INFECTION AND IMMUNITY, Feb. 1980, p. 501-5120019-9567/80/02-0501/12$02.00/0 Vol. 27, No. 2

Pathology of Chickens Infected with Avian NephroblastomaVirus MAV-2(N)

SUSAN L. WATTSt AND RALPH E. SMITH*Department ofMicrobiology and Immunology, Duke University Medical Center, Durham, North Carolina

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A nephroblastoma-inducing myeloblastosis-associated virus, MAV-2(N), de-rived from avian myeloblastosis virus was characterized with respect to biochem-ical composition and avian pathogenesis. Purified fibroblast-grown virus con-tained the same size 35S ribonucleic acid and the same relative amounts of viralpolypeptides as another myeloblastosis-associated virus inducing predominantlyosteopetrosis MAV-2(0). Plaque-purified MAV-2(N) induced a 76 to 93% inci-dence of nephroblastoma and a 3 to 50% incidence of osteopetrosis in SPAFASand line 15 x 7 chickens: the oncogenic spectrum and the onset of nephroblastomavaried with the line of chicken and the route of injection. Renal neoplasms weremanifest in chickens older than 2 months and grew to a massive size. Furthermore,29% of control chickens housed with MAV-2(N)-infected chickens demonstratednephroblastoma. MAV-2(N)-infected chickens had growth rates and blood packedcell volumes comparable to those of uninfected chickens. Infected chickens 2months of age had increased kidney, liver, and spleen weights; tumor-bearingchickens 3 to 4 months of age had increased liver, lung, brain, pancreas, and boneweights. The concentration of albumin was decreased and the concentration ofgamma globulin was increased in the serum of MAV-2(N)-infected chickens.Analysis of the sera of nephroblastoma-bearing chickens for virus and antibodyshowed that three states existed: (i) high levels of neutralizing antibody, (ii) highlevels of virus, and (iii) simultaneous presence of both at low levels. The patho-logical and virological features of MAV-2(N) which distinguish it from MAV-2(0)are discussed.

The standard strain of avian myeloblastosisvirus (AMV), BAI strain A, primarily inducesmyeloblastosis, but also induces osteopetrosis,nephroblastoma, lymphoid leukosis, and otherneoplasms in infected chickens (4). Efforts toselect for the induction of a single type of neo-plasm led to the isolation of several viral speciesfrom standard AMV: a subgroup A myeloblas-tosis-associated virus MAV-1 inducing a highincidence of osteopetrosis (45); a subgroup Bleukosis virus inducing predominantly osteope-trosis MAV-2(0) (45); a subgroup B leukosisvirus inducing primarily nephroblastoma (25,35,50); and leukemogenic viruses of subgroups Aand B, respectively, AMV-A and AMV-B (22,32).Evidence suggests that the nephroblastoma-

and osteopetirosis-inducing leukosis viruses arenondefective helper viruses providing glycopro-tein envelope components for the replication-defective leukemogenic viruses in standardAMV (30). Although the leukemogenic compo-nents have been shown to transform yolk sac

t Present address: Department of Microbiology, Universityof Minnesota, Minneapolis, MN 55455.

cells, bone marrow cells, and macrophages invitro, they cannot replicate in chicken embryofibroblast (CEF) cultures (6, 31). However,nephroblastoma and osteopetrosis viruses canreplicate in CEF and cause plaques characteris-tic of subgroup B leukosis viruses (17, 35, 45).Yet morphological transformation of any celltype in vitro has not been demonstrated by thelatter type of viruses. In vivo standard AMVinduces myeloblastosis within 2 weeks after in-fection (4), whereas nephroblastoma, osteope-trosis, or lymphoid leukosis become manifest inchickens infected with standard AMV or withmyeloblastosis-associated viruses weeks ormonths after infection (10, 35, 36, 45).MAV strains primarily inducing osteopetrosis

were isolated by endpoint dilution of superna-tant fluids obtained from cell cultures of stan-dard AMV-induced chicken tumors of standardAMV-treated fibroblasts (45). Subgroup B MAVstrains inducing a high incidence of nephroblas-toma were isolated by two routes: (i) passage ofstandard AMV-induced nephroblastoma ex-tracts in vivo (50) and (ii) transfection ofhamsterfibroblasts (25) or CEF (35) in vitro with stan-dard AMV-induced leukemic myeloblast deoxy-

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502 WATTS AND SMITH

ribonucleic acid (DNA). The virus strain isolatedby Ogura et al. (35, 36) has been cloned in ourlaboratory by plaque purification: the presentinvestigation reports the biochemical and path-ogenetic characterization of this cloned virusMAV-2(N).Avian nephroblastoma is a renal tumor of

both epithelial and mesenchymal cell types man-ifesting distorted morphology and abnormal dif-ferentiation. The avian tumor morphology iscomparable to that of Wilms' tumor of humans(33) as well as that of nephroblastomas found inrabbits, swine, cattle, sheep, dogs, and cats (7,14, 24, 38). Histopathological studies of aviannephroblastoma note the presence of all com-ponents of normal kidney in various states oforganization and differentiation as well as occa-sional evidence of cartilage, bone, keratinizedepithelium, and sarcoma. Thus, an embryonicrest residual in the adult kidney has been sug-gested as the origin of the nephroblastoma (5,21). However, the target cell and mechanism ofthe viral-induced transformation are unknown.The present study was undertaken to elucidatethe pathological manifestations of MAV-2(N)infection by investigating biological characteris-tics of MAV-2(N)-infected chickens.

(This investigation is part of a dissertationsubmitted by S.L.W. to the Department of Mi-crobiology and Immunology, Duke UniversityMedical Center, for the Ph.D. degree.)

MATERIALS AND METHODSEggs. Fertile chicken eggs certified free of chicken

helper factor and avian leukosis virus group-specificantigen were obtained from SPAFAS, Inc., Norwich,Conn. Fertile eggs of line 15 x 7 were obtained fromthe Regional Poultry Research Laboratory, East Lan-sing, Mich. Eggs were incubated at 37.50C in a humid-ified incubator.

Chickens-infection and maintenance. SPA-FAS and line 15 x 7 chickens were infected with 105plaque-forming units (PFU) of MAV-2(N) (0.1 ml)produced in tissue culture or mock-infected with 0.1ml of tris(hydroxymethyl)aminomethane (Tris) buffer(0.14 M NaCl, 0.025 M Tris-hydrochloride, pH 7.4, 5mM glucose, 100 U of penicillin per ml, 100 ig ofstreptomycin per ml, 0.7 mM Na2HPO4) containing10% (vol/vol) calf serum by one of two routes: (i)infection of a chorioallantoic membrane vein of 12-day-old embryos (2), or (ii) intraperitoneal infection of1-day-old hatched chicks. MAV-2(0) was adminis-tered at 104 PFU per embryo [a lower dose than MAV-2(N)] to the same lines of chickens, because 105 PFUofMAV-2(0) per embryo led to high embryo mortalityand early death of the hatched chick due to severeosteopetrosis. One day before hatching, eggs weretransferred to humidified hatchers maintained at37.50C, separate hatchers being used for infected andcontrol eggs. Birds infected with different viruses weremaintained in separate rooms of an animal isolation

facility. Infected and uninfected birds in the sameroom were kept in separate cages. Birds were fedPurina Growena Chow and water ad libitum. Forexperimental purposes, or when incapacitated by tu-mor or other disease, chickens were sacrificed byexsanguination. All chickens were subjected to post-mortem examination.Chicken body weight and organ weight deter-

minations. Birds were weighed on a Harvard labo-ratory balance. Organs excised from birds sacrificed byexsanguination were weighed on a Mettler analyticalbalance.

Extraction of virus from infected chicken or-gans. Extracts from organs excised from infectedchickens were prepared as follows: Organs werewashed with Tris buffer or phosphate-buffered saline(0.15 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mMNa2HPO4, 0.9 mM CaCl2, 0.5 mM MgCl2.6H20), thenminced with a scalpel and spatula or homogenizedwith a Virtis "23" homogenizer in 0.1 to 0.5 ml of Trisbuffer per g. Cells were sedimented by centrifugationat 800 x g for 15 min at 40C, and the supernatant fluidwas frozen at -70°C until used for virus titration byplaque assay.Blood packed cell volume (PCV). Blood samples

were collected in heparinized 50-,l microhematocrittubes and centrifuged in a Clay Adams microhemato-crit centrifuge. Packed cell volume for each samplewas calculated by comparison to a standard scale.Serum protein electrophoresis. Serum protein

electrophoretic analyses were performed for all sam-ples in triplicate by using a Beckman Microzone elec-trophoresis system, model R-101 (3). Serum samplesof 0.25 pl were applied to cellulose acetate membranesand subjected to 4.5 mA at 250 V for 20 min in sodiumbarbital (B-2) buffer, pH 8.6. Membranes were stainedin Ponceau red S, then cleared in dilute glacial aceticacid and ethanol. Membrane scans were performed byusing a Quick-Scan, Jr., interfaced with a Digital PDP-11/10 computer. Areas under the peaks were inte-grated and expressed relative to the total area scanned.Serum protein concentration was determined by themethod of Lowry et al. (27).Immunization of chickens with virus. Unin-

fected normal adult chickens were injected intrave-nously with 0.1 to 0.5 ml of virus in Tris buffer con-taining 10% calf serum and reinjected by the sameprocedure 3 to 4 weeks later. Approximately 2 monthsafter the primary injection, the chickens were sacri-ficed by exsanguination, and the serum was stored at-70°C for titration of serum antibody by the neutral-ization assay of Ishizaki and Vogt (23).

Statistical analysis. Values were reported asmean ± standard error for each determination. Levelsof significance (P) were calculated by Student's two-tailed t test. P values refer to the difference in param-eters between MAV-2(N)-infected and uninfectedchickens of the same age.

Virus sources and derivations. A nephroblas-toma-inducing virus derived by transfection ofCEF inculture with DNA from AMV-induced leukemic mye-loblasts (35) was received from Heinz Bauer, Institutfur Virologie, Justus-Liebig Universitat, Giessen, W.Germany. This virus, cloned three times by plaquepurification and propagated in our laboratory in SPA-

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AVIAN NEPHROBLASTOMA VIRUS PATHOLOGY 503

FAS CEF (C/E), will hereafter be referred to as MAV-2(N), denoting that it is a myeloblastosis-associatedvirus of subgroup B inducing nephroblastoma. MAV-2(0) (45) has been well characterized in our laboratory(3).

All other viruses were obtained as follows: PragueB strain of Rous sarcoma virus (PR.RSV-B) from R.R. Friis, Institut fur Virologie, Justus-Liebig Univer-sitat, Giessen, W. Germany; Prague C strain of Roussarcoma virus (PR.RSV-C), and ring-necked pheasantvirus (RPV) from P. K. Vogt, University of SouthernCalifornia, Los Angeles; and AMV from J. W. Beard,Life Sciences, Inc., St. Petersburg, Fla. MAV-2(N) andMAV-2(0) pseudotypes of Rous sarcoma virus (RSV)were produced by infection of Bryan strain RSV(-)CEF with MAV-2(N) or MAV-2(0). Cells transformedby RSV(-) were a generous gift of H. Hanafusa,Rockefeller University, New York, N.Y.

Cell culture. Primary CEFs were prepared from11-day-old SPAFAS embryos as described by Vogt(49) and propagated in supplemented Ham F10 me-

dium (42). Secondary CEFs seeded into tissue culturedishes or roller bottles were infected for virus produc-tion at an input multiplicity of infection of 0.01 to 0.10PFU or focus-forming units per cell.

Plaque assays were performed by the method ofGraf (17). The focus assay of Vogt (49) was used forassaying transforming avian sarcoma viruses (ASVs).Antiviral antibody was assayed by plaque or focusneutralization as described by Ishizaki and Vogt (23).Complete neutralization was scored as 90% reductionin plating efficiency.

Large-scale virus production. Infected cells weregrown in roller bottles for production of large amountsof virus for ribonucleic acid (RNA) and protein isola-tion. Roller bottles containing cells infected withMAV-2(N), MAV-2(0), and PR.RSV-C were fed andharvested every 2 h on a Bellco Smith-Kozoman Au-toharvester (41, 44).

Virus concentration, purification, and label-ing. Virus was concentrated by pelleting and purifiedon sucrose gradients as previously described (41). Vi-rus pellets were suspended in buffer and stored at-70°C. Viral RNA was labeled with 10 mCi per rollerbottle of [5,6-3H]uridine (40 to 50 Ci/mmol) by addingthe radioisotope in culture medium to confluent rollerbottle cultures. Supernatant fluids were harvested 24h after addition of radioisotope and every 12 h there-after. Virus was labeled with carrier-free [32P]ortho-phosphate as previously described (46).

Viral polypeptide polyacrylamide gel electro-phoresis. Viral polypeptides were analyzed by elec-trophoresis in slab gels of 10% (wt/vol) acrylamide-0.1% (wt/vol) sodium dodecyl sulfate (SDS) (8, 48).Gels stained with Coomassie brilliant blue were

scanned on a Quick-Scan, Jr., interfaced with a digitalPDP-11/10 computer. The peaks of optical densityobtained in the scans were integrated by the computerrelative to the total stained material and the amountof protein applied to the gel.

Viral RNA purification. Viral RNA was purifiedby SDS-pronase treatment and phenol extraction of

virus pellets (11, 13). Viral 70S RNA was isolated byrate-zonal gradient centrifugation in gradients of 15 to

30% (wt/vol) sucrose (13).

Polyacrylamide gel electrophoresis of viralRNA. Viral 35S RNA was analyzed by electrophoresisin 2.1% (wt/vol) acrylamide-0.6% (wt/vol) agarose-6M urea gels (15). Gel slices were solubilized overnightat 370C in Protosol/toluene/water (9:10:1, vol/vol),and radioactivity in each sample was determined in atoluene-based liquid scintillation cocktail.

RESULTSBiochemical composition of MAV-2(N).

The polypeptide composition of purified MAV-2(N) relative to that of other avian retroviruseswas investigated by SDS-polyacrylamide gelelectrophoresis. Electrophoretically separatedMAV-2(N) polypeptides comigrated with thegp85, gp37, p27, p19, p15/plO, and p12 of MAV-2(0), AMV, and PR.RSV-C. Although thesepolypeptides were not further characterized,their identity was assumed on the basis of elec-trophoretic mobility. A comparison of the rela-tive amounts of polypeptides in each virus wasachieved by using gel scans of Coomassie bril-liant blue-stained protein. The ratio of gp85/p27provided a further index of the relative amountsof gp85 and p27 polypeptides per virion. Therelative amounts ofMAV-2(N) polypeptides cor-responded completely with the values obtainedfor MAV-2(0) polypeptides. MAV-2(N), as wellas MAV-2(0) (48), contained more stained ma-terial identifiable as gp85 than did AMV orPR.RSV-C (Table 1).The size of MAV-2(N) 35S RNA was com-

pared to that of MAV-2(0) by electrophoresison the same gel. The 70S RNA from each viruswas labeled, extracted, heat denatured, and sub-

TABLE 1. Comparison of the polypeptidecomposition ofMAV-2(N) with that of other avian

oncovirusesa% of total polypeptideb

Polypeptideposition MAV-2- MAV-2- AMV PR.RSV-

(N) (0) C

gp85 8.68 10.04 2.60 2.26gp37 2.24 2.43 3.37 3.21p27 22.43 23.37 26.08 22.11p19 14.59 14.02 16.27 15.27p12 14.72 14.77 15.93 15.25

p15, plO 9.28 7.73 12.80 12.95gp85/p27C 0.39 0.43 0.10 0.10a SDS-polyacrylamide gel electrophoresis analyses

of purified PR.RSV-C, MAV-2(N), MAV-2(0), andAMV were performed in triplicate. Gels were stainedwith Coomassie brilliant blue and scanned, and peakareas were determined by computer integration.

b The mean value of percent stained material mi-grating in each polypeptide position relative to thetotal stained material on the gel is given for each virus.

c The ratio of gp85 to p27 was calculated as an indexof the relative amounts of these polypeptides in eachvirus.

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504 WATTSAND SMITH INFECT.IMMUN.~~~.ifjected to electrophoresis as described in Mate- 4_ o0rials and Methods. The MAV-2(N) 35S RNApeak comigrated precisely with that of MAV- > 2 c2(0).Pathology of chickens infected with 4 0 >

MAV-2(N). Chickens infected with plaque-pu- to L_ t-rifled CEF-grown MAV-2(N) manifested an in- c

ocidence of nephroblastoma greater than 75% andan incidence of osteopetrosis less than 50%, and _ o 00

none of the other neoplasms associated with 0oAMV (myeloblastosis, lymphoid leukosis) (Ta- C ticble 2). The nephroblastomas induced by MAV- S n

0 W MLo2(N) appeared identical by gross and histological W f >.examination to those described after induction

CC +1 +I+-H-by AMV or AMV-derived virus strains (21, 26, z36). The nephroblastomas consisted of abnor- CD 00

mally differentiated kidney tissue containing ele- S t ° oments of sarcoma, carcinoma, chondroma, os- .q .cV.teoid, and keratinized tissue. These noninvasive Z oneoplasms were multifocal in origin and pro-gressed to form massive, thickly encapsulated 3J 0N cstructures. The tumors varied in their composi- 2! V M D - -Otion of fluid-filled cysts, solid tissue, necrotic 0 oareas, and hemorrhagic areas. The largest ob- c5 _served tumor (369 g) exceeded one-fifth the body

Q-

LO_c

weight of the chicken. The osteopetrosis ob- ;CCserved with MAV-2(N) infection was of a later 4n0

onset and a more peripheral body distribution, X C

but otherwise was identical to that observed 4oafter infection with MAV-2(0) (3, 45). 14 rC cMAV-2(N)-infected chickens manifested sev- - s O o

eral other abnormalities, some of which could be W_

2'C- S o .attributed to virus infection or to the presence S o 0 CC

of nephroblastoma. Chickens dying before age .c00 _60 days died of cannibalism, anemia, other infec- ;: la - . .tions, intra-abdominal hemorrhage, or unknown 02 M q Lccauses (Table 2). Chickens dying after age 60 2

0oo

days also demonstrated a large incidence of can- O. . oJ >, t 3 3nibalism and were frequently observed to man- 1' z

05 -< 0o

ifest intradermal petechial hemorrhages of un- 2c.4^ p0known significance. Cannibalism was rarely ob- 3 oserved in uninfected chickens and chickens in- 0Ho > ._ofected with other viruses, indicating that feeding, olight, and confinement conditions did not con- _ _0- otribute to the high rate of cannibalism seen in in o 4 5MAV-2(N)-infected chickens. The incidence of

W o -cannibalism was recorded as a manifestation of 8 O

unknown etiology associated with MAV-2(N) .2infection. When the size of the nephroblastoma I= U , N 0 °caused abdominal obstruction, chickens mani- o0 4fested ascites, diarrhea, intra-abdominal hem- 0 co ow,orrhage, and paralysis. O= .-

Effect of chicken strain and route of in- o: +1->-. 3fection on the oncogenic spectrum of MAV- cc

C> C.0 =0) >2(N). The relative incidence of nephroblastomaO C/X .C t6i 0 _.g s

and osteopetrosis varied with the chicken line .cand route of injection used (Table 2). The inci-

a-xdence of nephroblastoma was approximately a co o

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AVIAN NEPHROBLASTOMA VIRUS PATHOLOGY 505

80% in SPAFAS chicks and approximately 90%in line 15 x 7 chickens. The similarity of neph-roblastoma incidence in chickens infected withMAV-2(N) as 12-day-old embryos or as 1-day-old hatched chicks suggests that the target cellfor nephroblastoma is present at both stages ofdifferentiation. Line 15 x 7 chickens infectedintravenously manifested the highest incidenceof osteopetrosis (50%). The incidence of osteo-petrosis was comparable for line 15 x 7 chickensinfected intraperitoneally and SPAFAS chick-ens infected intravenously (30 and 20%, respec-tively). However, SPAFAS chicks infected intra-peritoneally manifested only 3% incidence ofosteopetrosis.Influence of host age and route of infec-

tion on the onset of nephroblastoma andosteopetrosis. Chickens infected as embryosby the intravenous route manifested an earlieronset of nephroblastoma than those infected ashatched chicks by the intraperitoneal route (Ta-ble 2). This difference was especially evident ininfected SPAFAS chicks; the average age ofchickens dying or sacrificed with nephroblas-toma was approximately one month younger forintravenously infected embryos than for intra-peritoneally infected hatched chickens. A slightdifference in the age of chickens dying or sacri-ficed with osteopetrosis was apparent only withrespect to strain, with SPAFAS chicks showinga slightly earlier onset than did line 15 x 7chicks.Contact transmission of MAV-2(N) infec-

tion. A total of 34 uninfected SPAFAS and 15x 7 chickens were hatched and raised in thesame room with MAV-2(N)-infected chickens.Ten (29%) of the uninfected chickens demon-strated nephroblastoma. The mean age of con-tact-infected chickens manifesting nephroblas-toma (111 ± 9 days) was slightly older than thatfor experimentally infected chickens. None ofthe contact-infected chickens demonstrated os-teopetrosis.Growth rate of MAV-2(N)-infected chick-

ens. To determine whether infection with MAV-2(N) altered the growth rate of the chicken, thebody weight of intraperitoneally infected SPA-FAS chicks was measured every third day for 2months after hatching. Both infected and unin-fected chickens grew at a rate of 6.33 g per day.Therefore, no stunting of infected chickens wasobserved during this time period.Organ weights of MAV-2(N)-infected

chickens. Organ weights of MAV-2(N)-infectedchickens at 30, 60, and 90 to 130 days of age wereanalyzed to determine whether systemic organchanges associated with MAV-2(N) infectioncould be detected. Organ weights were expressed

relative to the total body weight of each bird(Table 3). At 30 days of age, the only significantdifference between infected and uninfected birdswas a 1.4-fold increase in relative spleen weight.At 60 days, when the earliest evidence of neph-roblastoma appeared in 3 of 12 birds, significantincreases were present in the weights of thekidney, the liver, and the spleen. Chickens sac-rificed during the interval of 90 to 130 days ofage manifested increases in pancreas, bone, lung,liver, and brain weights, and a decrease in thy-mus weight relative to values for organs of un-infected chickens. All 90- to 130-day-old infectedbirds possessed large nephroblastomas, and theirmean body weight was half that of uninfectedchickens.Virus in organs of MAV-2(N)-infected

chickens. Results of electron microscope stud-ies of nephroblastoma-bearing chickens infectedwith AMV (20) and a nephroblastoma-inducingisolate (51) indicate that virus particles are pres-ent in all organs with a large accumulation inthe kidney. Consequently, organ extracts fromMAV-2(N)-infected tumor-bearing chickenswere prepared to quantitate infectious virus.The results demonstrated that a correlation ex-isted between the virus titer in the organs andthe specificity of the virus for neoplasms of boneand kidney (Fig. 1). Nephroblastoma, lung,bones, and pancreas show high virus titers rela-tive to that of serum, whereas liver, spleen,bursa, thymus, heart, and brain show progres-sively lower relative titers. The statistical eval-uation of these results suffers from the low num-ber of chickens involved and the difficulties ofpreparing extracts, particularly from bone.Evidence of anemia in MAV-2(N)-in-

fected chickens. The association of anemiawith infection by subgroup B avian leukosisviruses has been well documented (3, 19, 37).Thus, the PCV of MAV-2(N)-infected chickensrelative to the PCV of uninfected chickens wasmonitored for 2 months after hatching. SPAFASchickens infected by the intravenous route didnot manifest significant anemia. Only on day 11(P < 0.02) was the decrease in PCV of infectedchickens statistically significant with respect tothe PCV of uninfected chickens. The PCV ofchickens bearing nephroblastomas (60 to 130days old) was also comparable to normal values.Serum chemistry of MAV-2(N)-infected

chickens. Serum protein electrophoresis pro-files of uninfected and MAV-2(N)-infectedchickens revealed significant differences in al-bumin and gamma globulin concentrations. At30 and 60 days of age, serum albumin concentra-tion of infected birds was 69 and 83%, respec-tively, of that of uninfected birds. Sera from

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INFECT. IMMUN.506 WATTS AND SMITH

TABLE 3. Relative organ weights ofMA V-2(N)-infected chickensRelative organ wt

Age No. ex- Body wtb p -

(days) amined' tomac Kidney Liver Lungs Brain Pan- Bursa Thymus Spleen Bonecrease

13 C 1.04; Absent 1.04; 1.01; 1.06; 0.97; 0.89; 1.02; 0.80; 1.37; 1.07;30 11 I NSd NS NS NS NS NS NS NS <0.05 NS

12 C 1.09; Present 1.23; 1.13; 1.08; 0.92; 1.08; 0.84; 1.20; 1.79; 1.04;12 I NS <0.01 <0.01 NS NS NS NS NS <0.01 NS

0 6 C 0.54; Present Absente 1.57; 1.46; 2.02; 1.35; 0.57; 0.27; 0.80; 1.38;90-130 9 I <0.01 <0.01 <0.01 <0.01 <0.01 NS <0.01 NS <0.01

a Number of animals sacrificed and examined at the times indicated. C, Uninfected SPAFAS chicks; I, day-old hatched chicks injected intraperitoneally with 105 PFU of MAV-2(N).

b Second value in each pair represents level of significance. NS, Not significant.'Mean nephroblastoma weight at 30 days was 0.7% of the chicken body weight, and at 90 to 130 days, 17.5%

of the body weight. Tumors were not observed in the contact controls used for this experiment.d Organ weight was determined by weighing on a Mettler analytical balance immediately after sacrifice. The

relative organ weight = [mean (organ weight of infected animal/body weight of infected animal)]/[mean (organweight of uninfected animal/body weight of uninfected animal)].

'Normal kidney tissue was almost nonexistent in these animals.

MAV-2(N)-infected chickens at 30 days of agehad serum gamma globulin levels 1.47-foldhigher than those of uninfected birds. Thegamma globulin level in serum from infectedbirds of 60 days of age increased 1.18-fold overthat of uninfected birds. Infected and uninfectedchicken sera showed no differences in the levelsof a and M globulins. The ratio of albumin tototal globulin at 30 and 60 days of age was 1.61and 1.85, respectively, for uninfected birds, andwas 0.87 and 1.49, respectively, for infected birds.Neutralizing antibody in the serum of

MAV-2(N)-infected and -immunized chick-ens. The presence of neutralizing antibody inthe serum of tumor-bearing chickens infectedwith MAV-2(N) was assayed to serve as anindication of how the immune response of thechicken might reflect or influence the course ofinfection. The neutralizing antibody titers ofMAV-2(N)-infected chicken sera did not corre-late with the extent of disease in the chickens(Table 4). Titration of neutralizing antibody andvirus present in the serum of 10 MAV-2(N)-infected tumor-bearing chickens demonstrateda complete inverse correlation between thesetwo factors (Table 5). Most chickens had in theirserum either no virus and a high neutralizingantibody titer, or no antibody and a high titer ofvirus in their serum. Animals were observed tohave neutralizing antibody in the serum and tobear nephroblastoma simultaneously (Tables 4and 5).The virus-specific nature of serum neutraliz-

ing antibody to serum MAV-2(N), serum MAV-2(0), CEF MAV-2(N), CEF MAV-2(0), RSV[MAV-2(N)], RSV [MAV-2(0)], PR.RSV-B,and RPV in the serum of MAV-2(N)-infected,MAV-2(N)-immunized, MAV-2(O)-immunized,and RPV-immunized chickens is summarized in

Table 4. These assays indicated that serum an-tibody may be type-specific as well as subgroup-specific for the virus in individual MAV-2(N)-infected or -immunized chickens. The titers ofantibody to MAV-2(N) compared with those toMAV-2(0) in certain MAV-2(N)-infected or-immunized chickens (790, 851, 1584, 1586, 1078,1079, 1082, 1084, 1086, 1898, 1915, and 1443)showed specificity for MAV-2(N), and in somecases, a specificity for MAV-2(N) grown in CEF

'5

10

N Lu Bo P Li S Bu T H Br

FIG. 1. Virus in MAV-2(N)-infected chicken or-gans relative to serum. SPAFAS chicks were infectedintraperitoneally I day after hatching. Six chickenswith tumors were sacrificed by exsanguination. Or-gans were removed and weighed, extracts were pre-pared as described in the text, and virus was assayedby plaque formation. Relative virus titer was calcu-lated by the following formula. [Virus in organ, ex-pressed as PFU per milliliter x (volume of mediaused for organ extraction)/organ weight in grams]/(serum virus titer expressed as PFU per milliliter.Virus in each organ relative to that in serum for eachchicken was determined, and then mean and stan-dard error values were calculated. N, Nephroblas-toma; Lu, lungs; Bo, bones; P. pancreas; Li, liver; S,spleen; Bu, bursa; T, thymus; H, heart; Br, brain.

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AVIAN NEPHROBLASTOMA VIRUS PATHOLOGY 507TABLE 4. Serum neutralizing antibody titers

Neutralizing antibody titerbDescription of Serum

chicken' (chicken Serum Serum CEF CEF RSV RSV PR.RSVno.) MAV- MAV- MAV- MAV- [MAV- [MAV- V RPV2(N)c 2(O)c 2(N)d 2(0)d 2(N)] 2(0)] B

MAV-2(N)-im- 790 500 50 5,000 500 5,000 500 500 Nonemunized 851 50 5 500 50 500 50 50 None

1584 50 50 500 50 50 50 5 None1585 500 500 5,000 500 500 50 500 None1586 50 50 500 50 500 50 5 None

MAV-2(0)-im- 1195 500 50 500 50 50 50 50 Nonemunized 1215 500 500 500 500 500 500 50 None

RPV-immunized 1197 None None None None None None None 5001200 None None None None None None None 5001201 None None 5 5 None 5 None 500

MAV-2(N)-in-fected

NE 1076 None None None None None None None NoneNO 1078 5 5 50 None 5 5 None NoneNE 1079 5 None 50 5 50 None 5 NoneNE 1082 None None 5 5 5 None None NoneNE 1084 None None 5 5 5 None None NoneNE 1086 None None 5 None 5 None None NoneNE + OS 1087 None None None None None None None NoneNO 1093 None None None None None None None NoneNE + OS 1098 None None None None None None None NoneNE 1898 50 50 500 50 50 50 50 NoneNE 1915 500 500 5,000 500 500 500 500 NoneNE 1443 500 50 500 500 5,000 500 500 Nonea Pathological manifestations of MAV-2(N)-infected chickens were scored as nephroblastoma (NE), nephro-

blastoma plus osteopetrosis (NE + OS), or normal (NO; no pathology at sacrifice).'Sera were analyzed for the presence of neutralizing antibody by plaque or focus neutralization assay as

described in the text. Neutralizing antibody titers are expressed as the reciprocal of the highest antiserumdilution giving greater than 90% reduction in plaques or foci. The titer of sera having no detectable neutralizingantibody is denoted "none."'Serum MAV-2(N) and serum MAV-2(O) denote virus present in infected chicken sera.d CEF MAV-2(N) and CEF MAV-2(O) denote virus obtained from infected chicken embryo fibroblast

cultures.

TABLE 5. Relationship of serum neutralizingantibody titer to virus titer in MAV-2(N)-infected

chickensNeutralizing anti- Serum virus titer

Chicken no." body titer' (PFU/ml)1431 <5 3 X 1051443 500 <10'1898 500 <10'1902 50 <10'1903 5 3 X 1041905 <5 2 X 1061915 5,000 <10'1916 <5 1051918 <5 3 X 1062287 <5 4 X 106

'All chickens were infected intraperitoneally asday-old hatched chicks and were 60 days of age orolder at sacrifice. All chickens had nephroblastomas.

b Neutralizing antibody titers are expressed as thereciprocal of the highest antiserum dilution givinggreater than 90% reduction in plaques. MAV-2(N)used for neutralization was grown in CEF.

(Table 4). A similar pattern of type-specific neu-tralizing antibody was not observed in a limitednumber of MAV-2(0)-immunized chickens. Theneutralization titer for RSV pseudotypes corre-lated generally with the neutralization titer forMAV-2(N) and MAV-2(0). The subgroup relat-edness of MAV-2(N), MAV-2(0), and PR.RSV-B was confirmed by the specificity of the anti-sera, with MAV-2(N) and MAV-2(0) evidentlybeing more closely antigenically related withinthe B subgroup. The different subgroup specific-ity of RPV (subgroup F) was detected also.

DISCUSSIONInvestigation of the polypeptides and RNA of

MAV-2(N) indicates a composition identical tothat of MAV-2(0) by the procedures used. Pu-rified MAV-2(N) contained approximately thesame relative amounts of polypeptides gp85,gp37, p27, p19, p15/plO, and p12 by polyacryl-amide gel electrophoresis as did MAV-2(0). The

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increase in the gp85 content of MAV-2(0) rela-tive to that of AMV and PR.RSV-C (48) wasparalleled by MAV-2(N). However, this in-creased gp85 content may be characteristic ofmany avian leukosis viruses: gels of RAV-O andRPV polypeptides likewise have shown consid-erable amounts of Coomassie blue staining ma-terial migrating in the position of gp85 (E.Schmidt and R. Smith, unpublished data). The35S RNA of MAV-2(N) comigrated precisely ina single peak with that of MAV-2(0). Thus, thegenome of MAV-2(N), like that of MAV-2(0)(43), may be slightly larger than or the same sizeas that of other nontransforming oncovirusesbut is definitely smaller than that of avian sar-coma viruses. The similarity in structural com-position of MAV-2(N) and MAV-2(0) may re-flect a common evolutionary origin.

Results of the infection of chickens withplaque-purified MAV-2(N) both associate thisvirus with related avian oncoviruses and estab-lish its distinctiveness with regard to the path-ological features induced in infected chickens.By gross and histological examination, nephro-blastomas induced by cloned MAV-2(N) werecomparable to those induced: by AMV, BAIstrain A (20, 21); by transplanted homogenatesofAMV-induced nephroblastomas (50); by aviannephroblastomas virus line DNV (26); by theoriginal subgroup B nephroblastoma-inducingvirus isolated by transfection (36); and by MAV-2(0). The only neoplasm induced by MAV-2(N)other than nephroblastoma was an osteopetrosissimilar or identical to that induced by MAV-2(0).Whether MAV-2(N) and MAV-2(0) are mon-

opotent or pluripotent with respect to oncogenicspectrum is an unresolved question. The wideoncogenic spectrum of AMV results from theconstitution of standard strains of AMV withdefective leukemogenic viruses and helper oste-petrosis and nephroblastoma viruses (45). How-ever, isolation of MAV-2(N) and MAV-2(0) byplaque formation presumes their nondefective-ness for replication. Thus, the overlap in onco-genic spectrum of MAV-2(N) and MAV-2(0) isa problem which likely cannot be explained bycontamination of cloned virus stocks with theother strain or by virus defectiveness. However,MAV-2(N) and MAV-2(0) may be pluripotentvirus strains having been selected for specificoncogenicity by their isolation. However, com-parative nucleic acid studies have indicated thatthe genomes of MAV-2(N) and MAV-2(0) areindeed different (S. Watts and R. Smith, manu-script in preparation).The incidence and onset of nephroblastoma

induced by MAV-2(N) are entirely comparableto those indexes found with infection by other

avian nephroblastoma viruses. The DNV virusof Lacour et al. (25) induced greater than 70%incidence of nephroblastoma with an onset of 45to 120 days in 1-day-old chicks injected intra-peritoneally. The latter virus strain also inducedsome osteopetrosis. The virus of Ogura et al.(36), in contrast, induced no osteopetrosis wheninjected intraperitoneally into 1-day-old line 15or Shaver chickens or intravenously into 11-day-old embryos. Approximately 55 to 75% of chick-ens infected with the latter virus strain devel-oped nephroblastomas with an onset from 60 to120 days of age. Ogura et al. (36) also notedcontact transmission of nephroblastoma virus touninfected hatchmates. A difference in the strainof chickens used and the route of infection maybe sufficient to explain some of the differencesin oncogenic spectrum of the original nephro-blastoma virus isolate and plaque-purifiedMAV-2(N). An analogous virus isolated fromCEF transfected with AMV myeloblast DNA orline DNV DNA when injected intraperitoneallyinto 1-day-old chicks also induced nephroblas-tomas with a similar onset (16).

Variation in the incidence of osteopetrosis inSPAFAS chickens with the route of MAV-2(N)infection (Table 2) seems analogous to the vari-ation in the incidence of erythroblastosis versussarcomas upon injection of avian erythroblasto-sis virus strain ES4 by the intravenous routeversus the intramuscular route (18). That is, ifMAV-2(N) has the potential to induce bothnephroblastoma and osteopetrosis, infection bythe intraperitoneal route may promote the ap-pearance of nephroblastoma over osteopetrosis.Such an enhancement must be related only toincidence and not to onset, because both SPA-FAS and line 15 x 7 chickens infected withMAV-2(N) demonstrated an earlier onset ofnephroblastoma when infected intravenously asembryos than intraperitoneally as hatchedchicks (Table 2).The pathological characteristics associated

with MAV-2(N) infection are of significance inconsidering the course of disease and in compar-ing the biological effects of MAV-2(N) andMAV-2(0). These characteristics may be relatedto the process of oncogenesis or may only reflectvirus infection and replication. Although osteo-petrotic chickens grew at a rate 26% of the rateof uninfected chickens (3), MAV-2(N)-infectedchickens through 2 months of age grew at a ratecomparable to that ofuninfected chickens. How-ever, the mean body weight of MAV-2(N)-in-fected chickens sacrificed at 90 to 130 days ofage was 54% of that of uninfected chickens (Ta-ble 3). The latter result may reflect debilitationresulting from tumor mass or stunting due todecreased growth rate.

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Organs of 4-week-old osteopetrotic chicksdemonstrated a decrease in lymphoid organweight and increases in the weights of heart,alimentary tract, bones, liver, kidneys, and brain(3). However, MAV-2(N)-infected chicksshowed an increase in spleen weight at 1 and 2months of age (Table 3). At greater than 3months of age, MAV-2(N)-infected chickensdemonstrated increases in the relative weightsof liver, lungs, brain, pancreas, and bones as wellas a large decrease in thymus weight. In MAV-2(0)-infected chicks, the decrease in lymphoidorgan weight has been associated with immu-nosuppression (47), whereas the increase inweight of other organs was attributed to edema(3). Specific viral proteins have been associatedwith lymphoid organ atrophy and immuno-suppression upon infection of kittens with felineleukemia virus (1, 28). The consequences oflymphoid organ atrophy in MAV-2(N)-infectedchickens have not been examined but edemamay be involved in the increase in weight ofsome organs of MAV-2(N)-infected chickens.However, an apparent increase in relative organweight may result from a decrease in bodyweight due to debilitation and wasting of thenephroblastoma-bearing chicken.

Unlike chickens infected with MAV-2(0) (37),chicken embryos infected intravenously withMAV-2(N) did not develop significant anemiaafter hatching. However, other investigationshave indicated that some chickens infected in-travenously at 8 to 9 days of age with MAV-2(N)developed a transient anemia (R. Paterson andR. Smith, unpublished data). Thus, the associ-ation of anemia with MAV-2(N) infection seemsto be ambiguous at this time. Anemia inducedby MAV-2(0) was accompanied by a thrombo-cytopenia (37). That MAV-2(N)-infected chick-ens had petechial hemorrhages might suggest ablood disorder promoting capillary fragility.Petechial hemorrhages may therefore lead tothe excess of cannabilism observed in MAV-2(N)-infected chickens.The changes in albumin and gamma globulin

levels detected in the serum of MAV-2(N)-in-fected birds parallel those seen in MAV-2(0)-infected chickens (3). Sanders et al. (39) alsonoted an increase in a y globulin-associated com-ponent and a decrease in the albumin/total glob-ulin (A/G) ratio for sera from leukosis-affectedchickens relative to uninfected chickens. Also,the increased serum gamma globulin level mayalso be a consequence of chronic virus infection.The virus titer in organ extracts of nephro-

blastoma-bearing chickens was greater than thatin serum (Fig. 1). The latter observation corre-lates with the finding of budding virus by elec-tron microscopy in all organs of chickens in-

fected with nephroblastoma-inducing viruses(20, 51). Results of electron microscopic studieshave indicated that the number of budding virusparticles was greatest in the target organs, bone,and kidney after infection with a nephroblas-toma virus (51) or with MAV-2(0) (9, 52; E.Schmidt and R. Smith, unpublished data). Thelatter phenomenon seems to be confirmed bythe titration of infectious virus in extracts oforgans of MAV-2(N)-infected chickens. The pri-mary lymphoid organs, the bursa and thymus,contained relatively little virus compared withthe nephroblastoma, bone, lung, and pancreas(Fig. 1). These results suggest that the replica-tion of MAV-2(N) in cells of the lymphoid or-gans is limited. The present study did not ad-dress the question of whether MAV-2(N) wascapable of transforming cells of the bursa. Allinfected chickens died or were sacrificed (129days [Table 2]) before sufficient time hadelapsed for the manifestation of lymphoid leu-kosis. Nevertheless, elevated virus titers in thenephroblastoma and bone suggest that replica-tion may play a role in organ-specific oncogen-esis.The presence of neutralizing antibody and

nephroblastoma in certain infected chickens(Tables 4 and 5) requires comment. First, thepresence of neutralizing antibody indicates thatthe birds were infected under conditions whichdid not permit tolerance to the virus. It is pos-sible that infection of the day-old chick by theintraperitoneal route permitted the infection ofcertain target cells, such as those of the mesen-chymal rest (5), and once infected, these cellswere destined to become transformed, despitethe presence of antibody. Second, it seems clearthat the presence of viremia and the absence ofneutralizing antibody did not accelerate the ap-pearance of nephroblastoma. It seems thereforelikely that there is little recruitment of newtarget cells by virus after the initial infection.This is in contrast to the situation in MAV-2(0)infection, in which administration of neutralizingantibody leads to an arrest of disease (R. E.Smith and J. Ivanyi, manuscript in preparation),suggesting a continuous recruitment of new tar-get cells by MAV-2(0). It is possible that alimited number of transformable target cells ex-ists for MAV-2(N), and that these cells remainsusceptible to virus transformation for a rela-tively long time during development. Furtherexperiments are required to elucidate this point,particularly those employing the infection ofembryos with MAV-2(N) followed by the admin-istration of neutralizing antibody.

Neutralizing antibody titers reported forMAV-2(N)-infected chickens indicate that thepresence of nephroblastoma was not always as-

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510 WATTS AND SMITH

sociated with virus-specific immunoglobulin.Furthermore, preliminary results indicated thattreatment of chickens with cyclophosphamidedid not prevent nephroblastoma induction byMAV-2(N). Nowygrod et al. (34) likewise foundthat growth of AMV-induced nephroblastomasdid not require the presence of circulating anti-body. However, the level of serum antibodydemonstrated a striking inverse correlation withthe level of serum virus in MAV-2(N)-infectedchickens. Thus, the antibody response in in-fected chickens may play a significant role inlimiting viremia, even though not related tonephroblastoma induction. As shown with RSV-induced sarcomas, virus immunity may have norelationship to or may enhance tumor growth(29). Furthermore, virus-antibody complexesmay be infectious (40).Moreover, as shown by the titration of serum

antibody to MAV-2(N) and related viruses (Ta-ble 4), in some chickens the humoral immuneresponse detectable by neutralization assaymanifested specificity for the infecting virus. Asshown by competition radioimmunoassay (48),MAV-2(0) virus or MAV-2(0) gp85 isolatedfrom CEF cultures and MAV-2(0) from theserum of osteopetrotic chickens showed differentantigenic reactivities. In correlation, the neu-tralizing activity of some of the MAV-2(N)-in-fected or -immunized chicken sera characterizedin Table 4 also demonstrated preference for CEFversus serum virus antigens. Virus neutralizationpatterns of some chicken sera also indicated aclose antigenic relatedness between MAV-2(N)and MAV-2(0)-closer than the relatedness be-tween either or these two viruses and PR.RSV-B. Lack of neutralization ofRPV with sera fromchickens infected or immunized with subgroupB virus reflected the subgroup-specific nature ofvirus antigenicity associated with the envelopeglycoprotein. Likewise, the type-specific reactiv-ities of these sera also may be associated withthe glycoprotein determinants of the virus (23).The present study presents a description of

the pathological manifestations of chickens in-fected with an avian nephroblastoma virus. Theresults of this investigation indicate that themodel system employed would be useful to ad-dress several fundamental questions of RNAtumor virus pathogenesis. First, the number andpersistence of target cells in the host duringdevelopment could be investigated by infectingboth embryos and adult animals. The numberof target cells could be estimated by microscopicenumeration of transformed foci present in thekidney at an early stage of tumor formation (i.e.,60 days postinfection) and observation of in-fected animals for nephroblastomas for a periodof at least 6 months. It is proposed that injection

of adults with a high dose of virus will lead toinfection and ultimately to transformation oftarget cells, if present, in spite of the develop-ment of neutralizing antibodies. Second, the ge-netics of disease induction could be investigated.Lymphoid leukosis induction is influenced by atleast one gene other than that coding for asurface receptor for virus (12). Since diseaseinduction in nephroblastoma is relatively rapid(compared with lymphoid leukosis) it would beof interest to determine whether several inbredlines of chickens differ in susceptibility to neph-roblastoma induction. Genetic analysis could im-plicate the same genetic loci for lymphoid leu-kosis and nephroblastoma. Third, the role ofimmunological competence could be addressedwith the nephroblastoma model. Infection ofbursectomized adult chickens could be used toinvestigate the role of antibody in the diseaseprocess. If nephroblastomas are observed in bur-sectomized chickens, the role of antibody in thedisease process will be eliminated. A preliminaryexperiment with a limited number of cyclophos-phamide-treated chickens indicated that theseanimals would develop nephroblastomas. How-ever, more complete experiments are planned,in which chicks which have been treated withboth testosterone and cyclophosphamide will beemployed, and treated chicks will be tested foragammaglobulinemia before administration ofvirus. If no tumors are observed in bursecto-mized chickens, passive antibody administrationto infected chickens might lead to the appear-ance of nephroblastomas if antigen-antibodycomplexes are involved in the disease process.The role of lymphocytes can be investigated bythe adoptive transfer of immune and nonim-mune lymphocytes from histocompatible do-nors. The experiments proposed should allowdefinitive answers to several interesting biologi-cal questions.

ACKNOWLEDGMENTSThe technical contributions of T. Wheeler and S. Nebes

provided indispensable support for this investigation. A. J.Banes gave advice and support during the inception andanalysis of these experiments. We wish to especially thankHeinz Bauer for generously providing the original isolate ofnephroblastoma virus.

This research was supported by Public Health Servicegrants R01-CA-12323 and P01-CA-14236 from the NationalCancer Institute. S.L.W. was a recipient of a National ScienceFoundation Predoctoral Fellowship HES-7422381.

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