INFECTION AND IMMUNITY, Apr. 1978, p. 307-309
Vol. 20, No. 1
0019-9567/78/0020-0307$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Direct Syncytial Assay for the Quantitation of Bovine Leukemia Virus C. V. BENTON,* A. E. SORIA, AND R. V. GILDEN Viral Oncology Program, National Cancer Institute Frederick Cancer Research Center, Frederick, Maryland 21701
Received for publication 14 November 1977
A direct in vitro tissue culture method is described for the quantitation of bovine leukemia virus, utilizing a feline S+L- cell line.
Several quantitative plaque assays have been described for type C and D retroviruses. Mixedculture cytopathogenicity assays have been developed by using Rous sarcoma virus-transformed cells of rat (XC) (8, 15) and human (KC) (12, 14) origin. A direct assay for type C viruses exploits their ability to complement and rescue defective murine sarcoma virus from sarcomapositive leukemia-negative (S'L-) cells (1). The present investigation describes a quantitative bioassay for bovine leukemia virus (BLV) (9) that is direct, similar to described S'L- assays, yet manifested by cell fusion, as previously seen in mixed-culture assays. F81, a feline S'L- cell line (4), was obtained from J. Derge, Frederick Cancer Research Center (FCRC). The feline embryo fibroblast cell line, FEA, was obtained from S. Rasheed, University of Southern California; the feline osteosarcoma cell line, F100, came from R. McAllister, Children's Hospital of Los Angeles; the feline whole-fetus cell line AG-581, was obtained from W. Nelson-Rees, Naval Medical Research Laboratory, Oakland, Calif.; and the bovine embryonic spleen cell line, BES, came from M. Van der Maaten, U.S. Department of Agriculture, Ames, Iowa. All feline lines were grown in minimal essential medium with Earle salts supplemented with 10% fetal bovine serum. The BES cells were grown in the same medium with 20% fetal bovine serum. All cell lines were negative for mycoplasma as tested by R. Del Giudice, FCRC. The F81 cells were seeded in 60-mm plasticgridded (2 mm) petri dishes (Lux Scientific Corp., Newbury Park, Calif.), at 6 x 105 cells per dish in 4 ml of medium containing 2 ,ug of polybrene (Aldrich Chemical Co., Milwaukee, Wis.) per ml, and incubated overnight at 370C in a humidified atmosphere of 5% CO2. AG-581, FEA, and F100 cells were seeded at 2 x 105 cells per dish. Twenty-four hours after seeding, the medium was aspirated and 0.5 ml of the virus preparation, diluted in complete tissue culture
medium (CTCM), was added to the plates. The plates were incubated for 1 h at 370C with constant gentle rocking and then fed with 4 ml of CTCM. The plates were incubated at 370C and fed every 48 h. On day 16 postinfection, the plates were fixed and stained with 0.67% methylene blue and 0.33% carbol fuchsen in absolute methanol. BLV was produced in fetal lamb kidney (FLK) cultures as previously described (17). These cultures and purified BLV were nonreactive in tests for bovine syncytial virus and a Visna-like virus of bovine origin (5). Virus utilized in assays was twice semi-isopycnically banded in sucrose, pelleted, and suspended in 0.01 M tris(hydroxyethyl)aminomethane (pH 7.2)-0.1 M NaCl0.001 M ethylenediaminetetraacetic acid at 1,000x concentration (C. V. Benton et al., In Vitro, in press). Using this assay system, BLV inoculation resulted in large, multinucleated syncytia that retained less stain than the surrounding monolayer. These fused cells were evenly distributed throughout the monolayer and did not occur in groups (Fig. 1). Titration of the virus resulted in a linear decrease in syncytia directly proportional to the virus dilution, consistent with a one-hit response (Fig. 2). Reproducibility was observed inasmuch as titers for different samples of one virus preparation ranged from 1.2 x 105 to 2.7 x 105 syncytia-inducing units (SIU) per ml and, for another preparation, from 1.2 x 104 to 4.3 x 104 SIU/ml, each from four independent assays. Variability among 10 purified BLV preparations ranged from 1.2 x 104 to 54.0 x 104 SIU/ml; this variation was generally proportional to the total number of virus particles in these preparations (5 x 109 to 100 x 109/ml). The question of specificity was approached in several ways. BLV propagated in fetal canine thymus cells, Cf2TH (Nelson-Rees et al., In Vitro, in press), resulted in 10- to 50-fold less virus than from the FLK line as measured by
Mg2t-dependent
307
reverse
transcriptase activity
308
INFECT. IMMUN.
NOTES _
^
,.Ai
FIG. 1. BLV-induced syncytium in feline F81 S+L- cells. x300. 1,0O
1Ct0
-
,
(n
1I0
1
3.g.104) 1 io-5
10-4
10-3
10-2
BLV Dilution Factor
FIG. 2. Titration of three BL Vpreparations in F81 cells demonstrating syncytial levels proportional to virus dilutions. Undilute titers, based on these titrations, in parentheses.
(5) and p24 complement-fixation titers (5). A similar reduction was observed in SIU titers. To determine whether this effect was a generalized phenomenon in feline cells, BLV (from FLK cells) was inoculated onto F100, AG-581, and FEA cells; no morphological changes were observed. Next, viruses from several subprimate (Rauscher murine leukemia virus [MuLV]; mink cell focus-inducing MuLV; amphotropic-MuLV, mouse mammary tumor virus) and primate (baboon leukemia virus, M7; Mason-Pfizer monkey virus) species were tested. These viruses were propagated by the Viral Resources Laboratory, FCRC, and have been described (2, 6, 7, 11, 16, 18). None of these viruses produced a morpho-
logical change like that described for BLV. However, mink cell focus-inducing MuLV and amphotropic-MuLV did produce foci of clustered, round cells, similar to those previously described with xenotropic MuLV on F81 cells (4). Additionally, BLV assayed on KC, XC, and murine S'L- cells did not result in morphological changes in these cells. Since syncytia formation was quantitative with BLV from both FLK and Cf2TH cells, it was unlikely that this effect was mediated by contaminating cellular components or by-products in the BLV preparations. Assays of normal BES cells and cell-free supernatants did not result in syncytia in the F81 cells, thus further supporting this contention. Blocking experiments were performed to determine whether preparations of disrupted BLV could inhibit F81 cell fusion (13). One milliliter of a 1,000X concentrate of BLV (2.2 x 104 SIU) was sonically treated on ice for three 30-s intervals, using a Bronwill Biosonik IV sonicator (fitted with a microtip) at maximum output. The sonically treated preparation was diluted to 30 ml in CTCM and centrifuged at 90,000 x g for 45 min at 4VC in a Beckman type 30 rotor. Both the supernatant and pelleted material (resuspended in 1 ml of CTCM) were assayed directly for residual SIU. No activity was detected in the supernatant; the pelleted material contained less than 4% of the original infectivity. Two-tenths milliliter of sonically treated supernatant, equivalent to 140 SIU based on the residual titer corrected for removed residual activity, was
VOL. 20, 1978
NOTES
309
This work was supported by the Virus Cancer Program contract NO1-CO-25423 with the National Cancer Institute. LITERATURE CITED 1. Bassin, R. H., N. Tuttle, and P. J. Fischinger. 1971. Rapid cell culture assay technique for murine leukemia viruses. Nature (London) 229:564-566. 2. Chopra, H. C., and M. M. Mason. 1970. A new virus in a spontaneous mammary tumor of a rhesus monkey. Cancer Res. 30:2081-2086. 3. Ferrer, J. F., and C. A. Diglio. 1976. Development of an in vitro infectivity assay for the C-type bovine leukemia virus. Cancer Res. 36:1068-1073. 10,000 1,000 10 100 4. Fischinger, P. J., P. T. Peebles, S. Nomura, and D. K. Reciprocal of Antiserum Dilution Haapala. 1973. Isolation of an RD-114-like oncornavirus from a cat cell line. J. Virol. 11:978-985. 5. Gilden, R. V., C. W. Long, M. Hanson, R. Toni, H. P. FIG. 3. Neutralization of BL V by bovine anti-BL V Charman, S. Oroszlan, J. M. Miller, and M. J. Van serum. Three separate titrations. der Maaten. 1975. Characteristics of the major internal protein and RNA-dependent DNA polymerase of boadded to plates and incubated 1 h at 37°C with vine leukemia virus. J. Gen. Virol. 29:305-314. constant gentle rocking. Two-tenths milliliter of 6. Hartley, J. W., and W. P. Rowe. 1976. Naturally occurring murine leukemia viruses in wild mice: characteriBLV (containing 50 SIU) was then added, and zation of a new "amphotropic" class. J. Virol. 19:19-25. the plates were incubated an additional 1 h as 7. Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. before; plates were fed and the assay was comRowe. 1977. A new class of murine leukemia virus associated with development of spontaneous lymphopleted as described above. In two separate exmas. Proc. Natl. Acad. Sci. U.S.A. 74:789-792. periments, inhibition of fusion was observed, 8. Klement, V., W. P. Rowe, J. W. Hartley, and W. E. with disrupted BLV-treated plates exhibiting 30 Pugh. 1969. Mixed culture cytopathogenicity: a new and 39% fewer syncytia than untreated plates. test for growth of murine leukemia viruses in tissue culture. Proc. Natl. Acad. Sci. U.S.A. 63:753-758. Neutralization assays were performed with 9. Miller, J. M., L. D. Miller, C. Olson, and K. G. Gillette. normal and immune bovine sera supplied by H. 1969. Virus-like particles in phytohemagglutin-stimuwere sera Before all FCRC. assaying, Charman, lated lymphocyte cultures with reference to bovine lymheat inactivated at 56°C for 30 min. Equal volphosarcoma. J. Natl. Cancer Inst. 43:1297-1305. 10. Ogura, H., J. Paulsen, and H. Bauer. 1977. Crossumes of virus (50 SIU) and serum dilutions, neutralization of ovine and bovine C-type leukemia made in CTCM, were mixed and incubated 1 h virus-induced syncytia formation. Cancer Res. at 25'C with shaking every 15 min, and then 37:1486-1489. assayed as previously described. Results with 10 11. Owens, R. B., and A. J. Hackett. 1972. Tissue culture studies of mouse mammary cells and associated viruses. normal sera and 9 immune sera indicated that, J. Natl. Cancer Inst. 49:1321-1332. at a 1:40 dilution, all immune sera demonstrated J., and F. MacIntyre. 1968. Long term culture significant neutralizing activity, whereas none of 12. Ponten, of normal and neoplastic human glia. Acta Pathol. the normal sera did. One of these immune sera, Microbiol. Scand. 74:465-468. V-34, neutralized 50% of the BLV at a 1:200 to 13. Rand, K., J. Davis, R. V. Gilden, S. Oroszlan, and C. Long. 1975. Fusion inhibition: bioassay of a type C viral 1:400 dilution in three separate assays (Fig. 3). protein. Virology 64:63-74. High-titer goat anti-Rauscher MuLV serum ex- 14. Rand, K., and C. Long. 1972. Syncytial assay for the hibited no neutralizing capability in these asputative human type C virus, RD-114, utilizing human cells transformed by Rous sarcoma virus. Nature (Lonsays. don) New Biol. 240:187-190. Collectively, these data indicate the developW. P., W. E. Pugh, and J. W. Hartley. 1970. ment of a direct, specific, reproducible, and sen- 15. Rowe, Plaque assay techniques for murine leukemia viruses. This BLV. sitive assay system for quantitating Virology 42:1136-1139. assay does not have the disadvantages of the 16. Todaro, G. J., C. J. Sherr, R. E. Benveniste, M. M. Lieber, and J. L. Melnick. 1974. Type C virus of requirement for mixed-cell culture (10) or unbaboons: isolation from normal cell cultures. Cell certain reproducibility (3) associated with pre2:55-61. viously described BLV infectivity assays. This 17. Van der Maaten, M. J., J. M. Miller, and A. D. Boothe. method will be useful in future studies requiring 1974. Replicating type-C virus particles in monolayer cell cultures of tissues from cattle with lymphosarcoma. either accurate quantitation of virus or titration J. Natl. Cancer Inst. 52:491-497. of neutralizing antibody. 18. Wright, B. S., P. A. O'Brien, G. P. Shibley, S. A. Mayyasi, and J. C. Lasfargues. 1967. Infection of an established mouse bone marrow cell line (JLS-V9) with We acknowledge the excellent technical assistance of B. Rauscher and Moloney murine leukemia viruses. CanBohn and the personnel of the Viral Resources Laboratory, cer Res. 27:1672-1677. Frederick Cancer Research Center. z
Vol. 20, No. 1
0019-9567/78/0020-0307$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Direct Syncytial Assay for the Quantitation of Bovine Leukemia Virus C. V. BENTON,* A. E. SORIA, AND R. V. GILDEN Viral Oncology Program, National Cancer Institute Frederick Cancer Research Center, Frederick, Maryland 21701
Received for publication 14 November 1977
A direct in vitro tissue culture method is described for the quantitation of bovine leukemia virus, utilizing a feline S+L- cell line.
Several quantitative plaque assays have been described for type C and D retroviruses. Mixedculture cytopathogenicity assays have been developed by using Rous sarcoma virus-transformed cells of rat (XC) (8, 15) and human (KC) (12, 14) origin. A direct assay for type C viruses exploits their ability to complement and rescue defective murine sarcoma virus from sarcomapositive leukemia-negative (S'L-) cells (1). The present investigation describes a quantitative bioassay for bovine leukemia virus (BLV) (9) that is direct, similar to described S'L- assays, yet manifested by cell fusion, as previously seen in mixed-culture assays. F81, a feline S'L- cell line (4), was obtained from J. Derge, Frederick Cancer Research Center (FCRC). The feline embryo fibroblast cell line, FEA, was obtained from S. Rasheed, University of Southern California; the feline osteosarcoma cell line, F100, came from R. McAllister, Children's Hospital of Los Angeles; the feline whole-fetus cell line AG-581, was obtained from W. Nelson-Rees, Naval Medical Research Laboratory, Oakland, Calif.; and the bovine embryonic spleen cell line, BES, came from M. Van der Maaten, U.S. Department of Agriculture, Ames, Iowa. All feline lines were grown in minimal essential medium with Earle salts supplemented with 10% fetal bovine serum. The BES cells were grown in the same medium with 20% fetal bovine serum. All cell lines were negative for mycoplasma as tested by R. Del Giudice, FCRC. The F81 cells were seeded in 60-mm plasticgridded (2 mm) petri dishes (Lux Scientific Corp., Newbury Park, Calif.), at 6 x 105 cells per dish in 4 ml of medium containing 2 ,ug of polybrene (Aldrich Chemical Co., Milwaukee, Wis.) per ml, and incubated overnight at 370C in a humidified atmosphere of 5% CO2. AG-581, FEA, and F100 cells were seeded at 2 x 105 cells per dish. Twenty-four hours after seeding, the medium was aspirated and 0.5 ml of the virus preparation, diluted in complete tissue culture
medium (CTCM), was added to the plates. The plates were incubated for 1 h at 370C with constant gentle rocking and then fed with 4 ml of CTCM. The plates were incubated at 370C and fed every 48 h. On day 16 postinfection, the plates were fixed and stained with 0.67% methylene blue and 0.33% carbol fuchsen in absolute methanol. BLV was produced in fetal lamb kidney (FLK) cultures as previously described (17). These cultures and purified BLV were nonreactive in tests for bovine syncytial virus and a Visna-like virus of bovine origin (5). Virus utilized in assays was twice semi-isopycnically banded in sucrose, pelleted, and suspended in 0.01 M tris(hydroxyethyl)aminomethane (pH 7.2)-0.1 M NaCl0.001 M ethylenediaminetetraacetic acid at 1,000x concentration (C. V. Benton et al., In Vitro, in press). Using this assay system, BLV inoculation resulted in large, multinucleated syncytia that retained less stain than the surrounding monolayer. These fused cells were evenly distributed throughout the monolayer and did not occur in groups (Fig. 1). Titration of the virus resulted in a linear decrease in syncytia directly proportional to the virus dilution, consistent with a one-hit response (Fig. 2). Reproducibility was observed inasmuch as titers for different samples of one virus preparation ranged from 1.2 x 105 to 2.7 x 105 syncytia-inducing units (SIU) per ml and, for another preparation, from 1.2 x 104 to 4.3 x 104 SIU/ml, each from four independent assays. Variability among 10 purified BLV preparations ranged from 1.2 x 104 to 54.0 x 104 SIU/ml; this variation was generally proportional to the total number of virus particles in these preparations (5 x 109 to 100 x 109/ml). The question of specificity was approached in several ways. BLV propagated in fetal canine thymus cells, Cf2TH (Nelson-Rees et al., In Vitro, in press), resulted in 10- to 50-fold less virus than from the FLK line as measured by
Mg2t-dependent
307
reverse
transcriptase activity
308
INFECT. IMMUN.
NOTES _
^
,.Ai
FIG. 1. BLV-induced syncytium in feline F81 S+L- cells. x300. 1,0O
1Ct0
-
,
(n
1I0
1
3.g.104) 1 io-5
10-4
10-3
10-2
BLV Dilution Factor
FIG. 2. Titration of three BL Vpreparations in F81 cells demonstrating syncytial levels proportional to virus dilutions. Undilute titers, based on these titrations, in parentheses.
(5) and p24 complement-fixation titers (5). A similar reduction was observed in SIU titers. To determine whether this effect was a generalized phenomenon in feline cells, BLV (from FLK cells) was inoculated onto F100, AG-581, and FEA cells; no morphological changes were observed. Next, viruses from several subprimate (Rauscher murine leukemia virus [MuLV]; mink cell focus-inducing MuLV; amphotropic-MuLV, mouse mammary tumor virus) and primate (baboon leukemia virus, M7; Mason-Pfizer monkey virus) species were tested. These viruses were propagated by the Viral Resources Laboratory, FCRC, and have been described (2, 6, 7, 11, 16, 18). None of these viruses produced a morpho-
logical change like that described for BLV. However, mink cell focus-inducing MuLV and amphotropic-MuLV did produce foci of clustered, round cells, similar to those previously described with xenotropic MuLV on F81 cells (4). Additionally, BLV assayed on KC, XC, and murine S'L- cells did not result in morphological changes in these cells. Since syncytia formation was quantitative with BLV from both FLK and Cf2TH cells, it was unlikely that this effect was mediated by contaminating cellular components or by-products in the BLV preparations. Assays of normal BES cells and cell-free supernatants did not result in syncytia in the F81 cells, thus further supporting this contention. Blocking experiments were performed to determine whether preparations of disrupted BLV could inhibit F81 cell fusion (13). One milliliter of a 1,000X concentrate of BLV (2.2 x 104 SIU) was sonically treated on ice for three 30-s intervals, using a Bronwill Biosonik IV sonicator (fitted with a microtip) at maximum output. The sonically treated preparation was diluted to 30 ml in CTCM and centrifuged at 90,000 x g for 45 min at 4VC in a Beckman type 30 rotor. Both the supernatant and pelleted material (resuspended in 1 ml of CTCM) were assayed directly for residual SIU. No activity was detected in the supernatant; the pelleted material contained less than 4% of the original infectivity. Two-tenths milliliter of sonically treated supernatant, equivalent to 140 SIU based on the residual titer corrected for removed residual activity, was
VOL. 20, 1978
NOTES
309
This work was supported by the Virus Cancer Program contract NO1-CO-25423 with the National Cancer Institute. LITERATURE CITED 1. Bassin, R. H., N. Tuttle, and P. J. Fischinger. 1971. Rapid cell culture assay technique for murine leukemia viruses. Nature (London) 229:564-566. 2. Chopra, H. C., and M. M. Mason. 1970. A new virus in a spontaneous mammary tumor of a rhesus monkey. Cancer Res. 30:2081-2086. 3. Ferrer, J. F., and C. A. Diglio. 1976. Development of an in vitro infectivity assay for the C-type bovine leukemia virus. Cancer Res. 36:1068-1073. 10,000 1,000 10 100 4. Fischinger, P. J., P. T. Peebles, S. Nomura, and D. K. Reciprocal of Antiserum Dilution Haapala. 1973. Isolation of an RD-114-like oncornavirus from a cat cell line. J. Virol. 11:978-985. 5. Gilden, R. V., C. W. Long, M. Hanson, R. Toni, H. P. FIG. 3. Neutralization of BL V by bovine anti-BL V Charman, S. Oroszlan, J. M. Miller, and M. J. Van serum. Three separate titrations. der Maaten. 1975. Characteristics of the major internal protein and RNA-dependent DNA polymerase of boadded to plates and incubated 1 h at 37°C with vine leukemia virus. J. Gen. Virol. 29:305-314. constant gentle rocking. Two-tenths milliliter of 6. Hartley, J. W., and W. P. Rowe. 1976. Naturally occurring murine leukemia viruses in wild mice: characteriBLV (containing 50 SIU) was then added, and zation of a new "amphotropic" class. J. Virol. 19:19-25. the plates were incubated an additional 1 h as 7. Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. before; plates were fed and the assay was comRowe. 1977. A new class of murine leukemia virus associated with development of spontaneous lymphopleted as described above. In two separate exmas. Proc. Natl. Acad. Sci. U.S.A. 74:789-792. periments, inhibition of fusion was observed, 8. Klement, V., W. P. Rowe, J. W. Hartley, and W. E. with disrupted BLV-treated plates exhibiting 30 Pugh. 1969. Mixed culture cytopathogenicity: a new and 39% fewer syncytia than untreated plates. test for growth of murine leukemia viruses in tissue culture. Proc. Natl. Acad. Sci. U.S.A. 63:753-758. Neutralization assays were performed with 9. Miller, J. M., L. D. Miller, C. Olson, and K. G. Gillette. normal and immune bovine sera supplied by H. 1969. Virus-like particles in phytohemagglutin-stimuwere sera Before all FCRC. assaying, Charman, lated lymphocyte cultures with reference to bovine lymheat inactivated at 56°C for 30 min. Equal volphosarcoma. J. Natl. Cancer Inst. 43:1297-1305. 10. Ogura, H., J. Paulsen, and H. Bauer. 1977. Crossumes of virus (50 SIU) and serum dilutions, neutralization of ovine and bovine C-type leukemia made in CTCM, were mixed and incubated 1 h virus-induced syncytia formation. Cancer Res. at 25'C with shaking every 15 min, and then 37:1486-1489. assayed as previously described. Results with 10 11. Owens, R. B., and A. J. Hackett. 1972. Tissue culture studies of mouse mammary cells and associated viruses. normal sera and 9 immune sera indicated that, J. Natl. Cancer Inst. 49:1321-1332. at a 1:40 dilution, all immune sera demonstrated J., and F. MacIntyre. 1968. Long term culture significant neutralizing activity, whereas none of 12. Ponten, of normal and neoplastic human glia. Acta Pathol. the normal sera did. One of these immune sera, Microbiol. Scand. 74:465-468. V-34, neutralized 50% of the BLV at a 1:200 to 13. Rand, K., J. Davis, R. V. Gilden, S. Oroszlan, and C. Long. 1975. Fusion inhibition: bioassay of a type C viral 1:400 dilution in three separate assays (Fig. 3). protein. Virology 64:63-74. High-titer goat anti-Rauscher MuLV serum ex- 14. Rand, K., and C. Long. 1972. Syncytial assay for the hibited no neutralizing capability in these asputative human type C virus, RD-114, utilizing human cells transformed by Rous sarcoma virus. Nature (Lonsays. don) New Biol. 240:187-190. Collectively, these data indicate the developW. P., W. E. Pugh, and J. W. Hartley. 1970. ment of a direct, specific, reproducible, and sen- 15. Rowe, Plaque assay techniques for murine leukemia viruses. This BLV. sitive assay system for quantitating Virology 42:1136-1139. assay does not have the disadvantages of the 16. Todaro, G. J., C. J. Sherr, R. E. Benveniste, M. M. Lieber, and J. L. Melnick. 1974. Type C virus of requirement for mixed-cell culture (10) or unbaboons: isolation from normal cell cultures. Cell certain reproducibility (3) associated with pre2:55-61. viously described BLV infectivity assays. This 17. Van der Maaten, M. J., J. M. Miller, and A. D. Boothe. method will be useful in future studies requiring 1974. Replicating type-C virus particles in monolayer cell cultures of tissues from cattle with lymphosarcoma. either accurate quantitation of virus or titration J. Natl. Cancer Inst. 52:491-497. of neutralizing antibody. 18. Wright, B. S., P. A. O'Brien, G. P. Shibley, S. A. Mayyasi, and J. C. Lasfargues. 1967. Infection of an established mouse bone marrow cell line (JLS-V9) with We acknowledge the excellent technical assistance of B. Rauscher and Moloney murine leukemia viruses. CanBohn and the personnel of the Viral Resources Laboratory, cer Res. 27:1672-1677. Frederick Cancer Research Center. z
