Pathology of Chickens Infected with Avian Nephroblastoma Virus MAV-2(N)
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INFECTION AND IMMUNITY, Feb. 1980, p. 501-512 Vol. 27, No. 2
0019-9567/80/02-0501/12$02.00/0
Pathology of Chickens Infected with Avian Nephroblastoma
Virus MAV-2(N)
SUSAN L. WATTSt AND RALPH E. SMITH*
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina
27710
A nephroblastoma-inducing myeloblastosis-associated virus, MAV-2(N), de-
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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 viral
polypeptides as another myeloblastosis-associated virus inducing predominantly
osteopetrosis 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 SPAFAS
and line 15 x 7 chickens: the oncogenic spectrum and the onset of nephroblastoma
varied with the line of chicken and the route of injection. Renal neoplasms were
manifest in chickens older than 2 months and grew to a massive size. Furthermore,
29% of control chickens housed with MAV-2(N)-infected chickens demonstrated
nephroblastoma. MAV-2(N)-infected chickens had growth rates and blood packed
cell volumes comparable to those of uninfected chickens. Infected chickens 2
months of age had increased kidney, liver, and spleen weights; tumor-bearing
chickens 3 to 4 months of age had increased liver, lung, brain, pancreas, and bone
weights. The concentration of albumin was decreased and the concentration of
gamma globulin was increased in the serum of MAV-2(N)-infected chickens.
Analysis of the sera of nephroblastoma-bearing chickens for virus and antibody
showed that three states existed: (i) high levels of neutralizing antibody, (ii) high
levels 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 myeloblastosis cells, bone marrow cells, and macrophages in
virus (AMV), BAI strain A, primarily induces vitro, they cannot replicate in chicken embryo
myeloblastosis, but also induces osteopetrosis, fibroblast (CEF) cultures (6, 31). However,
nephroblastoma, lymphoid leukosis, and other nephroblastoma and osteopetrosis viruses can
neoplasms in infected chickens (4). Efforts to replicate in CEF and cause plaques characteris-
select for the induction of a single type of neo- tic of subgroup B leukosis viruses (17, 35, 45).
plasm led to the isolation of several viral species Yet morphological transformation of any cell
from standard AMV: a subgroup A myeloblas- type in vitro has not been demonstrated by the
tosis-associated virus MAV-1 inducing a high latter type of viruses. In vivo standard AMV
incidence of osteopetrosis (45); a subgroup B induces myeloblastosis within 2 weeks after in-
leukosis virus inducing predominantly osteope- fection (4), whereas nephroblastoma, osteope-
trosis MAV-2(0) (45); a subgroup B leukosis trosis, or lymphoid leukosis become manifest in
virus inducing primarily nephroblastoma (25,35, chickens infected with standard AMV or with
50); and leukemogenic viruses of subgroups A myeloblastosis-associated viruses weeks or
and B, respectively, AMV-A and AMV-B (22, months after infection (10, 35, 36, 45).
32). MAV strains primarily inducing osteopetrosis
Evidence suggests that the nephroblastoma- were isolated by endpoint dilution of superna-
and osteopetirosis-inducing leukosis viruses are tant fluids obtained from cell cultures of stan-
nondefective helper viruses providing glycopro- dard AMV-induced chicken tumors of standard
tein envelope components for the replication- AMV-treated fibroblasts (45). Subgroup B MAV
defective leukemogenic viruses in standard strains inducing a high incidence of nephroblas-
AMV (30). Although the leukemogenic compo- toma were isolated by two routes: (i) passage of
nents have been shown to transform yolk sac standard AMV-induced nephroblastoma ex-
tracts in vivo (50) and (ii) transfection ofhamster
t Present address: Department of Microbiology, University fibroblasts (25) or CEF (35) in vitro with stan-
of Minnesota, Minneapolis, MN 55455. dard AMV-induced leukemic myeloblast deoxy-
501502 WATTS AND SMITH INFECT. IMMUN.
ribonucleic acid (DNA). The virus strain isolated facility. Infected and uninfected birds in the same
by Ogura et al. (35, 36) has been cloned in our room were kept in separate cages. Birds were fed
laboratory by plaque purification: the present Purina Growena Chow and water ad libitum. For
investigation reports the biochemical and path- experimental purposes, or when incapacitated by tu-
mor or other disease, chickens were sacrificed by
ogenetic characterization of this cloned virus exsanguination. All chickens were subjected to post-
MAV-2(N). mortem examination.
Avian nephroblastoma is a renal tumor of Chicken body weight and organ weight deter-
both epithelial and mesenchymal cell types man- minations. Birds were weighed on a Harvard labo-
ifesting distorted morphology and abnormal dif- ratory balance. Organs excised from birds sacrificed by
ferentiation. The avian tumor morphology is exsanguination were weighed on a Mettler analytical
comparable to that of Wilms' tumor of humans balance.
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(33) as well as that of nephroblastomas found in Extraction of virus from infected chicken or-
rabbits, swine, cattle, sheep, dogs, and cats (7, gans. Extracts from organs excised from infected
14, 24, 38). Histopathological studies of avian chickens were prepared as follows: Organs were
washed with Tris buffer or phosphate-buffered saline
nephroblastoma note the presence of all com- (0.15 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM
ponents of normal kidney in various states of Na2HPO4, 0.9 mM CaCl2, 0.5 mM MgCl2.6H20), then
organization and differentiation as well as occa- minced with a scalpel and spatula or homogenized
sional evidence of cartilage, bone, keratinized with a Virtis "23" homogenizer in 0.1 to 0.5 ml of Tris
epithelium, and sarcoma. Thus, an embryonic buffer per g. Cells were sedimented by centrifugation
rest residual in the adult kidney has been sug- at 800 x g for 15 min at 40C, and the supernatant fluid
gested as the origin of the nephroblastoma (5, was frozen at -70°C until used for virus titration by
21). However, the target cell and mechanism of plaque assay.
the viral-induced transformation are unknown. Blood packed cell volume (PCV). Blood samples
The present study was undertaken to elucidate were collected in heparinized 50-,l microhematocrit
tubes and centrifuged in a Clay Adams microhemato-
the pathological manifestations of MAV-2(N) crit centrifuge. Packed cell volume for each sample
infection by investigating biological characteris- was calculated by comparison to a standard scale.
tics of MAV-2(N)-infected chickens. Serum protein electrophoresis. Serum protein
(This investigation is part of a dissertation electrophoretic analyses were performed for all sam-
submitted by S.L.W. to the Department of Mi- ples in triplicate by using a Beckman Microzone elec-
crobiology and Immunology, Duke University trophoresis system, model R-101 (3). Serum samples
Medical Center, for the Ph.D. degree.) of 0.25 pl were applied to cellulose acetate membranes
and subjected to 4.5 mA at 250 V for 20 min in sodium
MATERIALS AND METHODS barbital (B-2) buffer, pH 8.6. Membranes were stained
in Ponceau red S, then cleared in dilute glacial acetic
Eggs. Fertile chicken eggs certified free of chicken acid and ethanol. Membrane scans were performed by
helper factor and avian leukosis virus group-specific using a Quick-Scan, Jr., interfaced with a Digital PDP-
antigen were obtained from SPAFAS, Inc., Norwich, 11/10 computer. Areas under the peaks were inte-
Conn. Fertile eggs of line 15 x 7 were obtained from grated and expressed relative to the total area scanned.
the Regional Poultry Research Laboratory, East Lan- Serum protein concentration was determined by the
sing, Mich. Eggs were incubated at 37.50C in a humid- method of Lowry et al. (27).
ified incubator. Immunization of chickens with virus. Unin-
Chickens-infection and maintenance. SPA- fected normal adult chickens were injected intrave-
FAS and line 15 x 7 chickens were infected with 105 nously with 0.1 to 0.5 ml of virus in Tris buffer con-
plaque-forming units (PFU) of MAV-2(N) (0.1 ml) taining 10% calf serum and reinjected by the same
produced in tissue culture or mock-infected with 0.1 procedure 3 to 4 weeks later. Approximately 2 months
ml of tris(hydroxymethyl)aminomethane (Tris) buffer after the primary injection, the chickens were sacri-
(0.14 M NaCl, 0.025 M Tris-hydrochloride, pH 7.4, 5 ficed by exsanguination, and the serum was stored at
mM glucose, 100 U of penicillin per ml, 100 ig of -70°C for titration of serum antibody by the neutral-
streptomycin per ml, 0.7 mM Na2HPO4) containing ization assay of Ishizaki and Vogt (23).
10% (vol/vol) calf serum by one of two routes: (i) Statistical analysis. Values were reported as
infection of a chorioallantoic membrane vein of 12- mean ± standard error for each determination. Levels
day-old embryos (2), or (ii) intraperitoneal infection of of significance (P) were calculated by Student's two-
1-day-old hatched chicks. MAV-2(0) was adminis- tailed t test. P values refer to the difference in param-
tered at 104 PFU per embryo [a lower dose than MAV- eters between MAV-2(N)-infected and uninfected
2(N)] to the same lines of chickens, because 105 PFU chickens of the same age.
of MAV-2(0) per embryo led to high embryo mortality Virus sources and derivations. A nephroblas-
and early death of the hatched chick due to severe toma-inducing virus derived by transfection of CEF in
osteopetrosis. One day before hatching, eggs were culture with DNA from AMV-induced leukemic mye-
transferred to humidified hatchers maintained at loblasts (35) was received from Heinz Bauer, Institut
37.50C, separate hatchers being used for infected and fur Virologie, Justus-Liebig Universitat, Giessen, W.
control eggs. Birds infected with different viruses were Germany. This virus, cloned three times by plaque
maintained in separate rooms of an animal isolation purification and propagated in our laboratory in SPA-VOL. 27, 1980 AVIAN NEPHROBLASTOMA VIRUS PATHOLOGY 503
FAS CEF (C/E), will hereafter be referred to as MAV- Polyacrylamide gel electrophoresis of viral
2(N), denoting that it is a myeloblastosis-associated RNA. Viral 35S RNA was analyzed by electrophoresis
virus of subgroup B inducing nephroblastoma. MAV- in 2.1% (wt/vol) acrylamide-0.6% (wt/vol) agarose-6
2(0) (45) has been well characterized in our laboratory M urea gels (15). Gel slices were solubilized overnight
(3). at 370C in Protosol/toluene/water (9:10:1, vol/vol),
All other viruses were obtained as follows: Prague and radioactivity in each sample was determined in a
B strain of Rous sarcoma virus (PR.RSV-B) from R. toluene-based liquid scintillation cocktail.
R. Friis, Institut fur Virologie, Justus-Liebig Univer- RESULTS
sitat, Giessen, W. Germany; Prague C strain of Rous
sarcoma virus (PR.RSV-C), and ring-necked pheasant Biochemical composition of MAV-2(N).
virus (RPV) from P. K. Vogt, University of Southern The polypeptide composition of purified MAV-
California, Los Angeles; and AMV from J. W. Beard, 2(N) relative to that of other avian retroviruses
Life Sciences, Inc., St. Petersburg, Fla. MAV-2(N) and was investigated by SDS-polyacrylamide gel
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MAV-2(0) pseudotypes of Rous sarcoma virus (RSV) electrophoresis. Electrophoretically separated
were produced by infection of Bryan strain RSV(-)
CEF with MAV-2(N) or MAV-2(0). Cells transformed MAV-2(N) polypeptides comigrated with the
by RSV(-) were a generous gift of H. Hanafusa, gp85, gp37, p27, p19, p15/plO, and p12 of MAV-
Rockefeller University, New York, N.Y. 2(0), AMV, and PR.RSV-C. Although these
Cell culture. Primary CEFs were prepared from polypeptides were not further characterized,
11-day-old SPAFAS embryos as described by Vogt their identity was assumed on the basis of elec-
(49) and propagated in supplemented Ham F10 me- trophoretic mobility. A comparison of the rela-
dium (42). Secondary CEFs seeded into tissue culture tive amounts of polypeptides in each virus was
dishes or roller bottles were infected for virus produc- achieved by using gel scans of Coomassie bril-
tion at an input multiplicity of infection of 0.01 to 0.10 liant blue-stained protein. The ratio of gp85/p27
PFU or focus-forming units per cell.
Plaque assays were performed by the method of provided a further index of the relative amounts
Graf (17). The focus assay of Vogt (49) was used for of gp85 and p27 polypeptides per virion. The
assaying transforming avian sarcoma viruses (ASVs). relative amounts of MAV-2(N) polypeptides cor-
Antiviral antibody was assayed by plaque or focus responded completely with the values obtained
neutralization as described by Ishizaki and Vogt (23). for MAV-2(0) polypeptides. MAV-2(N), as well
Complete neutralization was scored as 90% reduction as MAV-2(0) (48), contained more stained ma-
in plating efficiency. terial identifiable as gp85 than did AMV or
Large-scale virus production. Infected cells were PR.RSV-C (Table 1).
grown in roller bottles for production of large amounts The size of MAV-2(N) 35S RNA was com-
of virus for ribonucleic acid (RNA) and protein isola- pared to that of MAV-2(0) by electrophoresis
tion. Roller bottles containing cells infected with
MAV-2(N), MAV-2(0), and PR.RSV-C were fed and on the same gel. The 70S RNA from each virus
harvested every 2 h on a Bellco Smith-Kozoman Au- was labeled, extracted, heat denatured, and sub-
toharvester (41, 44).
Virus concentration, purification, and label- TABLE 1. Comparison of the polypeptide
ing. Virus was concentrated by pelleting and purified composition of MAV-2(N) with that of other avian
on sucrose gradients as previously described (41). Vi- oncovirusesa
rus pellets were suspended in buffer and stored at % of total polypeptideb
-70°C. Viral RNA was labeled with 10 mCi per roller Polypeptide
bottle of [5,6-3H]uridine (40 to 50 Ci/mmol) by adding position MAV-2- MAV-2- AMV PR.RSV-
the radioisotope in culture medium to confluent roller (N) (0) C
bottle cultures. Supernatant fluids were harvested 24 gp85 8.68 10.04 2.60 2.26
h after addition of radioisotope and every 12 h there- gp37 2.24 2.43 3.37 3.21
after. Virus was labeled with carrier-free [32P]ortho- p27 22.43 23.37 26.08 22.11
phosphate as previously described (46). p19 14.59 14.02 16.27 15.27
Viral polypeptide polyacrylamide gel electro- p12 14.72 14.77 15.93 15.25
phoresis. Viral polypeptides were analyzed by elec- p15, plO 9.28 7.73 12.80 12.95
trophoresis in slab gels of 10% (wt/vol) acrylamide- gp85/p27C 0.39 0.43 0.10 0.10
0.1% (wt/vol) sodium dodecyl sulfate (SDS) (8, 48).
Gels stained with Coomassie brilliant blue were
a
SDS-polyacrylamide gel electrophoresis analyses
scanned on a Quick-Scan, Jr., interfaced with a digital of purified PR.RSV-C, MAV-2(N), MAV-2(0), and
PDP-11/10 computer. The peaks of optical density AMV were performed in triplicate. Gels were stained
obtained in the scans were integrated by the computer with Coomassie brilliant blue and scanned, and peak
relative to the total stained material and the amount areas were determined by computer integration.
b The mean value of percent stained material mi-
of protein applied to the gel.
Viral RNA purification. Viral RNA was purified grating in each polypeptide position relative to the
by SDS-pronase treatment and phenol extraction of total stained material on the gel is given for each virus.
c The ratio of gp85 to p27 was calculated as an index
virus pellets (11, 13). Viral 70S RNA was isolated by
rate-zonal gradient centrifugation in gradients of 15 to of the relative amounts of these polypeptides in each
30% (wt/vol) sucrose (13). virus.504 WATTS AND SMITH INFECT. IMMUN.
AND SMITH
504 WATTS IMMUN.~~~.if
INFECT.
jected to electrophoresis as described in Mate- _ 4 o0
rials and Methods. The MAV-2(N) 35S RNA
peak comigrated precisely with that of MAV- > 2 c
2(0).
Pathology of chickens infected with 4 0 >
MAV-2(N). Chickens infected with plaque-pu- o tL_ t-
rifled CEF-grown MAV-2(N) manifested an in- c o
cidence of nephroblastoma greater than 75% and
an incidence of osteopetrosis less than 50%, and _ o 00
none of the other neoplasms associated with o0
AMV (myeloblastosis, lymphoid leukosis) (Ta- tic C
ble 2). The nephroblastomas induced by MAV- S n W MLo
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0
2(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, z CD 00
36). The nephroblastomas consisted of abnor-
mally differentiated kidney tissue containing ele- S t ° o
ments of sarcoma, carcinoma, chondroma, os- .q .c V.
teoid, and keratinized tissue. These noninvasive Z o
neoplasms were multifocal in origin and pro-
gressed to form massive, thickly encapsulated 3J 0N c
structures. The tumors varied in their composi- 2! V M D - -O
tion of fluid-filled cysts, solid tissue, necrotic 0o
areas, 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- ; CC
served with MAV-2(N) infection was of a later 0 4n
onset and a more peripheral body distribution, X C
but otherwise was identical to that observed o 4
after infection with MAV-2(0) (3, 45). rC c14
MAV-2(N)-infected chickens manifested sev- -s O o
eral other abnormalities, some of which could be W _S o2 'C . -
attributed to virus infection or to the presence S o 0 CC
of nephroblastoma. Chickens dying before age
60 days died of cannibalism, anemia, other infec-
00
;: la
_ .c
tions, intra-abdominal hemorrhage, or unknown - . .
2 0 Mc q L
causes (Table 2). Chickens dying after age 60 2 oo
0
days also demonstrated a large incidence of can- O. . oJ >, t0 3 3
nibalism and were frequently observed to man- 1' z < 0
5- o
ifest intradermal petechial hemorrhages of un- ^ 2c. p0 4
known significance. Cannibalism was rarely ob- 3 o
served in uninfected chickens and chickens in- Ho > _o.0
fected with other viruses, indicating that feeding, o
light, and confinement conditions did not con- _ _0
- o
tribute to the high rate of cannibalism seen in in o 5 4
MAV-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) .2
infection. When the size of the nephroblastoma I= U N , 0 °
caused abdominal obstruction, chickens mani- o0 4
fested ascites, diarrhea, intra-abdominal hem- co 0 ow,
orrhage, and paralysis. O= .-
Effect of chicken strain and route of in-
fection on the oncogenic spectrum of MAV-
o: -> -.+1 3
cc
2(N). The relative incidence of nephroblastoma O C/X .C C> t6iC.00 .g=0) s> _
and osteopetrosis varied with the chicken line .c
and route of injection used (Table 2). The inci- -x
dence of nephroblastoma was approximately a co
a
oVOL. 27, 1980 AVIAN NEPHROBLASTOMA VIRUS PATHOLOGY 505
80% in SPAFAS chicks and approximately 90% relative to the total body weight of each bird
in line 15 x 7 chickens. The similarity of neph- (Table 3). At 30 days of age, the only significant
roblastoma incidence in chickens infected with difference between infected and uninfected birds
MAV-2(N) as 12-day-old embryos or as 1-day- was a 1.4-fold increase in relative spleen weight.
old hatched chicks suggests that the target cell At 60 days, when the earliest evidence of neph-
for nephroblastoma is present at both stages of roblastoma appeared in 3 of 12 birds, significant
differentiation. Line 15 x 7 chickens infected increases were present in the weights of the
intravenously manifested the highest incidence kidney, the liver, and the spleen. Chickens sac-
of osteopetrosis (50%). The incidence of osteo- rificed during the interval of 90 to 130 days of
petrosis was comparable for line 15 x 7 chickens age manifested increases in pancreas, bone, lung,
infected intraperitoneally and SPAFAS chick- liver, and brain weights, and a decrease in thy-
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ens infected intravenously (30 and 20%, respec- mus weight relative to values for organs of un-
tively). However, SPAFAS chicks infected intra- infected chickens. All 90- to 130-day-old infected
peritoneally manifested only 3% incidence of birds possessed large nephroblastomas, and their
osteopetrosis. mean body weight was half that of uninfected
Influence of host age and route of infec- chickens.
tion on the onset of nephroblastoma and Virus in organs of MAV-2(N)-infected
osteopetrosis. Chickens infected as embryos chickens. Results of electron microscope stud-
by the intravenous route manifested an earlier ies of nephroblastoma-bearing chickens infected
onset of nephroblastoma than those infected as with AMV (20) and a nephroblastoma-inducing
hatched chicks by the intraperitoneal route (Ta- isolate (51) indicate that virus particles are pres-
ble 2). This difference was especially evident in ent in all organs with a large accumulation in
infected SPAFAS chicks; the average age of the kidney. Consequently, organ extracts from
chickens dying or sacrificed with nephroblas- MAV-2(N)-infected tumor-bearing chickens
toma was approximately one month younger for were prepared to quantitate infectious virus.
intravenously infected embryos than for intra- The results demonstrated that a correlation ex-
peritoneally infected hatched chickens. A slight isted between the virus titer in the organs and
difference in the age of chickens dying or sacri- the specificity of the virus for neoplasms of bone
ficed with osteopetrosis was apparent only with and kidney (Fig. 1). Nephroblastoma, lung,
respect to strain, with SPAFAS chicks showing bones, and pancreas show high virus titers rela-
a slightly earlier onset than did line 15 x 7 tive to that of serum, whereas liver, spleen,
chicks. bursa, thymus, heart, and brain show progres-
Contact transmission of MAV-2(N) infec- sively lower relative titers. The statistical eval-
tion. A total of 34 uninfected SPAFAS and 15 uation of these results suffers from the low num-
x 7 chickens were hatched and raised in the ber of chickens involved and the difficulties of
same room with MAV-2(N)-infected chickens. preparing extracts, particularly from bone.
Ten (29%) of the uninfected chickens demon- Evidence of anemia in MAV-2(N)-in-
strated nephroblastoma. The mean age of con- fected chickens. The association of anemia
tact-infected chickens manifesting nephroblas- with infection by subgroup B avian leukosis
toma (111 ± 9 days) was slightly older than that viruses has been well documented (3, 19, 37).
for experimentally infected chickens. None of Thus, the PCV of MAV-2(N)-infected chickens
the contact-infected chickens demonstrated os- relative to the PCV of uninfected chickens was
teopetrosis. monitored for 2 months after hatching. SPAFAS
Growth rate of MAV-2(N)-infected chick- chickens infected by the intravenous route did
ens. To determine whether infection with MAV- not manifest significant anemia. Only on day 11
2(N) altered the growth rate of the chicken, the (P < 0.02) was the decrease in PCV of infected
body weight of intraperitoneally infected SPA- chickens statistically significant with respect to
FAS chicks was measured every third day for 2 the PCV of uninfected chickens. The PCV of
months after hatching. Both infected and unin- chickens bearing nephroblastomas (60 to 130
fected chickens grew at a rate of 6.33 g per day. days old) was also comparable to normal values.
Therefore, no stunting of infected chickens was Serum chemistry of MAV-2(N)-infected
observed during this time period. chickens. Serum protein electrophoresis pro-
Organ weights of MAV-2(N)-infected files of uninfected and MAV-2(N)-infected
chickens. Organ weights of MAV-2(N)-infected chickens revealed significant differences in al-
chickens at 30, 60, and 90 to 130 days of age were bumin and gamma globulin concentrations. At
analyzed to determine whether systemic organ 30 and 60 days of age, serum albumin concentra-
changes associated with MAV-2(N) infection tion of infected birds was 69 and 83%, respec-
could be detected. Organ weights were expressed tively, of that of uninfected birds. Sera from506 WATTS AND SMITH INFECT. IMMUN.
TABLE 3. Relative organ weights of MA V-2(N)-infected chickens
Relative organ wt
Age No. ex- Body wtb p -
(days) amined' tomac Pan- Bursa Thymus Spleen Bone
Kidney Liver Lungs Brain crease
13 C 1.04; Absent 1.04; 1.01; 1.06;
NS 0.97;
NS 0.89;NS 1.02; NS 0.80; 1.37; 1.07;
NSVOL. 27, 1980 AVIAN NEPHROBLASTOMA VIRUS PATHOLOGY 507
TABLE 4. Serum neutralizing antibody titers
Neutralizing antibody titerb
Description of Serum
chicken' (chicken Serum Serum CEF CEF RSV RSV PR.RSVV
no.) MAV- MAV- MAV- MAV- [MAV- [MAV- RPV
2(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 None
munized 851 50 5 500 50 500 50 50 None
1584 50 50 500 50 50 50 5 None
1585 500 500 5,000 500 500 50 500 None
1586 50 50 500 50 500 50 5 None
MAV-2(0)-im- 1195 500 50 500 50 50 50 50 None
munized 1215 500 500 500 500 500 500 50 None
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RPV-immunized 1197 None None None None None None None 500
1200 None None None None None None None 500
1201 None None 5 5 None 5 None 500
MAV-2(N)-in-
fected
NE 1076 None None None None None None None None
NO 1078 5 5 50 None 5 5 None None
NE 1079 5 None 50 5 50 None 5 None
NE 1082 None None 5 5 5 None None None
NE 1084 None None 5 5 5 None None None
NE 1086 None None 5 None 5 None None None
NE + OS 1087 None None None None None None None None
NO 1093 None None None None None None None None
NE + OS 1098 None None None None None None None None
NE 1898 50 50 500 50 50 50 50 None
NE 1915 500 500 5,000 500 500 500 500 None
NE 1443 500 50 500 500 5,000 500 500 None
a 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 antiserum
dilution giving greater than 90% reduction in plaques or foci. The titer of sera having no detectable neutralizing
antibody 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 neutralizing (Table 4). A similar pattern of type-specific neu-
antibody titer to virus titer in MAV-2(N)-infected tralizing antibody was not observed in a limited
chickens number of MAV-2(0)-immunized chickens. The
Neutralizing anti- Serum virus titer neutralization titer for RSV pseudotypes corre-
Chicken no." body titer' (PFU/ml) lated generally with the neutralization titer for
1431508 WATTS AND SMITH INFECT. IMMUN.
increase in the gp85 content of MAV-2(0) rela- avian nephroblastoma viruses. The DNV virus
tive to that of AMV and PR.RSV-C (48) was of Lacour et al. (25) induced greater than 70%
paralleled by MAV-2(N). However, this in- incidence of nephroblastoma with an onset of 45
creased gp85 content may be characteristic of to 120 days in 1-day-old chicks injected intra-
many avian leukosis viruses: gels of RAV-O and peritoneally. The latter virus strain also induced
RPV polypeptides likewise have shown consid- some osteopetrosis. The virus of Ogura et al.
erable amounts of Coomassie blue staining ma- (36), in contrast, induced no osteopetrosis when
terial migrating in the position of gp85 (E. injected intraperitoneally into 1-day-old line 15
Schmidt and R. Smith, unpublished data). The or Shaver chickens or intravenously into 11-day-
35S RNA of MAV-2(N) comigrated precisely in old embryos. Approximately 55 to 75% of chick-
a single peak with that of MAV-2(0). Thus, the ens infected with the latter virus strain devel-
genome of MAV-2(N), like that of MAV-2(0) oped nephroblastomas with an onset from 60 to
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(43), may be slightly larger than or the same size 120 days of age. Ogura et al. (36) also noted
as that of other nontransforming oncoviruses contact transmission of nephroblastoma virus to
but is definitely smaller than that of avian sar- uninfected hatchmates. A difference in the strain
coma viruses. The similarity in structural com- of chickens used and the route of infection may
position of MAV-2(N) and MAV-2(0) may re- be sufficient to explain some of the differences
flect a common evolutionary origin. in oncogenic spectrum of the original nephro-
Results of the infection of chickens with blastoma virus isolate and plaque-purified
plaque-purified MAV-2(N) both associate this MAV-2(N). An analogous virus isolated from
virus with related avian oncoviruses and estab- CEF transfected with AMV myeloblast DNA or
lish its distinctiveness with regard to the path- line DNV DNA when injected intraperitoneally
ological features induced in infected chickens. into 1-day-old chicks also induced nephroblas-
By gross and histological examination, nephro- tomas with a similar onset (16).
blastomas induced by cloned MAV-2(N) were Variation in the incidence of osteopetrosis in
comparable to those induced: by AMV, BAI SPAFAS chickens with the route of MAV-2(N)
strain A (20, 21); by transplanted homogenates infection (Table 2) seems analogous to the vari-
of AMV-induced nephroblastomas (50); by avian ation in the incidence of erythroblastosis versus
nephroblastomas virus line DNV (26); by the sarcomas upon injection of avian erythroblasto-
original subgroup B nephroblastoma-inducing sis virus strain ES4 by the intravenous route
virus isolated by transfection (36); and by MAV- versus the intramuscular route (18). That is, if
2(0). The only neoplasm induced by MAV-2(N) MAV-2(N) has the potential to induce both
other than nephroblastoma was an osteopetrosis nephroblastoma and osteopetrosis, infection by
similar or identical to that induced by MAV- the intraperitoneal route may promote the ap-
2(0). pearance of nephroblastoma over osteopetrosis.
Whether MAV-2(N) and MAV-2(0) are mon- Such an enhancement must be related only to
opotent or pluripotent with respect to oncogenic incidence and not to onset, because both SPA-
spectrum is an unresolved question. The wide FAS and line 15 x 7 chickens infected with
oncogenic spectrum of AMV results from the MAV-2(N) demonstrated an earlier onset of
constitution of standard strains of AMV with nephroblastoma when infected intravenously as
defective leukemogenic viruses and helper oste- embryos than intraperitoneally as hatched
petrosis and nephroblastoma viruses (45). How- chicks (Table 2).
ever, isolation of MAV-2(N) and MAV-2(0) by The pathological characteristics associated
plaque formation presumes their nondefective- with MAV-2(N) infection are of significance in
ness for replication. Thus, the overlap in onco- considering the course of disease and in compar-
genic spectrum of MAV-2(N) and MAV-2(0) is ing the biological effects of MAV-2(N) and
a problem which likely cannot be explained by MAV-2(0). These characteristics may be related
contamination of cloned virus stocks with the to the process of oncogenesis or may only reflect
other strain or by virus defectiveness. However, virus infection and replication. Although osteo-
MAV-2(N) and MAV-2(0) may be pluripotent petrotic chickens grew at a rate 26% of the rate
virus strains having been selected for specific of uninfected chickens (3), MAV-2(N)-infected
oncogenicity by their isolation. However, com- chickens through 2 months of age grew at a rate
parative nucleic acid studies have indicated that comparable to that of uninfected chickens. How-
the genomes of MAV-2(N) and MAV-2(0) are ever, the mean body weight of MAV-2(N)-in-
indeed different (S. Watts and R. Smith, manu- fected chickens sacrificed at 90 to 130 days of
script in preparation). age was 54% of that of uninfected chickens (Ta-
The incidence and onset of nephroblastoma ble 3). The latter result may reflect debilitation
induced by MAV-2(N) are entirely comparable resulting from tumor mass or stunting due to
to those indexes found with infection by other decreased growth rate.VOL. 27, 1980 AVIAN NEPHROBLASTOMA VIRUS PATHOLOGY 509
Organs of 4-week-old osteopetrotic chicks fected with nephroblastoma-inducing viruses
demonstrated a decrease in lymphoid organ (20, 51). Results of electron microscopic studies
weight and increases in the weights of heart, have indicated that the number of budding virus
alimentary tract, bones, liver, kidneys, and brain particles was greatest in the target organs, bone,
(3). However, MAV-2(N)-infected chicks and kidney after infection with a nephroblas-
showed an increase in spleen weight at 1 and 2 toma virus (51) or with MAV-2(0) (9, 52; E.
months of age (Table 3). At greater than 3 Schmidt and R. Smith, unpublished data). The
months of age, MAV-2(N)-infected chickens latter phenomenon seems to be confirmed by
demonstrated increases in the relative weights the titration of infectious virus in extracts of
of liver, lungs, brain, pancreas, and bones as well organs of MAV-2(N)-infected chickens. The pri-
as a large decrease in thymus weight. In MAV- mary lymphoid organs, the bursa and thymus,
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2(0)-infected chicks, the decrease in lymphoid contained relatively little virus compared with
organ weight has been associated with immu- the nephroblastoma, bone, lung, and pancreas
nosuppression (47), whereas the increase in (Fig. 1). These results suggest that the replica-
weight of other organs was attributed to edema tion of MAV-2(N) in cells of the lymphoid or-
(3). Specific viral proteins have been associated gans is limited. The present study did not ad-
with lymphoid organ atrophy and immuno- dress the question of whether MAV-2(N) was
suppression upon infection of kittens with feline capable of transforming cells of the bursa. All
leukemia virus (1, 28). The consequences of infected chickens died or were sacrificed (129
lymphoid organ atrophy in MAV-2(N)-infected days [Table 2]) before sufficient time had
chickens have not been examined but edema elapsed for the manifestation of lymphoid leu-
may be involved in the increase in weight of kosis. Nevertheless, elevated virus titers in the
some organs of MAV-2(N)-infected chickens. nephroblastoma and bone suggest that replica-
However, an apparent increase in relative organ tion may play a role in organ-specific oncogen-
weight may result from a decrease in body esis.
weight due to debilitation and wasting of the The presence of neutralizing antibody and
nephroblastoma-bearing chicken. nephroblastoma in certain infected chickens
Unlike chickens infected with MAV-2(0) (37), (Tables 4 and 5) requires comment. First, the
chicken embryos infected intravenously with presence of neutralizing antibody indicates that
MAV-2(N) did not develop significant anemia the birds were infected under conditions which
after hatching. However, other investigations did not permit tolerance to the virus. It is pos-
have indicated that some chickens infected in- sible that infection of the day-old chick by the
travenously at 8 to 9 days of age with MAV-2(N) intraperitoneal route permitted the infection of
developed a transient anemia (R. Paterson and certain target cells, such as those of the mesen-
R. Smith, unpublished data). Thus, the associ- chymal rest (5), and once infected, these cells
ation of anemia with MAV-2(N) infection seems were destined to become transformed, despite
to be ambiguous at this time. Anemia induced the presence of antibody. Second, it seems clear
by MAV-2(0) was accompanied by a thrombo- that the presence of viremia and the absence of
cytopenia (37). That MAV-2(N)-infected chick- neutralizing antibody did not accelerate the ap-
ens had petechial hemorrhages might suggest a pearance of nephroblastoma. It seems therefore
blood disorder promoting capillary fragility. likely that there is little recruitment of new
Petechial hemorrhages may therefore lead to target cells by virus after the initial infection.
the excess of cannabilism observed in MAV- This is in contrast to the situation in MAV-2(0)
2(N)-infected chickens. infection, in which administration of neutralizing
The changes in albumin and gamma globulin antibody leads to an arrest of disease (R. E.
levels detected in the serum of MAV-2(N)-in- Smith and J. Ivanyi, manuscript in preparation),
fected birds parallel those seen in MAV-2(0)- suggesting a continuous recruitment of new tar-
infected chickens (3). Sanders et al. (39) also get cells by MAV-2(0). It is possible that a
noted an increase in a y globulin-associated com- limited number of transformable target cells ex-
ponent and a decrease in the albumin/total glob- ists for MAV-2(N), and that these cells remain
ulin (A/G) ratio for sera from leukosis-affected susceptible to virus transformation for a rela-
chickens relative to uninfected chickens. Also, tively long time during development. Further
the increased serum gamma globulin level may experiments are required to elucidate this point,
also be a consequence of chronic virus infection. particularly those employing the infection of
The virus titer in organ extracts of nephro- embryos with MAV-2(N) followed by the admin-
blastoma-bearing chickens was greater than that istration of neutralizing antibody.
in serum (Fig. 1). The latter observation corre- Neutralizing antibody titers reported for
lates with the finding of budding virus by elec- MAV-2(N)-infected chickens indicate that the
tron microscopy in all organs of chickens in- presence of nephroblastoma was not always as-510 WATTS AND SMITH INFECT. IMMUN.
sociated with virus-specific immunoglobulin. of adults with a high dose of virus will lead to
Furthermore, preliminary results indicated that infection and ultimately to transformation of
treatment of chickens with cyclophosphamide target cells, if present, in spite of the develop-
did not prevent nephroblastoma induction by ment of neutralizing antibodies. Second, the ge-
MAV-2(N). Nowygrod et al. (34) likewise found netics of disease induction could be investigated.
that growth of AMV-induced nephroblastomas Lymphoid leukosis induction is influenced by at
did not require the presence of circulating anti- least one gene other than that coding for a
body. However, the level of serum antibody surface receptor for virus (12). Since disease
demonstrated a striking inverse correlation with induction in nephroblastoma is relatively rapid
the level of serum virus in MAV-2(N)-infected (compared with lymphoid leukosis) it would be
chickens. Thus, the antibody response in in- of interest to determine whether several inbred
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fected chickens may play a significant role in lines of chickens differ in susceptibility to neph-
limiting viremia, even though not related to roblastoma induction. Genetic analysis could im-
nephroblastoma induction. As shown with RSV- plicate the same genetic loci for lymphoid leu-
induced sarcomas, virus immunity may have no kosis and nephroblastoma. Third, the role of
relationship to or may enhance tumor growth immunological competence could be addressed
(29). Furthermore, virus-antibody complexes with the nephroblastoma model. Infection of
may be infectious (40). bursectomized adult chickens could be used to
Moreover, as shown by the titration of serum investigate the role of antibody in the disease
antibody to MAV-2(N) and related viruses (Ta- process. If nephroblastomas are observed in bur-
ble 4), in some chickens the humoral immune sectomized chickens, the role of antibody in the
response detectable by neutralization assay disease process will be eliminated. A preliminary
manifested specificity for the infecting virus. As experiment with a limited number of cyclophos-
shown by competition radioimmunoassay (48), phamide-treated chickens indicated that these
MAV-2(0) virus or MAV-2(0) gp85 isolated animals would develop nephroblastomas. How-
from CEF cultures and MAV-2(0) from the ever, more complete experiments are planned,
serum of osteopetrotic chickens showed different in which chicks which have been treated with
antigenic reactivities. In correlation, the neu- both testosterone and cyclophosphamide will be
tralizing activity of some of the MAV-2(N)-in- employed, and treated chicks will be tested for
fected or -immunized chicken sera characterized agammaglobulinemia before administration of
in Table 4 also demonstrated preference for CEF virus. If no tumors are observed in bursecto-
versus serum virus antigens. Virus neutralization mized chickens, passive antibody administration
patterns of some chicken sera also indicated a to infected chickens might lead to the appear-
close antigenic relatedness between MAV-2(N) ance of nephroblastomas if antigen-antibody
and MAV-2(0)-closer than the relatedness be- complexes are involved in the disease process.
tween either or these two viruses and PR.RSV- The role of lymphocytes can be investigated by
B. Lack of neutralization of RPV with sera from the adoptive transfer of immune and nonim-
chickens infected or immunized with subgroup mune lymphocytes from histocompatible do-
B virus reflected the subgroup-specific nature of nors. The experiments proposed should allow
virus antigenicity associated with the envelope definitive answers to several interesting biologi-
glycoprotein. Likewise, the type-specific reactiv- cal questions.
ities of these sera also may be associated with
the glycoprotein determinants of the virus (23). ACKNOWLEDGMENTS
The present study presents a description of The technical contributions of T. Wheeler and S. Nebes
the pathological manifestations of chickens in- provided indispensable support for this investigation. A. J.
Banes gave advice and support during the inception and
fected with an avian nephroblastoma virus. The analysis of these experiments. We wish to especially thank
results of this investigation indicate that the Heinz Bauer for generously providing the original isolate of
model system employed would be useful to ad- nephroblastoma virus.
dress several fundamental questions of RNA This research was supported by Public Health Service
grants R01-CA-12323 and P01-CA-14236 from the National
tumor virus pathogenesis. First, the number and Cancer Institute. S.L.W. was a recipient of a National Science
persistence of target cells in the host during Foundation Predoctoral Fellowship HES-7422381.
development could be investigated by infecting LITERATURE CITED
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