Brief Communic,ation: Chronic Herpesvirus saimiri Infection in an Owl Monkey 1,
2
Gary R. Armstrong,3,4 Thomas Orr,5 Dharam V. Ablashi,3 Gary R. Pearson,> Harvey Rabin,6 Josef tuetzeler,' Walter F. loeb,6 and Marion G. Valerio 6 SUMMARY-An adult owl monkey (Aotus trivirgatus) used for immunologie 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 eoeultivation of peripheral blood for over 1 year. No elinieal, gross, or histopathologie findings of malignaney were exhibited by the animal. The HVS isolate from the animal was indistinguishable biologieally and serologieally from the original HVS strain of Melimdez and from an isolate of an experimentally HVS-indueed tumor. Inoeulation of this isolate into 2 young white-Iipped marmosets (Saguinus fuscieollis) produeed typieal malignant lymphoma and Iymphoeytie leukemia. Our findings suggested that the virus from the ehronieally infeeted animal was oneogenie and that host faetors were primarily responsible for determining the disease manifestation of the virus infeetion. Another owl monkey ehronieally infected with HVS for over 2 years has remained asymptomatie.-J Natl Cancer Inst 56: 1069-1071, 1976.
Two independent reports on spontaneous malignant lymphoma andjor leukemia in owl monkeys (Aotus trivirgatus) suggest the horizontal transmission of Herpesvirus saimiri (HVS) from squirrel monkeys (Saimiri seiureus) (1~ 3). Though squirrel monkeys are the natural hosts of HVS, no tumors were reported in this species that could be related to HVS infection (4-6). However, inoculations of HVS into ow1 monkeys or marmosets have regularly induced tumors (4, 7-9). In this communication, we report on some biologie properties of an isolate from a chronically infected disease-free owl monkey. To our knowledge, in vitro and in vivo properties of an HVS isolate from a chronic infection in an owl monkey have not been detailed, but similar studies with nonhuman primate species that are not susceptible to HVS-related disease have been described (10~ 11). MATERIALS AND METHODS
An adult male owl monkey (636H), received by Litton Bionetics, Ine. and held in quarantine for 3-4 months in a wire cage with a Kirschner box with other owl monkeys, was used as a control in another program. This animal was transferred to a Landon cage within a biohazard area before receiving six weekly inoculations of 1.0 ml of virus-free VERO cellfree extract. Although it is possible that the monkey was mistakenly inoculated with HVS, it is also possible that it was inadvertently infected by contact. The virus isolation from the peripheral blood of infected animals was described in (4~ 7). For detection of antibodies to HVS early antigens (EA), late antigens (LA), and membrane antigens (MA), HVS-infected VERO cells were used according to procedures described in (4). For complement-fixation (CF) antibodies to HVS, the method of Ablashi et al. (12) was used. Serum neutralization (SN) tests with 102 and 103 mean tissue culture infective doses (TCID50)jml HVS were used. The goat anti-HVS serum, supplied by Dr. L~is Melend~z, Pan American J:Iealt~ Organization, Washmgton, D.C., was used for the identification of the HVS isolated from the owl monkey and
from 2 young female white-lipped marmosets (Saguinus fuseieollis, 8541 and 8421) receiving the HVS isolate from owl monkey 636H. The SN test was conducted by the use of equal amounts of heat-inactivated HVS antiserum mixed with different concentrations of HVS, incubated at 4° C overnight, and inoculated in owl monkey kidney (OMK) cells (7). Two marmosets were each inoculated with 105 TC1D50 in 1.0 ml of HVS isolated from owl monkey 636H. Blood was collected from each of the monkeys (636H, 8541, and 8421) before inoculation and at regular intervals thereafter for hematologic examination, virus isolation, and antibody determination. After the monkeys died or were killed, the tissues were processed for histopathology and virus isolation. RESUlTS
Before the study, owl monkey 636H had neither HVS nor HVS-directed antibodies. This was demonstrated by the absence of HVS when peripheral blood was cocultiva ted with OMK and VERO cells and by the absence of immunoßuorescent (IF) or neutralizing antibody in the preinoculation serum (table 1). These findings suggest that the animal was not exposed to HVS before the study. The monkeys demonstrated the presence of HVS infection by a) release of HVS from circulating lymphoeytes from the peripheral blood and b) development of humoral antibodies to various HVS antigens (table 1) throughout the experiment. After the onset of the experiment (table 1), the monkey developed antibodies to HVS antigens (as determined by 1F and CF); two serum sampIes collected at different times, i.e., the seed stock HVS supplied originally by Dr. Melendez and the one isolated from the animal's own blood, neutralized HVS. Thus far all the data on HVS serology indicate that HVS does not cross-react extensively with other oncogenic (13) herpesviruses, and we examined the HVS from 636H monkey by using more than one assay (table 1). Throughout the experiment, the hemograms of monkey 636H were essentially normal (text-fig. 1), and blood smears prepared at various times gave no indication of cell abnormalities. The monkey was infected with typical HVS, but lack of tumor induction suggested the possibility of in vivo attenuation of the virus. The animal was killed 19 months after the beginning of the study, and organs and tissues were collected for microscopic ex amReceived August 27, 1975; accepted November 21, 1975. 2Supported in part by Public Health Service contract NOI CP43224 within the Virus-Cancer Program of the National Cancer Institute. 3 Viral Leukemia and Lymphoma Branch, National Cancer In~ti tute (NCI), National Institutes of Health, Public Health Service, V.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 4 Address reprint requests to Mr. Armstrong, NCI-Frederick Cancer Research Center, Fort Detrick, Frederick, Md. 21701. 5 Mayo Clinic, Plummer Building, Room 442, Rochester, Minn. 55901. 6 Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Md. 20795. 7 Visiting Associate at the NCI from the Pathology Institute of Cologne, Germany. 1
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 56, NO. 5, MAY 1976
1069
1070
ARMSTRONG ET AL.
TABLE
l.-HVS isolation [rom, and humoral antibody response oj, owl monkey 636H
Observati on days
Virus isolation a from peripheral blood
01 18 54 61 118 353 572 (terminal)s
Humoral HVS antibodies EAc LAc MAc CF 0 0
0
+ + + + + +
+ + + + +
0
+ + + + + +
ND ND ND ND ND
d
e
0 ND ND
+ + + +
+ +
b
Neut
0 ND ND
ination and virus isolations. Cultures prepared at neeropsy from various lymph nodes eontained HVS, but eell-free extracts from these lymphatie tissues or ultrastruetural examination did not reveal any infeetious virus or virus particles, respeetively. Histopathologie examination of this animal failed to show any form of neoplasia. The possibility that the 636H isolate of HVS was nononeogenie was explored in a sensitive animal system. Two white-lipped marmosets (8411 and 8541) free of HVS and HVS antibodies (table 2) were inoeulated with this virus. Both responded similarly to inoeulation in beeoming viremic and developing antibodies to HVS; HVS could be isolated from their peripheral lymphoeytes. No signs of illness were c1inieally evident. Seventy and 42 days, respeetively, after inoeulation, there was a marked rise in lymphocytes with atypieal forms (text-fig. I), and 2 weeks later, the animals were frankly leukemic. The animals died 84 and 63 days, respeetively, after inoeulation. The 636H isolate of HVS and those isolated from the 2 marmosets showed identieal patterns of cytopathic effeet and infeetivity titers in VERO andjor OMK eells. The gross and mieroseopie lesions in both marmosets were indistinguishable from those deseribed in (9~ 14); however, the magnitude of lymphoeytic proliferation was greater in 8541 than in 8421. The HVS isolate from both animals was neutralized by goat antiserum to HVS, which eonfirmed the identity of the isolate.
+ ++ ++ +
a All virus isolation from either peripheral blood or tissue collected at necropsy was done by cocultivation with indicator celIs. b ND =not done.
• + = ~I:IO. d + = ~1:4. with 4 U antigen. • Serum neutralized the 636H isolate of HVS and the original HVS used for 636H inoculation. The degree of neutralizing (neut) is expressed as =serum dilution of I: 4 and =serum dilution of I: 16 which neutralized 10 2 and 10 3 TCID50 of HVS. f Day 0 serum was tested undilu ted and at a dilution of I: 2. 11 Tissues colIected at necropsy for virus isolation were lymphatic tissues from the lymphatic system.
+
++
LYMPHOCYTE COUNT
70,000
- - 636H
I
60,000
---·8411 _ . - 8541
I
I
50,000
DISCUSSION
!
40,000 -
30,000
In our previous experiments (7), 4 of 5 owl monkeys reeeiving one HVS inoeulation Iailed to develop evidenee of leukemia or lymphoma in 2-4 months. These animals were reinoeulated and later developed tumors. Similarly, 3 of 4 owl monkeys studied by Mclendez et al. (8) were also reinoeulated to induee tumors. A partial resistanee to HVS oneogenesis in owl monkeys was attributed to host faetors. More recently, HVS failed to induce tumors in Callithrix jacchus marmosets and cebus monkeys, but these animals established latent HVS infection (10~ 11). Thus the resistanee to HVS oneogenesis in squirrel monkeys, eommon marmosets, and eebus monkeys, partial
20,000
10,000
o
20 40
60 70 80 100 120 140 160 180 200 300
380
460
540
572
PERIOD OF INFECTION (days)
1.-Total lymphocyte counts of HVS-infected owl monkey 636H and white-lipped marmosets 8411 and 8451.
TEXT-FIGURE
TABLE
2.-HVS isolation [rom, and humoral HVS antibody reeponse of, marmosets inoculated with 636H isolate Virus isolation
Monkey No.
Period of infeetion, days
8421 8541
Oe
8421 8541
21
8421 8541
38
8421 8541
49
8421 8541 (neeropsy) 8421 (neeropsy)
63
83
Humoral HVS antibodies
a
Peripheral blood
Lymph nodes
EAb
LAb
MAb
CFc
Neut
0 0
ND ND
0 0
0 0
0 0
0 0
0 0
0
ND ND
0 0
0
ND ND
ND ND
ND ND
ND ND
+0
+ +
+ +
+ +
ND ND
+0
ND ND
ND ND
ND ND
ND
+0 +
+ + +
+ + +
++ ++ ++
+ + + + + + + +
+ +
+ + + + + + + +
a
Only lyrnph nodes were used for virus isolation either cocultivated with indicator cells or cultured in RPMI-1640.
b
+=~1:1O.
•+=
d
>1:4, with 4 U antigen. The serum neutralized the virus from the inoculated animals' 636H isolate and original HVS inoculated in 636H owl monkey. The degree of neutralization (neut) is expressed as =serum dilution of I: 4 and =8erum dillution of I: 16 which neutralized 10 2 and 10 3 TCID50 of HVS. • 0 =preincubation status of the animals, d
+
++
PERSISTENT HVS IN AN OWL MONKEY
resistance in owl monkeys, and lack of resistance in cotton-topped and white-lipped marmosets could probably be attributed to host factors and their response to this agent. The knowledge and identification of such factors require further investigation. Cicmanec et al. (15) reported that 20-25'% of inoculated owl monkeys did not develop neoplastic disease. Previously reported studies (16) indicated that a second owl monkey (791) intentionally inoculated with a high dose of HVS remained c1inically healthy for over 2 years yet showed c1ear evidence of persistent infection. This owl monkey was inoculated at the same time with apreparation of HVS identical to that given to 3 other owl monkeys, all of which developed lymphoma or lymphoma and leukemia (17). Our studies and those of Molendez et al. (18) 19), Falk et al. (20), and Didier et al. (21) suggest that HVS attenuated either in vitro or in vivo at higher or lower temperatures, or, in another cell system, i.e., dog embryo lung, did not protect the cotton-topped marmosets against the oncogenic effect of HVS; however, the incubation per iod of the disease was prolonged. Generally, attenuation of a virus is referable to the species of the animal or the cell type used. Thus one might have astrain of a virus adapted to or attenuated for a certain species that could be lethai for another. This certainly is the case with HVS in squirrel monkeys and in C. jacchus marmosets, where HVS is nononcogenic; however, it induced tumors in marmosets (10). To assure the safety of a product to be used as a possible protective vaccine against virus-induced neoplasia, one is cautioned to use the most susceptible test system. Thus far, marmosets have been the most suitable animals for testing the safety and potency of HVS products for prevention of oncogenesis (22). The attenuation of herpesviruses, i.e., Marek's disease herpesvirus and mouse cytomegalovirus, in cell culture systems has led to the prevention of tumors or disease (23) 24). The immunity induced by this type of attenuated virus may or may not protect animals inoculated with HVS-induced tumor cells. Killed HVS fails to confer immunity against subsequent challenge with HVSinduced tumor cells (25). The possibility that the 636H isolate may be nononcogenic in owl monkeys or in other susceptible species of nonhuman primates still prevails; however, its oncogenicity and that of other isolates of HVS from owl monkeys (3) calls for caution in their use for development of protective vaccine.
(6)
(i)
(8) (9) (10)
(11) (12) (13) (14)
(15)
(16)
(1i)
(18)
(19)
(20)
(21)
REFERENCES (1) HUNT RD, GARCIA FG, BARAHONA HH, et al: Spontaneous Herpesvirus saimiri lymphoma in owl monkeys. J Infect Dis 127:7~3-725, 1973 (2) HUNT RD, BARAHONA HH, KING NW, et al: Naturally occurring Herpesuirus saimiri leukemia in owl monkeys (Aotus trivirgatus). In Abstracts of Sixth International Symposium on Comparative Leukemia Research. 1973, p 75 (3) RABIN H, NEUBAUER RH, PEARSON GR, et al: Spontaneous lymphoma associated with Herpesvirus saimiri in owl monkeys. J Natl Cancer lost 54:499-502, 1975 (4) ABLASHI DV, PEARSON GR: Animal models: Herpesvirus saimiri, a nonhuman primate model for herpesvirus associated neoplasia of man. Cancer Res 34:1232-1236, 1974 (5) FALK LA, NIGIDA S, DEINHARDT F, et al: Oral excretion of Herpesvirus saimiri in captive squirrel monkeys and inci-
(22) (23)
(2-1)
(25)
1071
dence of infection in feral squirrel monkeys. J Natl Cancer Inst 51:1987-1989,1973 FALK LA, WOLFE LG, DEINHARDT F: Epidemiology of Herpesvirus saimiri infection in squirrel monkeys. In Medical Primatology. ProceeeIings of the Third Conference on Experimental Medical Surgery on Primates, Lyon, 1972 (Goldsmith EI, Moor-jankowski J, eds.), part III. Basel, Karger, 1972, pp 151-158 ABLASHI DV, LOEB WF, VALF.RIO Me, et al: Malignant lymphoma with lymphocytic leukemia in owl monkeys induced by Herpesvirus saimiri. J Natl Cancer Inst 47:837-855, 1971 MELENDEZ LV, HUNT RD, DANIEL MD, et al: Acute Iymphoeyte leukemia in owl monkeys inoculated with Herpesvirus saimiri. Science 171:1161-1163, 1971 WOLFE LG, FALK LA, DEINHARDT F: Oncogenieity of Herpesvirus saimiri in marmoset monkeys. J Natl Cancer Inst 47: 1145-1162, 1971 LAUFS R, STEINKE H, STElNKE G, et al: Latent infeetion and malignant lyrnphoma in marmosets (Callithrix jacchus) after infeetion with two oncogenic herpesviruses from primates. J Natl Cancer Inst 53: 195-199, 1974 RABIN H, PEARSON GR, WALLEN WC, et al: Infection of capuchin monkeys (Cebus albitrons) with Herpesvirus saimiri. J Natl Cancer lost 54:673-677, 1975 ABLASHI DV, ARMSTRONG GR, BLACKHAM EA: Certain characteristies of Herpesvirus saimiri cultured in subhuman primate ceIl cultures. Am J Vet Res 33:1689-1694, 1972 FALK LA: Oncogenic DNA viruses of nonhuman primates. Lab Anim Sei 24:182-192, 1974 HUNT RD, MELENDEZ LV, KING NW, et al: Morthology of a disease with features of malignant lymphoma in marmosets and owl monkeys inoculated with Herpesvirus saimiri, J Natl Cancer Inst 44:447-465, 1970 CICMANEC JL, LOEB WF, VALERIO MC: Lymphoma in owl monkeys (Aotus trivirgatus) inoculated with Herpesvirus saimiri: Clinical, hematologic and pathologie findings. J Meel Primatol 3:8--17, 1974 WALLEN WC, RABIN H, NEUBAUER RH, et al: Depression in lymphocyte response to general mitogens by owl monkeys infected with Herpesvirus saimiri. J Natl Cancer Inst 54:679-685, 1975 PEARSON GR, RABIN H, WALLEN WC, et al: Immunologieal and virological investigations of owl monkeys infected with Herpesvirus saimiri, J Med Primatol 3:54-67, 1974 MELENDEZ LV, HUNT RD, GARCIA FG, et al: Herpesvirus saimiri attenuated by serial passage in dog fetal lung cell line as a vaccine against malignant lymphoma in owl monkeys tAotus trioirgatuss. In Abstracts of Eleventh International Cancer Congress, Florence, 1974 MELf.NDEZ LV, CADWALLADER J, JACKMAN D, et al: In vitro attenuation of Herpesvirus saimiri by serial passage in dog fetal lung (DFL) continuous cultures. In Oncogenesis and Herpesviruses 11 (de-Thc G, Epstein MA, zur Hausen H, cds.). Lyon, France, International Agency for Research on Cancer, 1975 FALK LA, WRIGHT J, DEINHARDT F, et al: Experimental infection of marmoset and squirre1 monkeys with attenuated HVS. Cancer Res 36:707-710, 1976 DIDIER ML, ABLASHI DV, OIE HK, et al: Some biological properties of Herpesvirus saimiri from chronically infected monolayer and suspension cultures. In Oncogenesis and Herpesviruses II (de-Thc G, Epstein MA, zur Hausen H, eds.) , part 1. Lyon, France, International Agency for Research on Cancer, 1975, pp 491-495 LAUFS R: Immunization oE marmoset monkeys with a killed oncogenic herpesvirus. Nature 249:571-572, 1974 CHURCHILL AE, PAYNE LN, CHUBB RC: Immunization against Marek's disease using a live attenuated virus. Nature 211:744-747, 1969 EASTON JM, ABLASHI DV, SIMMONS A, et al: Murine cytomegalovirus: Inhibition of splenomegaly induced by Rauscher plasma virus in mice. In Proceedings of the American Society for Mierobiology, 1970, p 159 LAUFS R, STEINKE H: Vaccination of nonhuman primates with killed oncogenic herpesviruses, Cancer Res 36:704-706, 1976
2
Gary R. Armstrong,3,4 Thomas Orr,5 Dharam V. Ablashi,3 Gary R. Pearson,> Harvey Rabin,6 Josef tuetzeler,' Walter F. loeb,6 and Marion G. Valerio 6 SUMMARY-An adult owl monkey (Aotus trivirgatus) used for immunologie 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 eoeultivation of peripheral blood for over 1 year. No elinieal, gross, or histopathologie findings of malignaney were exhibited by the animal. The HVS isolate from the animal was indistinguishable biologieally and serologieally from the original HVS strain of Melimdez and from an isolate of an experimentally HVS-indueed tumor. Inoeulation of this isolate into 2 young white-Iipped marmosets (Saguinus fuscieollis) produeed typieal malignant lymphoma and Iymphoeytie leukemia. Our findings suggested that the virus from the ehronieally infeeted animal was oneogenie and that host faetors were primarily responsible for determining the disease manifestation of the virus infeetion. Another owl monkey ehronieally infected with HVS for over 2 years has remained asymptomatie.-J Natl Cancer Inst 56: 1069-1071, 1976.
Two independent reports on spontaneous malignant lymphoma andjor leukemia in owl monkeys (Aotus trivirgatus) suggest the horizontal transmission of Herpesvirus saimiri (HVS) from squirrel monkeys (Saimiri seiureus) (1~ 3). Though squirrel monkeys are the natural hosts of HVS, no tumors were reported in this species that could be related to HVS infection (4-6). However, inoculations of HVS into ow1 monkeys or marmosets have regularly induced tumors (4, 7-9). In this communication, we report on some biologie properties of an isolate from a chronically infected disease-free owl monkey. To our knowledge, in vitro and in vivo properties of an HVS isolate from a chronic infection in an owl monkey have not been detailed, but similar studies with nonhuman primate species that are not susceptible to HVS-related disease have been described (10~ 11). MATERIALS AND METHODS
An adult male owl monkey (636H), received by Litton Bionetics, Ine. and held in quarantine for 3-4 months in a wire cage with a Kirschner box with other owl monkeys, was used as a control in another program. This animal was transferred to a Landon cage within a biohazard area before receiving six weekly inoculations of 1.0 ml of virus-free VERO cellfree extract. Although it is possible that the monkey was mistakenly inoculated with HVS, it is also possible that it was inadvertently infected by contact. The virus isolation from the peripheral blood of infected animals was described in (4~ 7). For detection of antibodies to HVS early antigens (EA), late antigens (LA), and membrane antigens (MA), HVS-infected VERO cells were used according to procedures described in (4). For complement-fixation (CF) antibodies to HVS, the method of Ablashi et al. (12) was used. Serum neutralization (SN) tests with 102 and 103 mean tissue culture infective doses (TCID50)jml HVS were used. The goat anti-HVS serum, supplied by Dr. L~is Melend~z, Pan American J:Iealt~ Organization, Washmgton, D.C., was used for the identification of the HVS isolated from the owl monkey and
from 2 young female white-lipped marmosets (Saguinus fuseieollis, 8541 and 8421) receiving the HVS isolate from owl monkey 636H. The SN test was conducted by the use of equal amounts of heat-inactivated HVS antiserum mixed with different concentrations of HVS, incubated at 4° C overnight, and inoculated in owl monkey kidney (OMK) cells (7). Two marmosets were each inoculated with 105 TC1D50 in 1.0 ml of HVS isolated from owl monkey 636H. Blood was collected from each of the monkeys (636H, 8541, and 8421) before inoculation and at regular intervals thereafter for hematologic examination, virus isolation, and antibody determination. After the monkeys died or were killed, the tissues were processed for histopathology and virus isolation. RESUlTS
Before the study, owl monkey 636H had neither HVS nor HVS-directed antibodies. This was demonstrated by the absence of HVS when peripheral blood was cocultiva ted with OMK and VERO cells and by the absence of immunoßuorescent (IF) or neutralizing antibody in the preinoculation serum (table 1). These findings suggest that the animal was not exposed to HVS before the study. The monkeys demonstrated the presence of HVS infection by a) release of HVS from circulating lymphoeytes from the peripheral blood and b) development of humoral antibodies to various HVS antigens (table 1) throughout the experiment. After the onset of the experiment (table 1), the monkey developed antibodies to HVS antigens (as determined by 1F and CF); two serum sampIes collected at different times, i.e., the seed stock HVS supplied originally by Dr. Melendez and the one isolated from the animal's own blood, neutralized HVS. Thus far all the data on HVS serology indicate that HVS does not cross-react extensively with other oncogenic (13) herpesviruses, and we examined the HVS from 636H monkey by using more than one assay (table 1). Throughout the experiment, the hemograms of monkey 636H were essentially normal (text-fig. 1), and blood smears prepared at various times gave no indication of cell abnormalities. The monkey was infected with typical HVS, but lack of tumor induction suggested the possibility of in vivo attenuation of the virus. The animal was killed 19 months after the beginning of the study, and organs and tissues were collected for microscopic ex amReceived August 27, 1975; accepted November 21, 1975. 2Supported in part by Public Health Service contract NOI CP43224 within the Virus-Cancer Program of the National Cancer Institute. 3 Viral Leukemia and Lymphoma Branch, National Cancer In~ti tute (NCI), National Institutes of Health, Public Health Service, V.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 4 Address reprint requests to Mr. Armstrong, NCI-Frederick Cancer Research Center, Fort Detrick, Frederick, Md. 21701. 5 Mayo Clinic, Plummer Building, Room 442, Rochester, Minn. 55901. 6 Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Md. 20795. 7 Visiting Associate at the NCI from the Pathology Institute of Cologne, Germany. 1
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 56, NO. 5, MAY 1976
1069
1070
ARMSTRONG ET AL.
TABLE
l.-HVS isolation [rom, and humoral antibody response oj, owl monkey 636H
Observati on days
Virus isolation a from peripheral blood
01 18 54 61 118 353 572 (terminal)s
Humoral HVS antibodies EAc LAc MAc CF 0 0
0
+ + + + + +
+ + + + +
0
+ + + + + +
ND ND ND ND ND
d
e
0 ND ND
+ + + +
+ +
b
Neut
0 ND ND
ination and virus isolations. Cultures prepared at neeropsy from various lymph nodes eontained HVS, but eell-free extracts from these lymphatie tissues or ultrastruetural examination did not reveal any infeetious virus or virus particles, respeetively. Histopathologie examination of this animal failed to show any form of neoplasia. The possibility that the 636H isolate of HVS was nononeogenie was explored in a sensitive animal system. Two white-lipped marmosets (8411 and 8541) free of HVS and HVS antibodies (table 2) were inoeulated with this virus. Both responded similarly to inoeulation in beeoming viremic and developing antibodies to HVS; HVS could be isolated from their peripheral lymphoeytes. No signs of illness were c1inieally evident. Seventy and 42 days, respeetively, after inoeulation, there was a marked rise in lymphocytes with atypieal forms (text-fig. I), and 2 weeks later, the animals were frankly leukemic. The animals died 84 and 63 days, respeetively, after inoeulation. The 636H isolate of HVS and those isolated from the 2 marmosets showed identieal patterns of cytopathic effeet and infeetivity titers in VERO andjor OMK eells. The gross and mieroseopie lesions in both marmosets were indistinguishable from those deseribed in (9~ 14); however, the magnitude of lymphoeytic proliferation was greater in 8541 than in 8421. The HVS isolate from both animals was neutralized by goat antiserum to HVS, which eonfirmed the identity of the isolate.
+ ++ ++ +
a All virus isolation from either peripheral blood or tissue collected at necropsy was done by cocultivation with indicator celIs. b ND =not done.
• + = ~I:IO. d + = ~1:4. with 4 U antigen. • Serum neutralized the 636H isolate of HVS and the original HVS used for 636H inoculation. The degree of neutralizing (neut) is expressed as =serum dilution of I: 4 and =serum dilution of I: 16 which neutralized 10 2 and 10 3 TCID50 of HVS. f Day 0 serum was tested undilu ted and at a dilution of I: 2. 11 Tissues colIected at necropsy for virus isolation were lymphatic tissues from the lymphatic system.
+
++
LYMPHOCYTE COUNT
70,000
- - 636H
I
60,000
---·8411 _ . - 8541
I
I
50,000
DISCUSSION
!
40,000 -
30,000
In our previous experiments (7), 4 of 5 owl monkeys reeeiving one HVS inoeulation Iailed to develop evidenee of leukemia or lymphoma in 2-4 months. These animals were reinoeulated and later developed tumors. Similarly, 3 of 4 owl monkeys studied by Mclendez et al. (8) were also reinoeulated to induee tumors. A partial resistanee to HVS oneogenesis in owl monkeys was attributed to host faetors. More recently, HVS failed to induce tumors in Callithrix jacchus marmosets and cebus monkeys, but these animals established latent HVS infection (10~ 11). Thus the resistanee to HVS oneogenesis in squirrel monkeys, eommon marmosets, and eebus monkeys, partial
20,000
10,000
o
20 40
60 70 80 100 120 140 160 180 200 300
380
460
540
572
PERIOD OF INFECTION (days)
1.-Total lymphocyte counts of HVS-infected owl monkey 636H and white-lipped marmosets 8411 and 8451.
TEXT-FIGURE
TABLE
2.-HVS isolation [rom, and humoral HVS antibody reeponse of, marmosets inoculated with 636H isolate Virus isolation
Monkey No.
Period of infeetion, days
8421 8541
Oe
8421 8541
21
8421 8541
38
8421 8541
49
8421 8541 (neeropsy) 8421 (neeropsy)
63
83
Humoral HVS antibodies
a
Peripheral blood
Lymph nodes
EAb
LAb
MAb
CFc
Neut
0 0
ND ND
0 0
0 0
0 0
0 0
0 0
0
ND ND
0 0
0
ND ND
ND ND
ND ND
ND ND
+0
+ +
+ +
+ +
ND ND
+0
ND ND
ND ND
ND ND
ND
+0 +
+ + +
+ + +
++ ++ ++
+ + + + + + + +
+ +
+ + + + + + + +
a
Only lyrnph nodes were used for virus isolation either cocultivated with indicator cells or cultured in RPMI-1640.
b
+=~1:1O.
•+=
d
>1:4, with 4 U antigen. The serum neutralized the virus from the inoculated animals' 636H isolate and original HVS inoculated in 636H owl monkey. The degree of neutralization (neut) is expressed as =serum dilution of I: 4 and =8erum dillution of I: 16 which neutralized 10 2 and 10 3 TCID50 of HVS. • 0 =preincubation status of the animals, d
+
++
PERSISTENT HVS IN AN OWL MONKEY
resistance in owl monkeys, and lack of resistance in cotton-topped and white-lipped marmosets could probably be attributed to host factors and their response to this agent. The knowledge and identification of such factors require further investigation. Cicmanec et al. (15) reported that 20-25'% of inoculated owl monkeys did not develop neoplastic disease. Previously reported studies (16) indicated that a second owl monkey (791) intentionally inoculated with a high dose of HVS remained c1inically healthy for over 2 years yet showed c1ear evidence of persistent infection. This owl monkey was inoculated at the same time with apreparation of HVS identical to that given to 3 other owl monkeys, all of which developed lymphoma or lymphoma and leukemia (17). Our studies and those of Molendez et al. (18) 19), Falk et al. (20), and Didier et al. (21) suggest that HVS attenuated either in vitro or in vivo at higher or lower temperatures, or, in another cell system, i.e., dog embryo lung, did not protect the cotton-topped marmosets against the oncogenic effect of HVS; however, the incubation per iod of the disease was prolonged. Generally, attenuation of a virus is referable to the species of the animal or the cell type used. Thus one might have astrain of a virus adapted to or attenuated for a certain species that could be lethai for another. This certainly is the case with HVS in squirrel monkeys and in C. jacchus marmosets, where HVS is nononcogenic; however, it induced tumors in marmosets (10). To assure the safety of a product to be used as a possible protective vaccine against virus-induced neoplasia, one is cautioned to use the most susceptible test system. Thus far, marmosets have been the most suitable animals for testing the safety and potency of HVS products for prevention of oncogenesis (22). The attenuation of herpesviruses, i.e., Marek's disease herpesvirus and mouse cytomegalovirus, in cell culture systems has led to the prevention of tumors or disease (23) 24). The immunity induced by this type of attenuated virus may or may not protect animals inoculated with HVS-induced tumor cells. Killed HVS fails to confer immunity against subsequent challenge with HVSinduced tumor cells (25). The possibility that the 636H isolate may be nononcogenic in owl monkeys or in other susceptible species of nonhuman primates still prevails; however, its oncogenicity and that of other isolates of HVS from owl monkeys (3) calls for caution in their use for development of protective vaccine.
(6)
(i)
(8) (9) (10)
(11) (12) (13) (14)
(15)
(16)
(1i)
(18)
(19)
(20)
(21)
REFERENCES (1) HUNT RD, GARCIA FG, BARAHONA HH, et al: Spontaneous Herpesvirus saimiri lymphoma in owl monkeys. J Infect Dis 127:7~3-725, 1973 (2) HUNT RD, BARAHONA HH, KING NW, et al: Naturally occurring Herpesuirus saimiri leukemia in owl monkeys (Aotus trivirgatus). In Abstracts of Sixth International Symposium on Comparative Leukemia Research. 1973, p 75 (3) RABIN H, NEUBAUER RH, PEARSON GR, et al: Spontaneous lymphoma associated with Herpesvirus saimiri in owl monkeys. J Natl Cancer lost 54:499-502, 1975 (4) ABLASHI DV, PEARSON GR: Animal models: Herpesvirus saimiri, a nonhuman primate model for herpesvirus associated neoplasia of man. Cancer Res 34:1232-1236, 1974 (5) FALK LA, NIGIDA S, DEINHARDT F, et al: Oral excretion of Herpesvirus saimiri in captive squirrel monkeys and inci-
(22) (23)
(2-1)
(25)
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dence of infection in feral squirrel monkeys. J Natl Cancer Inst 51:1987-1989,1973 FALK LA, WOLFE LG, DEINHARDT F: Epidemiology of Herpesvirus saimiri infection in squirrel monkeys. In Medical Primatology. ProceeeIings of the Third Conference on Experimental Medical Surgery on Primates, Lyon, 1972 (Goldsmith EI, Moor-jankowski J, eds.), part III. Basel, Karger, 1972, pp 151-158 ABLASHI DV, LOEB WF, VALF.RIO Me, et al: Malignant lymphoma with lymphocytic leukemia in owl monkeys induced by Herpesvirus saimiri. J Natl Cancer Inst 47:837-855, 1971 MELENDEZ LV, HUNT RD, DANIEL MD, et al: Acute Iymphoeyte leukemia in owl monkeys inoculated with Herpesvirus saimiri. Science 171:1161-1163, 1971 WOLFE LG, FALK LA, DEINHARDT F: Oncogenieity of Herpesvirus saimiri in marmoset monkeys. J Natl Cancer Inst 47: 1145-1162, 1971 LAUFS R, STEINKE H, STElNKE G, et al: Latent infeetion and malignant lyrnphoma in marmosets (Callithrix jacchus) after infeetion with two oncogenic herpesviruses from primates. J Natl Cancer Inst 53: 195-199, 1974 RABIN H, PEARSON GR, WALLEN WC, et al: Infection of capuchin monkeys (Cebus albitrons) with Herpesvirus saimiri. J Natl Cancer lost 54:673-677, 1975 ABLASHI DV, ARMSTRONG GR, BLACKHAM EA: Certain characteristies of Herpesvirus saimiri cultured in subhuman primate ceIl cultures. Am J Vet Res 33:1689-1694, 1972 FALK LA: Oncogenic DNA viruses of nonhuman primates. Lab Anim Sei 24:182-192, 1974 HUNT RD, MELENDEZ LV, KING NW, et al: Morthology of a disease with features of malignant lymphoma in marmosets and owl monkeys inoculated with Herpesvirus saimiri, J Natl Cancer Inst 44:447-465, 1970 CICMANEC JL, LOEB WF, VALERIO MC: Lymphoma in owl monkeys (Aotus trivirgatus) inoculated with Herpesvirus saimiri: Clinical, hematologic and pathologie findings. J Meel Primatol 3:8--17, 1974 WALLEN WC, RABIN H, NEUBAUER RH, et al: Depression in lymphocyte response to general mitogens by owl monkeys infected with Herpesvirus saimiri. J Natl Cancer Inst 54:679-685, 1975 PEARSON GR, RABIN H, WALLEN WC, et al: Immunologieal and virological investigations of owl monkeys infected with Herpesvirus saimiri, J Med Primatol 3:54-67, 1974 MELENDEZ LV, HUNT RD, GARCIA FG, et al: Herpesvirus saimiri attenuated by serial passage in dog fetal lung cell line as a vaccine against malignant lymphoma in owl monkeys tAotus trioirgatuss. In Abstracts of Eleventh International Cancer Congress, Florence, 1974 MELf.NDEZ LV, CADWALLADER J, JACKMAN D, et al: In vitro attenuation of Herpesvirus saimiri by serial passage in dog fetal lung (DFL) continuous cultures. In Oncogenesis and Herpesviruses 11 (de-Thc G, Epstein MA, zur Hausen H, cds.). Lyon, France, International Agency for Research on Cancer, 1975 FALK LA, WRIGHT J, DEINHARDT F, et al: Experimental infection of marmoset and squirre1 monkeys with attenuated HVS. Cancer Res 36:707-710, 1976 DIDIER ML, ABLASHI DV, OIE HK, et al: Some biological properties of Herpesvirus saimiri from chronically infected monolayer and suspension cultures. In Oncogenesis and Herpesviruses II (de-Thc G, Epstein MA, zur Hausen H, eds.) , part 1. Lyon, France, International Agency for Research on Cancer, 1975, pp 491-495 LAUFS R: Immunization oE marmoset monkeys with a killed oncogenic herpesvirus. Nature 249:571-572, 1974 CHURCHILL AE, PAYNE LN, CHUBB RC: Immunization against Marek's disease using a live attenuated virus. Nature 211:744-747, 1969 EASTON JM, ABLASHI DV, SIMMONS A, et al: Murine cytomegalovirus: Inhibition of splenomegaly induced by Rauscher plasma virus in mice. In Proceedings of the American Society for Mierobiology, 1970, p 159 LAUFS R, STEINKE H: Vaccination of nonhuman primates with killed oncogenic herpesviruses, Cancer Res 36:704-706, 1976
