Progression of Infection and Tumor Developmentin Damselfish
C.E. Campbell,1 P.D.L. Gibbs,2 and M.C. Schmale2,*
1Centers for Disease Control and Prevention, Atlanta, GA 30333, U.S.A.2Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600
Rickenbacker Cswy., Miami, FL 33149, U.S.A.
Abstract: The bicolor damselfish (Stegastes partitus) is a tropical marine teleost naturally affected by multiple
neurofibromas and chromatophoromas on South Florida reefs. Damselfish neurofibromatosis is a transmissible
disease caused by a subcellular agent. Development of tumors is associated with the appearance of a series of
extrachromosomal DNAs ranging in size from 1.2 to 7 kb that appear to be the genome of a small virus-like
agent which we termed the damselfish virus-like agent (DVLA). This DNA was found at high copy number in
most spontaneous and experimentally induced tumors. An essentially identical pattern of DNA, but with lower
copy numbers, was observed in non–tumor-bearing tissue from diseased fish. Copy numbers of DVLA DNA
in tumors and nontumorous tissues increased as the disease progressed from early to late stages. In healthy fish
in which DVLA DNA was detected, the quantities were much lower than those in diseased fish. Healthy fish
from populations with a high prevalence of disease exhibited more infected tissues than fish from populations
with low levels.
Key words: neurofibroma, damselfish, tumor virus, schwann cell, chromatophore.
INTRODUCTION
Model systems involving peripheral nerve sheath tumors in
any vertebrate animal are especially valuable because mam-
malian models of these tumors are rare. Investigation of the
mechanisms controlling alterations of Schwann cells, peri-
neurial cells, and axons during the development of such
tumors would be greatly facilitated by the use of an animal
model in which these tumors and the neoplastic process
could be manipulated experimentally, both in vivo and in
vitro. Animal models also are needed to investigate the ori-
gins and the roles of hyperpigmention and mast cell infil-
trates often associated with these lesions.
Damselfish neurofibromatosis (DNF) is a disease af-
fecting bicolor damselfish (Stegastes partitus [previously
Pomacentrus partitus]) in coastal water of South Florida.
This disease consists of multiple, disseminated plexiform
neurofibromas, neurofibrosarcomas (malignant peripheral
nerve sheath tumors) and chromatophoromas (Schmale
et al., 1983, 1986; Schmale 1991). The histologic similarity
of these neurofibromas to tumors observed in humans
led us to propose this system as an animal model of path-
ogenesis of these tumors. Our previous studies showed
that DNF is caused by a transmissible, subcellular agent
(Schmale and Hensley, 1988; Schmale, 1995). Viruses able to
induce tumors have proved to be valuable tools for studying
the molecular mechanisms of neoplastic transformation.
The majority of tumors caused by DNA viruses and retro-
viruses are papillomas, sarcomas, lymphomas, and leuke-
Received January 31, 2001; accepted March 30, 2001.
*Corresponding author: telephone 305-361-4140; fax 305-361-4600; e-mail:
Mar. Biotechnol. 3, S107–S114, 2001DOI: 10.1007/s10126-001-00323
© 2001 Springer-Verlag New York Inc.
mias (Tooze, 1980; Fan, 1994). DNF is the only naturally
occurring, transmissible cancer affecting a neuroectodermal
cell type (Schwann cells and chromatophores) and thus may
provide a unique and important model for investigating
carcinogenesis in these cells.
Recent studies indicated that the etiologic agent of
DNF is a small DNA virus-like agent which we termed the
damselfish virus-like agent (DVLA). The genome of DVLA
can be detected in infected cells as a series of extrachromo-
somal DNAs ranging in size from 1.2 to 7 kb (M.C.
Schmale, in press). The goal of the present study was to
document the distribution of this DNA in selected tissues in
healthy fish from high-disease and low-disease populations
and in diseased fish as the disease progressed from early to
end stage.
MATERIALS AND METHODS
Bicolor damselfish, Stegastes (previously Pomacentrus) par-
titus, were collected and maintained in the laboratory as
described previously (Schmale, 1995). Healthy fish and fish
with spontaneous tumors were collected from reefs with a
low prevalence of disease (Fowey Rocks, 0.3% of adults
affected) or high-disease reefs (North Dry Rocks and Little
Grecian Reef, average prevalence of 20.5% of adults af-
fected; Schmale, 1991).
Fish with spontaneously occurring tumors were classi-
fied into disease stages based on the surface area of the fish
covered with tumors, as described previously (Schmale et
al., 1986). The stages used in this numerical rating system
can be summarized as follows. Fish having a single sign of
possible tumor development, such as a tiny spot on the fins
or scales, were classified as stage 1. Stage 1 fish were not
included in the present study because the presence of de-
finitive tumors could not be confirmed in these fish. Fish
having 1 or 2 possible areas of early tumor development
were considered as stage 2. Stage 3 were those fish with
small numbers of tumors on several regions of the body
(e.g., flanks, fins, and head). Fish that exhibited large num-
bers of tumors on several body areas were classified as stage
4. The most severe stage, stage 5, included fish with all body
surfaces heavily affected by tumors. For the purposes of the
present study, this stage was further subdivided into stages
5A and 5B where the latter group exhibited essentially con-
tinuous heavy coverage of the entire body with pigmented
tumors.
Tumors characteristic of DNF were induced in healthy
damselfish in the laboratory by intramuscular injection of
tumor homogenates or cultured cells derived from tumors
as described previously (Schmale, 1995). Briefly, 20 to 50 µl
of either tumor homogenate or cultured cells was injected at
each of 2 sites on opposite flanks at a volume based on the
size of the fish. Tumor homogenates were prepared by ho-
mogenizing tumor tissues in Hank’s balanced salt solution
without Ca2+ or Mg2+ (HBSS) or L-15 medium (without
serum or antibiotics) at a concentration of 80 mg/ml using
a glass homogenizer on ice (Schmale and Hensley, 1988).
Cultured cells were harvested from previously established
tumor-derived cell lines, counted, and resuspended in
HBSS at a concentration of approximately 5 × 106 cells/ml.
Fish were lightly anesthetized with MS-222 (tricaine meth-
anesulfonate, 120 mg/ml, pH 8) before being injected. Fish
developing tumors in these experiments were not classified
by degree or rate of tumor development. However, most of
those used in the present study exhibited relatively large
tumors (greater than 1% of body weight).
Eleven tissues, including fin, muscle, brain, liver,
spleen, gonad (ovary, testis, or unclassified gonadal tissue
from sexually immature fish), heart, pronephros, eye, red
blood cells, and serum, as well as tumors when present,
were collected from each fish following euthanasia with
MS-222. DNA was extracted as described in Sambrook et al.
(1989). Four primers were used to amplify DVLA DNA:
5BU-58-TCG TCA CCA CCC ACT AAT CTA TC; 5BU2-58-AGA AGA
AGT GTC CTC CTC CTC CGA; 5AU-58-CAG ACA TAG GTG GAG ATA
ATC GT; and 5AL-58-AAC GAT TAT CTC CAC CTA TGT CT. For
hemi-nested polymerase chain reaction (PCR), 1 µg of DNA
was amplified with the primers 5AU and 5BU for 30 cycles
(94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1
minute), following which 1 µl of this reaction was reampli-
fied using the primers 5AU and 5BU2 for 30 cycles. Two
negative control reactions, one without template DNA and
one with template DNA from a healthy fish previously
shown to be negative in these assays, were run in parallel
with each group of samples to detect contamination of re-
agents.
Slot blot hybridization was performed by an alkaline
transfer protocol (Sambrook et al., 1989). Primers 5BU and
5AL were used in a 40-cycle PCR to amplify a 1.3 kb prod-
uct from tumor cell line DNA to use as a probe for the blots.
Probe DNA was purified from agarose gels and labeled in a
random primed reaction using 32P-labeled dCTP. Each slot
was loaded with 5 µg total DNA. Membranes were rinsed in
2× SSC buffer and placed in hybridization solution con-
S108 C.E. Campbell et al.
taining 50% formamide. Blots were hybridized with probe
overnight at 42°C and washed to a final stringency of 0.1×
SSC, 0.1% sodium dodecylsulfate (SDS) at 65°C.
Quantities of DVLA DNA per sample, determined
from slot blots by visually comparing them with standards,
were grouped into 4 categories for analysis: below detection
limits, less than 0.02 copies per cell (or 1 copy in 50 cells);
low, 0.02 to 0.19 copies per cell; medium, 0.2 to 2 copies per
cell; and high, more than 2 copies per cell. Standards were
based on dilutions of DNA from the DNF tumor-derived
cell line 92-16 (Schmale et al., 1994) and dilutions of a
1.0 kb PCR product (primers 5AU and 5BU with 92-16
DNA as a template). All dilutions were made using juvenile
damselfish chromosomal DNA that was shown to be
DVLA-free. Standards were quantified using the Molecular-
Analyst program (Bio-Rad). Copies per cell were estimated
from the modal diploid genome size for fish of 2 pg (Hin-
egardner, 1976) as there are no data on genome size avail-
able for bicolor damselfish or closely related species.
RESULTS
The distribution of DVLA DNA was documented in 11
tissue types (and tumors, where present) from healthy fish
of low-disease and high-disease populations (as defined by
Schmale, 1991), from fish with spontaneous tumors, and
from fish with experimentally induced tumors. At the time
of collection, all tissues were inspected for gross evidence of
tumors. Tissues were scored as tumors if any evidence of
invasion by the tumor was found, and these tissues were
analyzed with other tumors rather than with normal tissue.
All samples were analyzed by slot blot to quantify the copy
number of DVLA DNA. Selected healthy fish in which no
homologous DNA was detectable by this technique also
were screened by hemi-nested PCR (one round of PCR did
not increase sensitivity significantly over that obtained by
slot blot). These assays included 51 healthy fish from low-
disease reefs (559 tissues), 12 healthy fish from high-disease
reefs (132 tissues), 59 fish with spontaneous tumors (598
tissues, 124 tumors), and 17 with induced tumors (145
tissues, 17 tumors) examined by slot-blot analysis. In addi-
tion, 9 healthy fish (99 tissues) from high-disease reefs and
10 healthy fish (110 tissues) from low-disease reefs were
examined by hemi-nested PCR.
The prevalence of DVLA DNA in tissues of healthy fish
was compared with that in grossly normal tissues from fish
with spontaneous and induced tumors (Figure 1). The
mean number of positive tissues per fish by slot blot analy-
sis was lowest in healthy fish from low-disease reefs. The
numbers of positive tissues were slightly, but not signifi-
cantly, higher in healthy fish from high-disease reefs. At all
stages, fish with spontaneous or induced tumors exhibited
significantly higher numbers of infected tissues than either
class of healthy fish (t test, P < .025). Fish in the most
advanced stages of disease development, stage 5A and 5B
with spontaneous tumors, were found to have significantly
more tissues infected than all other categories of fish (P <
.05), but these numbers did not differ significantly between
these stages. The mean number of tissues affected was sig-
nificantly different in the increasing order of spontaneous
tumor stages: 2 < 4, 5A, and 5B; 3 < 5A and 5B; 4 < 5A and
5B (all at P < .05). Fish with experimentally induced tumors
exhibited significantly more infected tissues than all healthy
fish and fish with stage 2 spontaneous tumors. These fish
did not differ significantly from fish with stage 3 and 4
spontaneous tumors and had significantly fewer infected
tissues than fish with stage 5A and 5B spontaneous tumors.
Figure 1. Mean number of grossly normal tissues per fish testing
positive for DVLA DNA out of a maximum of 11 tissues tested (±1
SD, N = sample size of number of fish per group). Analyses were
conducted by slot blot (A) or hemi-nested PCR (B). Fish with
spontaneous tumors (TF) are subdivided from early-stage (2) to
late-stage (5B) disease. Both TF and fish with experimentally in-
duced tumors (EF) exhibited a significantly larger number of in-
fected tissues than healthy fish (HF) from either low-disease-
prevalence reefs (HF-L) or high-disease-prevalence ones (HF-H)
by slot-blot analysis (t test, P < .05).
Tumor Development in Damselfish S109
All fish with induced tumors and 97% of those with spon-
taneous tumors were positive for DVLA DNA by slot blot.
Hemi-nested PCR was used to assess the presence of
DVLA DNA in a subset of healthy fish from both high-
disease and low-disease reefs, which were negative by slot
blot (Figure 1). This assay detected DVLA DNA in 6 (60%)
of 10 healthy fish from low-disease reefs and 9 (100%) of 9
healthy fish from high-disease reefs, indicating that very low
levels of this DNA were present in some tissues, below
detectable limits for the slot blot assay. In contrast, standard
40-cycle PCR did not show increased sensitivity relative to
slot blots. The mean number of tissues affected in healthy
fish from high-disease reefs was significantly larger than
that in healthy fish from low-disease reefs (P < .05).
Southern analysis of undigested genomic DNA from
tissues of fish with spontaneous tumors indicated that in-
fected tissues contained an identical pattern of DVLA to
that observed in the tumors themselves (Figure 2) and in
tumor-derived cell lines (M.C. Schmale, in press). This pat-
tern was consistent across tumors arising in a variety of
tissue types, including fin, jaw, mouth (buccal cavity), and
muscle on the flank of the fish. In addition, weaker signals,
indicating lower copy numbers of this DNA, were observed
in several grossly normal tissues, including the pronephros,
spleen, muscle, and serum.
A more detailed analysis was made of the quantities of
DVLA DNA in normal and tumor tissues to determine the
progression of this infection throughout all stages of this
disease (Figure 3). Over 97% of samples from healthy fish
were below detectable limits (less than 0.02 copies per cell,
or 1 copy in 50 cells), and no samples from any healthy fish
exhibited levels of this DNA at, or above, 0.2 copies per cell
(C.E. Campbell and M.C. Schmale, in press). In all stages of
the disease, copy number in tumors exceeded that in grossly
normal tissues from fish with spontaneous tumors, with
over 45% of tumors exhibiting over 2 copies per cell in the
earliest stages and increasing to over 80% in late stages.
Most non-tumor tissues followed a similar pattern of in-
crease in copy number with progression of the disease, al-
though at a lower level. In late disease stages, the proportion
of tissues with over 2 copies per cell of DVLA DNA varied
from 0% in serum to 43% in the eyes. Tumors typically
developed in all fins in the advanced stages of disease, such
that no grossly normal fin tissues could be obtained for
analysis from stage 5A or 5B fish. Other tissues, such as
muscle, pronephros, spleen, and gonad, were sometimes
Figure 2. Patterns of extrachromosomal
DNA distribution in tissues from one
fish with spontaneous tumors with stage
5B DNF. Southern analysis of
undigested genomic DNA demonstrated
the presence in tumors (collected from 4
sites) and several grossly normal tissues
(including pronephros, spleen, muscle,
and serum) of extrachromosomal DNA
forms homologous to those previously
found in tumor-derived cell lines
(reference DNA lane). Note that larger
fragments in the reference lane could
also be visualized in tumor and tissue
samples with longer exposure of the
autoradiograph and that some DNA
degradation occurred in tumor samples
owing, in part, to the presence of
necrotic tissue in some tumors. DNAs
were loaded at 10 µg per lane (except
serum at approx. 0.7 µg). The probe was
made from a 500 bp DVLA clone
isolated from DNA of a tumor cell line.
S110 C.E. Campbell et al.
affected by tumors (Schmale et al., 1986; Schmale and
McKinney, 1987), and thus the absence of grossly visible
tumors could not rule out microscopic areas of tumor in-
vasion in these tissues. However, other tissues such as the
eye, heart, brain, and liver were never seen to contain tumor
cells. Although obvious tumor growth was not observed in
blood vessels, no attempt was made to rule out the presence
of tumor cells in circulating blood.
Of 17 fish with experimentally induced tumors used in
this analysis, 16 were in an advanced stage of tumor devel-
opment, with tumors ranging from 1% to 15% of body
weight. Although 100% of induced tumor tissues exhibited
detectable levels (>0.02 copies per cell) of DVLA DNA, copy
numbers were generally lower than for spontaneous tu-
mors, with less than 20% of tumors having more than 2
copies per cell (Figure 4). In fish with induced tumors, the
proportion of normal tissues with detectable DVLA DNA
and with high levels of DNA was lower than that observed
in fish with late-stage spontaneous tumors. In only 2 tissues
in fish with induced tumors, eye and heart, did levels ever
exceed 2 copies per cell (in approximately 10% of fish). All
of the 8 serum samples collected from fish with induced
tumors were negative.
Analysis of the tissue distribution of DVLA DNA using
hemi-nested PCR in healthy fish with levels below those
detectable using slot blots indicated an increased prevalence
of infection in fish those from high-disease reefs compared
with those from low-disease reefs in all tissues except the
eyes and serum (Figure 5). However, significant differences
were observed in the prevalence of DVLA between the high-
disease and low-disease sites in only 3 tissues, the spleen,
gonad, and heart (P < .05; x2 analysis). The pattern of
positive samples was relatively evenly distributed among
tissue types, with no dominant patterns within either group,
except for a lack of positive serum samples and a very high
prevalence level in the hearts of the fish from high-disease
sites.
DISCUSSION
A strong correlation was observed between the presence of
DVLA DNA and the development of neurofibromatosis
Figure 3. Alterations in copy number of DVLA DNA in tissues of
fish with spontaneous tumors at different stages of disease. The
results for disease stages 2, 3, 4, 5A, and 5B are arranged in order
for each tissue (sample sizes as in Figure 1). Copy number was
measured (by slot blot) as the proportion of samples in each group
falling into the categories of high (>2 copies per cell), medium
(0.2–2 copies per cell), or low (0.02–0.19 copies per cell). Other
samples did not contain detectable DNA (<0.02 copies per cell).
All fins of stage 5A and 5B fish had grossly visible tumors, and thus
no normal fin tissues were available for analysis. In previous stud-
ies, muscle, pronephros, spleen, and gonad often were found to be
invaded by tumor cells as the disease progressed (Schmale et al.,
1983). Tissues types not previously reported to contain tumors are
denoted with an asterisk (*).
Figure 4. Copy numbers of DVLA DNA in fish with experimen-
tally induced tumors (N = 17; all fish had relatively large tumors).
The copy number was measured by tissue as in Figure 3.
Tumor Development in Damselfish S111
among bicolor damselfish on reefs in South Florida. The
hypothesis that DVLA is the etiologic agent of this disease is
supported by data demonstrating that DVLA DNA is pre-
sent in the vast majority of both spontaneous and induced
tumors, is typically at high copy numbers in tumors, and is
found in healthy fish only at extremely low copy numbers.
The pattern of increasing copy number in tumors with ad-
vancing stage of the disease also supports this relationship.
The presence of very low levels of DVLA DNA in
healthy fish, detectable in most fish only by hemi-nested
PCR analyses, suggests that one or more factors are most
likely affecting tumor development in these fish. In the
most parsimonious scenario, all infected fish would go on
to develop tumors, given sufficient time. However, as this
would result in much higher disease prevalence rates than
observed in the wild, other factors are likely to be impeding
the development of disease. These might include a stochas-
tic relationship between the dosage of the agent in a fish, the
likelihood of that agent encountering a susceptible cell, and
the probability that such an appropriate, infected cell will
undergo neoplastic transformation. In such a scenario, fish
with higher dosages of the agent would be more likely to
experience cell transformation during their lifetime. Dose-
response or threshold effects have been reported in many
viral diseases whereby some minimum amount of a patho-
gen is required to induce the disease (O’Neil et al., 1999).
Differences in viral genotype or expression could also
influence the outcome of the disease. Many families of vi-
ruses are known to produce particles with wide ranges of
virulence based on mutations in the viral genome (Tooze,
1980; Fan 1994). Initial studies of sequence variation in
DVLA have suggested a high degree of conservation of
DVLA sequences (M.C. Schmale, unpublished data). How-
ever, these results are very preliminary and do not include
the entire sequence of DVLA.
Expression levels of viral genes might also affect the
outcome. Such a scenario was proposed for the neoplastic
disease, dermal sarcoma, in walleye (Stizostedion vitreum) in
North American lakes. Essentially, all walleye were positive
for walleye dermal sarcoma virus (WDSV) DNA despite a
maximal disease prevalence in affected lakes of 30% (Poulet
et al., 1996). In these animals, levels of WDSV DNA were
similar in healthy and diseased fish and ranged from unde-
tectable (less than 1 copy in 20,000 cells, based on PCR
quantification) to 1 copy in 2 cells. These authors attributed
the lack of symptoms in the presence of WDSV DNA in
healthy fish to a lack of effective transcription of viral RNA
as measured by low or undetectable RNA levels in healthy
fish. Thus the viral genomes in healthy fish appeared to be
effectively silenced, which could explain the lack of tumors.
The hypothesis that expression of DVLA DNA might be
reduced or absent in healthy damselfish could not be tested
in the present study because DVLA RNA levels were not
measured in most samples.
Other factors influencing the outcome of DNF in in-
fected fish might include environmental and genetic differ-
ences. However, there is no evidence of any differences in
susceptibility to DNF in the laboratory between fish from
high-disease and low-disease populations (Schmale, 1995),
and it is unlikely that there are genetic differences between
populations of bicolor damselfish on Florida reefs (Lacson
and Morizot, 1991). A role for environmental factors in
accelerating or slowing the development or progression of
the disease has not been investigated and cannot be ruled
out.
Unlike the situation in tissues of healthy fish, in which
DVLA DNA levels were very low or undetectable, many
grossly normal tissues in fish with spontaneous or induced
tumors contained moderate to high levels with patterns of
extrachromosomal DNA similar to that seen in tumors. A
similar situation was reported in tumor-bearing walleye, in
which high levels of WDSV DNA were observed in tissues of
diseased walleye that never developed tumors (Poulet et al.,
1996). Tumor invasion is commonly seen in some of these
tissues in damselfish, such as fins, muscle, and pronephros,
Figure 5. Percentage of positive organs in healthy fish from low-
disease and high-disease reefs by hemi-nested PCR. Fish from the
latter exhibited a higher proportion of any given organ infected
with DVLA compared with healthy fish from low-disease reefs. All
of the fish used were negative in all tissues by slot blot analysis (N
= 10 low, 9 high).
S112 C.E. Campbell et al.
and less often in spleen and gonad (Schmale et al., 1986;
Schmale and McKinney, 1987). Thus it is possible, particu-
larly in the fish with advanced-stage tumors, that micro-
scopic tumors were present in these grossly normal tissues
and that the DVLA DNA observed there was localized to
tumor cells (these tumors always originate in either periph-
eral nerve sheaths or chromatophores and never from neo-
plastic transformation of other tissues). However, in tissues
in which tumor invasion has never been detected, such as
eye, heart, brain, and liver, this explanation would not be
applicable. An alternative source for this DNA might be
blood and nerve tissues present in these organs. The rela-
tively low levels of DVLA DNA observed in blood and se-
rum suggest that these compartments are not likely reser-
voirs of this DNA. Although peripheral nerves are the
source of neurofibromas in DNF, and these nerves are pre-
sent in essentially all tissues in the fish, their extremely small
size prevented us from preparing satisfactory, pure samples
for DNA analysis. Thus the role of nerves as reservoirs of
DVLA DNA in unaffected tissues has not been investigated.
The complex pattern of extrachromosomal DVLA
DNA seen in both tumor and normal tissues suggests the
presence of a viral genome with replicative intermediates.
This pattern is somewhat similar to that seen with small
DNA viruses, such as SV40, which exhibit a wide range of
partial and complete multimers of the viral genome visible
as a series of different size fragments of extrachromosomal
DNA (Ariga, 1984). This pattern in damselfish tissues, if
indicative of DNA replication, would suggest that, at a
minimum, this agent is capable of infecting and replicating
DNA in many types of tissues in which tumor development
does not occur. Although assay techniques to determine the
presence or titer of infectious particles in these nontumor
tissues are not currently available, the lack of obvious cy-
topathic effects in these tissues in DNF-affected fish sug-
gests that large-scale release of virus particles does not oc-
cur.
These observations suggest that the formation of tu-
mors involving the peripheral nervous system and pigment
cells is a function of effects of the virus infection on specific
cell types rather than simply a limitation on the range of cell
types susceptible to infection by DVLA. This might be ho-
mologous to the life cycle of infection of many of the small
DNA tumor viruses that produce tumors in cells which are
nonpermissive for complete viral replication. In these cases,
an aborted cycle of replication in nonpermissive cell types
results in the survival of the cell containing the viral ge-
nome. This state of continued, suspended infection then
predisposes the cell to neoplastic transformation by a con-
tinued expression of a combination of viral genes that
stimulate cell replication (Nevins and Vogt, 1996).
Another factor relevant to understanding the relation-
ship between DVLA and nontumorous but apparently in-
fected tissues in fish with spontaneous or induced tumors is
the type and level of viral RNA transcripts present. How-
ever, no data are yet available on the levels of RNA expres-
sion in tumors relative to grossly normal infected tissues.
Although transcriptional silencing has been proposed to
explain lack of tumor development in healthy walleye, as
described above, unaffected tissue types in diseased walleye
did exhibit normal transcription levels, suggesting a differ-
ent mechanism must avert tumorigenesis in these cells
(Poulet et al., 1996).
Difficulties in isolating and cultivating many subcellu-
lar agents often hinder conclusive demonstration of etiology
of diseases related to these agents by the traditional criteria
proposed as Koch’s postulates. In such cases, identifying
etiologic agents must often rely on determining the distri-
bution of microbial nucleic acid sequences rather than ma-
nipulating cultured microbes (Fredricks and Relman, 1996).
Perhaps the strongest evidence supporting the hypothesis
that DVLA is the etiologic agent of DNF is the presence of
high levels of this DNA in the tumors of fish with experi-
mentally induced DNF. The data presented here also dem-
onstrate that the appearance and progression of damselfish
neurofibromatosis is associated with increasing levels of
DVLA DNA in all tissues with at least a 10-fold higher level
in tumors. Because DNF is the only naturally occurring,
transmissible cancer affecting a neuroectodermal cell type
(Schwann cells and chromatophores in this disease), an im-
proved understanding of the properties and life cycle of
DVLA, as well as of how infection by this agent alters cel-
lular functions, may provide unique insights into mecha-
nisms of neoplastic transformation in these cells.
ACKNOWLEDGMENTS
Fish were collected under research permits issued by the
State of Florida, Department of Environmental Protection,
the Biscayne National Park (National Park Service), and the
Florida Keys National Marine Sanctuary (National Oceanic
and Atmospheric Administration). Thanks are extended to
the personnel of the Florida Keys National Marine Sanctu-
ary and the Biscayne National Park for logistical support.
This research was supported by Public Health Service grants
NS36998 and ES05705.
Tumor Development in Damselfish S113
REFERENCES
Ariga, H. (1984). Identification of the replicative intermediates in
SV40 DNA replication in vitro. Nucleic Acid Res 12:6053–6062.
Campbell, C. E., and Schmale, M.C. (in press). Distribution of a
novel infectious agent in healthy and tumored bicolor damselfish
in Florida and the Caribbean. Mar Biol.
Fan, H. (1994). Retroviruses and their role in cancer. In: The
Retroviridae, Levy, J.A. (ed.). New York, NY: Plenum Press, 3:313–
362.
Fredricks, D.N., and Relman, D.A. (1996). Sequence-based iden-
tification of microbial pathogens: a reconsideration of Koch’s pos-
tulates. Clin Microbiol Rev 9:18–33.
Hinegardner, R. (1976). The cellular DNA content of sharks, rays
and some other fishes. Comp Biochem Physiol 55B:367–370.
Lacson, J.M., and Morizot, D.C. (1991). Temporal genetic varia-
tion in subpopulations of bicolor damselfish (Stegastes partitus)
inhabiting coral reefs in the Florida Keys. Mar Biol 110:353–357.
Nevins, J., and Vogt, P. (1996). Cell transformation by viruses. In:
Fields Virology, 3rd ed., Fields, B.N., Knipe, D.M., and Howley,
P.M., (eds.). Philadelphia, PA: Lippincott-Raven, 301–344.
O’Neil, S.P., Mossman, S.P., Maul, D.H., and Hoover, E.A. (1999).
Virus threshold determines disease in SIVsmmPBj14-infected ma-
caques. AIDS Res Hum Retrovir 15:183–194.
Poulet, F.M., Bowser, P.R., and Casey, J.W. (1996). PCR and RT-
PCR analysis of infection and transcriptional activity of walleye
dermal sarcoma virus (WDSV) in organs of adult walleyes (Sti-
zostedion vitreum). Vet Pathol 33:66–73.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y.: Cold
Spring Harbor Laboratory Press.
Schmale, M.C. (1991). Prevalence and distribution patterns of
tumors in bicolor damselfish (Pomacentrus partitus) on South
Florida reefs. Mar Biol 109:203–212.
Schmale, M.C. (1995). Experimental induction of neurofibroma-
tosis in the bicolor damselfish. Dis Aquat Org 23:201–212.
Schmale, M.C., Gibbs, P.D.L., and Campbell, C.E. (in press). A
virus-like agent associated with neurofibromatosis in damselfish.
Dis Aquat Org.
Schmale, M.C., and Hensley, G.T. (1988). Transmissibility of a
neurofibromatosis-like disease in bicolor damselfish. Cancer Res
48:3828–3833.
Schmale, M.C., and McKinney, E.C. (1987). Immune responses in
the bicolor damselfish, Pomacentrus partitus, and their potential
role in the development of neurogenic tumors. J Fish Biol 31A:
161–166.
Schmale, M.C., Hensley, G.T., and Udey, L.R. (1983). Multiple
schwannomas in the bicolor damselfish, Pomacentrus partitus; a
possible model of von Recklinghausen neurofibromatosis. Am J
Pathol 112:238–241.
Schmale, M.C., Hensley, G.T., and Udey, L.R. (1986). Neurofibro-
matosis in the bicolor damselfish (Pomacentrus partitus) as a
model of von Recklinghausen Neurofibromatosis. Ann NY Acad
Sci 486:386–402.
Schmale, M.C., Gill, K.A., Cacal, S.M., and Baribeau, S.D. (1994).
Characterization of Schwann cells from normal nerves and from
neurofibromas in the bicolor damselfish. J Neurocytol 23:668–681.
Tooze, J. (ed.) (1980). DNA Tumor Viruses. Cold Spring Harbor,
N.Y.: Cold Spring Harbor Press.
S114 C.E. Campbell et al.
