[CANCER RESEARCH 34, 3504—3508,December 1974]

Brief Communication

Occurrence of Mason-Pfizer Monkey Virus in HealthyRhesus Monkeys'

Mumtaz Ahmed, George Schidlovsky, Wlo Korol, Gary Vidrine, and John L. Cicmanec

The John L. Smith Memorial for Cancer Research, Pfizer Inc., Maywood, New Jersey 07607 fM. A., G. S., W. K., I. G. V.J, and Litton-Bionetics,Inc., Kensington, Maryland20795 fJ. L. C.J

SUMMARY

The Mason-Pfizer monkey virus (M-PMV) was originallyisolated from a spontaneous mammary adenocarcinoma of afemale monkey. Searching for evidence of M-PMV in healthyrhesus monkeys has led to the recovery of similar isolates fromcultures derived from normal mammary, fetal, and placentaltissues. The viral morphology as well as the process of virusformation observed with the new isolates are identical to theones reported for M-PMV where the doughnut-shaped type-Aparticle is incorporated into the viral nucleoid at the time ofbudding from the cell surface. The major internal protein,MW. 27,000 daltons, of the prototype M-PMV and those ofnew isolates from normal monkeys were found to beserologically identical. Although the presence ofM-PMV or itsantigens has appeared to be of common occurrence in tissuespecimens, sera from normal monkeys failed to show anyspecific reactivity against M-PMV by microimmunodiffusion orimmunofluorescence tests.

INTRODUCTION

The M-PMV,2 a virus with morphological features similar toRNA-containing viruses, was isolated from breast tumor cellsof a female rhesus monkey (9, 16). Although the oncogenicityof M-PMV has not yet been demonstrated, it is known tocontain a single-stranded RNA with sedimentation values of 60to 70 S and an RNA-dependent DNA polymerase (20). Fine eta!. (10) described the ability of M-PMV to transform rhesusmonkey cells in vitro. Recently, several investigators havereported virus particles similar in morphology to M-PMV incell lines derived from human tumors (7, 11, 13, 14, 17, 23,24). This report describes studies we have conducted toinvestigate the incidence of M-PMV in healthy rhesusmonkeys. The incidence of M-PMV in normal rhesus monkeypopulations has been examined by testing sera for specific viralantibody and by analyzing monkey breast tissues, placentas,

1 This work was supported by Contracts NO1-CP-3-3239 and

NO1-CP-43224within the Virus Cancer Progranof the National CancerInstitute.

2 The abbreviation used is: M-PMV, Mason-Pfizer monkey virus.

Received August 23, 1974; accepted September 23, 1974.

fetuses, and milk samples for the presence of M-PMV and itsantigens.

MATERIALS AND METHODS

Sources of Monkey Tissues, Milk, and Sera. Fetuses (60 to90 days old), placentas (pre- and postparturition), biopsiesfrom lactating breast, and sera were obtained from normalrhesus monkeys (Macaca mu!atta) housed at Litton-Bionetics,Kensington, Md. Milk samples from rhesus monkeys werekindly supplied by Dr. A. Bogden and Dr. W. Cobb of MasonResearch Institute, Worcester, Mass.

Tissue Culture. Tantalum gauze (Codman and Shurtleff,Randolph, Mass.) cultures of the breast tissue and the spongyportion of placental tissue of normal healthy monkeys wereprepared as described by Stewart et a!. (21). Briefly, 5- to8-mm pieces of the tissue were placed on the tantalum gauze(1 sq cm), and 4 to 6 of such preparations were transferredinto a vessel. Roswell Park Memorial Institute 1640 growthmedium with 20% fetal calf serum and usual amounts ofantibiotics was added, and the vessels were incubated by 37°.Monkey fetal and placental tissues were also cultured by theconventional technique of seeding cells following trypsinization of minced tissues.

Treatment of Milk. Monkey milk samples were treated asdescribed for purifying the mouse mammary tumor virus frommouse milk (6), with some modifications. Milk samples werediluted with an equal volume of 0.15 M EDTA in 0.9% NaClsolution and incubated at 4°for 15 mm. The solution wascentrifuged at 3000 X g for 15 mm, and the supernatant wasplaced in a centrifuge tube containing a discontinuous sucrosegradient [bottom cushion, 66% (w/v); top layer, 20% (w/v)].Following centrifugation at 80,000 X g for 3 hi, the band ontop of the 66% sucrose cushion pad was collected and treatedwith 10 volumes of ether before serological tests wereconducted. In order to obtain parallel positive controls,various dilutions of M-PMV were deliberately added to cowand monkey milk samples.

M-PMV Antiserum. The methods for preparation andcharacterization of the rabbit anti-M-PMV serum have beenreported earlier (3 , 8).

Screening of Normal Monkey Sera. Microimmunodiffusion

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SpecimenTested forNo.positive!

no.testedTechniquesSeraViral

antibody0!2910!47MID―IFBreast

culturesVirus and viral antigen3!! 8EM, MID, andIFFetalculturesVirus

Viralantigen1!22 8,115EMMIDorIFPlacentalculturesVirus

Viral antigen4,'8 4!4EM MID andIFMilksamplesVirus and viral antigenOi'8EM and MID

M-PMV inRhesusMonkeys

and indirect immunofluorescence tests were conducted asdescribed previously (3). For microimmunodiffusion tests,undiluted heat-inactivated monkey sera were used. M-PMVantigen was prepared by purifying and concentrating the virusreleased from either the cocultivated monkey mammary tumor(CMMT) cell line or chronically infected human lymphoblastoid (NC-37) cell line (15). Preparations containing 5 X10' â€t̃o 2 X 10' 2 virus particles were treated with 10 volumesof ether to release the major internal antigen, M.W. 27,000,before conducting microimmunodiffusion tests. For immunofluorescence tests, the monkey sera were diluted 1: 10 inphasphate-buffered saline, pH 7.4, before reacting on acetonefixed CMMT or infected NC-37 cells. Appropriate controls ofnormal monkey and human cells or extracts were used inimmunofluorescence and microimmunodiffusion tests.

Electron Microscopy. Cell culture pellets and tissue specimens were examined by thin-section electron microscopy asdescribed prevously (18). Milk samples, following EDTAtreatment, were analyzed by negative-staining electron microscopy as well as by pelletizing the treated milk in BEEMcapsules at 120,000 X g (average gravity units calculated in thecapsule) for 1 hr in a 50.1 SW rotor with special adaptors andthen examining the pellets in thin sections.

RESULTS

Studies of Sera. We had found earlier that the serum fromthe mammary tumor-bearing monkey from which M-PMV wasisolated did not contain any detectable antibodies to the virus(3).AsshowninTable1,wehavescreened291normalrhesusmonkey sera by microimmunodiffusion test and 47 sera byimmunofluorescence test, but no specific reactivity againstM-PMV antigens has been observed. Furthermore, adultmonkeys immunized with repeated doses of sucrose densitygradient-purified M-PMV produced only very weak antibodyagainst M-PMV as detected by microimmunodiffusion andimmunofluorescence tests (3). Failure to fmd antibody toM-PMV in monkeys may indicate that the monkeys areexposed to the virus early in life and thus become tolerant tothe M-PMV antigens. It became apparent that a betterapproach might be to screen for virus and its antigen in orderto determine the incidence of M-PMV in monkey populations.

Studies of Breast Tissue. To date, 18 of 28 mammarybiopsies obtained from normal lactating rhesus monkeys havebeen successfully grown in explant cultures. Fifteen of these

cultures were destroyed within 4 weeks after initiation due tolatent simian foamy virus infection (19). However, 3 mammary cultures grew rapidly and showed intracytoplasmicM-PMV antigens and budding M-PMV-like particles within 3weeks after initiation of culture (1). Mammary biopsies, takenfrom the same 3 monkeys 8 to 10 weeks later, again yielded avirus similar to M-PMV. Later passages of 2 of these cultures,however, also showed a massive infection with simian foamyvirus. The 3rd mammary culture (X-381) has gone throughmore than 60 passages in which simian foamy virus is notevident but yields infectious M-PMV-like virus in the culturefluid. The karyotyping studies performed by Dr. H. E. Holden,Pfizer Central Research, Groton, Conn., indicate that theX-38l cell line contains characteristic marker chromosomes ofMacaca mu!atta. No chromosomes of human characteristicswere observed. A detailed description of this cell line and thebiological, serological, and biochemical characteristics ofX-38l virus, which are very similar to those of M-PMV, will bereported elsewhere.

Studies of Fetuses. To date, 22 monkey fetuses have beenscreened after culture for M-PMV particles. Only 1 has shownM-PMV-like particles by electron microscopy. However, 8 outof 15 fetal cultures, including the one that produced virusparticles, have demonstrated M-PMV antigens by microimmunodiffusion and/or immunofluorescence technique (Table1). Treatment of 3 immunofluorescence-positive fetal cultureswith 5-iododeoxyuridine (20 pg/ml) appeared to intensify thestaining by immunofluorescence technique but failed to causeproduction of complete virus particles as detectable byelectron microscopy. Cocultivation of these fetal cultures withhuman lymphoblastoid suspension (NC-37) cells also failed toproduce virions.

Studies of Placentas. We have reported the presence oftypical type-C particles in rhesus placentas (1 8). Virus particleswere most frequently observed on mesenchymal cells adjacentto fetal capillaries underlying the basement membrane of theplacental trophoblasts. They were also seen within syncytialtrophoblasts but not in the cellular trophoblasts.

Initial attempts to grow rhesus placentas yielded verydiscouraging results. Only 1 out of 8 placentas grew in culture.To increase the chances of recovering virus from the placentaltissue, 3 placentas were cocultivated with human NC-37 cellline. Fig. 1 shows a monolayer of monkey cells of polygonalmorphology that originated from a rhesus placenta (FTP-i),previously determined to contain type-C virus particles byelectron microscopy. FTP-i and all placentas cocultivated with

Table 1Detection ofM-PMV and its antigen and antibody in normal rhesus monkeys

a MID, microimmunodiffusion; IF, immunofluorescence; EM, electron microscopy.

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M. Ahmed et a!.

NC-37 cells demonstrated intense fluorescence when testedwith anti-M-PMV serum (Fig. 2). Electron microscopicexamination confirmed the presence of M-PMV in thesecultures (Fig. 3). When tested against M-PMV antiserum,FTP-l virus showed a line of identity with the precipitin lineproduced by the major internal M-PMV protein, M.W. 27,000daltons, in the microimmunodiffusion test (Fig. 4). The KCtest, which has been recently developed for detection ofM-PMV infection (2), was also found to be positive with theFTP-i culture. M-PMV antigen was also detected in theplacental tissue before culturing (Fig. 5).

Studies of Milk Samples. Following EDTA and ethertreatment, each of the milk samples from 8 different rhesusmonkeys failed to react in the microimmunodiffusion testagainst specific M-PMV antiserum. Experiments designed tostudy the sensitivity of the microimmunodiffusion test fordetecting M-PMV antigen in the milk showed that at least 7 Xl0@ virus particles per ml were needed to detect virus by thismethod.

DISCUSSION

M-PMV infections appear to be of common occurrence inrhesus monkey populations. By searching various tissues, wehave found virus particles and/or viral antigens not only inbreast biopsies but also in placentas and fetuses. The X-381virus isolated from normal breast tissue has several biologicaland biochemical features of M-PMV (1 , 22). Finding M-PMV inplacental and fetal tissues leads us to suspect that the agent istransmitted vertically ; whether this occurs in utero or genetically remains to be determined. Further evidence supportingvertical transmission is offered by data indicating that the virusis poorly antigenic in monkeys. Failure to detect antibodiesagainst M-PMV internal proteins in 291 normal monkey seratested by microimmunodiffusion or in 47 sera tested byimmunofluorescence also suggests that perhaps monkeys aretolerant to these antigens.

It must be mentioned that negative immunodiffusion testsreported here with rhesus milk do not necessarily indicate thatthe M-PMV or its antigen is not present in these samples.Studies designed to determine the sensitivity of the microimmunodiffusion test showed that at least 7 X i0@ particles perml of milk were needed to detect the precipitin line against theantiserum used.

It was of interest to note that the monkey placentas thatshowed type-C virus particles prior to culturing demonstratedpredominantly M-PMV-like particles after culturing. Thisphenomenon occurred with all 4 different placentas that weresuccessfully cultured. However, the M-PMV antigen, wasdetected before and after initiating cultures. It is likely thatthese types of viruses can coexist in the placental tissues of therhesus monkeys or, alternatively, they may represent 2morphologicalformsof the samevirus.

Viruses that are similar in morphology and description toM-PMV have been reported in cell lines transformed by humanleukemic blood (4) and in certain cell lines established fromtumors (7, 11, 13, 14, 17, 23, 24). M-PMV-like particles werealso observed in a human brain culture that was spontaneouslytransformed (12). Although a serological relationship wasobserved between human and monkey M-PMV isolates (5 , 17),

it is not as yet known whether they all possess identicalbiological activity. Future studies of human sera and tissuespecimens conducted to determine the incidence of theseM-PMV-like viruses in a human population might provideresults that further parallel the data we have presented here.

ACKNOWLEDGMENTS

The authors thank Dr. Robert Bassin and Dr. Jeffrey Schlom forhelpful suggestions and review of the manuscript. The technicalassistance of Susan Herles, Mary Manousos, and Helen Molnar is alsogratefully acknowledged.

REFERENCES

1. Ahmed, M., Korol, W., Schidlovsky G., Vidrine, J., and Mayyasi, S.Detection of Mason-Pfizer Virus and Its Antigens in NormalMonkey Mammary Tissue and Embryonic Cultures. Proc. Am.Assoc. Cancer Res., 14: 34, 1973,

2. Ahmed, M., Korol, W., Yeh, J., Schidlovsky, G., and Mayyasi, S. A.Detection of Mason-Pfizer Monkey Virus Infection with HumanKC Cells CarryingRous SarcomaVirus Genome. J. Nail. CancerInst., 53: 383—387,1974.

3. Ahmed, M., Mayyasi, S. A., Chopra, H. C., Zelljadt, I., and Jensen,E. M. Mason-Pfizer Monkey Virus Isolated from SpontaneousMammary Carcinoma of a Female Monkey. 1. Detection of VirusAntigens by Immunodiffusion, Immumofluorescence and VirusAgglutination Technique. J. Natl. Cancer Inst., 46: 1325—1334,1971.

4. Andzhaparidze, 0. G., Lotte, V. D., and Stepanova, L. G.Morphology and Morphogenesis of an Oncogenic RNA-ContairnngVirus (LPV strain) Isolated from Man in Human Cell Culture.Vopr. Virusol., 18: 36—38,1973.

5. Bauer, H., Doams, J. H., Watson, K. F., Molling,K., Gelderblam,H., and Schafer,W. Oncornavirus-likeParticlesin HeLaCells. II.Immunological Characterization of the Virus. Intern. J. Cancer, 13:254—261,1974.

6. Blair, P. B., Smoller, C., Bonhag, R. S., and Weiss, D. W.Immunology of the Mouse Mammary Tumor Virus (MTV).Electron Microscopic Studies of Interaction between MTV andSpecific Antibody in Immunodiffusion. J. Natl. Cancer Inst., 40:1325—1338,1968.

7. Bykovsky, A. F., Miller, G. G., Yershov, F. I., Ilyin, K. V., andZhadanov, V. M. B-Type Oncornaviruses Isolated from ContinuousHuman Cancer Cell Lines. Arch. Ges. Virusforsch., 42: 21—35,1973.

8. Chopra, H. C., Hooks, J. J., Walling,M. J., Gibbs, C. J. Morphologyof Simian Foamy Viruses, with Particular Reference to VirusIsolated from Spontaneous Tumor of a Rhesus Monkey. J. Nail.Cancer Inst.,48: 451—463,1972.

9. Chopra, H. C., and Mason, M. M. A New Virus in a SpontaneousMammary Tumor of a Rhesus Monkey. Cancer Res., 30:2081—2086,1970.

10. Fine, C. L., Pienta, R. J., Malan, L. B., Kubicek, M. T., Bennett, D.G., Candon,J. C., Valerio,M.G., West,D. M.,Fabrizio,D. A., andChopra, H. C. Biologic Characteristics of Transformed RhesusForeskin Cells Infected with Mason-Pfizer Monkey Virus. J. Natl.Cancerlnst.,52: 1135—1142,1974.

11. Gelderblom, H., Bauer, H., Ogura, H., Wigand, R., and Fischer, A.B. Detection of Oncornavirus-like Particles in HeLa Cells. I. FineStructure and Comparative Morphological Classification. Intern. J.Cancer, 13: 246—253,1974.

12. Hooks, J., Gibbs, C. J., Chopra, H. C., Lewis,M.,and Gajdusek, D.C. SpontaneousTransformationof HumanBrainCells GrownIn

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Vitro and Description of Associated Virus Particles.Science, 176:1420—1422,1972.

13. ilyin, K. V., Bykovsky, A. F., and Zhandov, V. M. AnOncomavirus Type-B from Human Larynx Carcinoma Cells. Vopr.Virusol., 17: 494, 1972.

14. ilyin, K. V., Bykovsky, A., and Zhdanov, V. M. An OncornavirusIsolated from Human Cancer Line. Cancer, 32: 89—96,1973.

15. Jensen, E. M., Zelljadt, I., Chopra, H. C., and Mason, M. M.Isolation and Propagation of a Virus from Spontaneous MammaryCarcinoma of a Rhesus Monkey. Cancer Res., 30: 2388—2393,1970.

16. Mason,M.M.,Bogden,A. E., Ilievski,V., Esber,H. J., Baker,J. R.,and Chopra, H. C. History of a Rhesus Monkey AdenocarcinomaContaining Virus Particles Resembling Oncogenic RNA Viruses. J.Nail. Cancer Inst.,48: 1323—1331,1972.

17. Parks, W. P., Gilden, R. V., Bykovsky, A. F., Miller, G. G.,Zhdanov, V. M., Soloviev, V. D., and Scolnick, E. M. Mason-PfizerVirus Characterization: A Similar Virus in a Human Amniotic CellLine. J. VitO!.,12: 1540—1547,1973.

18. Schidolovsky, G., and Ahmed, M. C-type Virus Particles inPlacentas and Fetal Tissues of Rhesus Monkeys. J. Natl. Cancer

Inst.,51: 225—233,1973.19. Schidlovsky, G., and Ahmed, M. Detection of Foamy Virus in

Normal Lactating Monkey Mammary Tissues. Proc. Am. Assoc.Cancer Res., 14: 60, 1973.

20. Schlom, J., and Spiegelman, S. DNA Polymerase Activities andNucleic Acid Components of Virions Isolated from a Spontaneous‘MammaryCarcinoma from a Rhesus Monkey. Proc. Natl. Acad.sal. U.S., 68: 1613—1617,1971.

21. Stewart, S. E., Lovelace,E., Whang,J. J., and Ngu, V. A. BurkittTumor: Tissue Culture, Cytogenetic and Virus Studies. J. Nati.Cancer Inst., 34: 319—327,1965.

22. Yaniv, A., Ohno, T., Kacian, D., Colcher, D., Witkin, S., Schlom,J., andSpiegelman,S. SerologicalAnalysisof ReverseTranscriptaseof the Mason-Pfizer Monkey Virus. Virology, 59: 335—338,1974.

23. Zhdanov, V. M., Soloviev, V. D., Bektemirov, T. A., Filatov, F. P.,and Bykovsky, A. F. Isolation of a Leukovirus from a ContinuousTumor Cell Line. Arch. Ges. Virusforsch., 39: 309—316,1972.

24. Zhdanov, V. M., Soloviev,V. D., Valetine, D., Bektemirov, Y. S.,Bekt, T. A., Ilyin, K. V., Bykovsky, A. F., Mazurenko, N. P., Iplin,I. S., and Yershov, F. I. Isolation of OncornavirusesfromContinuous Human Cell Cultures. Intervirology, 1: 19—26,1973.

Fig. 1. The monolayer cell culture initiated from the spongy portion of rhesus monkey placentas, FTP-i . Giemsa stain.Fig. 2. Acetone-fixed FTP-l monolayer cells stained with rabbit anti-M-PMV serum by immunofluorescence technique. The immunofluores

cence is restricted mainly to the cytoplasm.Fig. 3. Viral buds (thick arrows) and cytoplasmic type-A particles (thin arrows) in human NC-37 lymphoblastoid cells infected with FTP-i virus.

The process of virus formation observed here is identical to that reported for M-PMV,where the doughnut-shaped type-A particle is incorporatedinto the viral nucleoid at the time of budding from the cell surface. X90,000.

Fig. 4. Microimmunodiffusion test: i, ether-treated M-PMV;ii, purified M-PMV P-27; iii, ether-treated FTP-i virus; iv, ether-treated X-381 virus;and v, rabbit anti-M-PMVserum.

Fig. 5. FTP-i monkey placental tissue impression, prior to culturing, stained with M-PMV antiserum by immunofluorescence technique. Notethe cytoplasmic immunofluorescence, which is restricted to a few cells in the center.

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1974;34:3504-3508. Cancer Res   Mumtaz Ahmed, George Schidlovsky, Wlo Korol, et al.   MonkeysOccurrence of Mason-Pfizer Monkey Virus in Healthy Rhesus

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