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Journal of Virology, December 1999, p. 10320-10328, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Experimental Infection of Rhesus and Pig-Tailed
Macaques with Macaque Rhadinoviruses
Keith G.
Mansfield,
Susan V.
Westmoreland,
Colin D.
DeBakker,
Susan
Czajak,
Andrew A.
Lackner, and
Ronald C.
Desrosiers*
New England Regional Primate Research Center,
Harvard Medical School, Southborough, Massachusetts 01772-9102
Received 28 May 1999/Accepted 10 August 1999
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ABSTRACT |
The recognition of naturally occurring rhadinoviruses in macaque
monkeys has spurred interest in their use as models for human infection
with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8).
Rhesus macaques (Macaca mulatta) and pig-tailed macaques
(Macaca nemestrina) were inoculated intravenously with
rhadinovirus isolates derived from these species (rhesus rhadinovirus
[RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus
antibody-negative and two rhadinovirus antibody-positive monkeys were
used for these experimental inoculations. Antibody-negative animals
clearly became infected following virus inoculation since they
developed persisting antibody responses to virus and virus was isolated
from peripheral blood on repeated occasions following inoculation.
Viral sequences were also detected by PCR in lymph node, oral mucosa,
skin, and peripheral blood mononuclear cells following inoculation.
Experimentally infected animals developed peripheral lymphadenopathy
which resolved by 12 weeks following inoculation, and these animals
have subsequently remained free of disease. No increased pathogenicity
was apparent from cross-species infection, i.e., inoculation of rhesus
macaques with PRV or of pig-tailed macaques with RRV, whether the
animals were antibody positive or negative at the time of virus
inoculation. Coinoculation of additional rhesus monkeys with simian
immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived
rhadinovirus resulted in an attenuated antibody response to both agents
and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days; P < 0.019). Coinfected
and immunodeficient macaques died of a variety of opportunistic
infections characteristic of simian AIDS. PCR analysis of sorted
peripheral blood mononuclear cells indicated a preferential tropism of
RRV for CD20+ B lymphocytes. Our results demonstrate
persistent infection of macaque monkeys with RRV and PRV following
experimental inoculation, but no specific disease was readily apparent
from these infections even in the context of concurrent SIV infection.
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INTRODUCTION |
The identification of Kaposi's
sarcoma (KS) and Pneumocystis carinii pneumonia in
previously healthy homosexual men led to the first case definitions of
AIDS (7, 8). In the years that followed, KS was recognized
as the most common malignancy in human AIDS patients. Epidemiologic
evidence suggested the presence of a second agent or additional
cofactors which acted in conjunction with the human immunodeficiency
virus (HIV) to produce the neoplasm (2, 33). In 1994, gammaherpesvirus DNA sequences were detected in KS tissues obtained
from human AIDS patients by representational difference analysis
(13). The virus that was subsequently identified has been
named KS-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8).
Viral sequences have been detected in KS patients whether positive or
negative for HIV (5, 14, 30), in body-cavity-based lymphomas
(a rare form of non-Hodgkin's lymphoma) (9, 10), and in
multicentric Castleman's disease (MCD) (22, 35).
We recently recognized a distinct new gammaherpesvirus in rhesus
macaques (21). The presence of the virus was first suspected on the basis of immunoreactivity to herpesvirus saimiri antigens detected in serologic surveys of colony monkeys. The virus was isolated
from peripheral blood mononuclear cells (PBMC) and could be grown
lytically and to high titer in primary rhesus monkey fibroblast cells.
Sequencing of the complete DNA polymerase and glycoprotein B genes
revealed a closer relatedness to KSHV than to herpesvirus saimiri,
Epstein-Barr virus, or any other herpesvirus (21).
Furthermore, an interleukin-6 (IL-6) gene homolog, analogous to a
virus-encoded IL-6 homolog found in KSHV but not in any other viruses,
was identified following open reading frame 11. The recent completion
of the DNA sequence of an independent isolate in Oregon revealed
similarity in organization and sequence with KSHV across the full
length of the genome (34). Collectively these findings demonstrated the presence of a distinct gammaherpesvirus, rhesus rhadinovirus (RRV), that is closely related to KSHV. Short stretches of
KSHV-like sequences have also been amplified from tissues of macaque
monkeys with retroperitoneal fibromatosis (3, 4, 32).
Serologic surveys of normal rhesus macaques housed at the New England
Regional Primate Research Center (NERPRC) and elsewhere showed that
greater than 90% of adult macaques are immunoreactive to RRV
(21). Rhesus macaques have not previously been screened for
the presence of this virus, and the vast majority of animals that have
been used in biomedical research have been potentially infected with
this agent. The effects of the virus on the pathogenesis of spontaneous
disease and on previous experimental findings need to be critically
evaluated. Attempts to correlate infection by RRV or closely related
viruses with lymphoma and retroperitoneal fibromatosis (3, 32,
34) are complicated by the high rates of natural infection among
normal animals. Controlled studies utilizing RRV-free animals and
well-defined inocula should facilitate the investigation of disease
causation. Furthermore, it is important to document the characteristics
of primary infection and subsequent persistence as a potential model
for KSHV infection of humans. Reported here are the results of
experimental inoculation of normal and immunodeficient macaques with
two distinct rhadinovirus isolates from nonhuman primates.
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MATERIALS AND METHODS |
Animals and housing.
All macaques (Macaca
mulatta and M. nemestrina) were housed at the NERPRC in
accordance with standards of the American Association for Accreditation
of Laboratory Animal Care and Harvard Medical School's Internal Animal
Care and Use Committee. Normal macaques were serologically negative for
simian T-lymphotropic virus, type D retrovirus, herpesvirus type B, and
simian immunodeficiency virus (SIV) (16, 19). Macaques
experimentally inoculated with SIV and/or nonhuman primate-derived
rhadinoviruses were individually housed in biolevel 2 and 3 containment
facilities as previously described (28, 36a).
Immunodeficient animals had previously been inoculated intravenously
with one of two strains of SIV (SIVmac251 or SIVmac239), the history,
preparation and in vivo and in vitro properties of which have been
described extensively (17, 18, 20, 23, 26). These animals
were included in a variety of infectivity and pathogenesis studies and
received neither antiretroviral agents nor antimicrobial prophylaxis.
Viral inoculations.
Five pig-tailed macaques (M. nemestrina) and 16 rhesus macaques (M. mulatta) were
inoculated intravenously with 5 × 105 50% tissue
culture infective doses of the H26-95 isolate of RRV and H19545 isolate
of pig-tailed rhadinovirus (PRV) prepared as previously described
(21). The serologic status to previous nonhuman primate
rhadinovirus infection was established prior to inoculation.
Same-species and cross-species transmissions of the respective
rhadinoviruses were carried out in seronegative and seropositive
animals. Species, animal number, and rhadinovirus and SIV serology
status prior to inoculation for 21 animals are listed in Table
1.
Antibody responses.
Macaque rhadinovirus was purified from
productively infected rhesus monkey fibroblast cell cultures, lysed,
coated onto plates, and used for the detection of reactive antibodies
by enzyme-linked immunosorbent assay (ELISA) as previously described
(21).
Clinical evaluation, biopsies, and blood samples.
Following
experimental rhadinovirus inoculation, all animals were examined daily
and body temperature and clinical data were recorded with an implanted
microchip and transponder (Bio Medic Data Systems, Maywood, N.J.). Oral
mucosal, skin, and lymph node biopsies and PBMC were obtained prior to
inoculation and at 2, 4, and 12 weeks postinoculation. Tissues were
fixed in 10% neutral buffered formalin and snap frozen at 
70°C.
Blood for viral antibody response, viral load, and complete blood count
were obtained at 0, 1, 2, 4, 8, and 12 weeks and monthly thereafter.
Viral isolation and viral load.
Viral isolation was
performed as previously described (21). Viral load in
infected macaques was estimated by quantitative cocultivation of PBMC
with primary rhesus fibroblasts. This technique was adapted from that
previously described to quantitate SIVmac viral load (20,
24). Briefly, PBMC were purified, counted in a hemocytometer, and
subsequently cocultured in various numbers from 106 to 152 PBMC via serial dilutions with 105 rhesus fibroblasts. The
presence of cytopathic effect was evaluated at 14 to 21 days, and the
number of PBMC required to recover RRV was calculated. Evaluation of
the levels of SIV RNA in plasma were also performed as previously
described (20, 36).
Histopathology and immunohistochemistry.
Formalin-fixed
paraffin-embedded and snap-frozen tissues were used in
immunohistochemical procedures to define the immunophenotype of cells
within lymphoid tissue as previously described (38). Briefly, tissue sections were fixed in 2% paraformaldehyde and immunostained with an avidin-biotin-horseradish peroxidase complex technique with diaminobenzidine chromogen. The primary antibodies used
were Snv71.1 (from C. Collignon and C. Thiriart, SmithKline Beecham
Biologicals, Rixensart, Belgium) for SIV gp120, B1 for CD20-positive B
lymphocytes, EBM11 (Dako Corp., Carpinteria, Calif.) for CD68-positive
macrophages, DK25 for CD8-positive T lymphocytes, Nu-Th/1 (from M. Yokoyama and Y. Matsuo, Nicheri Research Institute, Fukuoka, Japan) for
CD4-positive T lymphocytes, CR3/43 for HLA-DR, and MIB-1 (Immunotech,
Westbrook, Maine) for Ki67.
Detection of rhadinovirus sequences.
To examine biopsy and
necropsy tissues for the presence of RRV, DNA was extracted from fresh
frozen tissues, using a QIAmp tissue kit (Qiagen, Valencia, Calif.)
according to the manufacturer's instructions. DNA was eluted in 30 to
50 µl of sterile water treated with diethylpyrocarbonate, and PCR was
performed as described below. To analyze cell types harboring RRV, PBMC
were separated from whole blood from a rhesus macaque (270-97) 13 days
after inoculation with RRV, using standard Ficoll isolation techniques as instructed by the manufacturer (Organon Teknika, Malvern, Pa.). Isolated PBMC were washed in 1× phosphate-buffered saline with 1%
heat-inactivated fetal calf serum, resuspended in 2 ml of RPMI 1640 medium containing 5% fetal calf serum, and counted with a hemacytometer. Cells were pelleted at 500 × g,
resuspended at 10 × 106 cells/ml, aliquoted into
Falcon tubes with 5 × 106 to 10 × 106 cells/tube, and repelleted. Supernatant was decanted,
and pelleted cells were resuspended and labeled with 100 µl of
CD20-fluorescein isothiocyanate (Becton Dickinson [BD])
CD4-phycoerythrin (PE) (Ortho), CD8 peridinin chlorophyll protein (BD),
CD14-PE (BD), or CD3-PE (PharMingen). Cells were incubated on ice in
the dark for 30 min, then washed in 1× phosphate-buffered saline
pelleted, resuspended in 200 µl 2% paraformaldehyde, and stored at
4°C until analyzed. Cells were sorted in a Vantage flow cytometer
(BD). Sorted cells were then pelleted, decanted, and stored frozen at 80°C.
Genomic DNA from cells was isolated by using a Promega Wizard kit with
a modified protocol. Briefly, cells were lysed in 300
µl nuclei lysis
solution containing proteinase K and incubated
overnight at 37°C.
Protein precipitation solution (100 µl) was
added to the nuclear
lysate, which was then vortexed and centrifuged
at 14,000 ×
g. The supernatant was transferred to a new tube containing
300 µl of isopropanol to precipitate DNA. DNA was resuspended
in sterile
diethylcarbonate-treated H
2O at a concentration of
500, 1,000, or 2,000 cells/µl.
Nested PCR was performed on DNA isolated from PBMC and tissue as
previously described. Outer primers (RRV8758 [5'-GCC AAA
CCG TCT CTC
ATT CT] and RRV9767 [5'-CGA CCC CCA TCC CCA CAT AG])
were used to
produce a 1,029-bp product. Briefly, PCR was performed
with 0.2 µM
each primer, 100 µM deoxynucleoside triphosphates,
1.5 mM
MgCl
2, 2.5 IU of
taq DNA polymerase, and buffer
(50 mM
KCl, 10 mM Tris [pH 9.0]) in a 50-µl reaction volume.
Amplification
was performed at 94°C for 30 s, 56°C for 60 s, and 72°C for 45
s for a total of 30 cycles in a Perkin-Elmer
9600 thermocycler;
1 µl of this reaction was used as template for a
second reaction
utilizing internal primers (RRV8832 [5' CCC TCG CCA
CAC AAA ACC
AG] and RRV9009 [5' GGC GCG GAG TCT AAT GAA AA]).
Precautions
against PCR contamination including physical separation of
areas
used for DNA isolation, PCR, and post-PCR manipulations and use
of appropriate negative controls were utilized as previously described
(
25). PCR products were resolved in a 2% ethidium
bromide-stained
agarose
gel.
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RESULTS |
Experimental infection of immunocompetent macaques.
A
rhadinovirus was isolated from PBMC of a pig-tailed macaque (animal
19545). This virus was called PRV19545 for PRV isolate 19545, to
distinguish it from RRV isolates. Virus stocks of PRV19545 were
prepared as described previously for RRV (21). Sequencing of
the R1 gene (15) and gB gene (1) revealed
PRV19545 to be closely related to but distinct from RRV isolate 26-95 (21). Sequencing of full-length gB genes from nine
rhadinovirus isolates from three species of macaques has recently
revealed all to be rather closely related, with up to 7.2% divergence
at the amino acid level (1). However, phylogenetic analysis
demonstrated that the rhadinovirus isolates grouped according to
species of origin, not primate facility of origin (1). These
analyses suggested that RRV and PRV are closely related but distinct
viruses. RRV26-95 and PRV19545 were inoculated intravenously into a
total of 11 macaque monkeys in the absence of concurrent SIV infection (Table 1). Both rhesus and pig-tailed macaques were used for these
experimental inoculations in order to examine the effects of same-
versus cross-species infection on pathogenic potential (Table 1). Two
of the rhesus monkeys that were inoculated with PRV were antibody
positive to RRV at the time of inoculation. The other nine recipient
monkeys were antibody negative. Blood samples were obtained at periodic
intervals after inoculation and used for measurement of antibody
responses and virus recovery.
All monkeys inoculated with RRV seroconverted to positive anti-RRV
antibody status within the early weeks after inoculation
(Fig.
1A). The anti-RRV antibodies have
persisted at high levels
for as long as we have followed the animals.
The three antibody-negative
macaques that were inoculated with PRV
seroconverted to positive
anti-PRV antibody status within the early
weeks after inoculation
(Fig.
1B). The anti-PRV antibodies have also
persisted at high
levels for as long as we have followed the animals.
Animals infected
with RRV or PRV made antibodies that reacted strongly
with both
RRV and PRV antigens. Serologic cross-reactivity between RRV
and
PRV was extensive, with a tendency toward slightly increased
reactivity
to the homologous virus (data not shown). These results
demonstrate
consistent, persistent infection of naive macaques by RRV
and
PRV. The two RRV-antibody-positive rhesus monkeys (190-96 and
195-96) that were inoculated with PRV exhibited an increase in
antibody
reactivity to PRV following inoculation (Fig.
1C), suggesting
a
possible take of PRV in rhesus monkeys already infected with
RRV.
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FIG. 1.
Antibody responses following experimental inoculation
with RRV or PRV. (A) Antibody response to RRV by ELISA in macaque
monkeys inoculated with RRV. Mm, M. mulatta; Mn, M. nemestrina. (B) Antibody response to PRV by ELISA in macaque
monkeys inoculated with PRV. (C) Antibody response to PRV by ELISA in
rhesus monkeys 190-96 and 195-96 inoculated with PRV. Tests in panels A
and B were performed with a 1:20 dilution of plasma and 1:100
conjugate, and tests in panel C were performed with a 1:200 dilution of
plasma and 1:100 conjugates.
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Experimental infection of macaque monkeys with PRV and RRV was also
demonstrated by virus recovery from PBMC using rhesus
monkey fibroblast
cultures. RRV or PRV was recovered from the
majority of inoculated
animals at two or more time points. Attempts
to recover rhadinovirus
from RRV-seronegative animals in control
experiments have repeatedly
failed. RRV or PRV was recovered at
one or more time points from 11 of
the 11 monkeys used in these
studies. We also attempted to roughly
quantitate the numbers of
infectious cells in PBMC by performing RRV
and PRV recoveries
with serial threefold dilutions of cells starting at
10
6 PBMC in duplicate. Representative results from one set
of monkeys
inoculated at the same time with RRV are shown in Fig.
2. RRV
and PRV loads in this assay
appeared to peak 1 to 4 weeks following
the inoculation. The highest
loads reached a numerical score of
5, which corresponds to virus
recovery with 12,345 PBMC (Fig.
2). gB sequences from virus recovered
from 190-96 and 195-96 that
were already RRV positive when PRV19545 was
inoculated did not
correspond to the PRV19545 gB sequence (data not
shown).
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FIG. 2.
Semiquantitative recovery of RRV from PBMC of inoculated
monkeys. The numbers of infectious cells in PBMC were quantitated as
described in Materials and Methods. Code for PBMC load: 0, virus was
not recovered even when 106 PBMC were used; 1, virus was
recovered with an average of 106 but not fewer PBMC; 2, 333,333 PBMC; 3, 111,111 PBMC; 4, 37,037 PBMC; 5, 12,345 PBMC. Mm,
M. mulatta; Mn, M. nemestrina.
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Five of 11 animals had a febrile reaction, as defined by an increase
from baseline measurements of >2.0°C or absolute temperature
>104.0°C at any time point. Fever was recognized as early as 48
h following inoculation and persisted for up to 14 days. Of the
two
rhesus macaques seropositive to RRV prior to inoculation (Mm
190-96 and
195-96), neither became febrile. Of the animals that
became febrile,
two were rhesus macaques inoculated with RRV (same
species), two were
rhesus macaques inoculated with PRV (cross
species), and one was a
pig-tailed macaque inoculated with RRV
(cross
species).
The complete blood counts for each animal included leukocyte count,
absolute and relative counts of lymphocytes, neutrophils,
eosinophils,
basophils, and monocytes, hematocrit, platelet count,
erythrocyte
count, hemoglobin, mean corpuscular hemoglobin, mean
corpuscular
volume, and mean corpuscular hemoglobin concentration.
Values remained
within normal limits in all animals postinoculation.
While there were
minor fluctuations, there were no consistent
changes in any of these
values postinoculation. This was also
true when the group was examined
as a whole and when subgroups
(RRV versus PRV, seropositive versus
seronegative or
M. mulatta M. nemestrina) were
examined
individually.
Skin and oral biopsies showed no unusual features. There were no
clinically apparent cutaneous manifestations, and these animals
remained healthy and free of disease throughout the 437 days of
follow-up.
Rhadinovirus-associated lymphadenopathy in immunocompetent
SIV-negative macaques.
Clinically evident lymphadenopathy was
detected in 8 of the 11 animals as soon as 2 weeks after rhadinovirus
inoculation. Microscopic morphologic features were similar regardless
of the rhadinovirus inoculum. Histologically, the lymphadenopathy was characterized at 2 weeks by marked paracortical lymphocytic hyperplasia which effaced normal architecture (Fig.
3A). This paracortical expansion was
accompanied by an abundance of small arborizing vessels lined by
hypertrophied and hyperplastic endothelium (Fig. 3B). The expanded
paracortex contained increased numbers of immunoblasts, mitotic
figures, and histiocytes and moderate numbers of small lymphocytes.
Immunostaining revealed an increase in both CD20-positive B lymphocytes
and CD3-positive T lymphocytes within the expanded paracortical zone.
Dispersed within this diffuse paracortical expansion were occasional
regions of follicular hyperplasia. Mantle zones were variably
developed, and occasional follicles were confluent. An increased number
of small blood vessels were found between the developing follicles.
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FIG. 3.
Morphologic features associated with experimental
inoculation with RRV. (A) Marked paracortical lymphocytic hyperplasia 2 weeks following RRV inoculation in an immunocompetent rhesus macaque.
Hematoxylin-and-eosin (H&E) stain; magnification, ×53. (B)
Paracortical expansion accompanied by an abundance of small arborizing
vessels lined by hypertrophied and hyperplastic endothelium in an
immunocompetent rhesus macaque. H&E stain; magnification, ×106. (C)
Increased numbers of immunoblasts and mitotic figures 2 weeks after RRV
inoculation in an immunocompetent rhesus macaque. H&E stain;
magnification, ×315. (D) Marked follicular hyperplasia effacing normal
lymph node architecture 4 weeks after RRV inoculation in an
immunocompetent rhesus macaque. H&E stain; magnification, ×53. (E)
Germinal center surrounded by loosely concentric layers of lymphocytes
12 weeks after RRV inoculation in an immunocompetent rhesus macaque.
H&E stain; magnification, ×106. (F) Hyalinized germinal center 12 weeks after RRV inoculation in an immunocompetent rhesus macaque. (G)
Proliferative vasculitis 4 weeks after RRV inoculation of an
SIV-infected immunodeficient rhesus macaque. H&E stain; magnification,
×315.
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Lymphadenopathy was absent in the two rhesus macaques that were
seropositive at the time of PRV inoculation, 190-96 and 195-96.
Histologically these animals lacked the marked vascular changes
present
in most of the other animals. Mild to moderate paracortical
expansion
was present in animal 195-96.
The paracortical expansion in animals with lymphadenopathy was less
pronounced by 4 weeks postinoculation and had been replaced
by
extensive follicular hyperplasia. The most severe follicular
hyperplasia was seen in rhesus macaques 380-96, 282-96, and 266-97,
all
of which received PRV. In two animals (380-96 and 266-97),
follicular
hyperplasia had completely effaced the normal architecture
of the
medulla and cortex at 4 weeks after infection (Fig.
3D).
Regions of
vascular hyperplasia were still evident in the paracortex
and
surrounding follicles. Rhesus macaques 190-96 and 195-96 lacked
any
follicular
hyperplasia.
By 12 weeks postinoculation, clinically recognized lymphadenopathy had
resolved in all of the eight monkeys in which lymphadenopathy
was seen.
In all animals, there were increased numbers of involuted
follicles
characterized by small germinal centers and the presence
of periodic
acid-Schiff stain-positive material. These hyalinized
follicles were
occasionally penetrated by a single small blood
vessel and less
commonly surrounded by layers of loosely concentric
lymphocytes (Fig.
3E and F). These follicular changes, although
nonspecific, are unusual
in normal animals and share features
with the hyaline-vascular variant
of Castleman's disease in humans.
In animals in which enlargement of
the peripheral lymph nodes
was still present, there was a combination
of follicular hyperplasia
and continued paracortical expansion with
vascular
hyperplasia.
Rhadinovirus infection of immunodeficient macaques.
Six rhesus
monkeys that had been previously infected with SIV were also inoculated
with PRV or RRV (Table 1). Three of these six were already antibody
positive to RRV at the time of PRV inoculation (Table 1). Four
additional rhesus monkeys that were RRV negative and SIV negative were
coinoculated with RRV plus SIV or PRV plus SIV (Table 1).
Prior infection with SIV appeared to result in weaker and/or delayed
antibody responses to RRV or PRV (Fig.
1 and
4). Rhesus
monkeys 196-94, 181-90, and
229-91 were all SIV infected and RRV
negative at the time of RRV or PRV
inoculation, and all had weaker
or delayed antibody responses to RRV or
PRV (Table
1, Fig.
1,
and Fig.
4). Coinoculation with RRV and SIV or
PRV and SIV also
appeared to weaken or delay the antibody response to
PRV or RRV
in most animals (Fig.
5).
Despite the marginal or absent anti-PRV
antibody response in
coinoculated monkeys 84-96 and 91-96, PRV
was recovered from PBMC on
repeated occasions following the PRV-plus-SIV
inoculation, and thus
these animals were clearly infected by the
PRV inoculation.
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FIG. 5.
Antibody responses to RRV or PRV in M. mulatta (Mm) monkeys coinoculated with SIV and RRV or SIV and
PRV.
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Interpretation of the lymph node changes in immunodeficient macaques
was confounded by preexisting pathology in the lymph
nodes, including
follicular hyperplasia and dysplasia, findings
common in SIV-infected
macaques. Clinically there was enlargement
of lymph nodes
(lymphadenopathy) relative to the preinoculation
state in all animals
following rhadinovirus inoculation, and this
enlargement was most
severe in animals rhesus monkeys 229-91 and
168-94. In immunodeficient
RRV-negative rhesus macaques (229-91,
181-90, and 196-94) inoculated
with rhadinovirus, histologic changes
included paracortical expansion
and vascular hyperplasia accompanied
by florid follicular hyperplasia.
In seropositive animals, paracortical
expansion was seen to a lesser
degree and there was exacerbation
of follicular hyperplasia. The
specificity of these changes is
unknown.
Of the six rhesus macaques that were previously infected with SIV and
subsequently inoculated with RRV or PRV, four have died.
Three of these
four PRV/RRV-inoculated rhesus macaques that were
previously infected
with SIV died with thrombosis, vascular hypertrophy,
and nonsuppurative
vasculitis of pulmonary vessels. Aseptic proliferative
endocarditis was
present in two cases (Fig.
3G). These changes
are characteristic of SIV
arteriopathy, a condition of unknown
etiology recognized commonly in
SIV-infected macaques (
12).
Pulmonary lymphocytic
infiltrates were present in the three animals
with arteriopathy and
accompanied by renal lymphoid infiltrates
in two cases. These changes,
indicative of a lymphoproliferative
disorder recognized in SIV-infected
macaques (
11), are of unknown
etiology and a common
morphologic feature found at
necropsy.
Animals coinoculated with SIVmac251 and PRV or RRV developed a clinical
lymphadenopathy by 2 weeks postinoculation. Histologic
features in
these animals were similar to those seen in 8 of the
11 immunologically
normal macaques described above, including
extensive vascular
hyperplasia and paracortical expansion. The
degree of vascular
hyperplasia within lymph nodes was more florid
than that seen in
immunologically normal animals and persisted
in three animals (rhesus
macaques 121-96, 91-96, and 84-96) at
12 weeks
postinoculation.
Monkeys coinoculated with SIV and RRV or SIV and PRV also appeared to
have an attenuated antibody response to SIV (Fig.
6).
Coinfected animals not only had
weaker antibody responses but
also had shorter mean survival times than
40 SIVmac251-infected
controls studied previously (155 days versus 560 days;
P < 0.019).
Coinoculated monkeys developed SIV
RNA loads in plasma that were
about 10-fold higher than those found in
historical controls inoculated
with the same stock of SIVmac251 only
(Fig.
7). Differences at
weeks 1, 2, 4, and 12 were statistically significant by the Mann-Whitney
rank sum test
(
P = 0.020 to 0.048). Animals that were coinfected
with
SIV and RRV or PRV at the same time died with a variety of
opportunistic infections characteristic of simian AIDS, including
those
caused by
Cryptosporidium parvum,
P. carinii, and
enteropathogenic
Escherichia coli (Table
2). Pulmonary lymphocytic infiltrates
were present in two of four animals. In contrast to the animals
previously infected with SIV and then inoculated with rhadinovirus,
pulmonary vascular lesions were not identified in any of these
four
animals (Table
2).
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FIG. 6.
Anti-SIV antibody responses in M. mulatta
(Mm) monkeys inoculated with SIV and RRV, SIV and PRV, or SIV alone.
conj, conjugate.
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FIG. 7.
SIV RNA loads in plasma in monkeys inoculated with SIV
alone, or SIV and RRV, or SIV and PRV. Numbers in parentheses refer to
numbers of animals used for determining the average for each data
point.
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Detection of RRV in tissues and blood.
Lymph node, oral
mucosa, skin, and PBMC were obtained at 2, 4, and 12 weeks
postinoculation and tested for the presence of RRV by PCR (Table
3). Viral DNA was amplified from all
three tissues and from PBMC. Samples from RRV-inoculated, SIV-negative animals were PCR positive for RRV sequences by 2 weeks postinoculation in lymph node (four of four), oral mucosa (two of four), skin (one of
four), and PBMC (one of three). Virus persisted in the tissues to at
least 12 weeks postinoculation, as demonstrated by PCR in lymph node
(three of four), skin (two of four), and PBMC (three of four). Fewer
tissues were positive for RRV by PCR in two animals that were
coinoculated with RRV and SIVmac251.
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[in this window]
[in a new window]
|
TABLE 3.
Detection by PCR of RRV in lymph node, oral mucosa, skin,
and peripheral blood mononuclear cells following
experimental inoculation
|
|
To determine the cell type infected in peripheral blood, we performed
PCR using sorted cells from PBMC. PBMC were harvested
from pig-tailed
macaque 270-97 at 2 weeks after inoculation with
RRV. Cells were
stained for CD20, CD8, CD4, and CD14 and were
sorted by flow cytometry.
DNA was isolated from the sorted, pelleted
cells, and PCR was performed
to determine the primary cell type
infected with RRV. CD20
+
cells were positive at 15,000 cells per reaction and remained
positive
down to a dilution of 3,200 cells per reaction (Fig.
8). CD8
+ cells were positive
at 15,000 cells per reaction and were negative
at dilutions of 7,500 cells or less. CD4
+ and CD14
+ cells were PCR
negative for RRV. This dilution analysis indicated
that the
CD20
+ B lymphocyte is the primary cell type infected with
RRV in
M. nemestrina.
View larger version (38K):
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[in a new window]
|
FIG. 8.
RRV DNA in sorted cells. Products of PCR for RRV DNA in
sorted cells were visualized by ethidium bromide staining following
agarose gel electrophoresis. MW, molecular weight; FACS,
fluorescence-activated cell sorting.
|
|
| |
DISCUSSION |
Experimental inoculation of normal, seronegative, juvenile
macaques with RRV or PRV produced a clinical syndrome characterized by
a mild febrile reaction and moderate lymphadenopathy. The
lymphadenopathy was initially characterized by marked paracortical
expansion accompanied by vascular hypertrophy and hyperplasia which was
replaced by follicular hyperplasia in 4 to 8 weeks and which resolved
by 12 weeks postinoculation in most animals. These histologic features, while nonspecific, have been recognized in colony-housed animals with
clinical lymphadenopathy and spontaneous seroconversion to RRV (data
not shown). While qualitatively similar to changes observed in lymph
nodes following experimental inoculation of macaques with rhesus
lymphocryptovirus (rhesus Epstein-Barr virus) (29), the
rhadinovirus-associated lymphadenopathy was characterized by a greater
degree of vascular hyperplasia and hypertrophy and a lesser degree of
paracortical lymphocytic hyperplasia. Similar changes have been
described during the acute phase of HHV-8 infection of humans
(27).
All rhadinovirus-seronegative animals developed a strong antibody
response that persisted throughout the experimental period. Virus was
recovered from the peripheral blood, and viral DNA could be detected in
lymph node, skin, oral mucosa and PBMC by PCR as early as 2 weeks
postinoculation. The high rate of seropositivity noted in the NERPRC
colony indicates that the virus is readily transmitted among macaques,
and our ability to demonstrate viral sequences by PCR in oral mucosa
for up to 12 weeks postinoculation suggests that oral secretions may
play a role in viral transmission. Cross-species transmission of the
viruses was demonstrated and resulted in no observable difference in
the clinical manifestations of infection. However, the most severe
follicular hyperplasia was observed in monkeys inoculated with PRV.
The absence of a febrile reaction, clinical lymphodenopathy, and
follicular hyperplasia in animals 190-96 and 195-96 suggests that prior
exposure to RRV may have attenuated any infection following PRV
inoculation into these RRV-positive animals. The spike in anti-PRV
antibody levels following inoculation suggests that there may have been
a take of the PRV. The failure to detect PRV 19545 sequences in the
virus recovered during the weeks following inoculation indicates only
that the newly inoculated virus did not overcome the indigenous virus
to become the predominant species; this result is not surprising. More
sensitive tests using tagged viruses will be needed to investigate the
issue of superinfection more rigorously.
All experimentally infected, immunologically normal animals remained
healthy and have survived for greater than 437 days. These findings are
consistent with our observations of NERPRC colony animals that indicate
common spontaneous infection of animals greater than 1 year of age and
absence of obvious disease sequelae. Retroperitoneal fibromatosis, a
neoplastic proliferation of spindle cells resembling KS of humans, has
previously been associated by PCR analysis with a macaque
gammaherpesvirus related to RRV (4, 32). We found no
evidence for the ability of RRV or PRV isolates H26-95 and H19545 to
induce this condition. Whether other closely related viruses or
cofactors may play an etiologic role in the development of this or
other neoplasms remains to be determined.
Coinoculation of animals with SIVmac251 and macaque-derived
rhadinovirus resulted in an attenuated antibody response to both agents
and shorter mean survival compared to SIVmac251-inoculated controls
(155 days versus 560 days; P < 0.019). SIV infection of CD4+ T lymphocytes in conjunction with RRV infection may
thus result in an impaired humoral immune response to both viruses and
adverse clinical outcome. The mechanism responsible for producing this effect is unknown but may relate to direct infection of CD20-positive B
cells by the macaque rhadinovirus or to immune activation resulting from the rhadinovirus infection.
Experimental infection of macaques was associated with a clinical
lymphadenopathy characterized initially by paracortical hyperplasia and
vascular hypertrophy/hyperplasia that subsequently was replaced by
marked follicular hyperplasia. In the most severe cases, this
follicular hyperplasia obliterated medullary sinuses and completely
effaced the normal lymph node architecture. Similar changes have been
found in HIV-negative human patients with histologic features of
angioimmunoblastic lymphadenopathy and reactive lymphadenopathy in
which HHV-8 sequences could be detected by PCR (27). Our findings suggest that following infection these morphologic alterations may represent a continuum that occurs temporally and to differing degrees. B-cell proliferation is a feature common to MCD and
angioimmunoblastic lymphadenopathy and may result from expression
of the virus-encoded cytokine IL-6 (vIL-6). vIL-6 has been shown to
have proliferative activity on myeloma cells, and its expression has
been demonstrated in HIV-negative MCD (6, 31). The presence
of a vIL-6 homolog in RRV suggests a similar mechanism may operate
during histogenesis of the rhesus rhadinovirus-associated lymphadenopathy.
An arteriopathy has been found in 19 of 85 monkeys examined
retrospectively following death from SIV-induced immunodeficiency (12). This arteriopathy has never been seen outside the
setting of SIV-induced immunodeficiency in our macaque monkey colony, and a viral etiology has been suspected. A possible etiological role
for RRV in this lesion is intriguing because of fundamental similarity
of the vascular endothelial proliferation in this lesion and in
HHV-8-associated KS. The lesion is also similar to a large vessel
arteritis induced in mice by the murine gammaherpesvirus 68 (37). The occurrence of this arteriopathy in three of four monkeys sequentially infected with SIV and RRV is consistent with a
possible etiologic role. More detailed studies, including comparison of
the occurrence of lesions in RRV-positive versus RRV-negative animals
that die with SIV and the demonstration of RRV sequences in the lesions
by in situ hybridization, will be needed to draw meaningful
associations of RRV with this vascular proliferative syndrome.
Our results have not demonstrated any clear evidence for prolonged or
terminal disease induced by experimental infection with RRV or PRV
despite variation of a number of investigational parameters. These
include same- versus cross-species infection, prior antibody status,
and SIV coinfection. While pathologic conditions such as arteriopathy
and lymphoproliferative disease could still be caused, at least in
part, by RRV, they do not appear to be consistently present even in
SIV-plus-RRV/PRV coinfected animals. Nonetheless, the experimental
system described here still should prove valuable for modeling KSHV
infection. Since RRV and PRV can be grown lytically in cell culture, it
should be possible to construct a variety of gene knockouts, gene
substitutions, and point mutations within the viral genome. These can
be studied not only for their effects on viral replication and B-cell
persistence in cell culture but also for their effects on the acute
replication phase in monkeys, ability to persist, levels of
persistence, and sites of localization.
| |
ACKNOWLEDGMENTS |
We thank Jeff Lifson for the SIV plasma RNA analysis,
Daniel Silva, Allan McPhee, and Dong-Ling Xia for technical support, and Joanne Newton for manuscript preparation.
This work was supported by PHS grants AI 38131, RR07000, AI 42845, and RR00168.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: New England
Regional Primate Research Center, Harvard Medical School, One Pine Hill Dr., Box 9102, Southborough, MA 01772-9102. Phone: (508) 624-8042. Fax:
(508) 624-8190. E-mail:
ronald_desrosiers{at}hms.harvard.edu.
| |
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Abstract
Introduction
