White-lipped Marmosets Research Articles

Transplantable Primate Tumors Induced by Rous Sarcoma Virus. I. Induction of Tumors Transplantable Into Young Marmosets<xref ref-type="fn" rid="FN1">2</xref>

The tumors induced in white-lipped marmosets (Saguinus fuscicollis, S. nigricollis) by Rous sarcoma virus (RSV) of chicken origin (RSV-SR) were not transplantable to allogeneic hosts. In contrast, RSV rescued from these tumors (RSV-M) induced sarcomas that were transplantable to young but not to adult marmosets. The tumors induced by RSV-M and the transplants rapidly enlarged, metastasized to various organs, and killed the recipients 29-59 days post inoculation. Cell lines were readily established from all transplantable sarcomas. No virus expression was detected in transplantable tumor cell lines by electron microscopy or by biochemical and biological assays. However, RSV of the same subgroup as RSV-SR was rescued from both short-term and long-term tumor cell cultures by cocultivation with chicken embryo fibroblasts (CEF). The rescued viruses transformed marmoset cells 100-fold more efficiently than CEF cells, although CEF cells remained permissive for virus replication. Cytogenetic studies revealed extensive chromosome abnormalities in tumor transplants but not in RSV-M-induced sarcomas. All cell lines were hyperploid and contained structurally abnormal, large metacentric and telocentric chromosomes. Immunologic studies failed to detect group-specific (gs) antigen of the avian sarcoma-leukemia complex in either RSV-M-induced, transformed cells or tumor transplants. By complement-dependent cytotoxicity assays, with the use of marmoset anti-gs serum, RSV-associated antigen could be detected on the surfaces of tumor cells. No differences in the expression of this antigen existed between transplantable and nontransplantable marmoset sarcomas. All transplantable cell lines contained abnormal amounts of lipids and glycogen in comparison to RSV-SR-induced tumors and normal marmoset cell lines. The glycogen was associated with unique cytoplasmic membrane complexes and was surrounded by either single- or double-membraned vesicles.

Specifically unmethylated cytidylic-guanylate sites in Herpesvirus saimiri DNA in tumor cells.

The restriction endonucleases MspI (CCGG), HpaII (CCGG), FnuDII (CGCG), and HaeIII (GGCC) were used to study the methylation of Herpesvirus saimiri DNA in tumor cells taken directly from tumor-bearing animals. No evidence was found for methylation of the 5' terminal C in the sequence CCGG or of the internal C in the sequence GGCC, but extensive methylation of CG was detected. Fifteen HpaII sites and 17 FnuDII sites were detected in the unique DNA region of the H. saimiri strain used. Twenty-eight of the 32 sites were methylated in greater than 90% of the viral DNA molecules in tumor cells, but the remaining 4 sites were unmethylated in greater than 95% of the viral DNA molecules in tumor cells. The locations of the four specifically unmethylated sites were mapped and appeared to be identical in the four different induced leukemias examined (one owl monkey and three white-lipped marmosets). The nonproducer 1670 tumor cell line, in continuous passage for over 7 years, contained four similar specifically unmethylated sites. Possibilities for the physiological significance of the unmethylated sites are discussed.

Epstein-Barr virus-induced malignant lymphoma in a white-lipped marmoset.

One of six white-lipped marmosets inoculated with cell-free B95-8 virus developed diffuse malignant lymphoma. Epstein-Barr virus (EBV) DNA was detected in a pathologically enlarged mandibular lymph node by DNA-DNA hybridization. The affected animal at the time of killing had EBV antibody titers of 1:320 for viral capsid antigen (VCA) and 1:20 for early antigen (EA) while all non-diseased animals had less than or equal to 1:80 VCA antibody and no detectable EA antibody. This is the first report of lymphoma development in a white-lipped marmoset following EBV inoculation.

In vitro blastogenic responses of peripheral lymphocytes from marmosets to phytohemagglutinin and to autologous lymphocytes transformed by epstein-barr virus

White-lipped marmosets were evaluated for their cell mediated immune (CMI) response to EBV to determine the feasibility of CMI studies in marmoset models for EBV oncogenesis. The mitogen, cell concentrations, the length of incubation period and serum requirements were defined for in vitro lymphocyte stimulation tests. The level of response of each animal was dependent on the concentration of phytohemagglutinin-P (PHA-P) and was independent of cell densities employed. The rate of tritiated-thymidine incorporation by mononuclear cells due to PHA-P increased exponentially between 2–4 days. This test was reproducible for a given batch of PHA-P when the cells were cultured in the presence of 10% heat inactivated fetal bovine serum. The five white-lipped marmosets were seronegative for EBV antigens and did not show lymphocyte stimulation with EBV particles and EBV soluble antigen, but two of these animals exhibited significant stimulation with autologous lymphocytes transformed in vitro by B95-8 virus. Despite the limited amount of blood (3–4 ml) that could be obtained from each animal in a single bleeding, it was possible to perform multiple lymphocyte stimulation assays with the protocol used.

Thermal treatment and infectivity of hepatitis A virus in human feces

The susceptibility of white-lipped marmoset monkeys (Saguinus sp) to human hepatitis A virus (HAV) provides a system for evaluation of thermal inactivation of HAV in feces and contaminated shellfish. Intramuscular or oral administration of HAV derived from feces of four patients with acute hepatitis A induced hepatitis in 28--100% of the inoculated marmosets. A 10% (w/v) fecal pool (GBG-BM) prepared from two patients (GBG and GBM) induced hepatitis in marmosets (2/4 with 1 ml; 2/2 with 3 ml) when given orally as a 1 : 3 dilution. A HAV-baby food raw oyster mixture fed to fasted marmosets induced hepatitis in 1/4 and seroconversion in 2/4 animals. Two groups of oysters were injected with HAV (concentrated 3 : 1 by centrifugation of the GBG-BM pool); one group was treated at 140 degrees F for 19 minutes and the other served as an untreated control. In animals fed the untreated inoculum, 4/6 developed hepatitis and 6/6 seroconverted, whereas of those fed the heat-treated inoculum 1/7 developed hepatitis and 2/7 seroconverted. These data suggest that pasteurization methods could be developed that would eliminate shellfish-associated hepatitis A and retain the palatability of the shellfish.

Transformation of lymphocytes by herpesvirus papio

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.

Morphology of the GB hepatitis agent.

ATTEMPTS to isolate hepatitis A virus have been in progress for several decades. An important advance was the finding that the marmoset served as a permissive animal host for this virus1, but it became obvious that more than one agent was active. The pedigree of one agent was established as MS-1 (hepatitis A) isolate, while the other, which was demonstrably different by neutralisation and cross-challenge experiments, was designated GB. The GB infectious serum was obtained on the third day of jaundice from a surgeon in Chicago who developed a mild form of acute hepatitis. This serum induced hepatitis in all four marmosets inoculated. A pool of infectious GB serum marmoset (passage 11) was prepared from the total bleeding of nine white-lipped marmosets (Saguinus sp.) 19–24 d after inoculation and during the early acute phase of hepatitis as indicated by an increase of serum transaminase levels. This pool, designated H 205 GB pass 11, has an infectivity of 104.5 marmoset ID50. We have now examined the GB agent electron microscopically, and found that it is smaller and more fragile than the MS-1 strain2, and therefore also the identical CR326 isolate3 of hepatitis A, which have the morphological appearances of recognised, small cubic viruses measuring about 27 nm in diameter.

Chronic Herpesvirus saimiri Infection in an Owl Monkey 2

An adult owl monkey (Aotus tricirgatus) used for immunologic studies of Herpesvirus saimiri (HVS) developed early, late, membrane, and neutralizing antibodies to HVS approximately 3 weeks after the beginning of the experiment. HVS was isolated by the cocultivation of peripheral blood for over 1 year. No clinical, gross, or histopathologic findings of malignancy were exhibited by the animal. The HVS isolate from the animal was indistinguishable biologically and serologically from the original HVS strain of Meléndez and from an isolate of an experimentally HVS-induced tumor. Inoculation of this isolate into 2 young white-lipped marmosets (Saguinus fuscicollis) produced typical malignant lymphoma and lymphocytic leukemia. Our findings suggested that the virus from the chronically infected animal was oncogenic and that host factors were primarily responsible for determining the disease manifestation of the virus infection. Another owl monkey chronically infected with HVS for over 2 years has remained asymptomatic.

Experimental Infection of Marmosets with a Cytomegalovirus of Human Origin

Two adult and two neonatal cotton-topped marmosets and two neonatal white-lipped marmosets (Saguinus species) were inoculated with 10(7) plaque-forming units of cytomegalovirus (Colburn strain). No overt clinical disease developed in four marmosets during observation for eight months; one adult and one neonatal cotton-topped marmoset died from nonspecific causes 63 and 259 days after inoculation, respectively. By days 7-16 after inoculation, all marmosets developed plasma antibodies, which were detectable by neutralization and immunofluorescence assays (peak titers, 1:128-1:256 and 1:64-1:256, respectively). Attempts to isolate virus from whole blood, peripheral lymphocytes, oropharyngeal swabs, or vaginal swabs by cocultivation with permissive cell cultures were unsuccessful. Virus was recovered, however, by cocultivation from the kidney tissues of the adult marmoset that died. Immunosuppressive treatment with azathioprine resulted in a fourfold increase in antibody levels in plasma of two of three marmosets.

Effect of Bacillus Calmette-Guérin Immunization in Marmosets Infected Experimentally With <italic>Herpesvirus saimiri</italic><xref ref-type="fn" rid="fn2">2</xref>

Eight white-lipped marmosets immunized with BCG and 3 sham-immunized marmosets were studied after inoculation with Herpesvirus saimiri (HVS). BCG immunization had no significant influence on the incidence of infection by HVS, incidence of fatal malignant lymphoma, time of leukemia onset, development or titer of HVS antibodies, or average survival time. One BCG-immunized, HVS-infected marmoset failed to develop malignant lymphoma, whereas the remaining 10 HVS-infected marmosets died of malignant lymphoma. Prolonged survival occurred also in 1 marmoset immunized with BCG 100 days after HVS inoculation. The development and disappearance of lymphocyte reactivity to tuberculin were followed in 4 BCG-immunized marmosets.

Immunization of Rous Sarcoma Virus-Inoculated Marmosets With BCG and Transformed Allogeneic Cells2

The effects of specific immunotherapy with allogeneic cells transformed by Schmidt-Ruppin Rous sarcoma virus (SR-RSV), of treatment with BCG, and of surgery on the growth of SR-RSV-induced sarcomas in white-lipped marmosets were studied. Tumor incidence, tumor progression, and survival did not differ between control and treated animals. Animals immunized with BCG developed lymphocyte reactivity to tuberculin, which remained until the animals died. BCG was isolated from the spleen of one tumor-bearing animal.

Rous sarcoma virus-induced tumors in marmosets: characteristics of tumor cells and host cell-virus interrelationships.

Thirty-eight tumors were induced in white-lipped marmosets(Saguinus nigricollis, S. fuscicollis) by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV). They were analyzed for viral genome, virus-expressed antigen(s), chromosomal aberrations, and the production of acid mucopolysaccharides (AMPS). Tumors established in cell cultures had morphologic characteristics typical for Roustumor cells of fowl and other mammalian species. The viral genome was demonstrated in 7 of 18 tumors by cocultivation with live, X-irradiated, or disrupted chicken cells. No group-specific antigen was detected by complement fixation tests (COFAL) in tumors examined, and only a few animals with a long latent period developed COFAL antibodies. Although no specific chromosomal aberrations accompanied marmoset SR-RSV-induced tumors, chromosomes were often broken and rearranged, which wasattributed to inadequate cell nutrition in fast-growing and poorly vascularized tumors. Large quantities of AMPS were demonstrated in some tumors, and all tumors had some AMPS on the periphery of tumors infiltrating surrounding muscles. Infectious viruses isolated from marmoset SR-RSV-induced tumors had envelope antigens similar to either subgroup A or D of avian sarcoma-leukosis viruses, as did the original SR-RSV used for marmoset inoculation.

Characteristics of Herpesvirus saimiri-induced lymphoma cells in tissue culture.

Lymphoblastoid cells were cultured from twoHerpesvirus saimiri (HVS) inoculated white-lipped marmosets and from one HVS-inoculated owl monkey. Cells from all three animals grew clumped in suspension. The cells from both species were diploid in chromo-some number and showed no unusual chromosomal abnormalities. The marmoset cell line examined was chimaeric. The marmoset cells lacked HSV-associated antigens as determined by immunofluorescence, and no evidence for the presence of virus was found by either infectivity assays or electron microscopy. Cocultivation of these cells with Vero cells resulted in cytopathology and the recovery of complete, infectious virus. The owl monkey lymphoid cells were positive to a small degree for both viral antigens and infectivity. The cells were resistant to rechallenge with HVS. Cocultivation of these cells with Vero cells led to the development of cytopathology and an increased yield of virus.

Susceptibility to Herpesvirus saimiri and antibody development in old and new world monkeys.

Herpesvirus saimiri (HVS) induced malignant lymphoma in four cotton-topped (CT) marmosets (S. oedipus). The tumor of a CT marmoset was transplanted to a white-lipped (WL) marmoset (S. nigricollis) which developed malignant lymphoma. The inoculation of HVS and the transplantation of tumor cells did not induce malignant lymphoma in nine adultCallithrix jacchus monkeys and six adultCercopithecus aethiops monkeys. A 32-day-old Callithrix jacchus baby monkey died with malignant lymphoma after infection with HVS. All adult animals of the New World monkey species (S. oedipus, S. nigricollis, Callithrix jacchus) inoculated once with HVS or tumor cells developed equally high titers of neutralizing antibodies against HVS whether they developed a tumor or not. No neutralizing antibodies were detectable in the sera of the Old World monkeys (Cercopithecus aethiops) inoculated with HVS or tumor cells.

Occurrence of a Carrier State for Herpesvirus tamarinus in Marmosets

The ecology of herpesviruses in marmosets and other nonhuman primates is important today, for colonies of these animals are being established for biomedical research. This paper presents the first reported isolations of Herpesvirus tamarinus from throat swabs of a healthy white-lipped marmoset carrier (Saguinus nigricollis) during a 2-month period. Infectivity studies with this virus in both white-moustached (S. mystax) and white-lipped marmosets demonstrated that the virus is not lethal to white-moustached marmosets (perhaps a more resistant species) at 1,000 TCID(50). Which environmental conditions trigger the unmasking of herpesviruses in marmosets is not known. Hoever, intermittent H. tamarinus shedding may help explain spontaneous infections in established colonies as well as suggest an additional mechanism for transmission of virus between marmosets under natural conditions.

Occurrence of a carrier state for Herpesvirus tamarinus in Marmosets.

The ecology of herpesviruses in marmosets and other nonhuman primates is important today, for colonies of these animals are being established for biomedical research. This paper presents the first reported isolations of Herpesvirus tamarinus from throat swabs of a healthy white-lipped marmoset carrier (Saguinus nigricollis) during a 2-month period. Infectivity studies with this virus in both white-moustached (S. mystax) and white-lipped marmosets demonstrated that the virus is not lethal to white-moustached marmosets (perhaps a more resistant species) at 1,000 TCID(50). Which environmental conditions trigger the unmasking of herpesviruses in marmosets is not known. Hoever, intermittent H. tamarinus shedding may help explain spontaneous infections in established colonies as well as suggest an additional mechanism for transmission of virus between marmosets under natural conditions.