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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2090-2101
A Novel Epstein-Barr Virus-Like Virus, HVMNE, in a
Macaca Nemestrina With Mycosis Fungoides
By
E.D. Rivadeneira,
M.G. Ferrari,
R.F. Jarrett,
A.A. Armstrong,
P. Markham,
T. Birkebak,
S. Takemoto,
C. Johnson-Delaney,
J. Pecon-Slattery,
E.A. Clark, and
G. Franchini
From the National Cancer Institute, Basic Research Laboratory,
Bethesda, MD; the Leukaemia Research Fund Virus Centre, Department of
Veterinary Pathology, University of Glasgow, Glasgow, UK; the Advanced
Biotechnology Laboratory, Rockville, MD; the Department of Comparative
Medicine and the Washington Regional Primate Research Center,
University of Washington, Seattle, WA; and the Frederick Cancer
Research Development Center, National Cancer Institute, Frederick, MD.
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ABSTRACT |
Epstein-Barr virus (EBV) infection of humans has been associated
with the development of lymphoid malignancies mainly of B-cell lineage,
although occasionally T-cell lymphomas have been reported. We describe
here the characterization of a novel EBV-like virus (HVMNE)
isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II)
seronegative pigtailed macaque (Macaca nemestrina) with a
cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin
lesions demonstrated that the infiltrating cells were of the
CD3+/CD8+ phenotype. Two primary
transformed CD8+ T-cell lines were obtained from cultures
of peripheral blood mononuclear cells (PBMC) and skin, and, with time,
both cell lines became interleukin-2-independent and acquired the
constitutive activation of STAT proteins. Polymerase chain reaction
analysis of the DNA from the cell lines and tissues from the
lymphomatous animal demonstrated the presence of a 536-bp DNA fragment
that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HVMNE according to the existing nomenclature. In
situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8+ T-cell
lines as well as in the infiltrating CD8+ T cells of
skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from
the polymerase gene of HVMNE placed this virus within the
Lymphocryptovirus genus and demonstrated that HVMNE
is a distinct virus, clearly related to human EBV and other EBV-like
herpesviruses found in nonhuman primates.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MYCOSIS FUNGOIDES (MF) is a rare
cutaneous T-cell lymphoma (CTCL) that may involve lymph nodes and
viscera as well as skin.1,2 The Sézary syndrome (SS)
is the erythrodermic variant of MF characterized by the presence of
circulating tumor cells. The tumor cells in MF/SS are usually of the
CD4+ mature-cell lineage, although CD8+ lineage
has been described in a few cases.2 The diagnosis of MF is
based on clinical features and histopathological findings that include
infiltration of the dermis with lymphocytes with hyperconvoluted nuclei
and Pautrier's microabcesses.1 MF and SS are the most
frequent primary lymphomas involving the skin. Genetic predisposition,
alteration in cytokine profile, and viruses such as herpesviruses I and
II (HSV-I and HSV-II), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and human T-cell lymphotropic virus type I (HTLV-I) have been
suggested as possible causative agents.1
HSV-I/II-specific antigens and DNA have been found in lesions of
CTCL.3,4 One report describes the finding of HHV-6 in 1 of
30 patients with CTCL.5 EBV DNA has been found in patients with cutaneous lymphomas6-12 and, in several cases, viral
RNA expression has been demonstrated in the neoplastic tissue. Higher incidence of EBV seropositivity in CTCL patients with the consistent emergence of EBV in MF/SS-cultured lymphocytes has also been
reported.10 However, a direct causative role of EBV in CTCL
has been difficult to prove.13 In contrast, the importance
of EBV in the development of B-cell malignancies in human
immunodeficiency virus-infected individuals or iatrogenically
immune-suppressed patients is more broadly accepted.14
Although humans are the only known natural host for EBV, EBV-like
agents have been described in Old World nonhuman primates, including
chimpanzee,15-17 baboon,16,18-21 African green
monkey,22 gorilla,23 and macaque
species.24-29 Although the relative prevalence of these
viruses in animals in captivity or in the wild is
unknown,30 several studies suggest that it may be as high
as that in EBV in humans. In fact, the inability to generate models of
EBV-associated lymphomas in Old World monkeys with the human EBV
has been ascribed to the presence of cross-reactive immunity against
EBV in these species. In contrast, at least 2 New World monkey species,
including the cotton-top tamarin (Sanguinus oedipus
oedipus)31 and the owl monkey (Aotus
trivirgatus),32 develop B-cell lymphoma upon human EBV
exposure. We report here the occurrence of a rare case of
CD8+ T-cell MF in a pigtailed macaque and the isolation of
a novel EBV-like virus from 2 transformed CD8+ T-cell lines
obtained from the blood and the skin of the lymphomatous macaque.
These findings may help in the development of an animal model for
EBV-like virus-induced malignant proliferation of T cells.
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MATERIALS AND METHODS |
Establishment of macaque blood and skin T-cell lines.
Peripheral blood mononuclear cells (PBMC) were isolated by
density-gradient centrifugation on lymphocyte separation medium (LSM;
Organon Teknika Corp, Durham, NC) from anticoagulated blood obtained
from pigtailed macaque J94356 before death. The cell layers were washed
twice in Dulbecco's phosphate-buffered saline (DPBS; GIBCO BRL,
Gaithersburg, MD) and were suspended in RPMI (GIBCO BRL) with 10%
heat-inactivated (HI) fetal bovine serum (FBS; GIBCO BRL) with
penicillin/streptomycin (500 U/mL and 500 g/mL, respectively; GIBCO
BRL) and L-glutamine (2 mmol/L; GIBCO BRL) and stimulated with
phytohemagepletrinic (PHA; 5 g/mL; Murex Diagnostics, Norcross, GA). At
72 hours, the cells were washed twice in DPBS and resuspended in fresh
RPMI with 10% HI FBS, penicillin/streptomycin, L-glutamine, and
recombinant interleukin-2 (IL-2; 20 U/mL; Boehringer Mannheim,
Indianapolis, IN). Fresh skin biopsy tissues from diseased areas were
minced to release single cells. These were banded on LSM and placed in
culture as described above. All cells were incubated at 37°C with
5% CO2; fresh media were added once or twice per week, as
needed, to maintain adequate cell growth. After 6 weeks in culture,
both the PBMC and skin-derived cells showed evidence of increased
proliferation and clustering and the cells have been cultured for more
than 1 year. The concentration of IL-2 in the media in both cultures
was decreased in a stepwise manner from an initial concentration of 20 U/mL. Each cell line was maintained at a dose of IL-2 until it was
clear that cell growth was unimpaired. At the end of 5 months, both
J94356PBMC and J94356SKIN cells were growing
well, albeit at a slower rate than their IL-2-dependent counterparts.
DNA extraction, polymerase chain reaction (PCR) amplification, and
DNA sequence analysis.
Pellets of PBMC obtained by Ficoll gradient separation were incubated
for 1 hour at 37°C in a buffer containing 0.5% sodium dodecyl
sulfate (SDS), 10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA (pH 8.0), and
RNAse (20 g/mL; Boehringer Mannheim) before the addition of Proteinase
K (100 µg/mL; Boehringer Mannheim). After an overnight incubation at
37°C, DNA was extracted from the lysates with phenol and
chloroform. Frozen tissues, stored at  80°C until processing,
were thawed on ice and finely minced with sterile blades. The minced
tissue was resuspended in ice-cold DPBS and washed twice before lysis
and extraction as detailed above. DNAs were initially amplified with
the primer pools DFASA/GDTD1B and VYGA/GDTD1B described by Rose et
al,33 corresponding to a conserved region in the
herpesvirus family that codes for substrate binding sites within the
DNA polymerases.
PCR conditions were optimized using the Invitrogen PCR Optimizer Kit.
Optimal amplification was obtained with 500 ng of genomic DNA, 25 pmol
of each primer, 300 mmol/L Tris-HCl 5× buffer (pH 10) containing
2.5 mmol/L MgCl2, 75 mmol/L
(NH4)2SO4, 10 mmol/L dNTP, and 2.5 U Taq polymerase. The templates were subjected to a maximum of 45 cycles of amplification, each cycle consisting of 15 seconds at
94°C, 30 seconds at 60°C, and 15 seconds at 72°C (Perkin
Elmer GeneAmp PCR system 9600 thermal cycler; Perkin Elmer, Branchburg,
NJ). An aliquot of these amplification products was electrophoresed in
a 1.5% agarose gel, stained with 0.5 µg/mL ethidium bromide, and
visualized under UV transillumination. Amplification products of the
predicted size (~536 bp) were detected, and the resulting fragments
were cloned into Inv  F' bacterial cells using the TA cloning
kit from Invitrogen (Carlsbad, CA). White colonies were picked and
grown in LB broth in the presence of ampicillin at 37°C overnight.
Plasmids were isolated using the Promega Miniprep (Promega, Madison,
WI) and digested with EcoRI to confirm the presence of the
appropriately sized insert, and DNA sequencing was performed using the
T7 Sequenase v2.0 chain-termination method from Amersham Life Science
(Cleveland, OH) according to the manufacturer's instructions. Based on
the sequence of the DNA fragment first amplified, a primer set, EDR8/97
sense 5'-ATCTCTGTTATTCTACC-3', MGF2B antisense
5'-CCTGCAGCGTCA-3', was synthesized. An aliquot of the
first PCR products was amplified using these sequence-specific primers.
PCR amplification with the primer set was optimal at 5× buffer
(pH 8.5) containing 300 mmol/L Tris-HCl, 2.0 mmol/L MgCl2,
75 mmol/L (NH4)2SO4, 10 mmol/L
dNTP, and 2.5 U Taq polymerase. The templates were subjected to 45 cycles of amplification as described above. The nested PCR products
were analyzed by gel electrophoresis and subsequently cloned into Inv
cells, and sequence analysis was performed.
Other regions of the viral genome were amplified using the primer pairs
described by Ino et al25 and included the 2s/2as pair
covering IR1 region and the 11s/11as pair covering part of the EBV
BRRF-1 region.24 PCR products were analyzed by gel
electrophoresis and cloned into Inv cells, and the DNA sequence was
obtained as described above.
Southern blot analysis.
DNA (10 µg) of each sample was digested with Sau3AI or
Pvu II, electrophoresed in 0.8% agarose gels, and blotted onto
Nylon membranes (Nytran plus; Schleicher & Schuell, Keene, NH). The membranes were hybridized overnight at 42°C with the PCR-amplified EBV-like probe labeled using random-primer reaction (Boehringer Mannheim). Hybridization, washing, and detection were performed according to the manufacturer's instructions.
Phylogenetic analysis.
Phylogenetic analysis of the novel HVMNE pol-gene
fragment (465 bp) was performed using sequences from an analogous
strain from Macaca arctoides29 (HVMA) and the
following related strains published previously: human EBV: V01556;
retroperitoneal fibromatosis herpesvirus (RFHV) Macaca
nemestrina: AF005478; retroperitoneal fibromatosis herpesvirus
(RFHV)Macaca mulatta: AF005479; herpesvirus Saimiri (HVS):
M31122; Kaposi sarcoma herpesvirus (KSHV): AF005477; equine herpesvirus
2 (EHV-2): and U20824; herpesvirus Papio (HVPA).34 Sequences were aligned using ClustalX version
1.63B.35 Genetic distance estimates among all
pairs of pol sequences were estimated using the Tajima-Nei
model of substitution.36 Phylogenetic associations, using
the computer program Mega Version 1.01,37 were
ascertained by minimum evolution estimated by neighbor-joining (NJ) in
conjunction with the Tajima-Nei model of substitution. Bootstrap
analyses, consisting of 100 iterations, were performed to determine the consistency of the data in recapitulating the same tree for NJ analysis. Values of 70% or more were considered strong support for the
adjacent node.38
Electrophoretic mobility shift assay (EMSA).
PHA-stimulated human PBMC were cultured in the presence of IL-2 (20 U/mL) for 8 days and used as a control for this assay. In starvation
experiments, 1 × 107 stimulated PBMC and
IL-2-dependent and -independent J94356PBL were
resuspended in 20 mL of RPMI 1640 with 1% FBS after washing with
1× PBS twice and incubated for 21 hours at 37°C in 5%
CO2. Protein lysates were prepared in 20 mmol/L HEPES (pH
7.9), 450 mmol/L NaCl, 0.4 mmol/L EDTA, 0.5 mmol/L dithiothreitol
(DTT), 25% glycerol, 1 mmol/L
Na3VO4, 1 mmol/L AEBSF, 20 µg/mL aprotinin, and 20 µg/mL leupeptin. The binding reaction was performed by preincubating 5 µg of nuclear extracts with 1 µg of poly(dI-dC) in
a buffer containing 5.9 mmol/L HEPES (pH 7.9), 30 mmol/L KCl, 5.9 mmol/L Tris (pH 7.4), 0.7 mmol/L DTT, 0.6 mmol/L EDTA, 8.9% glycerol,
0.1 mmol/L Na3VO4, 1 mmol/L AEBSF, 20 µg/mL
aprotinin, and 20 µg/mL leupeptin in ice for 20 minutes. A
32P-labeled probe (20,000 cpm) corresponding to the MGF
binding site in the  -casein gene promoter
(5'-TAGATTTCTAGGAATTCG-3') was added to the reaction
mixture and incubated on ice for 30 minutes. For the supershift assay,
STAT5 antibody (N-20; Santa Cruz Biotechnology, Santa Cruz, CA) was
incubated with cell extracts on ice for 20 minutes after the addition
of radiolabeled probe. Complexes were resolved on 4.5% polyacrylamide gels.
Immunohistochemistry and EBV Epstein-Barr viral-encoded RNA (EBER)
in situ hybridization.
CD3 immunostaining was performed on formalin-fixed, paraffin-embedded
tissue using a rabbit polyclonal anti-CD3 serum (A0452; DAKO,
Carpinteria, CA) at 1:50 dilution followed by biotinylated antirabbit
secondary antibodies and ABC reagent (Vector Labs, Burlingame, CA) and
DAB substrate (Scytek Laboratories, Logan, UT). CD8 immunostaining was
performed on tissue sections frozen in OCT using a mouse anti-CD8
monoclonal antibody (clone Leu-2a; Becton Dickinson, San Jose, CA)
followed by a biotinylated antimouse Ig (ABC reagent; Vector Labs) and
DAB substrate (Scytek Laboratories). Each immunohistochemical analysis
included a test tissue assayed either using antibody against an
irrelevant antigen (from the appropriate species) or in absence of
primary antibody. Cell lines derived from peripheral blood and skin
were formalin-fixed, pelleted, and paraffin-embedded. Sections (3 to 5 µm) were cut onto glass slides and hybridized with a cocktail of
fluorescein isothiocyanate (FITC)-conjugated probes reactive with the
EBER RNAs, which are abundantly transcribed in all cells latently
infected with EBV (Novocastra Laboratories, Newcastle, UK).
Hybridization was detected using an alkaline-phosphatase-conjugated,
anti-FITC antibody (DAKO, High Wycombe, UK) and nitro-blue tetrazolium
as chromogenic substrate (DAKO). Subsequently, the EBER in situ
hybridization was performed using sections of skin biopsies obtained
from monkey skin affected with lymphoma. Hybridization using a poly-dT
probe (Novocastra) was used to confirm the integrity of the RNA in all
tissue sections.
Na-butyrate induction and electronmicroscopy (EM).
Cells (5 × 105) from the J94356PBMC cell
line were incubated in medium containing Na-butyrate (1 µmol/L) for
48 hours, fixed in 0.65% paraformaldehyde and 0.8% gluteraldehyde for
1 to 2 hours, and then incubated in 0.1 mol/L cacodylate buffer (pH 7.2 to 7.4) containing 0.1 mol/L sucrose. The cell pellet was postfixed
with 0.5% osmium fixative (pH 7.2 to 7.4) for 1 hour, processed
through an acetone gradient to 100% acetone, and then placed
sequentially in 1:1 and 1:3 acetone-resin mixes. The cells were
embedded into a pure resin capsule and cured in an oven at 60°C for
48 hours. The pellet was sliced with an Ultra Microtome (Leica
Microsystems UK, Milton Keynes, UK) at 50 to 60 nm, collected on copper
grids, and stained with uranyl acetate and lead citrate. The
preparations were analyzed on a Zeiss 109 Transmission Electron
Microscope (Cast Leiss, Welwyn Garden City, UK).
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RESULTS |
MF in animal J94356: Clinical presentation and histopathological
findings.
In May 1996, an 18-month-old pigtailed macaque (animal J94356), housed
at the Washington Regional Primate Research Center, developed a
unilateral eye infection with marked inflammation of the conjunctiva
and eyelids, which eventually spread to the opposite eye. Improvement
in the clinical manifestations was observed after treatment with
antihistamine and antibiotics, but inflammation of both conjunctivas
and eyelids recurred 8 days after treatment was discontinued. A small
unilateral corneal ulceration was found on examination and antibiotic
and antihistamine therapy was reinstated. At this time, an extensive
work-up to test for viral infections was performed. A serologic panel
of tests was run at a commercial laboratory. Tests were negative for
herpes B, HSV-1, measles, and simian immunodeficiency virus and were
positive for cytomegalovirus. In August 1996, the ulcerative lesions
recurred in the perioral area and new lesions involving the face,
trunk, forearms, and legs appeared. At this time, the serology was
negative for herpes B and STLV; however, EBV serology was positive and
the lesions improved on a 2-week course of acyclovir. Biopsy results of
skin lesions examined early in October 1996 were consistent with a multifocal cutaneous lymphoma. The animal was continued on antibiotic therapy with mild improvement of the lesions.
However, in mid-January 1997, the lesions dramatically worsened and,
after a course of steady deterioration, the animal was killed in
January 1997. At the time of death, animal J94356 displayed an
erythematous, hyperkeratotic rash, with alopecia and excoriated lesions
with prominent red borders and numerous plaque-like lesions on
extensive areas of the skin
(Fig 1A and B). Hematoxylin
Eosin staining of a skin-lesion sample showed profusely infiltrating mononuclear cells in the dermis and epidermis with prominence at the
dermal-epidermal junction in periadnexal tissue and in perivascular
tissue (Fig 1C). In the epithelium, the cells were arranged in small
solid clusters at the dermal-epidermal interface, resembling
Pautrier's microabcesses. The cells had moderately sized nuclei
containing 1 to 2 small nucleoli and up to 2 mitotic figures were
present per 400× field (Fig 1D). A few histiocytes and
eosinophils were admixed with the neoplastic cells. There were
multifocal erosions in the epithelium, which was acanthotic and
hyperkeratotic (Fig 1C). Immunohistochemical analysis of the skin
lesion showed CD3+ infiltrating T cells both in the dermis
(top left panel of Fig 2) and a small
portion of the epidermis. Coalescing clusters of CD3+ cells
were present along the dermal-epidermal junction and within the
epidermis. The lineage of the infiltrating cell population was further
characterized and found to be strongly CD8+ (right top and
bottom panels of Fig 2) and CD4 (not shown). No
staining was observed with an irrelevant antibody (left bottom panel of
Fig 2). Thus, clinical features of the skin lesions and the
histopathology were indicative of a CTCL with a CD8+ T-cell
phenotype.
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| Fig 1.
Macroscopic and histologic characteristics of a
skin lesion in the lymphomatous macaque. (A) and (B) show the
macroscopic appearance of the lesions present in pigtailed macaque
J94356. Note the extensive areas of involvement. The lesions are
hyperkeratotic, with alopecia, and with excoriated areas surrounded by
prominent borders. (C) shows a low-power magnification of the histology
of a skin lesion stained with Hematoxylin and Eosin (HE). Note the
profuse mononuclear cell infiltration of the dermis, dermal-epidermal
junction, epidermis, periadnexal tissue, and perivascular areas. (D)
shows a high-magnification view (400×) of the infiltrating cells
stained with HE. Numerous mitotic figures are present on a homogeneous
background. The cells have moderately sized nuclei with 1 or 2 nucleoli.
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| Fig 2.
Identification of the phenotype of neoplastic cells
infiltrating the skin. Top left panel (CD3) shows an area of affected
skin stained with monoclonal antibodies against CD3. Clusters of
CD3+ cells are present along the dermal-epidermal
junction and in the epidermis. Top and lower right panels (high-and
low-power magnification, respectively) demonstrate that the
infiltrating cells belong to the CD8+ T-cell subgroup.
Cells strongly positive for CD8 are present in clusters in the dermis
along the dermal-epidermal junction and in the epidermis. Lower left
panel is a control for antibody specificity.
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Infiltrates of mononuclear cells undergoing mitosis with a few
eosinophils and neutrophils were also observed in several organs, including the lungs and lymph nodes (data not shown). Occasional mitoses were present in the enlarged lymphoid follicles and germinal centers. The spleen contained prominent lymphoid nodules and hyaline material in the germinal centers, and the liver had a mild multifocal lymphocytic infiltrate in portal areas and subcapsular tissue. Although
staining with CD8 antibodies was not performed in the organs, it is
highly likely that these infiltrates were composed of the same
CD8+ T cells found in the skin.
Detection of an EBV-like virus in the transformed CD8+
T-cell lines from animal J94356.
Peripheral mononuclear cells obtained from whole blood at the time of
necropsy were cultured with PHA and recombinant IL-2. Skin-derived
cells were also placed in culture under similar conditions and, like
the PBMC culture, proliferated continuously in response to IL-2. Both
cell lines have been in culture for approximately 2 years. Giemsa
staining of cytospin preparations of these cells at 2.5 months after
initiation of culture of the cells indicated their pleomorphism with
myeloid-like features and multiple mitotic figures at higher
magnification (lower panels of
Fig 3). Cytogenetic analyses
of the chromosomes confirmed the simian origin of both cell lines (2n = 42; data not shown).
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| Fig 3.
Giemsa stains of cytospin preparations from the
2 cell lines derived from blood and skin of animal J94356
(J94356PBMC and J94356SKIN, respectively) after
2 months in culture in RPMI with 10% FBS and 20 U/mL of IL-2.
High-magnification views (lower panels) show the pleomorphic appearance
of these cells with myeloid-like features and presence of mitotic
figures.
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Electron-microscopic analysis of the cell lines under standard culture
conditions was negative even after an extensive search for any viral
particles, including retroviruses. In addition, the supernatants of
these cell lines were consistently negative for reverse transcriptase
activity. However, electron microscopic analysis performed 48 hours
after treatment with Na-butyrate allowed the detection of large
enveloped virions in the cytoplasm of cells derived from the PBMC of
animal J94356, consistent with the presence of a herpesvirus
(Fig 4A). To assess the genetic composition
of this herpesvirus, we used generic DNA primers previously
demonstrated to amplify efficiently genomes from several members of the
herpesvirus family,33 which amplified a DNA fragment of 536 bp from both CD8+ T-cell lines. This DNA fragment (p536)
was molecularly cloned and used as a probe to hybridize the DNA from
primary J94356 PBMC cultured for 46 days as well as from the J94356
skin culture at days 46, 226, and thereafter. After PCR amplification,
the expected DNA fragment of 536 bp was found in the macaque cell lines
but not in human PBMC (Fig 4B), suggesting that this putative
herpesvirus persisted in both cell lines originated from animal J94356.
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| Fig 4.
Detection of HVMNE in the cell lines
J94356PBMC and J94356SKIN by electron
microscopy and DNA PCR. (A) EM of cell line J94356PBMC
harvested 48 hours after induction with Na-butyrate (1 µmol/L).
Extensive EM search in the same cell line without Na-butyrate induction
failed to show viral particles (not shown). (B) Southern blot of PCR
products using probe 536. Detection of a 536-bp DNA fragment in the
J94356PBMC and J94356SKIN cell lines at 46 days
in culture and after 226 days in culture in line J94356SKIN
by PCR using the generic primer pair DFASA/GDTD1B (described by Rose et
al33).
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Genetic and phylogenetic characterization of the herpesvirus from
animal J94356.
To establish the genetic relationship of the putative viral fragment
found in cells from animal J94356 to other known nonhuman primate
herpesviruses, we obtained the DNA sequence of plasmid p536 and aligned
it to the equivalent polymerase region of the human KSHV and EBV as
well as EBV-like viruses or rhadinoviruses from various animal species
(Fig 5).29,33,34,39 Both DNA sequence alignment and the phylogenetic analysis by the NJ method (Fig 6) indicated that the herpesvirus in
J94356 cells clustered with the human EBV and the nonhuman primate
herpesviruses, HVMA and HVPA,34 and was distantly related
to the known rhadinoviruses.33,39,40
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| Fig 6.
Phylogenetic tree based upon 353 bp of the pol
gene. Shown is the minimum evolution tree estimated by NJ method using
distances among all pairs of sequences.36 Numbers on
branches indicate the percentage of sequence divergence. Numbers in
italics are bootstrap support (ME-NJ) for the adjacent node.
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Two other EBV-like viruses, SiIIA and HVMF-1, have been isolated from
Macaca fascicularis.26,41 To assess the genetic
relationship of HVMNE to SiIIA, we analyzed the
HVMNE DNA with primer sets for the BRRF-1 EBV-equivalent
region. As demonstrated in Fig 7, HVMNE appears to be distinct from SiIIA, because the DNA
sequence of this region was 85% identical to EBV and 90% identical to
the SiIIA strains. In the case of HVMF-1, the DNA sequence from this region is not available; therefore, HVMF-1 and HVMNE DNA
were compared by restriction enzyme and Southern blot analyses, which demonstrated differences in both restriction sites used and suggested genetic diversity between HVMNE and HVMF-1
(Fig 8). We therefore designated the virus
from animal J94356 as HVMNE for consistency with the common
name used for other EBV-like viruses found in nonhuman primates.
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| Fig 8.
Southern blot analysis of HVMNE and
HVMF-1-infected cell DNAs. Human PBMC DNA was used as negative
control. The molecular-weight complexes are indicated at the left of
each panel and the restriction enzyme used is indicated at the bottom
of each panel.
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JAK/STAT constitutive activation in CD8+ T cells infected
with HVMNE correlates with the acquisition of IL-2
independence.
Transforming viruses usually induce growth-factor independence by
interfering with intracellular signaling pathways,42-45 and this event occurs also in EBV-transformed B-cell lines.46
In addition, in human hematopoietic malignancies, including
Sézary syndrome, constitutive activation of JAK/STAT protein has
also been described.47-50
Both T-cell lines from animal J94356 acquired the ability to grow
independently from exogenous IL-2, and, to assess whether constitutive
activation of STAT5 protein also correlated with the acquisition of
IL-2 independence, cell extracts from normal human PHA-stimulated PBMC
and the J94356PBMC cell line, at 3 months and 17 months
from the start of culture, were analyzed after IL-2 withdrawal. At 3 months from initiation of culture, some degree of constitutive STAT5
binding to the MGF probe could be already observed in the absence of
IL-2 and serum (Fig 9C, lanes 7 and 8),
and, at 17 months, the cells in culture in absence of exogenous IL-2
displayed constitutive binding of activated STAT5 to the MGF probe (Fig
9C, lanes 9 through 12). Constitutive STAT1 and STAT3 binding was also
observed when the specific SIE probe was used (data not shown). Thus,
as in the case of HTLV-I-infected cells, the transition to IL-2
independence correlates with the acquisition of constitutive activation
of STAT proteins.43,44 To assess whether the resident
herpesvirus expression was contained in the IL-2-independent cells,
viral expression was analyzed using the human EBV EBER probe. As
demonstrated in Fig 9A and B, a clear nuclear signal was evident in
most HVMNE-infected cells, indicating also a good
conservation of this sequence in the HVMNE.
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| Fig 9.
The transition of the J94356PBMC expressing
HVMNE cell lines to IL-2 independence is associated with
STAT5 activation and HVMNE expression.
J94356PBMC cells hybridized with EBER RNA (A) or
incubated with hybridization buffer in the absence of probe (B). (C)
EMSA on control PBMC (lanes 1 through 4) and the J94356PBMC
cell line in the IL-2-dependent (lanes 5 through 8) and
IL-2-independent (lanes 9 through 12) status.
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Detection of EBV-like DNA in the tissues of animal J94356.
To investigate whether HVMNE found in the CD8+
T-cell lines from animal J94356 was also present in the infiltrating
CD8+ neoplastic T cells from the skin lesion as well as
other organs, the DNA from various tissues was analyzed using the same
primer set used to amplify the HVMNE polymerase gene from
the DNA of the J94356 cell lines.
As demonstrated in Fig 10A, viral DNA
sequences were found in the skin but not the PBMC obtained from animal
J94356 at the time of CTCL diagnosis (1996). However, at time of death
(1997), viral DNA was also found in the uncultured PBMC as well as in multiple skin specimens, lymph nodes, and muscles. The finding of viral
DNA in PBMC at time of death is consistent with the clinical finding of
an abnormal T-cell count (18,000/mL) in the blood of animal J94356.
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| Fig 10.
Detection of herpesvirus DNA by PCR and of EBV-specific
RNA by in situ hybridization in tissues of animal J94356. (A) Southern
blot of PCR products using probe p536. Detection of 536-bp and 236-bp
DNA fragments in tissues from animal J94356 by PCR using primer pairs
DFASA/GDTD1B (top) and VYGA/GDTD1B (bottom) at 2 time points. Lanes 1 and 2, PCR results on DNA obtained from noncultured PBMC and affected
skin approximately 7 weeks before death (1996). Lanes 3 through 8, PCR
products on DNA obtained from animal J94356 at time of necropsy (time
0). Lane 3, PBMC, noncultured; lane 4, affected skin; lane 5, lymph
node; lane 6, muscle; lane 7, normal skin. Lane 8, negative control
(DNA from normal human PBMC). EBV EBER in situ hybridization performed
on sections of affected skin biopsy at low (B) and high (C)
magnification. A large number of cells expressing EBV EBER RNA are
found infiltrating the dermis and epidermis in the lymphomatous
animal.
|
|
To assess whether the HVMNE sequences found in the tissues
were also detectable in the CD8+ neoplastic T cells in
vivo, the postmortem tissue specimens of animal J94356 were stained
with the EBER RNA probe. As demonstrated in the left panels of Fig 10B
and 10C, several cells in the skin expressed EBER RNA and the
distribution pattern closely mirrored the pattern observed with
immunohistochemical analyses using CD8-specific antibody (Fig 2); this
suggests that HVMNE was present in the CD8+ T
cells that infiltrated the dermis and epidermis of the lymphomatous animal.
| |
DISCUSSION |
Herpesviruses have been found in most animal species, and the family
Herpesviridae includes the 3 subfamilies Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. Within the
Gammaherpesvirinae subfamily, 2 genuses have been distinguished, the
lymphocryptoviruses (Epstein-Barr-like viruses) and rhadinoviruses
(Saimiri-ateles-like herpesviruses).51 Only members of the
Gammaherpesvirinae subfamily have been associated with human
malignancies. EBV causes B-cell lymphoma in immunodeficient individuals
and is epidemiologically associated with Burkitt's lymphoma,
Hodgkin's disease, and nasopharyngeal carcinoma.14
Similarly, human herpesvirus 8 has been epidemiologically linked to
Kaposi sarcoma40 and rare forms of
lymphoma.52-55 EBV induces B-cell lymphoma in previously
unexposed New World monkeys but not in Old World primates, presumably
because of preexisting cross-immunity against EBV, as demonstrated by
their frequent EBV seropositivity.56 However, after simian
immunodeficiency virus infection, approximately one third of the
infected macaques develop B-cell lymphoma, and this event has been
associated with the presence of an EBV-like virus in Macaca
fascicularis.57
We describe here the identification and partial characterization of
HVMNE from a pigtailed macaque with MF. HVMNE
is distinct from the known nonhuman primate EBV-like herpesviruses as
demonstrated by the phylogenetic analysis of the polymerase gene and
the BRRF-1 region. The finding of HVMNE in the
CD8+ infiltrating T cells in vivo as well as in transformed
CD8+ T-cell lines in vitro supports the notion that
HVMNE might target T cells and might have been involved in
the development and/or progression of MF in animal J94356.
EBV has been reported to induce B-cell lymphoma in New World monkeys
but not in rabbits,58 whereas other EBV-like strains from
nonhuman primates, such as HVMA (Macaca arctoides) and
EBV-related strains from cynomolgus monkeys, have been demonstrated to
induce B-cell lymphoma in rabbits.24,29 Conversely, the
pathogenicity of nonhuman-primate EBV-like viruses has not been
assessed in New World monkeys. Interestingly, HSV strains A and B cause
lymphoma in New World primates but not in New Zealand white
rabbits,58 suggesting that pathogenicity of different viral
strains varies in different species. Transfusion of blood from the
lymphomatous animal discussed in this study to other pigtailed macaques
did not result in lymphoma (data not shown), presumably because of a
preexisting infection of EBV-related virus in this species of Old World
monkey.30 It would be of interest to assess whether HVMNE induces lymphoma in the rabbit model. If so,
HVMNE may provide a model to study viral and cellular
determinants involved in oncogenesis. Lastly, the findings reported
here may prompt a further investigation on the possible role of human
EBV strains in T-cell lymphomas.
| |
ACKNOWLEDGMENT |
The authors thank Peter Biberfeld for helpful discussion and Steven
Snodgrass for editorial assistance. We thank June Freeland and Ross
Blackley, who performed the viral stimulation and EM studies.
| |
FOOTNOTES |
Submitted February 17, 1999; accepted May 19, 1999.
Supported in part by National Institutes of Health Grant No. RR00166
(Washington Regional Primate Research Center, Seattle, WA).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to G. Franchini, MD, Chief, Section of Animal
Models and Retroviral Vaccines, Basic Research Laboratory, Division of
Basic Sciences, National Cancer Institute, National Institutes of
Health, 41 Library Dr, Bldg 41, Room D804, Bethesda, MD 20892; e-mail:
veffa{at}helix.nih.gov.
| |
REFERENCES |
1.
Diamandidou E, Cohen PR, Kurzrock R:
Mycosis fungoides and Sezary syndrome.
Blood
88:2385, 1996[Free Full Text]
2.
LeBoit PE:
Lymphomatoid papulosis and cutaneous CD30+ lymphoma.
Am J Dermatopathol
18:221, 1996[Medline]
[Order article via Infotrieve]
3.
Duvic M, Magee K, Storthz KA:
In situ hybridization for herpes simplex virus in mycosis fungoides and alepecia areata.
J Invest Dermatol
92:423, 1989
4.
Lee LA, Huff JC, Edmond BJ, Weston WL, Norris DAI:
Identification of herpes simplex virus antigens and DNA in lesions of mycosis fungoides.
J Invest Dermatol
80:333, 1983 (abstr)
5.
Brice SL, Jester JD, Friednash M, Golitz LE, Leahy MA, Stockert SS, Weston WL:
Examination of cutaneous T-cell lymphoma for human herpesvirus by using the polymerase chain reaction.
J Cutan Pathol
20:304, 1993[Medline]
[Order article via Infotrieve]
6.
Anagnostopoulos I, Hummel M, Kaudewitz P, Korbjuhn P, Leoncini L, Stein H:
Low incidence of Epstein-Barr virus presence in primary cutaneous T-cell lymphoproliferations.
Br J Dermatol
134:276, 1996[Medline]
[Order article via Infotrieve]
7.
Angel CA, Slater DN, Royds JA, Nelson SN, Bleehen SS:
Absence of Epstein-Barr viral encoded RNA (EBER) in primary cutaneous t-cell lymphoma.
J Pathol
178:173, 1996[Medline]
[Order article via Infotrieve]
8.
d'Amore F, Johansen P, Houmand A, Weisenburger DD, Mortensen LS:
Epstein-Barr virus genome in non-Hodgkin's lymphomas occurring in immunocompetent patients: Highest prevalence in nonlymphoblastic T-cell lymphoma and correlation with a poor prognosis.
Blood
87:1045, 1996[Abstract/Free Full Text]
9.
Dreno B, Celerier P, Fleischmann M, Bureau B, Litoux P:
Presence of Epstein-Barr virus in cutaneous lesions of mycosis fungoides and Sezary syndrome.
Acta Derm Venereol
74:355, 1994[Medline]
[Order article via Infotrieve]
10.
Hopsu-Havu VK, Nikoskelainen J:
Epstein-Barr virus antibody titres in mycosis fungoides.
Acta Dermatovener
52:346, 1972[Medline]
[Order article via Infotrieve]
11.
Iwatsuki K, Ohtsuka M, Harada H, Han G, Kaneko F:
Clinicopathologic manifestations of Epstein-Barr virus-associated cutaneous lymphoproliferative disorders.
Arch Dermatol
133:1081, 1997[Abstract/Free Full Text]
12.
Peris K, Niedermeyer H, Cerroni L, Radaskiewicz T, Chimenti S, Hofler H:
Detection of Epstein-Barr virus genome in primary cutaneous T- and B-cell lymphomas and pseudolymphomas.
Arch Dermatol Res
286:364, 1994[Medline]
[Order article via Infotrieve]
13.
Slater D:
Epstein-Barr virus: An aetiological factor in cutaneous lymphoproliferative disorders?
J Pathol
165:1, 1991[Medline]
[Order article via Infotrieve]
14.
Rickinson AB, Kieff E:
Epstein-Barr virus, in
Fields BN,
Knipe DM,
Howley PM
(eds):
Virology. Philadelphia, PA, Lippincott-Raven, 1996, p 2397.
15.
Gerber P, Pritchett R, Kieff ED:
Antigens and DNA of a chimpanzee agent related to Epstein-Barr virus.
J Virol
19:1090, 1976[Abstract/Free Full Text]
16.
Gerber P, Kalter SS, Schidlovsky G, Peterson WD Jr, Daniel MD:
Biologic and antigenic characteristics of Epstein-Barr virus-related herpes viruses of chimpanzees and baboons.
Int J Cancer
20:448, 1977[Medline]
[Order article via Infotrieve]
17.
Landon JC, Ellis LB, Zeve VH, Frabrizio DP:
Herpes-type virus in cultured leukocytes from chimpanzees.
J Natl Cancer Inst
40:181, 1968
18.
Falk L, Deinhardt F, Nonoyama M, Wolfe LG, Bergholz C:
Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus.
Int J Cancer
18:798, 1976[Medline]
[Order article via Infotrieve]
19.
Heller M, Gerber P, Kieff E:
Herpes virus Papio DNA is similar in organization to Epstein-Barr virus DNA.
J Virol
37:698, 1981[Abstract/Free Full Text]
20.
Heller M, Kieff E:
Colinearity between the DNAs of Epstein-Barr virus and herpes virus Papio.
J Virol
37:821, 1981[Abstract/Free Full Text]
21.
Schätzl H, Tschikobava M, Rose D, Voevodin A, Nitschko H, Sieger E, Busch U, von der Helm K, Lapin B:
The Sukhumi primate monkey model for viral lymphomogenesis: High incidence of lymphomas with presence of STLV-I and EBV-like virus.
Leukemia
7:S86, 1993 (suppl 2)
22.
Bocker JF, Tiedmann KH, Bornkamm GW, zur Hausen H:
Characterization of an Epstein-Barr virus-like virus from African green monkey lymphoblasts.
Virology
101:291, 1980[Medline]
[Order article via Infotrieve]
23.
Neubauer RH, Rabin H, Strnad BC, Nonoyama M, Nelson-Rees WA:
Establishment of a lymphoblastoid cell line and isolation of an Epstein-Barr-related virus of gorilla origin.
J Virol
31:845, 1979[Abstract/Free Full Text]
24.
Hayashi K, Koirala TR, Ino H, Chen HL, Ohara N, Teramoto N, Yoshino T, Takahashi K, Yamada M, Nii S, Miyamoto K, Fujimoto K, Yoshikawa Y, Akagi T:
Malignant lymphoma induction in rabbits by intravenous inoculation of Epstein-Barr-virus-related herpesvirus from HTLV-II-transformed cynomolgus leukocyte cell line (Si-IIA).
Int J Cancer
63:872, 1995[Medline]
[Order article via Infotrieve]
25.
Ino H, Hayashi K, Yanai H, Teramoto N, Koirala TR, Chen HL, Oka T, Yoshino T, Takahashi K, Akagi T:
Analysis of the genome of an Epstein-Barr-virus (EBV)-related herpesvirus in a cynomolgus monkey cell line (Si-IIA).
Acta Med Okayama
51:207, 1997
26.
Li SL, Biberfeld P, Ernberg I:
DNA of lymphoma-associated herpesvirus (HVMF1) in SIV-infected monkeys (Macaca fascicularis) shows homologies to EBNA-1, -2 and -5 genes.
Int J Cancer
59:287, 1994[Medline]
[Order article via Infotrieve]
27.
Moghaddam A, Koch J, Annis B, Wang F:
Infection of human B lymphocytes with lymphocryptoviruses related to Epstein-Barr virus.
J Virol
72:3205, 1998[Abstract/Free Full Text]
28.
Rangan SR, Martin LN, Bozelka BE, Wang N, Gormus BJ:
Epstein-Barr virus-related herpesvirus from a rhesus monkey (Macaca mulatta) with malignant lymphoma.
Int J Cancer
38:425, 1986[Medline]
[Order article via Infotrieve]
29.
Wutzler P, Meerbach A, Farber I, Wolf H, Scheibner K:
Malignant lymphomas induced by an Epstein-Barr virus-related herpesvirus from Macaca arctoides: A rabbit model.
Arch Virol
140:1979, 1995[Medline]
[Order article via Infotrieve]
30.
Deinhardt F, Deinhardt J:
Comparative aspects: Oncogenic animal herpesvirus, in
Epstein MA,
Achong BG
(eds):
The Epstein-Barr Virus. Berlin, Germany, Springer-Verlag, 1979, p 373.
31.
Shope TC, Dechairo D, Miller G:
Malignant lymphoma in cotton-top marmosets after inoculation with Epstein-Barr virus.
Proc Natl Acad Sci USA
70:2487, 1973[Abstract/Free Full Text]
32.
Epstein MA, Hunt RD, Rabin H:
Pilot experiments with EB virus in owl monkey (Aotus trivirgatus) I. Reticuloproliferative disease in an inoculated animal.
Int J Cancer
12:309, 1973[Medline]
[Order article via Infotrieve]
33.
Rose TM, Strand KB, Schultz ER, Schaefer G, Rankin GW Jr, Thouless ME, Tsai CC, Bosch ML:
Identification of two homologs of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in retroperitoneal fibromatosis of different macaque species.
J Virol
71:4138, 1997[Abstract]
34.
Lapin BA, Timanovskaya VV, Yakovleva LA:
Herpesvirus HVMA: A new representative in the group of the EBV-like B-lymphotropic herpesviruses of primates, in
Neth R,
Gallo RC,
Greves MF,
Janka G
(eds):
Modern Trends in Human Leukemia VI. Berlin, Germany, Springer, 1985, p 312.
35.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG:
The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res
25:4876, 1997[Abstract/Free Full Text]
36.
Tajima F, Nei M:
Estimation of evolutionary distance between nucleotide sequences.
Mol Biol Evol
1:269, 1984[Abstract]
37.
Kumar S, Tamura K, Nei M:
MEGA (Molecular Evolutionary Genetics Analysis), version 1.01. University Park, PA, Pennsylvania State University, 1993.
38.
Hillis D, Bull J:
An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis.
Syst Biol
42:182, 1993
39.
Desrosiers RC, Sasseville VG, Czajak SC, Zhang X, Mansfield KG, Kaur A, Johnson RP, Lackner AA, Jung JU:
A herpesvirus of rhesus monkeys related to the human Kaposi's sarcoma-associated herpesvirus.
J Virol
71:9764, 1997[Abstract]
40.
Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, Moore PS:
Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.
Science
266:1865, 1994[Abstract/Free Full Text]
41.
Koirala TR, Hayashi K, Chen HL, Ino H, Kariya N, Yanai H, Choudhury CR, Akagi T:
Malignant lymphoma induction of rabbits with oral spray of Epstein-Barr virus-related herpesvirus from Si-IIA cells (HTLV-II-transformed Cynomolgus cell line): A possible animal model for Epstein-Barr virus infection and subsequent virus-related tumors in humans.
Pathol Int
47:442, 1997[Medline]
[Order article via Infotrieve]
42.
Danial NN, Pernis A, Rothman PB:
Jak-STAT signaling induced by the v-abl oncogene.
Science
269:1875, 1995[Abstract/Free Full Text]
43.
Migone TS, Lin JX, Cereseto A, Mulloy JC, O'Shea JJ, Franchini G, Leonard WJ:
Constitutively activated JAK-STAT pathway in T-cells transformed with HTLV-I.
Science
269:79, 1995[Abstract/Free Full Text]
44.
Ohashi T, Masuda M, Ruscetti SK:
Induction of sequence-specific DNA-binding factors by erythropoietin and the spleen focus-forming virus.
Blood
85:1454, 1995[Abstract/Free Full Text]
45.
Xu X, Kang SH, Heidenrich O, Okerholm M, O'Shea JJ, Nerenberg MI:
Constitutive activation of different Jak tyrosine kinases in human T-cell leukemia virus type 1 (HTLV-1) tax protein or virus-transformed cells.
J Clin Invest
96:1548, 1995
46.
Weber-Nordt RM, Egen C, Wehinger J, Ludwig W, Gouilleux-Gruart V, Mertelsmann R, Finke J:
Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines.
Blood
88:809, 1996[Abstract/Free Full Text]
47.
Gouilleux-Gruart V, Gouilleux F, Desaint C, Claisse JF, Capiod JC, Delobel J, Weber-Nordt R, Dusanter-Fourt I, Dreyfus F, Groner B, Prin L:
STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients.
Blood
87:1692, 1996[Abstract/Free Full Text]
48.
Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, Roifman CM:
Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor.
Nature
379:645, 1996[Medline]
[Order article via Infotrieve]
49.
Takemoto S, Mulloy JC, Cereseto A, Migone TS, Patel BKR, Matsuoka M, Yamaguchi K, Takatsuki K, Kamihira S, White JD, Leonard WJ, Waldmann T, Franchini G:
Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins.
Proc Natl Acad Sci USA
94:13897, 1997[Abstract/Free Full Text]
50.
Zhang Q, Nowak I, Vonderheid EC, Rook AH, Kadin ME, Nowell PC, Shaw LM, Wasik MA:
Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.
Proc Natl Acad Sci USA
93:9148, 1996[Abstract/Free Full Text]
51.
Murphy FA:
Virus taxonomy, in
Fields BN,
Knipe DM,
Howley PM
(eds):
Virology. Philadelphia, PA, Lippincott-Raven, 1996, p 15.
52.
Cesarman E, Chang Y, Moore PS, Said JW, Knowles DM:
Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.
N Engl J Med
332:1186, 1995[Abstract/Free Full Text]
53.
Gaidano G, Cechova K, Chang Y, Moore PS, Knowles DM, Dalla-Favera R:
Establishment of AIDS-related lymphoma cell lines from lymphomatous effusions.
Leukemia
10:1237, 1996[Medline]
[Order article via Infotrieve]
54.
Gessain A, Briere J, Angelin-Duclos C, Valensi F, Beral HM, Davi F, Nicola MA, Sudaka A, Fouchard N, Gabarre J, Troussard X, Dulmet E, Audouin J, Diebold J, de The G:
Human herpes virus 8 (Kaposi's sarcoma herpes virus) and malignant lymphoproliferations in France: A molecular study of 250 cases including two AIDS-associated body cavity based lymphomas.
Leukemia
11:266, 1997[Medline]
[Order article via Infotrieve]
55.
Luppi M, Torelli G:
The new lymphotropic herpesviruses (HHV-6, HHV-7, HHV-8) and hepatitis C virus (HCV) in human lymphoproliferative diseases: An overview.
Haematologica
81:265, 1996[Abstract/Free Full Text]
56.
Eberle R, Hilliard J:
The simian herpesviruses.
Infect Agents Dis
4:55, 1995[Medline]
[Order article via Infotrieve]
57.
Feichtinger H, Li SL, Kaaya E, Putkonen P, Grunewald K, Weyrer K, Bottiger D, Ernberg I, Linde A, Biberfeld G, Biberfeld P:
A monkey model for Epstein Barr virus-associated lymphomagenesis in human acquired immunodeficiency syndrome.
J Exp Med
176:281, 1992[Abstract/Free Full Text]
58.
Medveczky MM, Szomolanyi E, Hesselton R, DeGrand D, Geck P, Medveczky PG:
Herpesvirus Saimiri strains from three DNA subgroups have different oncogenic potentials in New Zealand white rabbits.
J Virol
63:3601, 1989[Abstract/Free Full Text]
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INTRODUCTION
