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J Virol, April 1998, p. 3484-3490, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Two Closely Related but Distinct Retroviruses Are
Associated with Walleye Discrete Epidermal Hyperplasia
Lorie A.
LaPierre,
Donald L.
Holzschu,
Greg A.
Wooster,
Paul R.
Bowser, and
James W.
Casey*
Department of Microbiology and Immunology,
College of Veterinary Medicine, Cornell University, Ithaca, New
York
Received 23 July 1997/Accepted 12 December 1997
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ABSTRACT |
Walleye discrete epidermal hyperplasia (WEH) is a
hyperproliferative skin disease that is prevalent on adult walleye fish throughout North America. We have identified two retroviruses associated with WEH, designated here as walleye epidermal hyperplasia virus type 1 and type 2 (WEHV1 and WEHV2), that are closely related to
one another (77% identity) and to walleye dermal sarcoma virus (64% identity) within the polymerase region. WEHV1 and/or WEHV2 viral
DNA was readily detected by PCR in hyperplastic tissue samples, but
only low levels of viral DNA were detected in uninvolved skin. Southern
blot analysis showed one to three copies of integrated WEHV2 viral DNA
in lesions but did not detect WEHV2 viral DNA in uninvolved skin from
the same fish. Northern blots detected abundant levels of WEHV1 and/or
WEHV2 virion RNA transcripts of approximately 13 kb in hyperplastic
tissue, but virion RNA was not observed in uninvolved skin and muscle.
These results suggest that WEHV1 and WEHV2 are the causative agents of
discrete epidermal hyperplasia.
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TEXT |
Four virus-associated skin
lesions have been observed on walleyes (Stizostedion
vitreum) inhabiting lakes throughout North America:
lymphocystis, dermal sarcoma, diffuse epidermal hyperplasia, and
discrete epidermal hyperplasia (reviewed in reference
30). These diseases can be found singly or in any
combination. Lymphocystis is caused by an iridovirus, and diffuse
epidermal hyperplasia is associated with a percid herpesvirus (12,
25). Walleye dermal sarcoma (WDS) is associated with a
retrovirus, walleye dermal sarcoma virus (WDSV) (13,
24). Walleye discrete epidermal hyperplasia (WEH), like WDS, is
thought to have a retroviral etiology based on the observation by
electron microscopy of retroviruslike particles in lesions (24,
29, 30).
Walleye discrete epidermal hyperplasia is a hyperproliferative skin
disease characterized by plaques of thickened epidermis that can be
found on any part of the body (24). The disease has been
observed on approximately 10% of adult breeding walleyes in Oneida
Lake, N.Y., and on up to 20% of the walleyes in some lakes in
Canada in a given year (3, 24, 30). Like many neoplastic
skin diseases of fish, including WDS, WEH is observed on sexually
mature fish and the lesions appear and regress on a seasonal basis
(1, 4, 17); they are present from fall through spring and
are absent in the summer months (3). This seasonality may be
caused by environmental factors such as water temperature that affect
host endocrine activity and immunocompetency (1).
Although 13 neoplastic diseases of fish are believed to have a
retroviral etiology (17), WDSV and the snakehead fish
retrovirus (SnRV) are the only piscine retroviruses that have been
molecularly cloned and sequenced (10, 11, 13). Furthermore,
WDSV is the only piscine retrovirus that is etiologically associated
with a neoplasia; it is a large complex retrovirus (12.7 kb) that
contains three open reading frames in addition to gag,
pol, and env, and it appears to have a complex
life cycle, i.e., dramatically differing levels of gene expression are
observed at different stages of disease (5, 11, 18). The
putative proteins encoded by the additional open reading frames may be
involved in the regulation of viral gene expression and the induction
of WDS. Although dermal sarcomas often appear to be malignant
histologically, the lesions regress, and neither local invasion nor
metastasis has been observed in feral fish (4, 15). However,
inoculation of 9-week-old walleye fingerlings with cell-free
filtrates from dermal sarcomas resulted in locally invasive tumors
(8).
Since retroviruslike particles have been observed in hyperplastic
lesions by electron microscopy (29), we were interested in
cloning and characterizing this virus and comparing it with WDSV. As a
step toward this goal, we cloned two retroviruses from walleye
hyperplasias, designated walleye epidermal hyperplasia type 1 and
type 2 (WEHV1 and WEHV2). These viruses are closely related to each
other and to WDSV. We report here the amino acid sequences of the
pro-pol open reading frames of these viruses as well as
evidence to suggest that they are the causative agents of WEH.
Cloning and characterization of retroviral pol
sequences.
Preliminary experiments showed that virion preparations
made from hyperplastic lesions had associated reverse transcriptase (RT) activity (data not shown). To amplify a segment of the
pol genes from the retroviruses present in hyperplastic
lesions, PCR amplification was accomplished with degenerate
pol primers as described by Donehower et al. (7).
These primers encode the amino acid sequences VLPQG
(5'-ctcggatccGTNYTNCCNCARGG-3') and YMDD
(5'-ctcgtcgacRTCRTCCATRTA-3') and generate an amplicon of approximately 135 bp (lowercase letters represent added restriction sites) from retroviral pol templates. Virion RNA from
sucrose gradient purifications or total RNA from hyperplastic tissue
was isolated with RNAzol (Tel-test Laboratories). The RNA was treated with 1 U of RNase-free DNase (Boehringer) in 1× first-strand synthesis buffer (Life Technologies) for 1 h at 37°C followed by
phenol-chloroform extraction. cDNA was prepared using 0.5 µg of the
YMDD primer or 1 µg of random hexamers (Boehringer) and 400 U of
Superscript II (Life Technologies). The products were digested with
BamHI and SalI and cloned into pBluescript II
SK
(Stratagene). Clones were manually sequenced with Sequenase
version 2.0 (U.S. Biochemicals) with T3 and T7 primers on
double-stranded DNA templates. Two 114-bp retroviral pol
segments were cloned from both RT PCR experiments (data not shown). The
sequences of the two cDNA clones were 78% identical, suggesting that
they were derived from two different but closely related retroviruses,
designated walleye epidermal hyperplasia virus type 1 and type 2 (WEHV1 and WEHV2, respectively).
To obtain larger segments of the viral DNA flanking the VLPQG-YMDD
interval in the pol gene, 5' and 3' rapid amplifications of cDNA ends (RACE) were employed with WEHV1- or WEHV2-specific primers. 5' RACE was carried out according to the manufacturer's directions (Life Technologies). Briefly, cDNA synthesis was
performed with the 3' YMDD primer and 1 µg of total RNA isolated from
hyperplastic tissue. After RNase H digestion, the cDNA was purified by
centrifugation and filtered through Millipore Ultrafree-MC 30,000 NMWL
filters. A poly(dC) tail was added onto the 3' end of the purified cDNA with terminal deoxynucleotidyltransferase. PCR amplification was performed with a 5' GI anchor primer and the 3' YMDD primer. A second
round of PCR was done with an adapter primer
(5'-GGCCACGCGTCGACTAGTAC-3') and 3' specific pol
primers for WEHV1 (5'-cattgaattcggatcCAAATTTCCGAAGTCAGAGA-3') or
WEHV2
(5'-ca ttgaattcggatccACATACTTCCGAAGTTAAGCT-3').
Template DNA was denatured for 5 min at 96°C, followed by the
addition of 2.5 U of Taq polymerase (Life Technologies). The
amplification program consisted of 30 s at 94°C, 30 s at
50°C, and 2 min at 72°C for 35 cycles. The WEHV1 and WEHV2 5' RACE
products were digested with BamHI/SalI and
EcoRI/SalI, respectively, and cloned into
Bluescript II SK (Stratagene). 5' RACE yielded products of 269 and
533 bp for WEHV1 and WEHV2, respectively (data not shown).
First-strand cDNA for 3' RACE was synthesized with an oligo(dT) primer
(5'-ggccacgcgtcgactagtacT
[12]-3') as the
anchor primer
and 1 µg of total RNA. 3' RACE was used in combination
with the
Elongase amplification system (Life Technologies) with
the
goal of obtaining the entire 3' end of the viral genomes.
Amplification
of the cDNA was done with the adapter primer (above)
and 5' specific
pol primers for WEHV1
(5'-cattgaattcggatccAATCTATCGCCAGATATGAC-3') or
WEHV2
(5'cattgaattcggatccAGGCAGTATACTTGGACAGTGT-3'). The PCR
program
consisted of 30 s at 94°C, 30 s at 55°C, and 6 min at 68°C for
35 cycles, yielding WEHV1 and WEHV2 products that
were smaller
than anticipated (1.9 and 1.6 kb, respectively). The 3'
RACE products
were cloned into pBluescript II SK
after digestion with
BamHI
and
SpeI. Subsequent DNA sequencing showed
that small 3' RACE
products were the result of the oligo(dT) primer,
used for cDNA
synthesis, annealing to an A-rich sequence within the
WEHV1 and
WEHV2
pol genes.
The 3' RACE fragments derived from WEHV1 and WEHV2 were
labeled with
32P by random priming (Boehringer) and used as
probes to isolate
genomic clones from a lambda library (Stratagene's
lambda DASH
II/
BamHI vector kit and Gigapak II XL packaging
extract). The
WEHV1 and WEHV2
pol sequences were determined
from overlapping
5' and 3' Bluescript SK II
subclones. The WEHV1
pol overlap consisted
of 2,922 bp and the WEHV2
pol overlap consisted of 1,517 bp.
Figure
1 shows an
alignment of the predicted amino acid sequences of the
pro-pol open reading frames of WEHV1 and WEHV2 compared
with
that of WDSV. The
pro-pol products consist of 1,200, 1,247,
and 1,169 amino acids, respectively. Sequence analysis of the
WEHV1
pol overlap region (amino acid positions 227 to 1200) showed
that the 5' and 3' subclones were identical. However, the WEHV2
5' and
3' subclones differed in the overlap region (636 to 1140)
by 18 nucleotides (1.2%), resulting in three amino acid substitutions:
K to
R (position 862), P to T (871), and L to M (889). The similarity
of the
overlapping regions in
pol for WEHV1 and WEHV2 suggests
that
their respective 5' and 3' subclones are derived from the
same strain
of virus, although a minor heterogeneity exists within
the population
of WEHV2 proviruses.
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FIG. 1.
Alignment of the predicted amino acid sequences of the
pro-pol open reading frame in WDSV, WEHV1, and WEHV2. The
seven conserved domains in RT are shown with brackets. LPQG and YMDD
are indicated by four asterisks. The five conserved regions of RNase H
are outlined in blocks labeled I through V. Identity with respect to
WDSV (-), gaps (.), and differences between WEHV1 and WEHV2 are in
bold-faced type. Amino acid changes between WEHV2 subclones are
indicated in parentheses.
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Like WDSV, the
pro and
pol genes of the
hyperplasia viruses are in the same open reading frame (Fig.
1).
Throughout the entire
pro-pol gene, WEHV1 and WEHV2 have
77% amino acid identity, and
each shares 64% identity with WDSV. The
seven conserved domains
in RT described by Xiong and Eickbush
(
28) are depicted in Fig.
1. Within this conserved region of
RT, WEHV1 and WEHV2 have 90%
amino acid identity and both have 70%
identity with WDSV. The
signature sequences for protease (LVDTG)
and RT (LPQG and YMDD)
are shown in Fig.
1 (
7,
19). The five
conserved regions of
RNase H, as described by McClure (
16),
are shown as blocks I
through V. Region I (FS/TDGS) is located at amino
acid position
646. The three
pol genes are the most
divergent in the region
located downstream of block V
(
GNAA
AD) in RNase H between amino
acids 784 and
950. This region contains a stretch of amino acid
residues in WEHV2
pol that is not found in WEHV1 or WDSV.
Phylogeny.
A phylogenetic analysis of WEHV1 and WEHV2 was
performed with the two previously described complex piscine
retroviruses, WDSV and SnRV (10), and with
representatives of the seven retroviral genera. The
pol sequences used in the analysis extended 132 amino acids, from domain 1 through the center of domain 5 (YXDD), as previously described (22), i.e., the conserved region of RT (28). This analysis was included so that the RT sequences of retroviral fragments isolated from two lower vertebrates, the lizard-like reptile tuatara (Sphenodon) and the poison
dart frog (Dendrobates ventrimaculatus), could be placed in
the alignment (22, 23). The yeast retrotransposon Ty3 was
chosen as an outgroup. The tree was generated with the Megalign
application in the DNAstar (Madison, Wis.) series of programs, in which
a modification of the neighbor-joining method is used (20).
The analysis showed that WEHV1, WEHV2, and WDSV have a common ancestor
(Fig.
2). WEHV1 and WEHV2 have 95% amino
acid identity
in this small region of RT, suggesting that they are
different
strains of the same virus or distinct species. We favor the
latter
possibility because (i) the amino acid sequences are only 77%
identical throughout the entire
pro-pol open reading frame,
(ii)
preliminary sequence analysis of WEHV1 and WEHV2 suggests that
they are significantly different in other regions of the genome,
and
(iii) the percentage of identity between WEHV1 and WEHV2 in
the
conserved region is similar to that in RT between feline leukemia
virus
and murine leukemia virus (MLV) (92% identity) (Fig.
2),
two viruses
that are considered to be distinct and cause similar
diseases in
different species. WEHV1 and WEHV2 have 83 and 81%
amino acid identity
with WDSV, respectively, over the same region
of
pol (Fig.
2). The relationship between the WEHVs and WDSV may
be analogous to
that of human T-cell leukemia virus types 1 and
2 (82% identity), two
viruses considered to be distinct that cause
different diseases in
humans (
9). The three walleye viruses
as a group are
distantly related to SnRV (41% identity) and to
members of the MLV
group of retroviruses (i.e., MLV, 45% identity).
Based on the limited
current database, it appears that the retroviruses
of fish, reptiles,
and amphibians may ultimately form their own
distinct genera within the
retrovirus family.
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FIG. 2.
Phylogenetic tree. The conserved RT domains spanning
domain 1 through the middle of domain 5 (YMDD) 132 residues
totalwere analyzed and compared among the viruses shown with software
from DNAstar. fiv, feline immunodeficiency virus; visna, visna virus;
eiav, equine infectious anemia virus; hiv-1, human immunodeficiency
virus type 1; siv agm, simian immunodeficiency virus of African green
monkeys; htlv-I, human T-cell leukemia type 1; blv, bovine leukemia
virus; mmtv, mouse mammary tumor virus; mpnv, Mason-Pfizer monkey
virus; felv, feline leukemia virus; galv, gibbon ape leukemia virus;
devl, Dendrobates ventrimaculatus; speV,
Sphenodon virus; hsrv, human spumaretrovirus.
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WEHV1 and/or WEHV2 DNA can be detected in walleye hyperplasias
by PCR and Southern blotting.
A preliminary screen for the
presence of WEHV1 and WEHV2 in hyperplastic lesions collected from 34 fish was done by PCR. To obtain sufficient material for analysis,
multiple lesions from individual fish were pooled to form individual
samples. Genomic DNA was isolated from approximately 100 mg of
hyperplastic tissue with DNA isolator reagent (Genosys). PCR was done
with 500 ng of DNA and the WEHV1- and WEHV2-specific pol
primer pairs described above (without restriction sites). PCR was
carried out as described for 5' RACE, except that a 60-s extension at
72°C was used. The WEHV1 and WEHV2 primer sets amplified products of
226 and 116 bp, respectively, and did not cross-amplify (data not
shown).
Representative PCR data are shown in Fig.
3A. Importantly, WEHV1 and WEHV2 were not
detected in sperm DNA (lane 11), indicating
that these viruses are
exogenous to walleye. Both WEHV1 and WEHV2
viral DNA were found in
30 hyperplasia samples represented in
lanes 2 to 10, whereas four
hyperplasia lesions contained only
WEHV2 viral DNA (data not shown).
WEHV1 viral DNA was not found
to be independent of WEHV2 viral DNA by
PCR. Some samples showed
an abundance of both viruses (lanes 4 and 7),
and others showed
an abundance of WEHV1 (lanes 3 and 8) or WEHV2 (lanes
5, 6, 9,
and 10). The weak PCR signal observed for WEHV1 and WEHV2 in
these
samples may represent amplification of low levels of viral DNA
due to background infection of the skin. To test this possibility,
hyperplasias and uninvolved skin from three affected animals and
skin
from two clinically normal fish were tested for both viruses
by PCR. As
shown in Fig.
3B, PCR signals for WEHV1 (lane 3) or
WEHV2 viral DNA
(lanes 3, 5, and 7) were strong in the diseased
tissue relative to the
signals for uninvolved skin samples (lanes
4, 6, and 8). Importantly,
WEHV1 and WEHV2 viral DNAs were not
detected in skin from normal fish
(lanes 9 and 10). Although only
semiquantitative, these PCR data
suggest that low levels of viral
DNA are present in the uninvolved skin
of diseased animals, whereas
high levels of viral DNA are present in
hyperplastic tissue. The
data also demonstrate that the two viruses are
commonly found
together in diseased fish.
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FIG. 3.
Detection of WEHV1 and WEHV2 in hyperplastic lesions.
(A) PCR of viral DNA from lesions (10 µl loaded). Positive control,
5' RACE clones (+); lesions (lanes 3 to 10); negative genomic DNA
control, walleye sperm DNA (Sp.); negative buffer control ().
std, standard. (B) PCR of viral DNA from lesions (H) and associated
uninvolved skin (U) from three diseased fish. Positive control (+);
negative buffer control (); H (lanes 3, 5, and 7); and U (lanes 4, 6, and 8). Skin from two clinically normal fish (N1 and N2) (lanes 9 and
10). std, standard. (C) Southern blot analysis. Positive control, pool
of lesions from four fish that were positive for both viruses by PCR
(+); undigested DNA from lesions (lanes 2 and 5);
BamHI-EcoRI (WEHV1)- or PstI
(WEHV2)-digested DNA from lesions (lanes 3 and 6); digested DNA from
uninvolved skin (lanes 4 and 7); copy-number controls of 1, 3, and 5 copies/cell (lanes 8, 9, and 10); negative control (), 5 copies of
WEHV1 or WEHV2 3' RACE clones, bottom and top, respectively.
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The finding that viral DNA could be amplified from uninvolved skin,
albeit at a low level, suggests that qualitative PCR alone
is not
sufficient to differentiate proviral DNA associated with
diseased
tissue and proviral DNA present in uninvolved skin. Therefore,
Southern
blot analysis was used to quantify viral DNA in these
tissues. Our
assumptions were that there would be at least one
proviral DNA copy per
cell in lesions if the viruses are etiologically
associated with
disease and that there would be a significantly
lower level in
uninvolved skin. Hyperplasias and uninvolved skin
from two of the three
fish that were positive for WEHV2 by PCR
(Fig.
3B, lanes 5 to 8) were
used for this analysis; the tissues
from the fish harboring both
viruses (lanes 1 and 2) were insufficient
to isolate a sufficient
amount of genomic DNA for this assay.
Genomic DNA was isolated from
hyperplasias and uninvolved skin
by standard procedures
(
21). DNA (15 µg) was electrophoresed
on a 0.9% gel,
blotted onto nitrocellulose (
21), and hybridized
with the
WEHV1- and WEHV2-specific
pol probes described above.
The
probes, which have only 67% nucleic acid identity, did not
cross-hybridize (Fig.
3C, lane 11 top and lane 11 bottom). WEHV1
and
WEHV2 were detected in a positive control sample made from
a pool of
several lesions previously found to be positive for
both viruses by PCR
(lane 1). WEHV2 DNA was detected at 1 to 3
copies per cell (lanes 3 and
6) and comigrated with uncut genomic
DNA, indicating that it is
integrated into the chromosome (lanes
2 and 5). In contrast to the PCR
results, WEHV2 DNA was not detected
in uninvolved skin by Southern
analysis (lanes 4 and 7). These
data imply that the weakly positive
results obtained by PCR from
uninvolved skin (Fig.
3B, lanes 6 and 8)
represent viral DNA that
is present at less than one copy per cell. We
infer that the same
is true for the weakly positive results for the
hyperplasias shown
above (Fig.
3A, lanes 5, 6, 9, and 10 for WEHV1 and
lanes 3 and
8 for WEHV2) and that only the more abundant species
is etiologically
associated with the lesions. From the PCR and Southern
data, we
conclude that WEHV1 and WEHV2 proviruses are abundant in
diseased
tissue, relatively rare in uninvolved skin, and undetectable
in
clinically normal fish.
The
PstI digest of WEHV2 genomic DNA resulted in two bands
of approximately 5.3 and 6.0 kb that hybridized with the WEHV2
pol probe (Fig.
3C, lanes 1, 3, and 6). The 5.3-kb band is
consistent
with that predicted from the sequence of the WEHV2 genomic
clone.
The larger band may result from restriction fragment length
polymorphism,
but this possibility needs to be investigated. Since
pools of
lesions from individual fish were used in the analysis, it is
possible that variants of WEHV2 with
PstI polymorphisms are
present
in the same sample, as suggested by the nucleic acid mutations
observed in the WEHV2
pol overlap sequence (above).
Restriction
fragment length polymorphisms are common among retroviral
isolates.
Full-length WEHV1 and/or WEHV2 RNAs are abundant in hyperplastic
lesions.
To determine if WEHV1 and WEHV2 genes are expressed in
hyperplastic lesions, 26 of the 34 samples analyzed by PCR were
analyzed by Northern blotting. Total RNA was isolated from hyperplastic lesions with RNAzol, and 10 µg was electrophoresed in formaldehyde gels and blotted onto nitrocellulose (21). The blots were
hybridized with WEHV1 and WEHV2 pol-specific probes to
detect full-length genomic viral RNA. As shown in Fig.
4, WEHV1 and WEHV2 genomic RNAs are
abundant and largely undegraded in spring lesions (lanes 1 to 4). The
apparent size of WEHV1 and WEHV2 full-length RNAs is approximately 13 kb, similar to that of WDSV (12.7 kb). Both WEHV1 and WEHV2 viral RNAs
were detected in 13 samples, whereas WEHV1 RNA was detected alone in
five samples and WEHV2 RNA was detected alone in six samples. A sample
in which only WEHV2 RNA was detected is shown in lane 2. Two samples
did not contain detectable levels of full-length viral RNA, perhaps due
to sample degradation. Importantly, no viral RNA was detected in
uninvolved skin or muscle of the infected fish (lanes 5 and 6, respectively), whose lesions were analyzed in lane 1.
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FIG. 4.
Representative sample of Northern blot analysis of total
RNA isolated from hyperplastic tissue. WEHV1 (top)- and WEHV2
(bottom)-specific pol probes were used for hybridization.
Hyperplastic tissue samples (lanes 1 to 4). Each sample contains two or
three lesions collected from an individual fish. RNA isolated from
uninvolved skin and muscle (lanes 5 and 6) from the fish lesion is
shown in lane 1.
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We have identified two related exogenous retroviruses, WEHV1 and WEHV2,
that are associated with walleye discrete epidermal
hyperplasia.
The sequences of the putative
pro-pol open reading
frames of
WEHV1 and WEHV2 show that they are closely related but
distinct from
each other and that they are related to WDSV (
11).
By
analogy with WDSV, WEHV1 and WEHV2 are presumed to be complex
retroviruses.
PCR was used as a preliminary screen to test for the presence of WEHV1
and/or WEHV2 in hyperplastic lesions. However, our
data suggest that
PCR was not ideal for these studies because
it did not unambiguously
discriminate between viral DNA associated
with diseased tissue and
proviruses present in uninvolved skin
at low copy numbers. Southern
blot analysis clearly showed that
the copy number and integrated status
of WEHV2 is consistent with
a retroviral etiology, i.e., at least one
proviral copy was present
per cell in lesions, whereas uninvolved skin
did not contain detectable
levels of viral DNA. Consistent with these
results, Northern blot
analysis showed that abundant levels of WEHV1
and/or WEHV2 genomic
RNA were present in lesions but not in uninvolved
tissues. Together,
these data provide evidence that WEHV1 and WEHV2 are
expressed
specifically in diseased tissue and are involved in
tumorogenesis.
The abundance of WEHV1 and WEHV2 viral RNA observed in spring
hyperplasias is similar to that of WDSV viral RNA observed in
spring
dermal sarcomas (
5,
17). In contrast, one to three
copies
per cell of proviral DNA were present in spring hyperplasias,
whereas
up to 50 copies per cell of unintegrated viral DNA (UVD)
were found in
spring dermal sarcomas (
13). This difference in
viral copy
number may reflect the conditions of the lesions at
the time of
sampling. Typically, spring dermal sarcomas collected
during the
spawning run in April are in the late stages of regression,
characterized by tumor shedding and necrosis (
15). The
hyperplasias
used for this analysis were also collected in April but
were not
visibly regressing. The regression of hyperplastic lesions has
not been documented, but it is assumed to occur because hyperplasias
are not seen in the summer (
3). WEHV1 and WEHV2 UVD may
accumulate
in cells of hyperplastic lesions as they begin to regress
later
in the season.
The high levels of UVD seen in spring dermal sarcomas may contribute to
tumor regression by inducing cell death, analogous
to the suggestion
that UVD contributes to the cytopathic effects
of avian leukosis virus
subgroups B, D, and F (
26,
27). However,
it has been shown
recently that the envelope proteins of avian
leukosis virus subgroups B
and D interact with a cellular receptor,
inducing apoptosis, possibly
explaining the cytopathology associated
with these viruses
(
6). Therefore, the effect of UVD on cell
viability in ALV
and also on that in walleye retrovirus systems
remains unclear.
In summary, our data strongly suggest that WEHV1 and WEHV2 are the
causative agents of discrete epidermal hyperplasia. This
hypothesis is
supported by a recent experimental transmission
of epidermal
hyperplasia to walleye fingerlings inoculated with
cell-free
filtrates of hyperplastic lesions. As with the WDSV
transmission model
(
14), the injected fingerlings developed
epidermal
hyperplasia in 20 to 24 weeks, and the WEHV1 and WEHV2
viral DNAs were
found in samples of the transmitted hyperplasias
which were assayed by
PCR (
2).
The discovery of two retroviruses associated with walleye epidermal
hyperplasia was unexpected. Although most of the fish
analyzed by PCR
harbored both viruses, the observation that some
hyperplasias contained
only WEHV2 viral DNA suggests that this
virus is capable of causing
disease independently of WEHV1. Because
we pooled multiple lesions from
individual fish, it is not clear
if both WEHV1 and WEHV2 are naturally
found together in the same
lesion or if they exist independently in
different lesions on
the same fish. To address this issue, individual
hyperplastic
lesions will be characterized to determine their virus
profiles.
Additionally, experimental transmission of epidermal
hyperplasia
to walleye fingerlings with WEHV1 or WEHV2 preparations
will be
necessary to demonstrate their ability to independently induce
disease. Further molecular characterization of WEHV1 and WEHV2
will
provide insight about the role they play in the pathogenesis
of
discrete epidermal hyperplasia.
Nucleotide sequence accession numbers.
The accession numbers
for the WEHV1 and WEHV2 pro-pol sequences are AF014792 and
AF014793, respectively.
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ACKNOWLEDGMENTS |
We thank R. Colesante and M. Babenzein of the New York State
Department of Environmental Conservation, Oneida Hatchery, for providing fish and V. Vogt and A. Eaglesham for critical reading of the
manuscript.
The work was supported in part by a USDA grant (93-37204-9214) to
P.R.B. and by funds from the USDA Animal Health and Disease Program
administered by the College of Veterinary Medicine, Cornell University,
to D.L.H. L.A.L. was supported by an NIH training grant (CA09682).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. Phone: (607) 253-3579. Fax: (607) 253-3384. E-mail: jwc3{at}Cornell.edu.
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J Virol, April 1998, p. 3484-3490, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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