Lack of Association between BK Virus and

.aU""'•••• v.vE;J

Editor-in-Chief: J. L. Melnìck. Houslon. Te..

Publishers: S. Karger. Basel Reprint (Printed in Switzerland)

Intervirology 15: 10-18 (1981)

Lack of Association between BK Virus and Ependyrnornas, Malignant Turnors of Pancreatic Islets, Osteosarcornas and Other Hurnan Turnors Maria Pia Grossi, Guerrino Meneguzzi, Nicole Chenciner, Alfredo Corallini, Ferruccio Poli, Giuseppe Altavilla, Saverio Alberti, Gabriele Milanesi, Giuseppe Barbanti-Brodano Istituto di Microbiologia, Università di Ferrara; Laboratorio di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche. Pavia,ltaly; Centre de Biochimie. Faculté des Sciences, N ice, France; Istituto di Anatomia ed Istologia Patologica, Università di Ferrara, Italy

Key Words. BK virus' Blot hybridization . DNA-DNA reassociation kinetics . BK virus T antigen . Human tumors Summary. BK virus (BKV) DNA sequences were not detected in 142 human tumors analyzed by DNA-DNA reassociation kinetics and blot hybridization. The investigation was focused mainly on those rare types of human tumors (ependymomas, choroid plexus papillomas, tumors of pancreatic islets and osteosarcomas) that are induced with highest frequency by BKV in experimental animals. In addition, other tumors of the urinary apparatus and of the centrai nervous system were analyzed. BKV tumor (T) antigen was not detected in neoplastic tissues, and BKV T antibodies were not found in sera and cerebrospinal fluids from patients with neoplasms. Sequences homologous to BKV DNA were found in normal tissue from a kidney carrying a carcinoma. The neoplastic tissue from the same organ, however, had no sequences homologous to BKV DNA. Such DNA does not belong to BKV but probably to another papovavirus related to BKV.

BK virus (BKV) is a human papovavirus first isolated from the urine of an immunosuppressed renal transplant recipient. Since then, BKV has been isolated frequently from the urinary tract and from the centraI nervous Address inquiries lo: Dr. Maria Pia Grossi.lnstitute of Microbiology, University of Ferrara, Via Luigi Borsari 46, 1-44100 Ferrara (ltaly)

Received: October 8,1980

system of immunosuppressed or immunodeficient patients [I]. BKV is related to simian virus 40 (SV40) by base and amino acid sequence homology [2]; itinduces a virus-specific intranuclear tumor (T) antigen that immunologically cross-reacts with SV40 T antigen [I]. N umerous studies carried out during the past few years have established the main epidemiological and pathogenic features of BKV: (i) BKV is common and widespread in the normal human population [I], with a notable percent-

[

BK Virus and Human Tumors

age of cases showing long-lasting, persistent infections (3]; (ii) BKV seems to replicate in visceral organs and mainly in the kidney, since it is causally related to inflammatory complications of the donor's ureter in renal transplants (4]; (iii) BK V or BK V DNA readily transform hamster, mouse, rat, rabbit, monkey and human cells in vitro (5-11]; (iv) BKV is highly oncogenic in hamsters and mice, displaying a remarkable neurotropism. When it is injected either intracerebrally or intravenously, tumors observed with the highest frequency are ependymomas, followed by choroid plexus papillomas, malignant tumors of pancreatic islets, and osteosarcomas (12-16]. Allthesecharacteristics indicate BK Vas a possible candidate in the etiology or human neoplasia. Three studies have been carried out by n ucleic acid hybridization on the presence of BKV DNA in the mostcommon and frequent human tumors. The results were conflicting: Fiori and Di Mayorca [17] found BK V DNA sequences in 5 of 12 human tumors and 3 of 4 human tumor celliines, whereas other investigators (18,19] reported completely negative results. Since none of those studies analyzed the rare types of human tumors most frequently induced by BK V in experimental animais, we analyzed such tumors (2 ependymomas, 1 choroid plexus papilloma, 4 tumors or pancreatic islets and 7 osteosarcomas) for the presence of BKV DNA by DNA-DNA reassociation kinetics and blot hybridization. Moreover, our investigation was extended to 128 tumors or the urinary apparatus and of the centrai nervous system. The study was completed by looking for BK V T antigen in tumors as well as for antibodies to BK V T and capsid antigens in sera and cerebrospinal fluids (CSF) rrom patients with neoplasms. As a control, normal human lissue from different organs was included in alltests.

1\

Materials and Methods Tumors alld Normal Tissues Tumors and normal tissues were obtained during

surgery and quickly frozen al - 80'. None of Ihe palienls wilh neoplasms had beon trealed wilh anliblaslic chemotherapy or irradialion before removal of Ihe turnor lissue used in these teSIS.

C~I/s

alld Virus Primary cultures explanled from 6 urinary bladder papillomas and 17 meningiomas were grown in Eagle's basai medium with 2 x amino acids and vitamins,

supplemenled wilh 10% felal bovine serum. Vero cells and a human meningioma celliine were cultured in the same medium supplemented with S% calf serum.

BK V was grown in Vero cells and purified onlo a cushion of a saluraled KBr Solulion, followed by IwO cycles of equilibrium densily gradieol centrifugation in

CsCI. DNA-DNA Reassol'ialioll Killelics amI Blol Hybridizolion Cellular DNA was exlracted from lumors Iysed wilh 0.5% SDS and 50 fLg/ml proleinase K (Merck AG, Darmsladl, FRG). After Ihree phenol-chloroform extraclions, RNA was degraded by Irealmenl wilh 40 fLg/ mi ribonuclease A (Worthinglon Biochemical Corp., Freehold, N.l.) al 37' for I h followed by prolcinase K digestion and IWO more phenol-chloroform exlraelions. Virai DNA was extracled by disruplion of purified virions wilh SDS al 50', followed by digeslion wilh proleinase K. Superhelical BK V DNA was Ihen purified in a CsCl-elhidium bromide densily gradienl. In vilro labeling of Ihe BK V DNA probe with 32p

was carried out in a reacrion mixturc containing

20 mM Tris-HCI, pH 7.5, IO mM MgCl2, IO mM Ilmercaploethanol, 20 fLg bovine serum albumin, 80 fLCi each of the four [lX-32 P)-deoxyribonucleoside Iriphosphales (The Radiochemical Ceni re, Amersham, Buckinghamshire, UK; spec. acl. 350 Ci/mmol), 4 fLg of superhelical BK V DNA, and 20 unils of Escherichia coli DNA polymerase t (Grade \, Boehringer GmbH, Mannheim, FRG). Incubalion was al 18' for 45 min and was lerminaled by addilion of EDTA. Incorporaled radioaclivily (35-40% of inpuI) was separaled from residua I Iriphosphales by Sephadex G-75 (Pharmacia, Piscalaway, N.l.) gel filtralion. Thc 3~P-labeled DNA probe had a ,pecific aClivily of I x IO' lO 2 x IO'

12

Orossi/Meneguui/Chenciner/Corallini/Poli/Allavilla/AlbertilMilanesi/Barbanli-Brodano

cpm!l.lg of input DNA on the day of synthesis. For some renaturation kinetics experiments, BK V DNA

was labeled in vitro wilh [3H]-deoxyribonucleoside lriphosphales by lhe nick Iranslalion melhod of Rigby el al. [20}, using ["H)-dCTP and [3H)-dTTP (New England Nuclear, Boston, Mass.; spec. acl. 25 and 56 CiI 010101, respeclively). The reaction mixture (150 f'1) contained 50 mM potassium phosphate buffer, pH 7.5, 5 mM MgCI2, 100 f'M dATP, 100 f'M dOTP, 25 f'M [3H)-dCTP, 25 f'M [3H}-dTTP, 15 f'g/ml superhelical BK V DNA, and IO ng/ml pancrealic deoxyribonuclease (Worlhinglon). The synthelic reaclion was begun by adding J00 unils of DNA polymerase I followed by incubalion al 14' for 2 h. Afrer Sephadex gel fillralion, Ihe 3H-labeled BKV DNA probe had a specific activily of 6.6 x IO. cpm/f'g ofinput DNA. For DNA-DNA renaluralion kinetics 32p_ or 3H_ labeled BK V DNA, as well as unlabeled BKV, cellular or salmon sperm DNAs, were fragmented by heating in a walerbath al 97' for 15 min in 0.3 M NaOH and were neulralized wilh HCI. The fragmenled virai and cellular DNAs had a similar size of 300-400 nucleotides (6-7S) as determined by analysis in alkaline sucrose

nitrocellulose membrane was then washed at 60' with gentle shaking in 500 mi of 2 x SSC-O.I % SDS, followed by three more washings of 2 h each in 2 x SSC al 60'. The nilrocellulose membrane was Ihen airdried, mounted on filler paper, laid against a preflashed Kodak Royal X-O-Mal film in the presence ofa CaWO. inlensifying screen (liford Fast Tungslale), and exposed al -70'. Serological Tec/miques To lesI for BK V T anligen, 20% homogenales of tumors and normal tissues were assayed by complemenI fixation with a specifìc seru", obtained from

hamslers bearing lumors induced by BK V-Iransformed hamsler cells. Due lo scarcity of malerial for complemeni fixation. 34 urinary bladder papillomas were cul in a cryostal and sections were stained for T antigen

by indirect immunofluorescence using the same specific hamster serum and nuorescein-eonjugated goat anti-

hamsler IgO (Anlibodies Inc., Davis, CaliL). Likewise, cell5 from primary cultures of 6 urinary bladder papillomas and from Ihe second culture passage of 17 meningiomas were assayed for T antigen by indirect im-

gradients. Jmmediately before the renaluration reac-

munofluorescence. T antibodies were searched for in

tion, DNAs were denalured by boiling for IO min. Hybridizalion mixtures (100 f'1) were incubaled at 6S' and contained 6 mg/ml cellular or salmon sperm DNA, 3.S nglml radioaclive BK V DNA, and various amounts of unlabeled BKV DNA in 0.5 M NaCI and 0.12 M polassium phosphale buffer, pH 6.S. Portions of the

sera from patiencs wich neoplas01s by indirect immunof1uorescence on a conlinuous celi line derived from a BK V-induced hamster osteosarcoma expressing

BKV T anligen. Antibodies lo BKV capsid anligens were detected in sera by hemagglutinalion inhibition

reaction mixtures were rcmoved al various lime inter-

(Hl) as described previously [21]. T and Hl antibodies were also searched for in CSF from palienls wilh tu-

vals, and DNA reassociation was monitored wilh SI

mors of the centraI nervous system.

nuclease.

For blot hybridization experiments, cellular DNA was used undigested and afler digestion wilh lhe reslriclion endonucleases EcoRI, Hilldll, HÙ,dlIl and Hpol (Miles Laboratories, Elkhart, Ind.). Afler digeslion al 37' for 4 h, DNA was subjecled lo eleclrophoresis in O.S% agarose gels. Afrer eleclrophoresis, DNA was denalured in the gels and Iransferred to nilrocellulose membranes(type B-6; Schleicher & Schuell, Keene, N.H.) which were Ihen healed al SO' for 2 h. For hybridization the nitrocellulose membrane was preincubated

al6S' in 40 mi ofO.9 M NaCl, 90 mM sodium cilrale (6 x SSC)conlainingO.02% Ficoll (Pharmacia), 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin in a sealed box for 4 h. Heal-denatured 32P_BKV DNA probe (5 x IO. cpm) logelher wilh 150 f'g of codenalured salmon sperm DNA was then added, and incubalion was conlinued for 60 h. The hybridized

Results Searchfor BKV DNA Seqllences ;11 Tlll110rs alld Normal Tissues

No BK V DNA sequences were detected by DNA-DNA reassociation kinetics in any ofthe tumors or norma I tissues tested (table I), since hybridization of cellular DNA to the radioactive BK V DNA probe was neverhigher than that exhibited by the unrelated sa\mon sperm DNA (fig. Il. Consistent wilh these dala was lhe absence of bands hybridizable to BK V DNA in ali the tumor and norma\


13

T.ble I. Cases of human tumors and normal subjects showing no relationship lO BKV by DNA-DNA hybridization and by search for T antigen and antibodies Tumors

Number

Normal tissues

Number of specimens tested

Kidney Urinary bladder

6

of specimens

lested Kidney carcinoma Urinary bladder carcinoma Urinary bladder papilloma Carcinoma of suprarenal glands Ependymoma Choroid plexus papilloma Meningioma 2 Astrocyloma Glioma and glioblastoma Oligodendroglioma Neurinoma Medulloblastoma Spongioblasloma Cerebral metastasis of carcinoma Other brain tumors Tumors of pancreatic islets3 Osteosarcoma

18 7 I 2 2 I 35 18 24 2 5 I 2 5 8 4

Pancreas

Nervous tissue White blood cells t Spermalozoa

I 2

8 I

I

7

2

Pool of cells from the blood of 13 di!ferenl persons. Includes I human meningioma celiline.

3

2 adenomas and 2 malignant carcinomas. Ali 4 tumors were secreting and releasing insulin. A carcinoma

produced hepatic melasta5OS.

DNAs when they were assayed by blot hybridization (table I). The tumor DNA samples exposed to 32P-BK V DNA resulted in negative lanes, similar to the T lanes shown in figure 2. The bio t hybridization technique, however, showed the presence of a specific band in one human DNA sample extracted from the normal portion of a kidney carrying a carcinoma. On the other hand, the neoplastic tissue from the same organ had no detectable sequences homologous to BK V DNA. Figure 2 shows the results of an experiment in which DNAs from the norma I and the neoplastic tissue of Ihe alfected kidney from patient L. M. were digested with severa I restriction endonucleases

and analyzed in parallel by blot hybridization. A specific hybridization band was always visible in the normal (N) tissue but not in the tumor (T) tissue. This band was located in the region of control BK V DNA form Il (nicked circular) and form III (Iinear) which in these 0.8% agarose gels migrated quite close to each other. The mobility of the band was unchanged when uncut cellular DNA or DNA restricted with either Hindll or Hpal (two endonucleases that do not cut BK V DNA) were electrophoresed and blot-hybridized (fig.2), indicating that the sequences homologous to BK V DNA were not integrated into the celi genome. Incubation with Hindll re-

14

Grossi/ Meneguzzi/Chenciner/Cora Ili ni/Poli/AIlavi Ila/Alberti/M i1anesi/ Barbanli· Brodano

sulted in a second band located in the region of BKV DNA form III. The mobility of the hybridizable band did not change even when cellular DNA was digested with enzymes that cut BK V DNA, such as EcoRI (one cut) or Hilldlll (four cuts) (fig. 2). This result suggests that, in spite of the homology demonstrated by hybridization, the free DNA sequences present in this sample were not identical to prototype BK V DNA. When a.p_ labeled SV40 DNA was used as the hybridization probe, the band in sample N was barely detectable (data not shown), indicating that homology to this virus was lower than to BK V. Densitometric evaluation ofthe amount ofviral DNA in the normal kidney tissue suggested that it was present in less than one copy per celI. However, this is a minimum estimate, since the extent of homology to BKV DNA is unknown. Not enough DNA of this preparation was available to test it by DNA-DNA reassociation kinetics. Attempts to grow such a virus by transfection of cultured human embryonic fibroblasts and Vero cells with cellular DNA were unsuccessful.

"

HOUIIS

Fig.!. Reassocialion kinetics Of32p·BKV DNA in Ihe presence of various cellular DNAs. A = Salmon sperm DNA alone; B, C = salmon sperm DNA mixed with unlabeled BKV DNA al ralios of 0.5 and 2.5 genome equivalents per diploid celi genomc, respectively; D, F, H = meningioma; E = osteosarcoma;

G = choroid plexus papilloma; I = urinary bladder papilloma; L, M, N, Q = kidney carcinoma; O = normal while blood cells; P = normal kidney. Portions of the reactioo mixtures were removed al various lime

intervals and assayed by the SI nuclease melhod for Ihe fraction of labeled DNA remaining single-stranded (fss ). The linear plolling represenls Ihe minimum squares slraighl lines obtained from at leasl eighl in· dependently determined experimental points.

Searchlor T Anligell andlor Anlibodies lO T and Capsid Anligells Search for BK V T antigen was negative in ali tumors and normal tissues tested by complement fixation (table I) and in 34 urinary bladder papillomas (not listed in table I) assayed by immunofluorescence. Likewise, no BKV T antigen was detected by immunofluorescence in cultured cells from 6 urinary bladder papillomas and 17 meningiomas (not listed in table I). No BK V T antibodies were found in the sera and CSF from patients with neoplasms (table I). H I antibodies to BK V capsid antigens were present in 68.8% of sera from patients with neoplasms; 33.2% of the positive sera had a titer of 512 or greater. CSF


15

a uncut I

EcoRI I I

I

b Hp.1 i

Hin dIII

uncut i l EcoR1

I

1i

Hind"il

I Oj ... j

,

'

FII

~ FIII~

FI

~

....

BK

N

T

N

TNTBI(NT'

Fig. 2. Biol hybridizalion of 32p_BK V DNA to DNA from Ihe normal (N) and tumor (T) kidney tissue of patienl L. M. wilh renal carcinoma. Aliquols (20 l'g) of tissue DNA, uncut or digested with Ihe indicated enzymes, were blot-hybridized to 32P_BKV DNA. Lanes marked BK contained 50 pg of circular BKV DNA [predominanlly form Il (FII) with traces of form J (FI)] and 50 pg of EcoRl-linearized BKV

from 2 patients with tumors of the centrai nervous system had an Hl antibody titer of 8. Discussion The correlation of papovaviruses of the SV40-human papovavirus subgroup with human tumors is stili open for discussion. Besides the results of Fiori and Di Mayorca [17], Tabuchi et al. [22] reported finding papovavirus T antigen, detected by the immunoperoxidase technique, in 2 of 39 human brain

BI(

N

T

N

T

N

T

DNA (Fili). Next to flindlll-digested samples, 50 pg of HÙ,dlll-digesled BKV DNA were also run as a control; A, B, C and D label the bands corresponding lo Ihe four Hindlll fragments. In samples with uncul DNA, Iransfer was poor and considerable trapping ofhybridizable sequences occurred,leading lO bands of low and variable intensity. a and b were separate experiments.

tumors. Jnterestingly, the 2 pOSItive tumors were both of ependymal origin - a malignant ependymoma and a choroid plexus papilloma. Weisset al. [23] detected papovavirus Tantigen by immunofiuorescence in human meningiomas. This result was confirmed in a recent study [24]. Scherneck et al. [25] reported isolation of a papovavirus from a glioblastoma multiforme, and Meinke et al. [26] detected papovavirus DNA sequences by DNA-DNA reassociation kinetics in another glioblastoma multiforme. On the contrary, Greenlee et al.

16

Grossi/Meneguzzi/Chenciner/Corallini/Poli/Altavilla/Alberti/Milanesi/Barbanti-Brodano

[27] reported negative results after testing 80 human brain tumors, including 6 ependymomas and l choroid plexus papilloma by immunoftuorescence for the presence of papovavirus T antigen. Likewise, no papovavirus T antigen was found by immunoftuorescence on cells cultured from a number of human urogeni tal cancers (28). The present experiments, employing two nucleic acid hybridization techniques, indicate that BK V DNA was not present in the DNA from 142 human tumors. Reconstruction experiments with known amountsofBK V DNA mixed with salmon sperm DNA showed that both DNA-DNA renaturation kinetics and blot hybridization could detect I copy per celi of 10% of the viraI genome. Transformation with endonuclease-restricted fragments of BKV DNA showed that large T antigen is necessary for the induction and maintenance of the transformed phenotype [Barballli-Brodallo. manuscript in preparation]. Therefore, since BK V-induced tumors should contain at least I copy per celi of approximately 50% of the virai genome (corresponding to the early region coding for large T antigen), it is unlikely that BK V DNA sequences present in cellular DNA were missed for lack ofsensitivity ofthe hybridization techniques. The results of hybridization experiments are consistent with serological data demonstrating lack ofT antigen in tumor tissues and in cultured tumor cells as well as absence ofTantibodies in sera and eSF from patients with neoplasms. Also, the percentage of sera from these patients with Hl antibodies and with high Hl titers was comparable to that found in a norma I human population in Italy (21). H I by eSF from 2 patients with neoplasms was probably not dependent on BK V antibodies since the two Hl-positive samples did not react by immunofluorescence with BK V

capsid antigens on BK V-infected Vero cells. On the whole the foregoing results suggest, in agreement with previous studies [18,19], that BK V is related neither to the most common human tumors nor to those types of rare human tumors which are readily induced by the virus in experimental ani maIs. The sequences homologous to BKV DNA present in the normal portion of the kidney of patient L. M. most probably belong to another papovavirus related to BK V that was present in the urinary tract of this patient. The fact that virai sequences were not integrated in the cellular genome is suggestive of productive infection. The DNAs from other known human papovaviruses, such as BK V (M M) or le virus and its variants, are incompatible with the electrophoretic pattern observed, since they ali have three Hindlll restriction sites [29,30). It see ms, therefore, that other yet undiscovered papovaviruses may circuiate in the human population, producing an acute or persistent infection. The lack of viraI sequences in the tumor of patient L. M. is at present unclear, although it is possible that the virus was unable to replicate in neoplastic tissue.

Acknowledgments This work was supported by 'Consiglio Nazionale delle Ricerche - Progetto Finalizzato Virus', grants 78.00338.84,79.00367.84,80.00577.84 and 20412\/84/ 8\821. We lhank Wi/ma Rossi-Pusinanti and C..are Mussi for excellent technical assistance. Wc are inde bIed lO G. Bocci. G. Federspil. G. Gaisl, A. Migliore. G. C. Nico/a. P. Pao/elli, S. Pezzolla, and G. Pizzo for providing neoplaslic and norma! tissues.

References Padgett, B. L.: Walker, D. L.: New human papovaviruses. Prog. med. Virol., voI. 22, pp. \-35 (Karger, Basel 1976).


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17

hamsters with BK virus, a human papovavirus. Lab. Invest. 36: 636-641 (1977). 15 Corallini, A.; Altavilla, G.; CeccheUi, M.G.; Fabris, G.; Grossi, M.P.; Balboni, P.G.; Lanza, G.; Barbanli-Brodano, G.: Ependymomas, malig. nant tumors of pancreatic islets, and osteosarcomas induced in harnslers by BK virus, a human papovavirus. J. natn. Caneer Inst. 61: 875-883 (1978). J6 Uchida, S.; Watanabe, S.; Aizawa, T.; Furuno, A.;

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M.; Borgalli, M.: High incidenee ofependymomas induced by BK virus, a human papovavirus. J. natn. Caneer Insl. 59: 1561-1563 (1977). 14 Greenlee, J. E.; Narayan, O.; lohnson, R. T.; Herndon, R. M.: Induction of brain tumors in

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Muto, T.: Polyoncogenicity and insulinoma-induc· ing abilily of BK virus, a human papovavirus, in Syrian golden harnsters. J. natn. Caneer Inst. 63: 119-126 (1979). Fiori, M.; Di Mayorca, G.: Occurrenee of BK virus DNA in DNA obtained from eertain human tumors. Proc. natn. Acad. Sci. USA 73: 4662-4666 (1976). Wold, W.S.M.; Mackey, J.K.; Brackmann, K.H.; Takemori, N.; Rigden, P.; Green, M.: Analysis of human tumors and human malignant eelliines for BK virus-specific DNA sequences. Proc. natn. Acad. Sci. USA 75: 454-458 (1978). Israel, M.A.; Martin, M.A.; Takemoto, K.K.; Howley, P.M.; Aaronson, S.A.; Solomon, D.; Khoury, G.: Evalualion ofnormal and neoplastic human lissue for BK virus. Virology 90: 187-196 (1978). Rigby, P. W.J.; Dieckmann, M.; Rhodes, C.; Berg, P.: Labeling deoxyribonucleic acid to high specific aClivity in vilro by nick translation with DNA polymerase I. J. molec. Biol. JJ3: 237-251 (1977). Portolani, M.; Marzocchi, A.; Barbanli·Brodano, G.; La Placa, M.: Prevalenee in Italy of antibodies to a new human papovavirus (BK virus). J. med. Microbio!. 7: 543-546 (1974). Tabuchi, K.; Kirsch, W. M.; Low, M.; Gaskin, D.; Van Burskirk, J.; Maa, S.: Screening of human brain tumors far SV4o-reiated T antigen. Int. J. Caneer 21: 12-17 (1978). Weiss, A. F.; Portmann, R.; Fischer, H.; Simon,J.; Zang, K. D.: Simian virus 4o-related antigens in three human meningiomas with defined chromosome loss. Proc. natn. Acad. Sci. USA 72: 609-613 (1975). May, G.; Fischer, H.; Zang, K.D.: SV4o-reiated T antigen expression in humao meningiomas with normal and G-22-monosomic karyotype. J. gen. Viro!. 43: 697-700 (1979). Scherneck, S.; Rudolph, M.; Geissler, E.; Vogel, F.; Lubbe, L.; Wahlte, H.; Nisch, G.; Weickmann,

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Grossi/Meneguzzi/Chenciner/Cora1lini/Poli/Allavilla/Alberti/Milanesi/Barbanti-Brodano

F.; Zimmennann, W.: Isolalion of a SV40-like papovavirus from a human glioblastoma. Inl. 1. Cancer 24: 52l-531 (1979). 26 Meinlce, W.; Goldstein, D.A.; Smith, R.A.: Simian virus 4O-related DNA sequences in a human brain lumor. Neurology 29: 159(}-1594 (1979). 27 Greenlee, 1. E.; Beclcer, L. E.; Narayan, O. ; 10hnson, R.T.: Failure lo demonslrate papovavirus lumor antigen in human cerebral neoplasms. Ann. Neurol. 3: 479-481 (1978). 28 Shah, K.V.; Daniel, R.W.; Stone, K.R.; Elliol,

A. Y.: Invesligalion of human urogenilal lract turnors for papovavirus eliology. 1. natn. Cancer Inst. 60: 579-582 (1978). 29 Osbom, I.E.; Robertson, S.M.; Padgett, B.L.; Wallcer, D.L.; Weisblum, B.: Comparison of lC and BK human papovaviruses with simian virus 40: DNA homology sludies. 1. Virol. 19: 675--684 (1976). 30 Yang, R.C.A.; Wu, R.: BK virus DNA: cleavage map and sequence analysis. Proc. naln. Acad. Sci. USA 75: 2150-2154 (1978).