The Lucke Frog Kidney Tumor and its Herpesvirus

AMER. ZOOL., 13:97-114 (1973).

The Lucke Frog Kidney Tumor and its Herpesvirus

ROBERT G. MCKINNELL

Department of Zoology, University of Minnesota, Minneapolis, Minnesota 55455

SYNOPSIS. Northern leopard frogs are afflicted with a spontaneous malignant neoplasmof the mesonephros. A herpesvirus is invariably associated with tumors obtained fromfrogs hibernating 30 days or longer and in tumors of frogs taken from breeding ponds.Tumors obtained from prehibernating frogs do not have viruses as detected by elec-tron microscopy but virus particles are found in some tumors within 7 days after theonset of hibernation. Tumors of frogs maintained at warm temperature in the labora-tory do not have viruses and tumors of frogs maintained at cold temperatures in thelaboratory do contain viruses. However, the production of viruses in the laboratoryfollows a distinctly slower chronology than that which occurs in nature. Injection ofcell fractions containing the herpesvirus into frog embryos induces tumors near thetime of metamorphosis in many experimental animals. Embryos injected with tumorextracts not containing herpesviruses do not develop tumors. Environmental andlaboratory observations are discussed which may relate to natural transmission of thispresumed viral oncogenic agent.

The cell biology correspondent of Na-ture recently stated: "The Lucke' adeno-carcinoma, however, still offers what seemsto be the best opportunity to test the onco-genic potential of a herpes virus" (Anony-mous, 1971). What follows is a review ofthe Lucke herpesvirus and the renal tu-mor with which it is thought to be relatedetiologically. A historical note is in order:The renal tumor of the leopard frog wasthe first neoplasm thought to be caused bya herpesvirus.

What are herpesviruses?

Herpesviruses contain a double-strandedDNA core that is enclosed within a capsid

This study was supported by Grant 990-CT fromthe Damon Runyon Memorial Fund for CancerResearch, Inc., New York, N. Y., and Grant GB-29482 from the National Science Foundation. Theelectron micrographs were made by Mrs. VirginiaL. Ellis. The author expresses his appreciation toLyle M. Steven, Jr. and David C. Dapkus, gradu-ate students at the University of Minnesota, andto Dr. William E. Boernke, Nebraska WesleyanUniversity, for their help in collecting frogs onfield trips and for their stimulating discussions.Mark Halverson and Gerry Geisbauer, undergradu-ates of the University of Minnesota, were the scubadivers of this study. Officer Miles Pooler of theState of Minnesota Department of Natural Re-sources, Division of Enforcement and Field Ser-vices provided valuable information and assistancein the collection of frogs.

of icosahedral symmetry composed of 162hollow capsomeres. The core and the cap-sid together are known as the nucleocap-sid. The nucleocapsid measures about 100nm diameter and the mature virion, en-veloped in a membrane, is 150 to 250 nmdiameter. The taxonomy of viruses includ-ing herpesviruses has been presented byWildy (1971) and Melnick (1971, 1972).Herpesviruses afflict a variety of animalsthat include frogs, snakes, chickens, pigs,dogs, cats, and man. Current informationabout this widely distributed and abundantvirus group has been reviewed by Roizman(1969, 1971, 1972). Although viral onco-genesis has a history exceeding 60 years(Rous, 1911; Gross, 1970; Howatson, 1971),there is renewed interest in herpesvirusesbecause several have been linked with spon-taneous or "natural" cancer in animals andman (Goodheart, 1970; Green, 1970; Ep-stein, 1971; Klein, 1972a). A symposiumwas held recently at Christ's College, Cam-bridge, England, concerning herpesvirusesassociated with cancer (Biggs et al., 1972).The northern leopard frog, known as Ranapipiens (Schreber) (see Brown, 1972, forinformation concerning this species group)has been shown to be the host to two dif-ferent herpesviruses. One of the viruses isthe probable etiological agent of the frogrenal adenocarcinoma. The other virus has

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ROBERT G. MCKINNELL

an unknown relationship, if any, to thetumor of leopard frogs.

The frog renal adenocarcinoma

Leopard frogs from the Northern UnitedStates are susceptible to an extremely com-mon (McKinnell, 1955) renal tumor (Fig.1). The tumor was initially described bySmallwood (1905), who, while describingand illustrating a mitotically active renaladenocarcinoma, reputed it to be of adre-nal origin. The histology of Smallwood'stumor is better illustrated by Murray(1908) who stated, "Histologically the cellsdo not present the slightest trace of thecharacteristic granules of the adrenal tis-sue. . . ." Lucke again illustrated Small-wood's tumor and concluded that it, as wellas his large series of frog tumors, was ofrenal origin (Lucke, 1934). Thus, Luckewas not the first to describe the commonrenal neoplasm but his first and subsequentpapers were important in two significantways. First, he correctly identified the tu-mor as renal in origin, and second, he ex-amined a sufficiently large series of tumorssuch that he could report on nuclear in-clusions, an observation Smallwood andMurray were not likely to have made ina mitotically active tumor. Lucke described

! ! l II

FIG. 1. Kidney of northern leopard frog with sev-eral tumorous nodules greater than 5 mm diame-ter (from McKinnell, 1965).

intranuclear inclusions in the epitheliumof certain tumors that were similar to theintranuclear inclusions of herpes and otherknown virus diseases (Lucke, 1934, 1938,1952).

The frog kidney tumor seems to be moreprevalent during cold seasons than duringthe warm time of year. This observationwas initially made when comparing tumorprevalence in commercially obtained frogsbought during the cold months with tumorprevalence in frogs collected in the fieldduring warm weather (McKinnell, 1967).Later, in a study of frogs collected entire-ly in the field, we reported finding onetumor from among 1,554 frogs collected inJune, July, and August contrasted with 18renal tumors from 491 frogs collected dur-ing the month of April when frogs wereleaving their overwintering lakes (McKin-nell and McKinnell, 1968). I reported thattumor prevalence may be as high as 10.5%in Otter Tail County, Minnesota (McKin-nell, 1969). The reports of prevalence oftumors in my studies have been by obser-vation of kidneys in dissected fresh speci-mens. I used fluorescent lighting and alow power dissecting lens to detect tumorsat autopsy. Accordingly, I feel that it is un-likely that a tumor of i/£ mm or greaterescaped my detection. However, it is quiteobvious that tumors smaller than i/2 mmexist. Indeed, a recent histological studyrevealed that up to 99.5% of commerciallyobtained South Dakota leopard frogs haverenal tumors (Marlow and Mizell, 1972).The Marlow and Mizell study indicates theremarkable susceptibility of leopard frogsto tumor formation. It is unfortunate,therefore, that the precise locality of frogcollection was not reported in their papernor did they indicate how long the frogshad been stored in the dealer's facilitiesprior to shipment to them. Prevalence ofgrossly detectable tumors and tumor finestructure is affected by temperature andstorage (Zambernard and Vatter, 1966a;Rafferty, 1962, 1963b). Accordingly, itwould be useful to know these conditionsfor their tumor-prone sample of frogs. Mystudents and I are currently studying tu-

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THE LUCRE TUMOR AND ITS HERPESVIRUS 99

mor prevalence, as detected by histology,of unstored frogs collected by us near LakeKampeska at Water town, South Dakota,because of the stimulating paper of Mar-low and Mizell.

Distribution and prevalence studieswould be of value in interpreting pro-posed modes of transmission of the tumorvirus. Unfortunately, exceedingly little isknown of tumor distribution. It is knownthat frog renal tumors are found in Min-nesota (McKinnell, 1969; McKinnell andEllis, 1972rt), and Vermont (Auclair, 1961).Frogs collected from intermediate placesbetween South Dakota and New Englandare susceptible to tumor formation if storedand maintained in the laboratory for sev-eral months (Rafferty, 1967). We were un-able to find renal tumors among 932 NorthDakota and 466 Louisiana leopard frogsat autopsy despite careful histological ex-amination of all suspicious appearing areasin the kidneys. Both populations of leop-ard frogs were shown to be susceptible totumors when treated under tumor promot-ing conditions (McKinnell and Duplan-tier, 1970).

The herpesvirus of the Lucke renaladenocnrcinoma

Despite the early observations of Luckdconcerning the similarity of the Luck£ tu-mor nuclear inclusions to inclusions seenin herpes infections, it was not until 1956that the presence of viruses in frog tumorcells was confirmed by direct electron mi-croscopic observation (Fawcett, 1956). Faw-cett observed "virus-like" particles in in-clusion-containing tumor cells that bore astriking resemblance to the particles de-scribed in cells known to be infected withherpes simplex (Morgan et al., 1954). Faw-cett's viruses varied in diameter between95 to 110 nra and were composed of adense core enclosed within either a singleor double membraned capsid. The parti-cles were found to be invested with an ad-ditional membrane or envelope on the out-side of the cell.

Virus particles of the frog tumor were

first isolated by homogenization followedby equilibrium centrifugation. Negativestaining of virus particles prepared in thismanner and examined with the electronmicroscope revealed a capsid with triangu-lar facets structured of five shared capso-meres per edge with edges of a facet en-closing three central capsomeres. A capsidso structured may be calculated to contain162 capsomeres with the facets forming apolygon of icosahedral symmetry (Lunger,19646). The Luck£ tumor virus was thusshown to have viral architecture identicalto other herpesviruses (Wildy et al., I960;Home and Wildy, 1961).

Herpesviruses are by definition virusescontaining DNA. What do we know aboutthe DNA of the Lucke1 tumor herpesvirus?The first unequivocal demonstration thatthe core of the virus was indeed DNA wasthe digestion study of Zambernard andVatter (19666). Trypsin, pepsin, andRNase failed to digest cores of virus par-ticles but DNase completely removed thestructures from thin sections. Fortunatelyviruses are abundant in spontaneous tu-mors such that they can be readily isolatedfor study of DNA. It has been estimatedthat there is an average of 5 X 1011 virusparticles per gram of algid tumor (Toplinet al., 1969). The abundant virus in thetumor made possible characterization of itsDNA and comparison of its DNA withother herpesviruses (Wagner et al., 1970;Gravell, 1971; Bachenheimer et al., 1972).

Thin section electron microscopy of in-clusion-containing Lucke1 tumors has nowbeen studied in a number of laboratories(Fawcett, 1956; Lunger, 1964o,fr; Lunger

et al., 1965; Zambernard and Mizell, 1965;Zambernard et al., 1966; Stackpole, 1969).The studies are in essential agreement. Thevirus is found in different developmentalstages (Fig. 2). Nuclear virus particle typesinclude single and double membraned cap-sids lacking cores (Fig. 3a), single mem-braned capsids with cores (Fig. 3b); freecores, and double membraned capsids withcores (Fig. 3c). The cytoplasm containsdouble membrane particles with a core andthe same type particle but enveloped in a

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100 ROBERT G. MCKINNELL

FIG. 2. Renal tumor cell showing productive viralinfection. Tumor detected at autopsy of frog col-

lected 26 March 1971 from a river in southeastPope County, Minnesota. X 14,000.

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loosely fitting membrane (Fig. 3d). Final-ly, only fully mature (i.e., enveloped) par-ticles are found extracellularly (Fig. 3e).The virus particles described are found intumors of hibernating and post-spawningfrogs. Tumors of prehibernating frogs donot have virus particles detectable with theelectron microscope (Fig. 4).

With such a variety of assembly stages, itwould seem that an ontogeny of virion syn-thesis would have been proposed andwould have been shown to be essentiallycorrect after so many years. Ontogeneticsequences have indeed been proposed(Fawcett, 1956; Stackpole, 1969), but I con-cur with Lunger et al. (1965) who wrote:"The interpretation of viral maturationevents derived from thin-section studies of'spontaneous' tumors must be undertakenwith particular caution, because unless atime sequence system is used (as may beafforded by tissue culture), it is difficult todetermine the precise sequence of viralmaturation events and the direction of par-ticle movement within the cell."

The relevance of environmental studies toviral ontogeny

The studies of particle assembly thus farhave been of tumors obtained commercial-ly. It has been known for some time nowthat the morphology of leopard frog renaltumors is exceedingly vulnerable to fluctu-ations in temperature as revealed by lightmicroscopy (Roberts. 1963) and electronmicroscopy (Zambernard and Vatter,1966a). Maintenance of tumor-bearingfrogs for as few as 3 days at laboratory tem-perature (20 to 22 C) results in the lysisof the majority of inclusion-containing tu-mor cells. Commercially obtained frogs aretransported from the field, stored in col-lectors' facilities, trucked to the dealer,stored again under crowded conditions,flown to the buyer's city, and retrucked totheir final destination where another stor-age period of variable length may occur(Gibbs et al., 1971). During the time thatelapses from collection to fixation for elec-

tron microscope examination, the tumor-bearing frog may experience a number oftemperature changes and other less wellknown factors that may alter the fine struc-ture of its tumor cells. Since frogs of com-mercial origin have an unknown history,it seemed reasonable to me that a study of"spontaneous" tumors collected in the fieldwould be useful in interpreting proposedsequences in viral assembly.

Chronology of virus replication in nature

We showed several years ago that tu-mors obtained from frogs collected in coldwater invariably contained virus particles(McKinnell and Zambernard, 1968). Short-ly thereafter, we reported that calid tumorscollected in the late summer and autumnwere devoid of viruses as detected by elec-tron microscopy (Zambernard and Mc-Kinnell, 1969). It would seem, therefore,that viral replication must have its onsetafter the host frogs go into hibernation andviruses must disappear from the tumorssometime after the emergence of frogs fromthe lakes but prior to late summer. Thisis indeed the case. We examined frogs withtumors immediately prior to entering thelakes for hibernation and 4, 7, 13, and 16days after the frogs were in cold water.Mature herpesviruses were observed insome tumors of frogs that had been in lakewater for 7 days (McKinnell et al., 1972).All tumors of frogs hibernating for 1month or more contained virus particles(McKinnell and Ellis, 1972a). We still donot know precisely when the last virusesleave tumors with the onset of hot weath-er, but we do know that they persist for atleast 52 days after the emergence of frogsfrom the overwintering lakes and for atleast 42 days after fertilization of eggs (Mc-Kinnell and Ellis, 1972&).

Studies of the chronology of virus pro-duction in tumors obtained from frogs col-lected in nature and never stored in thelaboratory nor shipped by a dealer produceunexpected results. The first surprise thatwe noted was the short time after the onset

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l i t ROBERT G. MCKINNELL

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of hibernation that viral replication be-gins. It should be noted that tumors main-tained as eye-chamber transplants in thelaboratory for less than 9 weeks did notshow a productive infection (Mizell et al.,1969«) and viruses cannot be detected priorto 7 weeks in tumors explanted in vitroand maintained at temperatures that variedfrom 4 C to 11.5 C (Breidenbach et al.,1971; Skinner and Mizell, 1972). Note thatwe reported viruses in all stages of replica-tion in as few as 7 days after the onset of hi-bernation (McKinnell et al., 1972). At 20to 22 C, tumor cells with nuclear inclusionsbegin lysis within 1 day and no inclusionsare found after 1 week (Zambernard andVatter, 1966a). However, in the warm Min-nesota spring, mature herpesviruses arefound in frogs as long as 42 days afterspawning (McKinnell and Ellis, 1972&).These observations suggest that cautionshould be exercised in extending observa-tions from the laboratory to what may ormay not occur in nature.

Is the Lucke herpesvirus the etiologicalagent of the renal adenocarcinoma?

In a recent review on herpesviruses andoncogenesis, Klein (1972a) makes the judg-ment that a herpesvirus is the "most prob-able" etiological agent of the frog tumor.I concur with this judgment for the fol-lowing reasons. Tadpoles injected withpreparations containing the Lucke herpes-virus frequently develop renal tumors atmetamorphosis (Tweedell, 1967, 1969,1972; Mizell et al., 1969&; Toplin et al.,1971; McKinnell and Tweedell, 1970;Naegele and Granoff, 1972). It is especiallysignificant that as high as 92% of embryosinjected with Lucke1 herpesvirus-containingcell fractions develop tumors, but "virus-

free" material derived from non-inclusiontumor cells has no oncogenous potentialwhen injected into embryos (Tweedell,1967). While caution should be used in in-terpreting these results (because of thepossibility that the cell fractions may con-tain other unidentified viruses; Green,1970), I am inclined to accept these resultsin support of a working hypothesis of her-pesvirus etiology.

The other kind of evidence that supportsviral etiology of the tumor is epidemiologi-cal (Goodheart, 1970). Although there is avariable expression of viruses and nuclearinclusions in commercially obtained frogswith tumors (Fawcett, 1956; Marlow andMizell, 1972), one does not expect nor doesone find a capricious relationship betweenthe tumor and its presumed etiologicalagent when one examines frogs with tu-mors taken directly from their natural habi-tat. The herpesvirus is found to be associ-ated invariably with the tumor during hi-bernation of the host (McKinnell and El-lis, 1972a), after emergence from hibernat-ing (McKinnell and Zambernard, 1968),and in tumors of frogs from breedingponds (McKinnell and Ellis, 1972b). Theonly time in the life cycle of afflicted frogswhen tumors are devoid of viruses as de-tected by electron microscopy is immedi-ately prior to hibernation (Zambernardand McKinnell, 1969; McKinnell et al.,1972). There is emerging an impressive ar-ray of observations that argue that the tu-mors lacking a productive virus infectionhave a latent virus.

Laboratory observations are in essentialharmony with environmental observations,i.e., tumors held at high temperature seemto be bereft of viruses and tumors held atlow temperature manifest virus production(Collins and Nace, 1970; Mizell et al., 1968;

FIG. 3. Stages of assembly of Luck£ herpesvirus.a. Single and double membraned capsids. X76.0Q0.b. Single membraned capsid with DNA core. X76,-000. c. Double membraned capsids with DNA cores(nucleocapsids). X46.000. d. Mature virion in cyto-plasm. X 76,000. e. Extracellular mature virion withloose envelope. X 76,000. The viruses of 3a, b, d,

and e were from a tumor found at autopsy of afrog collected 26 March 1971 from southeast PopeCounty, Minnesota. Viruses in 3c were from a frogtumor collected 1 December 1970 from a streambelow Lake Calhoun, Kandiyohi County, Minne-sota.

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ROBERT G. MCKINNELL

FIG. 4. "Virus-free" calid tumor obtained from a jaceut to Diamond Lake, Kandiyohi County, Mill-prehibernating frog collected 18 October 1970 ad- nesota. X 17,600.

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Mizell et al., 1969a; Stackpole, 1969; Mor-ek, 1972; Breidenbach et al., 1971; Skinnerand Mizell, 1972). Antiserum preparedagainst purified Lucke herpesvirus revealsthe presence of viruses in the cold-tempera-ture tumors but fails to react with calidtumor or normal tissue (Paul, S. M., etal., 1972). The question that seeks an an-swer, then, is whether or not the warmtemperature tumor in the laboratory andin nature contains a latent herpesvirus.Green (1969, 1970) suggested that the an-swer to this question is important in es-tablishing the viral etiology of the Luck6tumor.

Multiple viral genome equivalents havebeen found in "virus-free" human tumorsthought to be caused by a herpesvirus (zurHausen and Schulte-Holthausen, 1970,1972; zur Hausen et al., 1970). Thus, asuspected etiological agent can indeed per-sist in a masked form in a tumor cell. Isthere a covert herpesvirus in the non-pro-ductive Lucke summer tumor? Detectionof viral DNA or viral specified mRNA incalid ("virus-free") tumors would fulfillthe first of Koch's postulates.

Mizell (1972) described the unpublishedstudies of Collard and others who reportthe detection of a covert infection in warmtumors by presenting evidence of tran-scription of herpesvirus genetic informa-tion following the procedure of Fujinagaand Green (1968). Thus, it seems thatKoch's first postulate, which requires thepresence of the etiological agent in all le-sions of the disease, is fulfilled in the frogrenal tumor. An unidentified skeptic, how-ever, has stated: "That Lucke adenocarci-noma cells are infected with a persistentherpesvirus does not, however, prove any-thing" (Anonymous, 1971). However, de-spite the still unproven etiology of the her-pesvirus, it seems that in the renal adeno-carcinoma of leopard frogs, we already havea tumor system where virus replication istemperature-sensitive and tumorigenesis oc-curs at a temperature that does not allowfor a productive infection, a situationwhich has been laboriously sought in othertumor systems (Klein, 1972&).

Concerning the integrity of the tumor cellgenome and the proposed viral etiology

Some somatic nuclei of selected fully dif-ferentiated adult plants contain genetic in-formation that is competent to programfor the production of another entire plant(Steward, 1968). What can be said for somenormal plant cell nuclei can be extendedto at least one plant tumor. Thus, tobaccocrown gall tumor cells, when properlymanipulated, can be provoked into form-ing normal tobacco plants (Braun, 1959,1972). Two generalizations are possiblefrom these experiments with plant cells.First, there is direct evidence that genomicintegrity is maintained throughout devel-opment in at least some normal cell typesin plants; and second, whatever the natureof the change in nuclei of one plant tumor,the change is a functional one and involvesno developmentally significant loss or al-teration of DNA nucleotide sequence. Ani-mal biologists and medical scientists havebeen chided for being unaware of these ex-periments (Stonier, 1972).

The Luck^ renal adenocarcinoma isunique among animal tumor systems, inthat so far, it alone has been characterizedby nuclear transplantation. The amphibiannuclear transplantation procedure is atechnique that is designed to reveal thenature of the genome of developing andmature amphibians. The technique in-volves the removal of the maternal chro-mosomes from unfertilized eggs, eithermanually with a microneedle (Porter,1939) or with a microbeam ruby laser (Mc-Kinnell et al., 1969&) and the insertion ofa somatic nucleus into the cytoplasm bymeans of a micropipette (King, 1967).Whereas nuclei of wild-type embryos(Briggs and King, 1952, 1955) and nucleiof mutant embryos (McKinnell, 1960, 1964)will direct recipient cytoplasm to form nor-mal frogs that can be reared to sexual ma-turity (McKinnell, 1962), normal in vitrocultured nuclei of genuinely adult frogshave at best the capacity to promote onlylimited development to an early tadpolestage (Lasky and Gurdon, 1970). Prior cul-ture in vitro is not a prerequisite for nu-

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clear transplantation of Lucke tumor cells.Indeed, tadpoles that will live for severalweeks have developed from the transplan-tation of primary renal tumor nuclei intoenucleated eggs (King and McKinnell,1960; King and DiBernardino, 1965; Mc-Kinnell et"al., 1969a).

What do the tumor nuclear transplan-tation studies reveal to us concerning theintegrity of the tumor cell genome and theproposed viral etiology? First, we may con-clude that the tumor cell genome is large-ly, if not entirely, intact. It is clear thatthe portion of the genome that controlsthe production of skin, heart, cilia, blood,muscle, nervous system, etc., that charac-terize the swimming tadpoles is intact andfunctional. It is as functional as the pre-sumed intact genome of normal culturedcells from adults because the latter pro-gram for no further development than dotumor cell nuclei. It remains for future ex-periments to demonstrate that the genomeof adult cells, either malignant or normal,contain all of the genetic information thatis present in a zygote nucleus.

The demonstrated pluripotency of thetumor cell genome may be significant inthe interpretation of the biological prop-erties of a covert viral infection. Is there alatent virus in the tumor nuclear trans-plant tadpole? This question can be an-swered (McKinnell and Ellis, 1972a). Ifthere is indeed a herpesvirus genome inthe tadpole produced by tumor nucleartransfer, then it suggests that reversion tonormal, or near normal, differentiative ac-tivities is not impeded by the virus andthat presence of the virus, per se, does notconvey a malignant state. Further, just asthe demonstration of viral genome activityin calid tumors relates to the etiology ofthe tumor (Green, 1969), presence of viralgenome in the highly differentiated cellsof the tumor nuclear transplant tadpolewould suggest an intimate association ofthe virus with the tumor nucleus.

Is transmission of the Lucke herpesvirusvertical or horizontal?

Horizontal transmission refers to the

spread of an etiological agent among mem-bers of a single generation. The period oftime from exposure to the agent to the de-velopment of the disease is characteristical-ly brief. Vertical transmission refers to con-tagion from one generation to another. Along latent period is encountered betweentime of exposure and the manifestation ofthe disease. Transmission of the mousemammary carcinoma agent (Bittner, 1957)is from the mother to her young via milkand is accordingly a model for verticaltransmission (Gross, 1944, 1949, 1970). Thequestion posed by the caption of this sec-tion cannot be answered at the presenttime. However, it seems reasonable fromwhat is known about the biology of thefrog renal adenocarcinoma and other tu-mor systems to postulate that transmissionof the frog tumor agent is vertical.

Is there any evidence to support whatwe deem the less likely spread of the Luck£tumor virus by horizontal transmission?For transmission to occur, we need thepresence of susceptible hosts in close prox-imity to an infectious agent with an ap-propriate route for the latter to transmitto the former.

Northern leopard frogs overwinter inlakes and rivers that freeze over with athick blanket of ice. They survive in thewater below the ice cover. Despite the num-ber and size of Minnesota lakes, frogs maybe crowded during hibernation. Brecken-ridge (1944) described the sand bottom ofa stream below a dam in the winter as"literally paved with hundreds of closelycrowded leopard froes." I have made simi-lar observations. Minnesota conservationofficer Dan Pooler and I collected 194 largefrogs in approximately one hour in astream below Lake Cynthia in Scott Coun-ty, Minnesota, on February 20, 1971.Ninety-four large leopard frogs were col-lected in about 10 minutes in the streambelow the dam of Lake Calhoun in Kandi-vohi County on November 27, 1970 (Fig.5). Frogs sometimes accumulate in greatnumbers where streams enter lakes. Theinflowing water causes a small area ofmelted ice to be maintained and it is

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T H E LUCRE TUMOR AND ITS HERPESVIRUS WT

FIG. 5. The abundance of frogs that sometimesaccumulates in a stream below a lake is indicated

thought that frogs congregate at such placesbecause of higher oxygen tension (Fig. 6).I would like to suggest that such crowdingmay be unusual, however, because scubadivers (Fig. 7) working for my laboratorywere able to find only a few frogs on thebottom of Diamond Lake in KandiyohiCounty, Minnesota. The few frogs that thescuba divers brought up from under theice were found sitting individually on thesandy lake bottom, generally among reedsand other aquatic plants. Our underwaterobservations of hibernating frogs may becompared with those of Emery et al.(1972). It should be noted that DiamondLake harbors a population of frogs that isexceptionally tumor-prone but adults thus

by this single scoop of a net. Kandiyohi County,Minnesota.

far have not been found huddled in greatnumbers during hibernation. Althoughboth Cynthia and Calhoun Lakes havefrogs that are exceedingly crowded instreams below their dams, we have observeda paucity of tumors from the frogs obtainedin their streams.

If the relationship of the extent ofcrowding (and hence the likelihood ofadults encountering an oncogenic virus)is an unresolved matter, it may be usefulto inquire as to the susceptibility of adultsto viral infections leading to tumorigen-esis. It has long been known that neonatalmammals are more susceptible to viral on-cogenesis than are adult mammals (An-dervont et al., 1942; Gross, 1951; Eddy et

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*»••

FIG. 6. Melted area of a lake adjacent to an inletof a stream with an accumulation of hibernatingfrogs. Douglas County, Minnesota.

FIG. 7. Scuba divers preparing to enter lake insearch of hibernating frogs. See text for descrip-tion of frogs on lake bottom. Diamond Lake, Kandi-yohi County, Minnesota.

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al., 1958; Eddy, 1964; Law and Ting, 1965;McCoy et al., 1972). If anurans resemblemammals with respect to susceptibility toviral oncogenesis, one would predict thatearly developmental stages (tadpoles)would be more vulnerable to a virus-in-duced tumor infection than adult animals(frogs).

Is there any evidence to suggest thatadult frogs are susceptible to new infectionleading to tumor formation? Althoughboth Luck6 (1952) and Duryee (1956) re-ported that frogs injected with cell frac-tions prepared from tumors developedtumors, it should be noted that Roberts(1963) and Rafferty (1963a) reported nodifference in tumor incidence in frogs re-ceiving no inoculum or in those receivingextracts from normal kidney and kidneytumor. High incidences of tumor in un-inoculated frogs maintained in the labora-tory were reported by Rafferty and Raf-ferty (1961) and Rafferty (19636). DiBer-ardino and King (1965) reported a higherprevalence of tumors in laboratory frogsthat were crowded than in those that werenot crowded, but their data differ fromthose of Rafferty (19636) who reported thesame high tumor rate in isolated andcrowded frogs. Rafferty (1964) concluded:"These results appear to show that theinjection of tumor extracts acceleratestumor development somewhat for the firstfew months but does not change the finalincidence, provided observations are con-tinued for a sufficiently long period." Iwould add that it is clear that a relativelyhigh percentage of frogs develop tumorswith maintenance in the laboratory, butthere is at the present time little evidencethat the tumors are the result of exposureto a virus during the period of storage. Ithink that the best explanation of the hightumor frequency among both inoculatedand uninoculated frogs is the succumbingof the host frog to a latent viral infectionacquired extremely early in life, and ac-cordingly, I conclude that as yet we haveprecious little reason to believe that adultfrogs differ in their susceptibility to viraloncogens from adult mammals.

Before excluding the possibility of hori-

zontal transmission of the presumed viraloncogen of the frog renal tumor, it maybe well to consider the evidence of hori-zontal transmission of Marek's diseaseherpesvirus (Solomon et al., 1970; Witter,1972), evidence of horizontal transmissionof the turkey herpesvirus (Witter et al.,1970; Paul, P., et al., 1972), and the sug-gestion of horizontal transmission of theherpesvirus thought to be the etiologicalagent of Burkitt's lymphoma (Wright,1967).

If contagion of the Lucke" herpesvirus isvertical, where in the life cycle of the hostfrog is the agent passed from one genera-tion to the next? Transmission of theLucke herpesvirus may be via a latentvirus carried in either the eggs or sperm.It is certainly possible, by analogy withother tumor systems, that healthy adults(i.e., adults that do not manifest a tumor-ous kidney) carry the viral genome, notonly in the target kidney cell, but also ingametes. This would be similar to thesituation of vertical transmission describedby Gross (1970) who stated: "At the timeof actual transmission of the virus, the in-fecting carriers would be in perfect health,even though they would spread the dis-ease." The detection of latent viral DNAin gametes of otherwise healthy adult frogshas not been done, but it would seem thatit is at least technically feasible. The Ep-stein-Barr herpesvirus genome has been de-tected in "virus-free" tumor cells (zurHausen and Schulte-Holthausen, 1970,1972; zur Hausen et al., 1970).

If vertical transmission is not via a latentvirus but is accomplished by means of amature virion, when and where in the lifecycle is it most likely that the virus passesfrom generation to generation? In con-sidering this question, once again it isinstructive to contemplate susceptibility,proximity of individuals to an infectiousagent, and a route to deliver the agentfrom the infected individual to the sus-ceptible host. Eggs and embryos of Ranapipiens are susceptible to cell extracts con-taining the Lucke herpesvirus with the for-mation of massive renal tumors followinginjection (Tweedell, 1967, 1969; Mizell et

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al., 19696; Toplin et al., 1971; McKinnelland Tweedell, 1970; Naegele and Granoff,1972). If eggs and tadpoles are equally sus-ceptible to the oncogenic agent, is thereany evidence to suggest which become in-fected in nature? This question can be re-phrased to ask is infection preamplexus(i.e., does the agent infect oocytes or

sperm) or is it postamplexus (i.e., doescontagion occur in breeding ponds)? Fromthe information at hand, it would seemthat both alternatives are possible.

What observations support the view thatthe Lucke herpesvirus infects gametes?Oocytes of the northern leopard frog arefully formed at the time the animal enterswater for hibernation (mid to late Octoberin Minnesota). Mature, infectious herpes-viruses are found in the tumors of frogswithin a few days after the frogs enter thewater for hibernation (McKinnell et al.,1972). The frogs remain in hibernation fora period of approximately 6 months. Dur-ing that time, oocytes may be exposed tomature virions via the blood in the folliclewall. Both the kidney and the ovary arewell vascularized and the blood is knownto carry another unrelated virus (Bernardet al., 1969), therefore, by analogy, it couldcarry the Luck£ herpesvirus. Non-jelly cov-ered body cavity oocytes (i.e., eggs thathave been ovulated but have not yet passedthrough the oviduct) may be exposed toherpesvirus-containing ascites fluid andmay acquire a latent infection. Ascites fluidof tumor-bearing R. pipiens has beenshown to contain mature herpesvirus andthe fluid has been shown to be oncogenicto tailbud tadpoles (Naegele and Granoff,1972). Accordingly, with the informationpresently at hand, vertical transmission viagametes prior to amplexus is clearly pos-sible.

Is vertical transmission after amplexusequally possible? Frogs reproduce in shal-low breeding ponds or swamps. Adultleopard frogs collected in breeding ponds42 days after spawning have tumors andthe tumors contain large numbers of ma-ture herpesviruses as revealed by electronmicroscopy (McKinnell and Ellis, 1972/;).Herpesviruses have been observed in the

urine of tumor-bearing frogs (Granoff andDarlington, 1969). Tadpoles are found inabundance in breeding ponds. Tadpolesare susceptible to tumor formation wheninjected with virus-containing extracts ofLucke tumors (Tweedell, 1967, 1969;Mizell et al., 19696; McKinnell andTweedell, 1970; Toplin et al., 1971). Thereis no reason to preclude the possibility thattumor-bearing frogs release viruses via theurine to the water of the breeding pondand that these mature viruses infect thesusceptible tadpoles that are crowded inthe pond. There is, of course, no informa-tion at present to suggest what portal thevirion might enter the susceptible host, i.e.,does the virus enter the tadpoles throughthe gills, gut, or some other portion of itsbody? It should be further noted that whilemethods have been developed for recoveryof viruses from large volumes of naturalwater (Hill et al., 1971; Berg et al., 1971),the Lucke herpesvirus has not yet beendetected in pond water.

Breeding ponds may contain many pairsof frogs in amplexus. Indeed, the intensityof reproductive activity in some Otter TailCounty, Minnesota, breeding ponds is suchthat I have on several occasions observedfrog menages a trois and a quatre and evena cinq. Post-amplexus transmission allowsfor infection of progeny other than thoseof the parents. Given the possibility thatinfectious virions may be acquired by tad-poles from the water, the progeny of un-afflicted adults would be exposed to virionsproduced by tumorous adults who are com-patriots of the unafflicted parents in thepond's conjugal congress. Rafferty (1964)suggested the possibility of transmission inbreeding ponds. Recovery of an active on-cogenic virion from the water should besought.

The other frog herpesvirus.

The probable etiological relationship ofthe Lucke renal adenocarcinoma and theherpesvirus associated with it is compli-cated by the fact that R. pipiens has atleast one other herpesvirus. The other her-pesvirus was originally isolated from

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pooled urine of tumor-bearing frogs (Raf-ferty, 1965) and has been known either asthe "tumor urine virus" (Lunger, 1966) orfrog virus 4 (FV-4) (Rafferty, 1967). Her-pesvirus FV-4 does not cause tumors wheninjected into tadpoles (Granoff et al.,1969) or into adults (Rafferty, 1967). Her-pesvirus FV-4 was propagated in an R.pipiens normal embryo cell line (producedby Dr. Jerome Freed, Institute for CancerResearch, Philadelphia) and was shown toreplicate in the nucleus, have a capsidstructured of 162 capsomeres with a diam-eter of 95-100 nm, and some extracellularparticles were enveloped. Hence, the FV-4is clearly a herpesvirus (Gravell et al.,1968). The DNA of the Lucke herpesvirusand FV-4 are quite different however. TheLuckd herpesvirus contains 45-47% gua-nine + cytosine (G -f- C) while FV-4 con-tains 54-56% G -f- C. The herpesviruses al-so differ imm,unologically (Gravell, 1971;Granoff, 1972). It is clear, therefore, thatFV-4 and the Lucke herpesviruses are dif-ferent. What then is its effect, if any, on thehealth of frogs? Rafferty (1967) reportedthat FV-4 caused massive edema in post-metamorphic frogs that had been exposedto the virus as tadpoles. Whether or notsimilar pathology occurs in nature is un-known.

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The Lucke Frog Kidney Tumor and its Herpesvirus

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