Experimental infection of rhesus monkeys with type D retrovirus.
ABSTRACT The naturally occurring immunodeficiency syndrome of macaque monkeys is an important animal model for the acquired immunodeficiency syndrome in humans. A new type D retrovirus, distinct from Mason-Pfizer monkey virus, has been isolated from affected animals at the New England Regional Primate Research Center. We now report the results of experimental infection of macaques with retrovirus D/New England after 13 months of study. Inoculated macaques developed lymphadenopathy without follicular hyperplasia, profound neutropenia, and a transient decrease in peripheral blood lymphocyte blastogenic responsiveness. Despite our varying the strain of virus, the manner in which the virus was grown, the size of the inoculum, and the age of the inoculated animals, infected macaques have not developed opportunistic infections or profound, prolonged loss of T cell function, key features of the macaque immunodeficiency syndrome. Therefore, experimental infection of naive macaques with D/New England has not reproduced the naturally occurring macaque immunodeficiency syndrome.
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ABSTRACT: We haveisolated a molecular cloneofthefull-length integrated provirus ofsimianacquired immune deficiency syndrome retrovirus serotype 1(SRV-1) froma fatal caseofsimianacquired immunedeficiency syndrome inajuvenile rhesus macaque.An integrated SRV-1provirus was cloned, sequenced, andfoundto contain fourlarge openreading framesencoding gag-precursorprotein, protease, polymerase, andenvelope. Theproviral clone was transfected intoD17canine osteosarcoma cells andfoundtoproduce infectious virus. A comparison ofthesequencesofthisclone witha noninfectious cloneshowed20differences, resulting in10 aminoacidchanges. Also, a cluster ofexchanges, shortinsertions, anddeletions inthe5'leader sequences resulted inextension ofthetRNALYSprimer-binding sitefrom14to19nucleotides. Virusisolated from transfected cells was showntobeinfectious andpathogenic, resulting indisease thatfollowed thesame time courseandmortality asdisease induced byuncloned, invitro cultivated virus isolated fromdiseased animals. Theseresults unequivocally showthat atypeD retrovirus (SRV-1) causesafatal immunosuppressive syndrome inrhesus monkeys. A typeD retrovirus, simian acquired immunedeficiency syndrome (SAIDS) retrovirus serotype 1(SRV-1), hascon- sistently beenassociated withSAIDSoccurring spontane- ously inrhesus monkeys (Macacca mulatta) attheCalifornia Primate Research Center (19, 20,21). Thisfatal immunosup- pressive disease wasexperimentally transmitted torhesus monkeyswithtissue culture-derived virus. Animals with terminal disease hadavariety ofclinical signs ofSAIDS,
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ABSTRACT: Simian retrovirus type D (SRVD) is a naturally occurring betaretrovirus in nonhuman primates of the genus Macaca. Infection can lead to a variety of clinical, hematologic, and histopathologic abnormalities. We report an unusual clinical presentation of facial paralysis and histologic lymphocytic neuritis in an SRVD type 2 (SRVD2)-infected rhesus macaque (Macaca mulatta) with a catheter-associated vena caval thrombus, anemia, thrombocytopenia, and multisystemic lymphoid hyperplasia. At initial presentation, a right atrial mass was detected by echocardiography. The macaque was clinically asymptomatic but had persistent anemia, thrombocytopenia, hyperglobulinemia, and later neutropenia. It was seropositive for SRV and PCR-positive for SRVD 2. Approximately 1 mo after initial presentation, the macaque developed right facial paralysis and was euthanized. Histologic lesions included lymphoplasmacytic aggregates affecting multiple organs, consistent with SRV-related lymphoid hyperplasia. The right facial nerve showed lymphoplasmacytic inflammation. The nerve itself was negative immunohistochemically for SRV antigen, but antigen was present infrequently in pericapillary lymphoid cells within the facial nerve and abundantly within lymphoid aggregates in the adjacent parotid salivary gland, bone marrow, and soft tissue. Known neurotropic viruses could not be identified. Given the widespread inflammation in this macaque, particularly in the area surrounding the facial nerve, lymphocytic neuritis and facial paralysis likely were an indirect effect of SRV infection due to local extension of SRV-related inflammation in the surrounding tissue.Comparative medicine 01/2011; 61(6):538-45. · 0.76 Impact Factor
Vol. 52. No. 2
JOURNAL OF VIROLOGY, Nov. 1984, p. 683-686
Copyright © 1984, American Society for Microbiology
Experimental Infection of Rhesus Monkeys with Type D Retrovirus
NORMAN L. LETVIN,* MUTHIAH D. DANIEL, PRABHAT K. SEHGAL, LAURA V. CHALIFOUX, NORVAL W.
KING, RONALD D. HUNT, WAYNE R. ALDRICH, KRISTINE HOLLEY, DIANE K. SCHMIDT,
AND RONALD C. DESROSIERS
Harvard Medical School, New England Regional Pri,nate Research Center, One Pine Hill Drive, Soluthborolugh,
Received 30 April 1984/Accepted 16 July 1984
The naturally occurring immunodeficiency syndrome of macaque monkeys is an important animal model for
the acquired immunodeficiency syndrome in humans. A new type D retrovirus, distinct from Mason-Pfizer
monkey virus, has been isolated from affected animals at the New England Regional Primate Research Center.
We now report the results of experimental infection of macaques with retrovirus D/New England after 13
months of study. Inoculated macaques developed lymphadenopathy without follicular hyperplasia, profound
neutropenia, and a transient decrease in peripheral blood lymphocyte blastogenic responsiveness. Despite our
varying the strain of virus, the manner in which the virus was grown, the size of the inoculum, and the age of
the inoculated animals, infected macaques have not developed opportunistic infections or profound, prolonged
loss of T cell function, key features of the macaque immunodeficiency syndrome. Therefore, experimental
infection of naive macaques with D/New England has not reproduced the naturally occurring macaque
The immunodeficiency syndrome of macaque monkeys
has many parallels to the acquired immunodeficiency syn-
drome in humans (7,11). Animals with this syndrome
develop profound, prolonged T lymphocyte dysfunction and
eventually die of lymphomas or opportunistic infections by a
spectrum of agents similar to those seen in human acquired
immunodeficiency syndrome. We have shown that the ma-
caque immunodeficiency syndrome can be transmitted to
previously healthy macaque monkeys by using filtrates of
lymphoma tissue from these animals (8. 10). Such studies
implicate an infectious agent in this syndrome.
Our recent isolation of a new retrovirus from macaques
with the immunodeficiency syndrome is therefore of ex-
treme interest (4). This type D retrovirus is related to, but
distinct from, Mason-Pfizer monkey virus, a retrovirus pre-
viously isolated from a rhesus monkey mammary tumor (3).
We have cloned replicative intermediate (Hirt supernatant)
DNA from this new type D retrovirus, derived detailed
restriction endonuclease maps, and compared these with
Mason-Pfizer monkey virus and squirrel monkey type D
retrovirus maps. Although some restriction endonuclease
sites are conserved, most sites are not (4).
The fact that this newly isolated virus is a retrovirus. a
member of a family of viruses capable of inducing immuno-
suppression and malignancies (1), makes it a plausible candi-
date as the etiological agent in the macaque immunodeficien-
cy syndrome. We have isolated this new type D retrovirus
from 13 macaques with clinical evidence of disease, but
isolation attempts from healthy macaques have repeatedly
failed (4). We now report the results of inoculations of this
newly isolated type D retrovirus into rhesus monkeys after
13 months of study.
Virus isolates. The type D retrovirus isolates used in these
inoculation studies included two (D398 and D184) isolated
from individual Macaca cyclopis with well documented,
naturally occurring macaque immunodeficiency syndrome.
These isolations were done by cocultivation of peripheral
blood lymphocytes (PBL) from these animals with Raji cells,
as previously described (4). Virus stocks frozen after less
than 1 month in culture were used to prepare fresh virus
stock for these inoculations. Another isolate (D225) was
derived from a thymus explant culture from a Macaca
mulatta with experimentally transmitted immunodeficiency
disease. This animal developed the macaque immunodefi-
ciency syndrome after receiving a filtrate of a lymphoma
tissue homogenate from a macaque with an experimentally
transmitted lymphoma. This isolate was never passaged in
Clinical and pathological studies. Macaques were exam-
ined clinically on a daily basis for the first month after
inoculation and then on a weekly basis. Peripheral venous
blood for hematological. immunological, and virological
testing was drawn 1, 2, 4, and 8 weeks after inoculation.
Such studies were done every 2 months thereafter.
The routine hematological studies reported here were
carried out in the clinical laboratory at the New England
Regional Primate Research Center. PBL were assayed for
their proliferative response to pokeweed mitogen, concanav-
alin A, and Candida antigen as previously described (10).
Mixed lymphocyte reactions with mitomycin C-treated Sa-
guinus oedipuis B cell lines as stimulator cells were carried
out as previously described (10). Staining and analysis of
PBL for cell surface antigens were carried out as previously
Lymph node biopsies were performed on all the macaques
4 weeks after virus inoculation. Hematoxylin-and-eosin-
stained sections were prepared from 10% neutral buffered,
Formalin-fixed tissue by standard histological techniques.
Lymph node tissue was stained with lymphocyte subset-
specific monoclonal antibodies as previously described (2).
Inoculation of macaques with type D retrovirus. Thirteen
monkeys were inoculated with D/New England during a 13-
month period. A number of parameters, including the partic-
ular virus isolate, the manner in which the virus was grown,
the quantity of virus inoculated, and the age of the inoculat-
ed animal, were varied to optimize the likelihood of inducing
disease in these monkeys (Table 1).
In spite of the many variables introduced into these
studies, the clinical responses of the rhesus monkeys to the
inoculation of the type D retrovirus have been remarkably
consistent. All of the macaques developed a fever and
axillary and inguinal lymphadenopathy 1 to 2 weeks after
TABLE 1. Animals inoculated with type D retrovirus
aMm, M. mulatta; Cj, C. jacchus. Virus in 1 to 5 ml of medium was inoculated intravenously, subcutaneously, intraperitoneally, and intranasally into each
bSee text for details.
cSyncytia-forming units determined by serial dilution onto Raji cells; for inoculation of >109 U, virus was concentrated 500-fold.
dAt time of death.
Thymus explant culture
Thymus explant culture
Thymus explant culture
Thymus explant culture
Thymus explant culture
Thymus explant culture
inoculation. This lymphadenopathy, although easily demon-
strable, was not dramatic, and was no longer present 4 to 6
weeks after inoculation.
Virus isolations. Rhesus monkeys were selected for use in
the study after the ability to isolate type D retrovirus from
their peripheral blood was assessed and their antibody
response to this virus was determined. Virus isolation was
attempted by cocultivation of PBL with Raji cells, and
antibody status was determined by an indirect immunofluo-
rescence test. The animals used in the study showed no
evidence of type D retrovirus infection before experimental
inoculation. Cocultivations of all the macaque PBL yielded
virus by 1 to 2 weeks after inoculation. Furthermore, type D
retrovirus has been repeatedly isolated from these macaques
as long as 9 months after inoculation. This type D retrovirus
was frequently recovered from the cell-free plasma of infect-
ed macaques, suggesting that these animals remained vire-
mic for a significant period.
Attempts to isolate other viruses from the macaques have
only been made routinely on the first four rhesus monkeys
listed in Table 1. Oral and anal swabs and PBL from these
animals were cultured on human embryonic lung and Vero
cell lines. These cultivations yielded only adenovirus iso-
Studies done over a 6-week period after inoculation pro-
vided no evidence that Callithrix jacchus can be infected
with this agent.
Hematological status of inoculated macaques. A consistent
hematological picture was seen in the macaques after inocu-
lation with the type D retrovirus. The data on the first seven
animals listed in Table 1, those which have been followed for
more than 270 days, indicate that all the monkeys developed
a significant neutropenia. By 10 to 23 days after inoculation,
these macaques had a mean total granulocyte count of 1,300
(range, 360 to 1,750). Moreover, this neutropenia persisted,
with all the animals having total granulocyte counts of
<2,000, and five of the seven animals occasionally having
counts of <700 during the entire postinoculation period. The
mean total granulocyte count of these animals before inocu-
lation was 3,530.
Although these animals also developed an anemia, it was
transient. By 7 to 30 days after inoculation, their mean
hemoglobin concentration was 10.6 g/dl (range, 9.6 to 11.3).
These values returned to normal in all the monkeys by 20 to
60 days after inoculation. None of the animals developed a
persistent monocytosis or circulating mononuclear cells with
prominent nucleoli and vacuolated cytoplasm (characteristic
findings in the macaque immunodeficiency syndrome).
Of the two macaques inoculated at 1 month of age, one
(353-83) developed a persistent neutropenia, but in the other
animal, which died (356-83), the neutrophil count remained
normal. Both macaques inoculated at 1 day old developed
neutropenia; however, in the one which died (28-84), the
neutrophil count was normal 10 days before death.
Immunological status of inoculated macaques. Although
minor variations in PBL blastogenic responses to mitogenic
and antigenic stimulation occurred when comparing one
inoculated macaque with another, the responses of the PBL
of the animals shown in Fig. 1 and 2 were characteristic of the
pattern seen in these animals. By 7 days after inoculation,
the proliferative responses of PBL from these animals were
decreased compared with those ofthe controls. The prolifer-
ative responses of PBL from the macaque shown in Fig. 1
normalized by 1 month after inoculation and have remained
normal since that time. This transient change was typical of
the immunological abnormalities seen in these inoculated
monkeys. The longer period of decreased PBL responsive-
ness shown by cells from another animal represented one of
the most abnormal immunological profiles seen in these
inoculated macaques (Fig. 2). However, in no case was the
PBL responsiveness of these monkeys profoundly de-
creased, nor did it remain depressed for a prolonged period.
The absolute number of circulating T4 (helper/inducer) and
T8 (suppressor/cytotoxic) T cells in the animals did not
change significantly throughout these studies.
Histological appearance of lymph nodes. Sheets of mature-
appearing lymphocytes containing occasional cells with an
immunoblastic or histiocytic appearance were seen on hema-
toxylin-and-eosin-stained sections of lymph nodes. There
was no evidence of the marked follicular hyperplasia or
lymphoid depletion characteristically seen at various stages
of the macaque immunodeficiency syndrome. Follicles were
noted when the sections were stained with monoclonal
antibodies specific for lymphocyte subsets. The overwhelm-
ing number of paracortical lymphocytes were T4 bearing
when assessed by this technique. T8-bearing cells predomi-
VOL. 52, 1984
FIG. 1. PBL from a D/New England retrovirus-inoculated rhesus
monkey, showing a transient decrease in blastogenic responsive-
ness. The proliferative responses of PBL from this animal to lectin
and antigen stimulation are expressed as a percentage of the
response shown by PBL from two age-matched control rhesus
monkeys assayed simultaneously.
nate in the paracortex of lymph nodes early in the course of
the macaque immunodeficiency syndrome (la).
Necropsy findings. Two animals, both under the age of 4
weeks at the time ofinoculation, died during this study. Both
had a terminal diarrheal illness without associated gastroin-
testinal histopathological changes. Thymic atrophy was not-
ed in both macaques, with an apparent secondary depletion
of lymphocytes in the spleen and lymph nodes of one of
them. One animal (28-84) had an aspiration pneumonia and
inclusion bodies in hepatocytes comparable to those previ-
ously associated with paramyxovirus and not infrequently
seen in immunologically normal monkeys (9).
Conclusions. The clinical picture seen in the rhesus mon-
keys inoculated with the retrovirus D/New England was
remarkably consistent in spite of variations in the age of the
animals, in the source of virus inoculum, and in the quantity
of virus inoculated. The most unusual change seen in these
inoculated animals was the profound neutropenia. Although
neutropenia is a common transient phenomenon in a variety
DAYS POST INOCULATION
FIG. 2. PBL from D/New England retrovirus-inoculated rhesus
monkey Mm 106-83, showing a more prolonged decrease in blasto-
genic responsiveness. The proliferative responses are expressed as
described in the legend to Fig. 1.
of viral syndromes, its persistence in these macaques was
quite striking. The other pathological changes caused by this
agent in macaques immediately after inoculation, the mor-
phological changes in the lymph nodes and the transient
depression of blastogenic responsiveness of PBL, are char-
acteristic of a variety of viral syndromes. Some of the
findings described here were also noted in a previous study
of newborn rhesus monkeys inoculated with Mason-Pfizer
monkey virus (5).
Inoculated rhesus monkeys showed no evidence of pro-
found, prolonged T lymphocyte dysfunction, no opportunis-
tic infections, and no apparent lymphomas or lymphoprolif-
erative abnormalities. The lymph nodes of these animals did
not show the depleted appearance characteristic of the
macaque immunodeficiency syndrome. These animals also
did not develop the persistent monocytosis or circulating
mononuclear cells with prominent nucleoli seen previously
in this syndrome. The two inoculated rhesus monkeys which
died showed none ofthe clinical or immunological abnormal-
ities characteristically seen in the macaque immunodeficien-
cy syndrome (11) and showed none of the characteristic
pathological features of this syndrome at necropsy (9).
Therefore, experimental infection of clinically healthy mon-
keys with the D/New England retrovirus has not reproduced
the macaque immunodeficiency syndrome. This virus still
may play a causative role in the macaque disease. Rapid in
vitro attenuation of the virus, variations in the virulence of
different virus strains, a critical age of the macaques at the
time of infection, or additional factors could make it difficult
to establish a relationship between the virus and the disease.
Furthermore, these infected macaques may develop the
macaque immunodeficiency syndrome or lymphomas after a
latency period of more than 1 year. It is possible, however,
that this type D retrovirus simply represents another of the
many opportunistic agents present in this syndrome.
Marx et al. (13) and Gravell et al. (6) have reported that
two offive and two of two inoculated macaques, respective-
ly, died after inoculation with a type D retrovirus isolated
from macaques at the California Primate Research Center in
Davis. Whether these animals developed opportunistic in-
fections and profound, prolonged loss of T-cell function,
however, is not clear.
Continued investigation is required to determine the spec-
trum of pathological changes caused by type D retroviruses
and what role they play, if any, in the macaque immunodefi-
We thank Martha Elliott for help in animal handling, Cynthia
Harris for assistance in the hematology studies, and Bettye-Jean
Roy for preparing the manuscript.
This work was supported by Public Health Service grants
RR00168 from the National Institutes of Health, Division of Re-
search Resources, R01-AI 20729-01 from the U.S. Public Health
Service, and CA34979-01 from the National Cancer Institute.
N.L.L. is a recipient of an American Cancer Society Junior Faculty
Research Award. R.C.D. is a Special Fellow of the Leukemia
Society of America.
Animals used in this study were maintained in accordance with
the guidelines of the Committee on Animals of the Harvard Medical
School and those prepared by the Committee on Care and Use of
Laboratory Animals of the Institute of Laboratory Animal Re-
sources, National Research Council (D.H.E.W. publication no.
[NIH] 78-23, revised 1978).
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