response primary infection herpesvirus immunosuppressed ...squirrel monkey igg or igm, respectively....

INFECTION AND IMMUNITY, Sept. 1975, p. 528-535Copyright © 1975 American Society for Microbiology

Vol. 12, No. 3Printed in U.S.A.

Response to Primary Infection with Herpesvirus saimiri inImmunosuppressed Juvenile and Newborn Squirrel Monkeys

LOUIS N. MARTIN* AND WILLIAM P. ALLENDivision of Pathobiology, Thuane University, Delta Regional Primate Research Center,

Covington, Louisiana, 70433Received for publication 10 February 1975

Immunosuppression of juvenile squirrel monkeys with combined azathioprine,prednisolone, and antilymphocyte globulin resulted in decreased antibodyresponses to viral antigens after primary infection with Herpesvirus saimiri(HVS). The virus was repeatedly isolated from the oropharynx of immunosup-pressed monkeys but not from untreated infected controls. Thus immune factorsare important in inhibiting shedding of HVS from the oropharynx. HVS could beisolated from the peripheral blood lymphocytes of infected control monkeys butnot from the lymphocytes of immunosuppressed monkeys. Immunosuppressedmonkeys also had decreased percentages of lymphocytes capable of formingrosettes with sheep erythrocytes. These results indicate that the immunosuppres-sive agents had inhibitory effects on lymphocytes (presumably thymus derived)capable of being latently infected with HVS. Antibody responses in newbornmonkeys infected with HVS were delayed compared with juvenile monkeys.Treatment of newborn monkeys with antilymphocyte globulin had no suppres-sive effect on antibody responses to HVS.

The squirrel monkey (Saimiri sciureus) ap-pears to be the natural host of Herpesvirussaimiri (HVS) (2, 10). Horizontal transmissionoccurs, and in colonies of captive animals thereis an age-dependent incidence of infection anddevelopment of antibody reactive with HVS (3,8). The incidence of infection is also high inferal animals (2). The virus causes a latentinfection of long duration within the peripherallymphocytes (4, 5). Although the humoral im-mune response is not capable of eliminating thelatent infection, the virus causes no apparentdisease in squirrel monkeys (5, 8).HVS produces cytopathology in a large num-

ber of cultured cell types (10) and induces inthese cells the formation of three antigensdistinguishable by fluorescent antibody assay(12, 14) that are reminiscent of the early antigen(EA), late antigen (LA), and membrane antigen(MA) of Epstein-Barr virus observed in cellscultured from patients with Burkitt's lym-phoma (6).HVS infection of marmosets (Saguinus

oedipus, S. fuscicollis, and S. nigricollis) andowl monkeys (Aotus trivirgatus) causes fatalreticulum cell lymphoma (7, 12) or lymphocyticleukemia (1, 11). The antibody response to bothEA and LA in owl monkeys and marmosets isconsiderably delayed compared with the squir-rel monkey, and it has been suggested that themore vigorous response of the squirrel monkey

could be protective against lymphoproliferativedisease (8).

MATERIALS AND METHODSAnimals. Colony-born squirrel monkeys were sepa-

rated from their mothers on the day of birth andhand-reared to maintain them HVS free until experi-mentally infected. Six HVS-free juvenile squirrelmonkeys, seronegative for antibody to HVS, were 13to 14 months old when experimentally infected. Sevennewborn squirrel monkeys were 2 to 20 days old at thetime of infection.

Immunosuppression. Commercially preparedALG (rabbit anti-rhesus lymphocyte globulin, Mi-crobiological Associates) was further purified in ourlaboratory by elution from diethylaminoethylcellulose(DE32, Whatman) in 0.01 M phosphate buffer, pH7.9, plus 0.08 M NaCl. This purified ALG preparationgave 50% inhibition of rosette formation betweensquirrel monkey lymphocytes and sheep erythrocytes(SRBC) when tested at a concentration of 0.016mg/ml by the method of Kreeftenberg and Leerling(9).

Three of the six juvenile monkeys were immuno-suppressed by daily subcutaneous injections of 17 mgof azathioprine per kg (Imuran powder, BurroughsWellcome Co.) plus 5 mg of prednisolone per kg and byintramuscular injections of 16 mg of ALG per kg threetimes weekly. Four of the seven newborn monkeyswere treated with 16 mg of ALG per kg three timesweekly.

Immunosuppressive treatments were initiated 7days before infection in the juvenile group and at thetime of infection in the newborn monkeys.

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SQUIRREL MONKEY RESPONSE TO HVS 529

Infection with HVS. HVS was obtained from D. V.Ablashi, National Cancer Institute, Bethesda, Md.,and passed twice in primary squirrel monkey kidneycells. Each juvenile monkey was inoculated intraperi-toneally with a cell suspension of HVS-infected squir-rel monkey kidney cells containing 5 x 1052 meantissue culture infective doses (TCID50), subcutane-ously with the same cell suspension containing 105 2TCID5O and intravenously with a filtered, cell-freesupernatant containing 7.5 x 105 TCID,o. The new-born monkeys received the same inocula intraperito-neally and subcutaneously but were not inoculatedintravenously.Hematology. Blood samples for hematological,

virological, and serological analysis were collectedfrom the femoral vein into ethylenediaminetetraa-cetic acid after the animals had been fasted overnight.Total leukocyte counts were determined electronicallywith a Coulter counter, model B (Coulter Electron-ics), standardized daily with Coulter 4C normal andabnormal control cells. Differential counts were ob-tained by counting 200 leukocytes in May-Gruen-wald-Giemsa-stained blood films.

Indirect fluorescent antibody assay. Slides usedfor the detection of antibody to LA by indirectimmunofluorescence were prepared with HVS-infected owl monkey kidney cells. Trypsinized sus-pensions of infected cells were placed in wells ofTeflon-coated slides (Roboz Surgical Instrument Co.),air dried, fixed in acetone, and stored at -70 C untilused (4, 12). For detection of antibody to EA, slideswere prepared from cultures infected in the presenceof 20 ug of cytosine arabinoside per ml (14). Theindirect fluorescent antibody assay was performed asdescribed by Pearson et al. (12), utilizing the cross-reactions of fluorescein-conjugated goat anti-humanimmunoglobulin G (IgG) (Hyland Laboratories) andgoat anti-human IgM (Meloy Laboratories) withsquirrel monkey IgG or IgM, respectively. The fixedcells were reacted first with dilutions of squirrelmonkey plasma for 30 min in a humid chamber at37 C. The slides were then washed three times inphosphate-buffered saline, pH 8.1, and were stainedby reaction with the fluorescein conjugates for another30 min. The slides were washed three times, mountedwith cover slips, and observed with a Zeiss Universalfluorescent microscope. Antibody titers were deter-mined by using serial twofold dilutions of squirrelmonkev plasma or serum and are expressed as thereciprocal of the highest dilution showing positivestaining.

Virus isolations. Peripheral lymphocytes from thejuvenile monkeys were separated from blood sampleson Ficoll-Hypaque gradients (15) and were co-cul-tivated on monolayers of susceptible cells for HVSisolation (1). We used a fibroblastic cell line (Cal E)initiated from the skin of a Callicebus moloch monkeyby S. R. S. Rangan of this center as our HVS-suscepti-ble monolayer. Routinely 4 x 105 lymphocytes wereplated in quadruplicate on Cal E monolayers grown inmultiple-well plates (16-mm diameter; Linbro Chem-ical Co.). Blood volumes sufficient for lymphocytepurification could not be obtained from the newbornmonkeys, and a whole-blood co-cultivation technique

(1) was used in attempts to isolate HVS. Cal E cellswere grown in minimal essential medium with Earlesalts (GIBCO), containing 10% fetal calf serum(FCS), 100 ug of streptomycin per ml, and 100 U ofpenicillin per ml. The medium was changed to RPMI1640 (GIBCO) plus 20% FCS at the time lymphocytesor blood specimens were inoculated. Oropharyngealsamples were taken with dry cotton-tipped swabs.The swabs were introduced into 1 ml of minimalessential medium containing 10% FCS, 500 gg ofstreptomycin per ml, 500 U of penicillin per ml, and50 Mg of Fungizone (E. R. Squibb and Sons) per ml.The samples were incubated for 1 to 2 h at 37 C toenhance the effect of the antibiotics, and 0.2 ml ofeach sample was inoculated into each of four wells of amulti-well plate containing Cal E monolayers. Theinocula were allowed to adsorb for 1 h before 1 ml ofmedium was added to each well.

Spontaneous rosette formation. The percentageof lymphocytes from juvenile monkeys forming spon-taneous rosettes with SRBC was tested by usinglymphocytes purified on Ficoll-Hypaque gradients(16). Lymphocytes and SRBC were washed in phos-phate-buffered saline and resuspended in dextrose-gelatin-Veronal buffer (GIBCO) at concentrations of107 cells/ml and 1%, respectively. Mixtures of 0.1 mlof lymphocytes and 0.2 ml of SRBC were incubatedfor 5 min at 37 C and centrifuged at 200 x g for 5 min.The pellets were then allowed to stand undisturbedfor 90 min at room temperature. Trypan blue, 0.1 mlat 0.1% in dextrose-gelatin-Veronal buffer, was thenadded. The pellets were gently resuspended and thepercentage of lymphocytes with three or more adher-ant SRBC was determined by hemocytometer count-ing.

RESULTSResponses to HVS in immunosuppressed

juvenile monkeys. Treatment of juvenile mon-keys with ALG + azathioprine + prednisolonecaused lymphopenia compared with untreatedcontrols. The average number of lymphocytesper cubic millimeter of blood decreased toapproximately 25% of control over the first 21days of treatment (14 days postinfection) andremained below 40% of control over a period of42 days (35 days postinfection). Subsequentlythe lymphopenia was not as severe, but lympho-cyte counts in treated animals were usuallybelow control values. An early lymphopenia didnot occur in other animals treated with thesame dosage of azathioprine + prednisolone(Martin and Allen, in preparation) and can beconsidered an effect of the ALG. The effects ofthe treatment on individual animals varied.Animals 3701 and 3702 were most severelydepressed, whereas 3695 demonstrated a greaterdegree of recovery after the initial period ofsevere depression.

Occasionally immature lymphocytes andlymphocytes with eccentric nuclei and cytoplas-

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530 MARTIN AND ALLEN

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mic projections were observed during routinedifferential counting of stained blood films fromthe untreated animals. These cells were seenduring a period ranging from 21 to 81 days afterinfection and were never more than 1% of thetotal lymphocytes. Such cells were never ob-served in blood films from the HVS-infectedimmunosuppressed animals. Others have re-ported the occurrence of similar cells in bloodfilms from leukemic HVS-infected owl monkeysand suggested they may be related to the motilelymphocytes observed in vitally stained prepa-rations from leukemic owl monkeys (1).Monkey 3702 died 36 days after the im-

munosuppressive treatments began (29 dayspostinfection). No evidence of lymphoma wasdetected in the gross or histological examina-tions. We were not able to recover HVS fromany of 21 post-mortem tissues co-cultured withCal E cells, but virus had been isolated frompharyngeal swabs taken on 3 successive weeksbefore death. The hematocrit, determined theday before death, was severely decreased, indi-cating that the cause of death was bone marrowdepression resulting from the cytotoxic drugtreatment.IgG antibody responses to LA and EA were

delayed and diminished as a result of theimmunosuppressive treatment (Tables 2 and 3).All three control animals had antibody to LA by14 days after infection. Only one of the immuno-suppressed monkeys (3695) had antibody to LAat this time, and the titer in that animal wassignificantly decreased. The monkeys demon-strating the longest delay in antibody produc-tion (3701 and 3702) were those with the great-est degree of lymphopenia. Animal 3701 re-mained seronegative for 28 days postinfection,and when antibody production did begin thetiters remained significantly lower than titers incontrol animals. IgG antibody responses to EAwere also delayed by the immunosuppressivetreatments (Table 3). Animals 3701 and 3702again were most severely depressed.IgM antibody to LA was determined by

indirect immunofluorescence using fluores-cein-labeled anti-human IgM (Table 4). Titersof IgM antibody were not determined, butresults scored on the basis of intensity offluorescent staining indicated that the IgMresponse was also delayed and decreased by theimmunosuppressive treatments.Attempts at virus isolations from pharyngeal

swabs were only successful in the two animalsdemonstrating the most severe immunosup-pression (monkeys 3701 and 3702). HVS isolateswere obtained 14 and 21 days postinfection from

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TABLE 2. IgG antibody response to late antigen in immunosuppressed and untreated juvenile squirrel monkeys

Days postinfectionAnimal no.

-7 0 7 14 21 28 35 42 49 62 69 73 80 87 94 108 115

Immunosuppressed3701 -a _ _ - - - 32b 8 32 16 8 8 - - 32 NDc 1283702 - - - - - 4 Dead3695 - - - 8 32 32 32 128 256 256 256 256 128 128 128 ND 256

Untreated3738 - - - 32 64 256 128 128 256 256 256 256 16 64 64 ND 1283739 - - 8 32 32 128 256 128 128 128 256 256 128 128 64 ND 2563703 - - - 32 32 128 128 256 256 256 256 256 128 64 128 ND 256

a Indirect fluorescent antibody titer less than 1:4.b Reciprocal of indirect fluorescent antibody titer.c ND, Not determined.

TABLE 3. IgG antibody response to early antigen in immunosuppressed and untreated juvenilesquirrel monkeys


-7 0 7 14 21 28 35 42 49 62 69 73 80 87 94 108 115

Immunosuppressed3701 -a _ _ _ _ 16b - - - 4 - - - NDc 323702 - - - - - - Dead3695 - - - - - 8 16 32 - 16 64 64 32 32 32 ND 32

Untreated3738 - - - 16 16 16 8 64 64 - 64 16 16 ND 32 ND 83739 - - - - - - 32 8 - 8 16 16 - 4 8 ND -3703 - - - 8 4 - 64 32 - 16 32 - - 16 4 ND -

a, b, c Footnotes as in Table 2.

TABLE 4. IgM antibody response to late antigen in immunosuppressed and untreated juvenile squirrel monkeys


-7 0 7 14 21 28 35 42 49 62 69 73 80

Immunosuppressed3701 _ +

3702 - - - - - - Dead3695 - - + ++ ++ ++ ++ - NDc ++ ++ +++

Untreated3738 - - - +++ ++ ++ ++ - ++ ND ++ ++ ++3739 - - +++ +++ +++ +++ ++ - ++ ++ ++ ++ +++3703 - - _ ++ ++ ++ + + + ++ ++ ++ ++

a Indirect fluorescent antibody titer less than 1:4.° Positive antibody reaction at 1:4 dilution scored for intensity detected by using fluorescent anti-human IgM.c ND, Not determined.

both animals and on the day preceding death(28 days postinfection) from monkey 3702(Table 5). Attempts at virus isolation frompharyngeal swabs of monkey 3701 were consist-ently negative after antibody production beganon day 35 postinfection. Monkey 3695, thesuppressed animal that began antibody produc-tion at the same time as the control group, didnot yield an isolate from pharyngeal swabs, nordid any of the control animals. These data show

that the immune response is important ininhibiting the shedding of virus from the oro-pharynx.Attempts at HVS isolation from peripheral

lymphocytes were consistently negative fromthe immunosuppressed animals but were usu-ally positive in the control animals from 14 to 62days after infection (Table 6). Since equivalentnumbers of lymphocytes were co-cultured ineach instance, significantly fewer latently in-

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TABLE 5. HVS isolation from pharyngeal swabs of immunosuppressed and untreated juvenile squirrel monkeys


-7 0 7 14 21 28

Immunosuppressed3701 - - - + + -

3702 - - - + + +3695 -

Untreated3738 - - - - - -3739 - - - - - -3703 - - - - - -

aHVS not recovered from pharyngeal swab.bHVS recovered from pharyngeal swab.

TABLE 6. HVS isolation from lymphocvtes of immunosuppressed and untreated juvenile squirrel monkevs


-7 0 7 14 35 42 49 60 62 80 94

Immunosuppressed3701 _ a _ - NDb3702 - - - - Dead3695 - - -

Untreated3738 + C + + +3739 - - - + + - - + - - -3703 - - - + + + - + - - -

a HVS not isolated after co-cultivation of 1.6 x 101 peripheral blood lymphocytes with susceptible Cal Emonolayers.

b ND, Not determined.c HVS isolated after co-cultivation of 1.6 x 106 lymphocytes.

fected lymphocytes were present in the immu-nosuppressed group.

Since it has been reported that HVS-infectedlymphocytes from leukemic owl monkeys ap-pear to be thymus derived and form spontane-ous rosettes with SRBC (16), we determined thepercentage of rosette-forming cells in lympho-cyte preparations from each monkey on day 49postinfection. The results, presented in Table 7,show that immunosuppression caused a de-crease in the percentage of thymus-derivedrosette-forming cells.Responses to HSV infection in ALG-

treated newborn monkeys. Antibody re-sponses to LA in ALG-treated newborn squirrelmonkeys are compared with untreated controlsin Table 8. All animals in this experiment hadseropositive mothers and had detectable anti-body, presumably passively derived by trans-placental transfer of maternal antibody at thetime of infection. Antibody levels decreased to aminimum 22 days after infection. Increasedantibody titers were not observed until 28 to 36days after infection. There were no differencesin titers between ALG-treated and untreated

monkeys. The active antibody response to LA innewborn monkeys was delayed and of lower titerthan responses in juvenile monkeys (Table 2).In addition, IgM antibody to LA was notdetectable by indirect immunofluorescence insera from the newborn monkeys at any timeafter infection. This difference in responsive-ness could result from inhibition of viral infec-tion by passively acquired antibody in neonatesor from an inhibitory effect by the maternalantibody on the initiation of active antibodyproduction. Alternatively, the neonatal animalsmay not have developed sufficient im-munocompetence for optimal responsiveness.Counts of total leukocytes and differential

counts of blood smears did not reveal anydifferences between infected ALG-treated anduntreated newborn monkeys. The numbers ofcirculating lymphocytes varied greatly betweenindividual untreated animals and between sam-ples from the same animal taken at differenttimes. This extreme normal variability ob-scured detection of any possible effect of ALGtreatment on blood lymphocyte numbers.We were unable to isolate HVS from the

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newborn monkeys by co-cultivation of periph-eral blood with susceptible Cal E monolayers,probably because the blood samples obtainablefrom such young animals were too small. Wewere occasionally successful in isolating HVSfrom pharyngeal swabs from five of the sixsurviving animals. These isolates were onlyobtained after 2 months of infection since ear-lier attempts caused vomiting and endangeredthe life of the infants. However, the isolationsthat were obtained from oral swabs and theantibody responses indicate that the animalswere actively infected.

Passively transferred maternal antibody toLA can be detected in newborn monkeys for avariable period of time (8), probably dependingon the titer in the maternal circulation. We wereable to detect antibody in some sera fromhand-reared monkeys as late as 48 days of age.This is an important problem in determining

TABLE 7. Percentage of rosette-forming cells inimmunosuppressed and untreated juvenile monkeys

Percentage of rosettes"

Animal no. Individualdetermi- Group avgnations

Immunosuppressed3701 22.6

28.23695 27.6

24.8 25.8Untreated

3738 4040

3739 4647

3703 49.541 43.9

a Percentage of lymphocytes with three or moreadherant SRBC.

the possible pathogenic effect of HVS in thenewborn squirrel monkey since the presence ofserum antibody may alter the course of infec-tion.

DISCUSSIONThese experiments were designed to test the

effect of general immunosuppression on theoutcome of primary HVS infection in squirrelmonkeys. The results indicate that altering theimmune response does change the course ofinfection. For example, in the juvenile monkeyspharyngeal isolates were only obtained from theimmunosuppressed group and only before anti-body could be detected.

Falk et al. (2) reported isolation of HVS frompharyngeal swabs from naturally infected squir-rel monkeys having circulating IgG antibodytiters to LA as high as 1:256, but the time oforiginal infection in these animals was unknownand antibody titers to EA were not reported (2).The present data revealed that inhibition ofpharyngeal shedding of HVS may be related tospecific IgG or IgM antibodies or to other factorsin the primary immune response. No sheddingwas detected when either IgG antibody to EA or

IgM antibody to LA was present in the serum.

The presence of these antibodies would not beexpected in the naturally infected animals fromwhich Falk et al. obtained pharyngeal isolatessince those animals probably harbored infec-tions of long duration. It is clear from theliterature and our own unpublished data thatantibody titers to EA and LA decline late afterinfecion and that most animals with naturalinfection of possibly long duration lack anti-body reactive with EA (8). Similarly, IgMantibody to LA must decline after the primaryresponse since we have not detected it innaturally infected squirrel monkeys. It is there-fore likely that after primary infection in nor-

TABLE 8. IgG antibody response to late antigen in newborn squirrel monkeys


-2 15 22 28 36 40 60 66 80

ALG treated4217 16a 16 - b 8 8 32 32 32 1284218 8 8 - 16 8 32 32 16 324219 8 8 - 4 8 32 32 32 644220 16 16 4 4 8 32 16 32 32

Untreated4243 32 16 - 4 8 16 32 16 164247 32 - 4 - 8 32 32 32 164245 32 16 Dead

a Reciprocal of highest serum dilution giving a positive indirect fluorescent antibody reaction."Indirect fluorescent antibody titer less than 1:4.

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mal animals the shedding of HVS from theoropharynx is inhibited by the vigorous primaryimmune response and only occurs after certainantibody levels or other immune factors de-crease.The question of the importance of IgA anti-

body for inhibiting pharyngeal shedding of HVSremains open, as does the question of theimportance of cell-mediated immunity. Ourresults indicate that primary infection of immu-nosuppressed squirrel monkeys would provide agood model for further studies of the cellularorigin of virus isolated from pharyngeal swabsand of the local immune factors involved ininhibiting pharyngeal shedding.Immunosuppression of the juvenile monkeys

by combined treatment with ALG, azathio-prine, and prednisolone was successful as indi-cated by the effects on lymphocytes and anti-body titers. However, the immunosuppressantsdecreased the number of latently infected cells,either by eliminating the target cells or by adirect effect on the HVS-infected cells.Treatment of the infant monkeys with ALG

had no effect on lymphocyte counts or antibodytiters and may have been ineffective. The infantmonkeys were actively infected since we wereable to obtain HVS isolates from the oro-pharynx of five of the six surviving animals. Thequestion of whether or not HVS could beoncogenic in newborn squirrel monkeys san onlybe answered when offspring from virus-freemothers become available.A comparison of antibody responses in differ-

ent monkey species after HVS infection gives noclear indication of whether or not antibody isprotective against oncogenesis. It has been sug-gested that the more rapid immune response insquirrel monkeys could be protective sincesquirrel monkeys produce antibody to LA andEA more rapidly than owl monkeys and marmo-sets (5, 8). However, occasional owl monkeysbegin antibody production as early as squirrel'monkeys (13) and yet go on to develop lym-phoma. In addition, capuchin monkeys (Cebusalbifrons) have a delayed antibody response toHVS similar to that in owl monkeys, yet do notdevelop lymphoma (H. Rabin, G. R. Pearson,W. C. Wallen, R. H. Neubauer, J. L. Cicmanec,and T. W. Orr, J. Natl. Cancer Inst., in press).In the present study disease was not producedin the one severely suppressed animal thatremained alive (animal 3701). This does notindicate that antibody is not important forprotection since we also demonstrated de-creased numbers of latently infected cells due tothe immunosuppressive treatments.

Since the immunosuppressants adversely af-

fected the thymus-derived lymphocytes whichbecome latently infected with HVS, other morespecific methods of suppressing antibody re-sponses must be used to determine the impor-tance of antibody for protection of squirrelmonkeys against lymphoma induction by HVS.

ACKNOWLEDGMENTS

This research was supported by the Special Virus CancerProgram, Public Health Service contract NO1-CP-33396 fromthe National Cancer Institute and Public Health Servicegrant RR-00164 to the Delta Regional Primate ResearchCenter from the Division of Research Resources.We are indebted to Robert H. Wolf and Thaddius Martin

for breeding and hand-rearing of squirrel monkeys, to A.Doskey, L. Trahan, W. McGehee, T. Hahn, and E. Perrin fortechnical assistance, and to M. LaCroix for typing the manu-script.

LITERATURE CITED1. Ablashi, D. V., W. F. Loeb, M. G. Valerio, R. H.

Adamson, G. R. Armstrong, D. G. Bennet, and U.Heine. 1971. Malignant lymphoma with lymphocyticleukemia induced in owl monkeys by Herpesuirussaimiri. J. Natl. Cancer Inst. 47:837-855.

2. Falk, L. A., S. Nigida, F. Deinhardt, R. W. Cooper, and J.I. Hernandez-Camacho. 1973. Oral excretion of Herpes-virus saimiri in captive squirrel monkeys and incidenceof infection in feral squirrel monkeys. J. Natl. CancerInst. 51:1987-1989.

3. Falk, L., L. Wolfe, and F. Deinhardt. 1972. Epidemiologyof Herpesvirus saimiri infection in squirrel monkeys, p.151-158. In E. I. Goldsmith and J. M. Jankowski (ed.),Medical primatology, part 3. S. Karger, Basel.

4. Falk, L. A., L. G. Wolfe, and F. Deinhardt. 1972. Isolationof Herpesvirus saimiri from blood of squirrel monkeys(Saimiri sciureus). J. Natl. Cancer Inst. 48:1499-1505.

5. Falk, L. A., L. G. Wolfe, and F. Deinhardt. 1973.Herpesvirus saimiri: experimental infection of squirrelmonkeys (Saimiri sciureus). J. Natl. Cancer Inst.51:165-170.

6. Henle, G., W. Henle, G. Klein, P. Gunven, P. Clifford, R.H. Morrow, and J. L. Ziegler. 1971. Antibodies to earlyEpstein-Barr virus-induced antigens in Burkitt's lym-phoma. J. Natl. Cancer Inst. 46:861-871.

7. Hunt, R. D., L. V. Melendez, N. W. King, C. E. Gilmore,M. D. Daniel, M. E. Williamson, and T. C. Jones. 1970.Morphology of a disease with features of malignantlymphoma in marmosets and owl monkeys inoculatedwith Herpesvirus saimiri. J. Natl. Cancer Inst.44:44 -465.

8. Klein, G., G. Pearson, A. Rabson, D. V. Ablashi, L. Falk,L. Wolfe, F. Deinhardt, and H. Rabin. 1973. Antibodyreactions to Herpesvirus saimiri (HVS)-induced earlyand late antigens (EA and LA) in HVS-infectedsquirrel, marmoset, and owl monkeys. Int. J. Cancer12:270-289.

9. Kreeftenberg, J. G., and M. F. Leerling. 1972. Evaluationof antihuman antilymphocyte serum with the rosetteinhibition test using human and cynomologous thymo-cytes. Transplantation 14:665-670.

10. Melendez, L. V., M. D. Daniel, F. G. Garcia, C. E. 0.Fraser, R D. Hunt, and N. W. King. 1969. Herpesvirussaimiri. 1. Further characterization studies of newvirus from the squirrel monkey. Lab. Anim. Care19:372-377.

11. Melendez, L. V., R. D. Hunt, M. D. Daniel, B. J. Blake,and F. G. Garcia. 1971. Acute lymphocytic leukemia inowl monkeys Aotus trivirgatus inoculated with Herpes-

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virus saimiri. Science 171:1161-1163.12. Pearson, G., D. Ablashi, T. Orr, H. Rabin, and G.

Armstrong. 1972. Intracellular and membrane immu-nofluorescence investigations on cells infected withHerpesvirus saimiri. J. Natl. Cancer Inst.49:1417-1424.

13. Pearson, G. R., T. Orr, H. Rabin, J. Cicmanec, D.Ablashi, and G. Armstrong. 1973. Antibody patterns toHerpesvirus saimiri-induced antigens in owl monkeys.J. Natl. Cancer Inst. 51:1939-1943.

14. Rabin, H., G. Pearson, G. Klein, D. Ablashi, W. Wallen,and J. Cicmanec. 1973. Herpesuirus saimiri antigens

and virus recovery from cultured cells and antibodylevels and virus isolations from squirrel monkeys. Am.J. Phys. Anthropol. 38:491-96.

15. Thorsby, E., and A. Bratlie. 1970. A rapid method forpreparation of pure lymphocyte suspensions, p.

655-656. In P. J. Teraski (ed.), Histocompatibilitytesting. The Williams & Wilkins Co., Baltimore.

16. Wallen, W. C., R. H. Neubauer, H. Rabin, and J. L.Cicmanec. 1973. Nonimmune rosette formation bylymphoma and leukemia cells from Herpesvirussaimiri-infected owl monkeys. J. Natl. Cancer Inst.51:967-975.

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response primary infection herpesvirus immunosuppressed ...squirrel monkey igg or igm, respectively....

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