6410 cells was observed when these cells were infected with doses of virus ranging from 1-250 virus particles per cell. In these experiments,

therefore, a clear dose response was observed. With lowermultiplicities of virus input as long as 3-4 weeks were requiredfor the appearance of immunofluorescence in 30-50% of the

cells. For further studies these investigators reasoned that sincevirus adsorption is the first step of infection, an assay for adsorption may help to determine the target cell capable of supporting viral replication. Following this approach, they furtherconfirmed biologic activity of HTV by successful infection of3 additional human cell lines, all of which were negative byimmunofluorescence and electron microscopy prior to infection.Their studies indicate that among the cells tested thus far byimmunofluorescence and electron microscopy, only human andmonkey lymphocytes or lymphoblast-like cells have the abilityto adsorb virus. All such immunofluorescent positive cells whenobserved with phase contrast microscopy were mononucleated,uniform in size, approximately 12 microns in diameter withthe nucleus occupying 75% of the cell. These and other findings seem to indicate an affinity of the virus for lymphocyticor lymphoblastic type cells of primate origin. These conclusionsappear to be supported by the work of Gerber and Birch (26),who by complement-fixation detected antibodies to HTV inhuman, chimpanzees, and 4 species of monkeys but not in rats,guinea pigs, cats, dogs, goats, sheep, pigs, horses, and cows.

DISCUSSION

Since no DNA virus has yet been found to induce leukemiaor lymphoma, and since all of the known leukemia and lymphoma viruses are of the same subgroup of RNA agents, itwould appear logical, from the animal prototype diseases, toconcentrate the search for a counterpart human agent onviruses of the medium-sized, membrane-bound, RNA variety.However, at the present stage of knowledge and experience,it would be unwise to exclude viruses of any type as potentialcandidates for leukemogenesis simply because no member ofthe group has thus far been discovered which induces the usualforms of leukemia or lymphoma. The facts that the medium-sized, membrane-bound, RNA viruses of avian leukemia cancause kidney carcinomas as well as leukemia and that fibrosar-comas of nearly identical gross and microscopic morphologycan be induced in hamsters with both polyoma (DNA) andRous sarcoma (RNA) viruses further caution against stereotyping all oncogenic virus-host interactions on the basis of theusually predictable tumor responses of laboratory animals tolaboratory strains of tumor viruses.

After successful propagation of a virus in sufficient quantitiesto support its further investigation, the next step in the searchfor viruses associated with human leukemia and lymphoma isthe demonstration of the capacity to induce neoplasia in somelaboratory test-animal or tissue culture system. Testing in animals of the homologous species is impractical. Although theproduction of neoplasia in other animals would not constituteproof for oncogenic potential in man, it would demonstratethat this basic property is possessed by the virus and wouldrepresent important evidence to support the possibility of asimilar action in man (6). In like manner, the induction ofin vitro cytoneoplasia (6) in human or other cell lines would

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provide valuable supporting evidence, but would not aloneconstitute proof for oncogenicity of the agent in man.

The oncogenic dose of Rous sarcoma virus is several ordersof magnitude higher for hosts of foreign species than for thenatural host (60, 61); this type of quantitative relationshipmay also hold for human candidate viruses tested in other animal species.

The problems of testing for oncogenicity in animal and tissue culture systems have been described by many others. Theinduction of leukemia in mice, inoculated when newborn withspecimens from human leukemia, has been reported by investigators from 3 different laboratories (4, 12, 64), but others(27, 52) have failed to confirm this finding. Approximately700 primates mostly rhesus monkeys, but including animals of9 species (cynomologous, African green, baboon, pig-tail macaque, stump-tail macaque, Galago, Bonnet, marmosets, andchimpanzees), have been inoculated when newborn with specimens from human leukemia and lymphoma by investigatorsboth at the National Cancer Institute and at other institutionscollaborating in the Special Virus-Leukemia Program of theNational Cancer Institute (F. J. Rauscher and A. J. Pallotta,unpublished data). No neoplastic responses have been observedthus far. However, only about 220 of these animals are morethan 1 year of age, and the oldest are only about 4 years old.If a leukemogenic agent is responsible for the age-peak whichoccurs at 3-4 years in human childhood leukemia, and if susceptible primates approach the human host in time-to-inductionof disease, the primate animals would have to be observed forseveral additional years before their responses could be interpreted as definitely negative.

Since most of the studies presented in this paper were basedon materials derived from patients with Burkitt's lymphoma,

it is important to note that herpes simplex (3, 65, 71), vaccinia(10), Bunyamwera (70), an unidentified agent (9), and Reoviruses (3, 70), in addition to HTV, have been detected inmaterials derived from Burkitt lymphoma patients. It is ofinterest and possibly significant that only HTV has been detected or isolated from American leukemia patients and fromBurkitt lymphoma patients studied in the United States. Ofthe above agents and in addition to HTV, Reo viruses, particularly type 3, appear to be most uniformly present in Burkittlymphoma patients. In one study, Reo viruses were isolatedfrom biopsy material from 25 of 90 cases of Burkitt's tumor in

Uganda and Tanzania, whereas this virus was isolated fromonly 1 of 21 other patients with non-Burkitt tumors (3).Neutralizing antibody studies using type 3 Reo virus haveshown that there is a relationship between this virus andBurkitt's tumor (2). Antibody was found in 53 of 72 cases ofBurkitt's tumor, but in only 12 of 65 controls consisting of a

variety of other tumor cases in normal children. Comparableresults were obtained using hemagglutination-inhibition testson another series of serum specimens (47). Stanley et al. (66)reported the induction of lymphomas in mice following theirinoculation with spleen cells from a mouse presenting withchronic lesions induced after neonatal Reo virus 3 infection.The disease appears to be a manifestation of an autoimmunedisease, although some features are reported as resemblingBurkitt's tumor (56). These preliminary results and the report

by Parker et al. (57) on the isolation of Reo 3 virus frommosquitoes, while suggestive of a more than casual relationshipwith Burkitt's tumor, must obviously await confirmation. It

seems clear from the studies of Kajima and Pollard (44) andHuebner (40) that mice of all strains carry viruses capable ofinducing leukemia and lymphoma. Since these leukemiaviruses are known to be helpers (33, 41) for at least one of theknown murine sarcoma viruses (32, 51), it would appecr unwarranted to ascribe an etiologic role to a human isolate of aReo virus and the induction of a lymphoma in a laboratorymouse.

The case for assigning an oncogenic role to HTV at thepresent time is certainly as tenuous as that concerned with otherviruses. Incrimination of HTV may be strengthened by theapparent relationship of a herpes virus to the induction ofrenal carcinomas in the frog, and by the recent report ofChurchill and Biggs (7) and Burmester (personal communication) that the agent of Marek's disease may be a herpes virus.

Similarly, cocarcinogenic properties have been reported forherpes simplex virus when inoculated intradermally into micein conjunction with multiple applications of 3-methylcholan-threne (69). While these and other studies presented in thispaper and at this symposium certainly do not detract from thehigh priority need for expanded studies on HTV, they obviously have not yet definitively determined whether HTVis (a) an international ubiquitous passenger virus or (6) etio-logically related, even indirectly, to human cancer.

REFERENCES

1. Armstrong, D., Henle, G., and Henle, W. Complement-fixationTests with Cell Linos Derived from Burkitt's Lymphoma and

Acute Leukemias. J. Bacteriol., 91: 1257-1262, 1966.2. Bell, T. M. The Chemotherapy of Burkitt's Tumor. Proceed

ings of a Conference, Kampala, Uganda, 1966. Sponsored bythe U.I.C.C., in press.

3. Bell, T. M., Massie, A., Ross, M. G. R., Simpson, D. I. H.,and Griffin, E. Further Isolations of Reovirus Type 3 fromCases of Burkitt's Lymphoma. Brit. Med. J., 1: 1514-1517,

1966.4. Bergoltz, V. M. Experimental Studies of the Etiology of Leu

kemia in Man. I. The Discovery in Leukemic Tissues fromMan of a Cell-free Factor Producing Leukemia in Mice.Probi. Hematol. Moscow, 8: 10-21, 1957.

5. Bryan, W. R., Calnan, D., and Moloncy, J. B. BiologicalStudies on the Rous Sarcoma Virus. III. The Recovery ofVirus from Experimental Tumors in Relation to InitiatingDose. J. Nati. Cancer Inst., 16: 317-335, 1955.

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