Int. J. Cancer: 17,785-788 (1976)
EPSTEIN-BARR VIRUS: EXPERIMENTAL INFECTION OF CALLITHRIX JACCHUS MARMOSETS Lawrence FALK', Friedrich
DEINHARDT
I,
Lauren WOLFEI , Donald JOHNSON
l,
Jo HILCERS ' and Guy
DE-THE
Departments of Microbiology, Rush-Presbyterian-St. Luke's and University of Illinois Medical Centers, Chicago, Illinois, USA; Department of Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; arid International Agency for Research on Cancer, Lyons, France
SUMMARY
Eight common marmoset monkeys (Callithrix jacchus) were inoculated with about lo4 transforming units of B95-8 virus; seven of the marmosets died 50I I I days post inoculation and all seven showed microscopic and/or macroscopic lesions compatible with a diagnosis of lymphoproliferafive disease. Low levels of anti- VCA antibodies were detected in plasma from six marmosets. Attempts failed to establish continuous EB V-carrying lyrnphoblastoid cell cultures by cultivation in vitro of circulating lymphocytes or minced lymphoid tissues obtained at necropsy.
Epstein-Barr virus (EBV) is the cause of heterophile-positive infectious mononucleosis and most likely an essential factor in the development of the majority of African Burkitt's lymphomas and nasopharyngeal carcinomas (for current summaries see zur Hausen, et al., 1975). The close association between EBV and these diseases has been based upon : ( I ) extensive seroepidemiological studies; ( 2 ) demonstration of EBV DNA in tumor cells; (3) presence of nuclear antigens and/or membrane antigens in the tumor cells; and (4) ability of EBV to transform human and simian lymphocytes in vitro into continuous lymphoblastoid cell lines. Furthermore, EBV produced by certain EBV-carrying lymphoblastoid cell cultures has induced lymphoproliferative disease after experimental infection of cotton-topped marmosets (Saguinus sp.) and owl (Aotus sp.) monkeys (Shope et al., 1973; Epstein et ol., 1973; Falk et al., 19746; Deinhardt et al., 19746; zur Hausen, personal communication). We describe here experimental infection of common marmosets (Callithrix jacchus) with EBV, strain B95-8. and discuss the lesions induced. MATERIAL A N D METHODS
Cell cultures and virus titration
The EBV-transformed cotton-topped marmoset cell line, B95-8 (Miller et al., 1972), was cultured in medium RPMI 1640 supplemented with 15% heatinactivated fetal calf serum, 100 IU penicillin and 50 pg streptomycin/ml. Supernatant fluids were
collected from cultured B95-8 cells when about 7-8 % of the cells were positive for viral capsid antigens (VCA) as determined by indirect immunofluorescence tests. The supernatants were clarified by centrifugation and a small amount of Escherichia coli was mixed with the supernatant which was then passed through 0.45 ,urn pore size filter (Nalge, Rochester, N.Y., USA) pretreated with bovine serum albumin. One ml samples were mixed with thioglycollate medium before and after filtration and assayed for bacterial growth. Filtered virus was stored in 1 ml aliquots at -70" C. Virus stock was assayed by the method of Miller and Lipman (1973) for ability to transform human umbilical cord blood lymphocytes; titers of virus stock were expressed as transforming units (TU). The virus stock used in the present experiments titered lo* TU/ml. A ninials
Eight adult common marmosets (Callithrix jacchus), born in the primate center at the Netherlands Cancer Institute, Amsterdam, were inoculated with about lo4TU of B95-8 virus [three cotton-topped marmosets (Saguinus oedipus), inoculated with this stock preparation, had developed malignant lymphoma (Falk et al., 19746; Deinhardt e f al., 1974b)J.Inoculations were given intravenously (femoral vein), intramuscularly (thigh) and intraperitoneally; the total inoculation volume per animal was about 1 ml distributed equally at the three inoculation sites. Four marmosets (see Table I) were immunosuppressed by daily injections of horse antihuman thymocyte serum (Upjohn Co., Kalamazoo, Michigan, USA) at a dose of 100 mg/kg for fourteen days following virus inoculation. Blood samples were collected into heparinized syringes before virus inoculation and at weekly intervals post inoculation (PI) for hematologic and serologic studies and for attempts to establish continuous lymphoblastoid cell cultures. Necropsies were performed on seven animals and representative sections of selected organs were fixed Received: December 29, 1975, and in revised form April 6, 1976.
786
FALK ET AL.
in 10% phosphate-buffered formalin. All tissues were embedded in paraffin, sectioned at 3-6pm and stained with hematoxylin and eosin. Stains and histochemical reactions used on selected tissues included Giemsa, periodic acid-Schiff and methyl green-pyronin. Serologic studies
Plasma samples obtained before and after virus inoculation were evaluated for antibodies to EBVspecified viral capsid antigens, EBV nuclear antigens and early antigens (VCA, EBNA and EA) by indirect immunofluorescence tests. To test for VCA antibodies acetone-fixed smears of HR-I cells were used. The anti-complement (ACIF) test described first by Reedman and Klein (1973) was used as modified by Henle et a/. (1974) for the detection of EBNA antibodies. The source of complement was human serum (VCA < 1 :4) diluted 1 :4 and the conjugate was goat anti-human C3 (B,C/B,A-globulin) (Hyland, Costa Mesa, Calif., USA), diluted 1 :20.The EA test was performed on Raji cells superinfected with HR-I virus according to the method of Henle e t a / . (1970). Serum samples were also examined for heterophile antibodies: sera were tested in serial two-fold dilutions, beginning at a 1:4 dilution, for sheep erythrocyte agglutinins. Cultivation of lymphocytes and tissues
Lymphocytes were separated from whole blood samples collected at weekly intervals PI ;whole blood was centrifuged over Ficoll-Hypaque and after washing the collected lymphocytes were cultured in medium RPMI 1640 (supplemented with 15% heatinactivated fetal calf serum, 100 IU penicillin and 50 pg streptomycinlml) in small plastic flasks. Tissues collected at necropsy (spleen, lymph nodes and thymus) were minced into small fragments and cultured in medium RPMI 1640 in small flasks. All cell cultures received 50% (v/v) renewal of culture medium at 4- to 5-day intervals. RESULTS
Seven of eight common marmosets inoculated with about lo4TU of B95-8 virus died 50-1 11 days PI; six of the marmosets died within 50-54 days PI (Table I). The one surviving marmoset has remained clinically well for over 1 year PI.
Microscopic lesions of a lymphoproliferative disease were found in all seven animals. Lymph nodes were characterized by diffuse hyperplasia, sometimes present simultaneously with follicular hyperplasia. The diffuse hyperplastic lesions were composed of a heterogeneous population of cells, namely small lymphocytes, lymphoblasts, immunoblasts and cells with a plasmacytoid appearance. The predominance of individual cell types was variable among animals and sometimes among nodes of the same animal. Numerous mitotic figures, sinusoidal filling with cells, and increased vascularity were constant features ; capsular and perinodal infiltrations were frequent findings. The degree of distortion of normal cytoarchitecture was variable depending on t h e intensity of the reaction. The normal cytoarchitecture of thymuses was replaced by a dense distribution of the same mixed population of cells as described for the lymph nodes; cellular infiltrates in thymic capsules and surrounding fat were more prominent than those associated with lymph nodes. Spleens were characterized by follicular hyperplasia, usually mild, and by the presence of a few lymphoblasts and plasmacytoid cells within sinusoids. Lymphoid infiltrates of variable intensity were consistent features in perivascular and interstitial tissues of kidney cortices and salivary glands and prominent surrounding bronchi, bronchioles and pulmonary vessels. Similar lesions, when present in other tissues, were relatively mild. The cause of death was attributed to pneumonia in two animals, enteritis in one animal and was undetermined in four animals. Evaluation of tissues in vitro
Tissues were collected at necropsy for cultivation, for preparation of impression smears for EBNA staining and for molecular hybridization studies. Minced tissues (thymus, spleen and lymph nodes) from five marmosets were cultivated in vitro for up to 3 months but failed to grow after this time. Impression smears of liver, kidney, spleen and lymph nodes were prepared from three marmosets and stained for EBNA but no specific staining was observed in any of the preparations. Tissues (heart, muscle, spleen, liver, lymph nodes and lung) for molecular hybridization studies were collected from two marmosets and results from these experiments will be reported later. Serologic studies
Pathologic findings
Peripheral lymphadenopathy, enlarged thymus and mild splenomegaly were consistent macroscopic features. Focal grayish mottling of kidney cortices and enlarged salivary glands were observed in two animals and pneumonia or enteritis was present in one animal.
Six of the eight marmosets developed anti-VCA antibodies which were first detected 30-46 days PI (Table I) ; five marmosets developed maximal titers of 1 :4 and marmoset No. 5794 which was killed 11 1 days PI had a VCA titer of 1 :16. Marmoset No. 5800 had an anti-VCA titer of 1:4, 46 days PI, that persisted for about 2 months, at which time no
787
EXPERIMENTAL EBV INFECTION IN MARMOSETS TABLE I INOCULATION A N D CLINICAL DATA OF CALLlTHRIX JACCHKJS INOCULATED WITH EBV. STRAIN B95-8 Marmoset number
4446 5794 5795 5796 5797 5798 5799 5800
Immunosuppression
Antibodies VCA
Yes
Yes Yes Yes No No No NO
EA
EBNA
EPSTEIN-BARR VIRUS: EXPERIMENTAL INFECTION OF CALLITHRIX JACCHUS MARMOSETS Lawrence FALK', Friedrich
DEINHARDT
I,
Lauren WOLFEI , Donald JOHNSON
l,
Jo HILCERS ' and Guy
DE-THE
Departments of Microbiology, Rush-Presbyterian-St. Luke's and University of Illinois Medical Centers, Chicago, Illinois, USA; Department of Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; arid International Agency for Research on Cancer, Lyons, France
SUMMARY
Eight common marmoset monkeys (Callithrix jacchus) were inoculated with about lo4 transforming units of B95-8 virus; seven of the marmosets died 50I I I days post inoculation and all seven showed microscopic and/or macroscopic lesions compatible with a diagnosis of lymphoproliferafive disease. Low levels of anti- VCA antibodies were detected in plasma from six marmosets. Attempts failed to establish continuous EB V-carrying lyrnphoblastoid cell cultures by cultivation in vitro of circulating lymphocytes or minced lymphoid tissues obtained at necropsy.
Epstein-Barr virus (EBV) is the cause of heterophile-positive infectious mononucleosis and most likely an essential factor in the development of the majority of African Burkitt's lymphomas and nasopharyngeal carcinomas (for current summaries see zur Hausen, et al., 1975). The close association between EBV and these diseases has been based upon : ( I ) extensive seroepidemiological studies; ( 2 ) demonstration of EBV DNA in tumor cells; (3) presence of nuclear antigens and/or membrane antigens in the tumor cells; and (4) ability of EBV to transform human and simian lymphocytes in vitro into continuous lymphoblastoid cell lines. Furthermore, EBV produced by certain EBV-carrying lymphoblastoid cell cultures has induced lymphoproliferative disease after experimental infection of cotton-topped marmosets (Saguinus sp.) and owl (Aotus sp.) monkeys (Shope et al., 1973; Epstein et ol., 1973; Falk et al., 19746; Deinhardt et al., 19746; zur Hausen, personal communication). We describe here experimental infection of common marmosets (Callithrix jacchus) with EBV, strain B95-8. and discuss the lesions induced. MATERIAL A N D METHODS
Cell cultures and virus titration
The EBV-transformed cotton-topped marmoset cell line, B95-8 (Miller et al., 1972), was cultured in medium RPMI 1640 supplemented with 15% heatinactivated fetal calf serum, 100 IU penicillin and 50 pg streptomycin/ml. Supernatant fluids were
collected from cultured B95-8 cells when about 7-8 % of the cells were positive for viral capsid antigens (VCA) as determined by indirect immunofluorescence tests. The supernatants were clarified by centrifugation and a small amount of Escherichia coli was mixed with the supernatant which was then passed through 0.45 ,urn pore size filter (Nalge, Rochester, N.Y., USA) pretreated with bovine serum albumin. One ml samples were mixed with thioglycollate medium before and after filtration and assayed for bacterial growth. Filtered virus was stored in 1 ml aliquots at -70" C. Virus stock was assayed by the method of Miller and Lipman (1973) for ability to transform human umbilical cord blood lymphocytes; titers of virus stock were expressed as transforming units (TU). The virus stock used in the present experiments titered lo* TU/ml. A ninials
Eight adult common marmosets (Callithrix jacchus), born in the primate center at the Netherlands Cancer Institute, Amsterdam, were inoculated with about lo4TU of B95-8 virus [three cotton-topped marmosets (Saguinus oedipus), inoculated with this stock preparation, had developed malignant lymphoma (Falk et al., 19746; Deinhardt e f al., 1974b)J.Inoculations were given intravenously (femoral vein), intramuscularly (thigh) and intraperitoneally; the total inoculation volume per animal was about 1 ml distributed equally at the three inoculation sites. Four marmosets (see Table I) were immunosuppressed by daily injections of horse antihuman thymocyte serum (Upjohn Co., Kalamazoo, Michigan, USA) at a dose of 100 mg/kg for fourteen days following virus inoculation. Blood samples were collected into heparinized syringes before virus inoculation and at weekly intervals post inoculation (PI) for hematologic and serologic studies and for attempts to establish continuous lymphoblastoid cell cultures. Necropsies were performed on seven animals and representative sections of selected organs were fixed Received: December 29, 1975, and in revised form April 6, 1976.
786
FALK ET AL.
in 10% phosphate-buffered formalin. All tissues were embedded in paraffin, sectioned at 3-6pm and stained with hematoxylin and eosin. Stains and histochemical reactions used on selected tissues included Giemsa, periodic acid-Schiff and methyl green-pyronin. Serologic studies
Plasma samples obtained before and after virus inoculation were evaluated for antibodies to EBVspecified viral capsid antigens, EBV nuclear antigens and early antigens (VCA, EBNA and EA) by indirect immunofluorescence tests. To test for VCA antibodies acetone-fixed smears of HR-I cells were used. The anti-complement (ACIF) test described first by Reedman and Klein (1973) was used as modified by Henle et a/. (1974) for the detection of EBNA antibodies. The source of complement was human serum (VCA < 1 :4) diluted 1 :4 and the conjugate was goat anti-human C3 (B,C/B,A-globulin) (Hyland, Costa Mesa, Calif., USA), diluted 1 :20.The EA test was performed on Raji cells superinfected with HR-I virus according to the method of Henle e t a / . (1970). Serum samples were also examined for heterophile antibodies: sera were tested in serial two-fold dilutions, beginning at a 1:4 dilution, for sheep erythrocyte agglutinins. Cultivation of lymphocytes and tissues
Lymphocytes were separated from whole blood samples collected at weekly intervals PI ;whole blood was centrifuged over Ficoll-Hypaque and after washing the collected lymphocytes were cultured in medium RPMI 1640 (supplemented with 15% heatinactivated fetal calf serum, 100 IU penicillin and 50 pg streptomycinlml) in small plastic flasks. Tissues collected at necropsy (spleen, lymph nodes and thymus) were minced into small fragments and cultured in medium RPMI 1640 in small flasks. All cell cultures received 50% (v/v) renewal of culture medium at 4- to 5-day intervals. RESULTS
Seven of eight common marmosets inoculated with about lo4TU of B95-8 virus died 50-1 11 days PI; six of the marmosets died within 50-54 days PI (Table I). The one surviving marmoset has remained clinically well for over 1 year PI.
Microscopic lesions of a lymphoproliferative disease were found in all seven animals. Lymph nodes were characterized by diffuse hyperplasia, sometimes present simultaneously with follicular hyperplasia. The diffuse hyperplastic lesions were composed of a heterogeneous population of cells, namely small lymphocytes, lymphoblasts, immunoblasts and cells with a plasmacytoid appearance. The predominance of individual cell types was variable among animals and sometimes among nodes of the same animal. Numerous mitotic figures, sinusoidal filling with cells, and increased vascularity were constant features ; capsular and perinodal infiltrations were frequent findings. The degree of distortion of normal cytoarchitecture was variable depending on t h e intensity of the reaction. The normal cytoarchitecture of thymuses was replaced by a dense distribution of the same mixed population of cells as described for the lymph nodes; cellular infiltrates in thymic capsules and surrounding fat were more prominent than those associated with lymph nodes. Spleens were characterized by follicular hyperplasia, usually mild, and by the presence of a few lymphoblasts and plasmacytoid cells within sinusoids. Lymphoid infiltrates of variable intensity were consistent features in perivascular and interstitial tissues of kidney cortices and salivary glands and prominent surrounding bronchi, bronchioles and pulmonary vessels. Similar lesions, when present in other tissues, were relatively mild. The cause of death was attributed to pneumonia in two animals, enteritis in one animal and was undetermined in four animals. Evaluation of tissues in vitro
Tissues were collected at necropsy for cultivation, for preparation of impression smears for EBNA staining and for molecular hybridization studies. Minced tissues (thymus, spleen and lymph nodes) from five marmosets were cultivated in vitro for up to 3 months but failed to grow after this time. Impression smears of liver, kidney, spleen and lymph nodes were prepared from three marmosets and stained for EBNA but no specific staining was observed in any of the preparations. Tissues (heart, muscle, spleen, liver, lymph nodes and lung) for molecular hybridization studies were collected from two marmosets and results from these experiments will be reported later. Serologic studies
Pathologic findings
Peripheral lymphadenopathy, enlarged thymus and mild splenomegaly were consistent macroscopic features. Focal grayish mottling of kidney cortices and enlarged salivary glands were observed in two animals and pneumonia or enteritis was present in one animal.
Six of the eight marmosets developed anti-VCA antibodies which were first detected 30-46 days PI (Table I) ; five marmosets developed maximal titers of 1 :4 and marmoset No. 5794 which was killed 11 1 days PI had a VCA titer of 1 :16. Marmoset No. 5800 had an anti-VCA titer of 1:4, 46 days PI, that persisted for about 2 months, at which time no
787
EXPERIMENTAL EBV INFECTION IN MARMOSETS TABLE I INOCULATION A N D CLINICAL DATA OF CALLlTHRIX JACCHKJS INOCULATED WITH EBV. STRAIN B95-8 Marmoset number
4446 5794 5795 5796 5797 5798 5799 5800
Immunosuppression
Antibodies VCA
Yes
Yes Yes Yes No No No NO
EA
EBNA
