Inhibition of Bovine Leukemia Virus Release 1,2 M. Onuma, C. Olson, and L. E. Baumgartener 3,4 SUMMARY-Sera from 3 cows with the adult form of lymphosarcoma inhibited release of leukemia virus from a cell line of fetal lamb spleen infected with bovine leukemia virus (BlV). Sera from 5 or 7 cattle experimentally infected with BlV also suppressed virus release. The inhibition of virus release was reversible. Sera from cattle with the calf form and the thymic form of lymphosarcoma and normal bovine control sera did not repress virus release.-J Natl Cancer Inst 54:1199-1202, 1975.
A BOVINE (C-TYPE) leukemia virus (BLV) can be demonstrated in short-term cultures of mitogenstimulated lymphoid cells of cattle with the adult form of lymphosarcoma (1) and from cattle experimentally and naturally infected with BLV (2). The virus is shed from the cells and can be seen extracellularly by electron microscopy (1) or shown by a precipitin reaction with specific antiserum in gel immunodiffusion (3) and single radial immunodiffusion (SRID) of an ether-treated antigen preparation in agar gel containing antibody. The antibody also can be demonstrated by an immunodiffusion (ID) test (4, 5), an immunofluorescence (IF) test (6), and a complement-fixation (CF) test (7). Virus and antibody may coexist in the adult form of bovine lymphosarcoma (1, 3). Although neither BL V nor its antibody can be demonstrated in the calf and thymic forms, experimental calves and lambs develop BLV infection after inoculation with material from such forms (3). Recently, Van Der Maaten et al. (8) established monolayer cultures of cells by co cultivation of fetal lamb spleen (FLS) and tumor tissue of bovine lymphosarcoma. These BLV -producing cells will be designated FLSv+. In preliminary trials, serum from adult cattle infected with BLV seemed to contain a factor inhibiting the production of virus in cell cultures as measured by electron microscopy. This inhibition has now been examined more quantitatively with the use of an FLSv+ cell line and SRID. Such serum was found to affect virus release by the cells. MATERIALS AND METHODS
Cell and cell culture.-The FLSv+ cell line was obtained from Dr. M. J. Van Der Maaten (National Animal Disease Center, Ames, Iowa) and maintained in passage by his methods (8). Approximately 3 XI 0 6 cells/ml were transferred to a sterile 100-ml culture flask containing 15 ml culture medium consisting of Eagle's minimum essential medium supplemented with 100 f-Lg penicillin/mI, 100 f-Lg dihydrostreptomycin/ ml, 100 J..Lg kanamycin/ml, and 2.5 f-Lg fungazone/ml plus 20% fetal calf serum (FCS). After 4 or 5 days' incubation at 37° C, monolayers were developed. Antigen preparation.-The supernatant media from the culture (15 ml) were concentrated to ~~5 the
volume by centrifugation at 66,000Xg for 90 minutes. After ultracentrifugation, the pellet was suspended in 0.2 ml phosphate-buffered saline (PBS) at pH 7.2; then 1.8 ml diethyl ether was added and agitated occasionally until it had evaporated. The suspension was examined for antigenic activity by the SRID test and the measurement of the diameter of the precipitin ring. An antigen was also prepared from the cells. The cells of the monolayer were washed twice with PBS, then removed with 0.02% EDTA (8), and centrifuged at 450Xg for 5 minutes. The pellet of cells (3X 10 7 cells) was suspended in 0.2 ml PBS and treated with ether. The antigenicity was then tested by SRID to measure comparatively the amount of viral antigen. Double immunodiffusion precipitin tests on sera were done with a routine method developed specifically for BLV antibody (4). CF tests on sera were done by Dr. J. M. Miller (National Animal Disease Center)
(7). SRID test.- This was a modification of that reported by Mancini et al. (9). Antiserum (0.25 ml) from an infected sheep (V-34) was added to 3.75 ml of 0.8% agarose in 0.05 M Tris buffer, pH 7.2, containing 8.5% NaCl. This mixture (4 ml) was placed into level 50-mm glass dishes. Two or three wells (10 mm in diameter) were placed equidistant from the center and periphery of the dish. The wells were filled with equal volumes of the prepared antigens (0.1 ml). The plates were incubated at room temperature in a moist chamber for 3 days. The diameter of the precipitin ring was measured as an expression of the amount of antigen. Sera.-Sera 3094, 3133, 3153, 3122, LS-15, 71-1, 72-2, 71-13,3084, and 70-7 were from cattle naturally infected with BL V. Sera 7028, 7082, 6971, 6293, 6284, and 5393 were from normal cattle (in a herd kept in isolation for 25 yr with no history of lymphosarcoma, persistent lymphocytosis, or antibodies to bovine C-type virus). All normal cattle sera and some naturally infected cattle sera (LS-15, 71-1, 72-2, 71-13, and 70-7) were obtained from Dr. J. M. Miller. Sera 526, 222, 221, 216, 515, 229, and 228 were from inoculated cattle. The condition of these cattle from which the sera were obtained is summarized in table 1. All these sera were filtered and inactivated at 56° C for 30 minutes before use. V RI test.-The monolayer FLSv+ cells were dispersed with 0.02% EDTA and transferred to three Received October 29,1974; accepted January 14, 1975. Supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, and in part by Public Health Service research grant CA 13628 from the National Cancer Institute. 3 Department of Veterinary Science, University of Wisconsin, Madison, Wis. 53706. 4 We gratefully acknowledge the technical assistance of Mrs. Sally Bertelson and Miss Marilyn Gill. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 54, NO.5, MAY 1975
1199
1200 TABLE
ONUMA, OLSON, AND BAUMGARTENER
I.-Identity and condition of animals from which sera were obtained, correlated with virus release inhibition (VRI) reaction type
Serum No.
3133 3153 3094 LS-15 71-1 72-2 3122 71-13 3084 70-7 526 222 221 216 515 229 228 7028 7082 6971 6293 6284 5393 a b
A BOVINE (C-TYPE) leukemia virus (BLV) can be demonstrated in short-term cultures of mitogenstimulated lymphoid cells of cattle with the adult form of lymphosarcoma (1) and from cattle experimentally and naturally infected with BLV (2). The virus is shed from the cells and can be seen extracellularly by electron microscopy (1) or shown by a precipitin reaction with specific antiserum in gel immunodiffusion (3) and single radial immunodiffusion (SRID) of an ether-treated antigen preparation in agar gel containing antibody. The antibody also can be demonstrated by an immunodiffusion (ID) test (4, 5), an immunofluorescence (IF) test (6), and a complement-fixation (CF) test (7). Virus and antibody may coexist in the adult form of bovine lymphosarcoma (1, 3). Although neither BL V nor its antibody can be demonstrated in the calf and thymic forms, experimental calves and lambs develop BLV infection after inoculation with material from such forms (3). Recently, Van Der Maaten et al. (8) established monolayer cultures of cells by co cultivation of fetal lamb spleen (FLS) and tumor tissue of bovine lymphosarcoma. These BLV -producing cells will be designated FLSv+. In preliminary trials, serum from adult cattle infected with BLV seemed to contain a factor inhibiting the production of virus in cell cultures as measured by electron microscopy. This inhibition has now been examined more quantitatively with the use of an FLSv+ cell line and SRID. Such serum was found to affect virus release by the cells. MATERIALS AND METHODS
Cell and cell culture.-The FLSv+ cell line was obtained from Dr. M. J. Van Der Maaten (National Animal Disease Center, Ames, Iowa) and maintained in passage by his methods (8). Approximately 3 XI 0 6 cells/ml were transferred to a sterile 100-ml culture flask containing 15 ml culture medium consisting of Eagle's minimum essential medium supplemented with 100 f-Lg penicillin/mI, 100 f-Lg dihydrostreptomycin/ ml, 100 J..Lg kanamycin/ml, and 2.5 f-Lg fungazone/ml plus 20% fetal calf serum (FCS). After 4 or 5 days' incubation at 37° C, monolayers were developed. Antigen preparation.-The supernatant media from the culture (15 ml) were concentrated to ~~5 the
volume by centrifugation at 66,000Xg for 90 minutes. After ultracentrifugation, the pellet was suspended in 0.2 ml phosphate-buffered saline (PBS) at pH 7.2; then 1.8 ml diethyl ether was added and agitated occasionally until it had evaporated. The suspension was examined for antigenic activity by the SRID test and the measurement of the diameter of the precipitin ring. An antigen was also prepared from the cells. The cells of the monolayer were washed twice with PBS, then removed with 0.02% EDTA (8), and centrifuged at 450Xg for 5 minutes. The pellet of cells (3X 10 7 cells) was suspended in 0.2 ml PBS and treated with ether. The antigenicity was then tested by SRID to measure comparatively the amount of viral antigen. Double immunodiffusion precipitin tests on sera were done with a routine method developed specifically for BLV antibody (4). CF tests on sera were done by Dr. J. M. Miller (National Animal Disease Center)
(7). SRID test.- This was a modification of that reported by Mancini et al. (9). Antiserum (0.25 ml) from an infected sheep (V-34) was added to 3.75 ml of 0.8% agarose in 0.05 M Tris buffer, pH 7.2, containing 8.5% NaCl. This mixture (4 ml) was placed into level 50-mm glass dishes. Two or three wells (10 mm in diameter) were placed equidistant from the center and periphery of the dish. The wells were filled with equal volumes of the prepared antigens (0.1 ml). The plates were incubated at room temperature in a moist chamber for 3 days. The diameter of the precipitin ring was measured as an expression of the amount of antigen. Sera.-Sera 3094, 3133, 3153, 3122, LS-15, 71-1, 72-2, 71-13,3084, and 70-7 were from cattle naturally infected with BL V. Sera 7028, 7082, 6971, 6293, 6284, and 5393 were from normal cattle (in a herd kept in isolation for 25 yr with no history of lymphosarcoma, persistent lymphocytosis, or antibodies to bovine C-type virus). All normal cattle sera and some naturally infected cattle sera (LS-15, 71-1, 72-2, 71-13, and 70-7) were obtained from Dr. J. M. Miller. Sera 526, 222, 221, 216, 515, 229, and 228 were from inoculated cattle. The condition of these cattle from which the sera were obtained is summarized in table 1. All these sera were filtered and inactivated at 56° C for 30 minutes before use. V RI test.-The monolayer FLSv+ cells were dispersed with 0.02% EDTA and transferred to three Received October 29,1974; accepted January 14, 1975. Supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, and in part by Public Health Service research grant CA 13628 from the National Cancer Institute. 3 Department of Veterinary Science, University of Wisconsin, Madison, Wis. 53706. 4 We gratefully acknowledge the technical assistance of Mrs. Sally Bertelson and Miss Marilyn Gill. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 54, NO.5, MAY 1975
1199
1200 TABLE
ONUMA, OLSON, AND BAUMGARTENER
I.-Identity and condition of animals from which sera were obtained, correlated with virus release inhibition (VRI) reaction type
Serum No.
3133 3153 3094 LS-15 71-1 72-2 3122 71-13 3084 70-7 526 222 221 216 515 229 228 7028 7082 6971 6293 6284 5393 a b
