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A line of ring-necked pheasant cells susceptible to infection by avian oncornaviruses
VIROLOGY
73, 548-552 (1976)
A Line of Ring-Necked
Pheasant Cells Susceptible Avian Oncornaviruses MAXINE
Department of Medicine, Division of Oncology, Washington 98195, and the Fred Hutchinson
to Infection
by
LINIAL
University of Washington School of Medicine, Cancer Research Center, Seattle, Washington
Seattle, 98104
Accepted May 4,1976 Ring-necked pheasant cells from a single embryo have been continuously cultured in this laboratory since March, 1975, and have been designated Ph 310. The established cell line grows as fibroblastic cells and has no properties associated with cell transformation. No viral-associated proteins were detected in these Ph 310 cells, although a subgroup F virus could be induced after infection with Bryan high titer Rous sarcoma virus. Ph 310 cells are highly susceptible to avian oncornavirus subgroups E and F but not A or C.
to subgroup F, called RAV-61 (2) or RSV(RPV) (3). Therefore, the Ph 310 cells were tested for the presence of viral proteins such as avian oncornavirus groupspecific (gs) antigens (4) and helper activity associated with envelop protein gp 85 (5). Complement fixation tests for gs antigen (6) were negative for this antigen in Ph 310 cells. Infection of Ph 310 with RSV (RAV-0) did not rescue a virus with subgroup F properties able to form foci on C/E or C/BE chicken cells, as would be expected if subgroup F ring-necked pheasant endogenous virus envelop proteins were present (2, 3). Further, no RNAdependent DNA polymerase activity was detected in Ph 310 supernatants. Therefore, Ph 310 cells appeared to be free of all subgroup F viral gene products by these measurements. However, Ph 310 cells do contain the genetic information encoding for subgroup F leukosis virus. A portion of the Ph 310 culture was infected with envelop defective Bryan high titer Rous sarcoma virus, BH RSV or RSV(-), using Sendai virusmediated fusion (7). One month after infection, RSV(-) Ph 310 cells were not producing infectious virus. After 3 months of culture, an infectious virus was found in the cell supernatant. This virus formed foci with equal efficiency on C/BE, Ph 310,
The study of avian oncornaviruses has been limited by the lack of nontransformed avian cell lines. This report describes the properties of a line of ringnecked pheasant cells (Phasimus colchicus torquatus) which has been in continuous culture since March, 1975. Ph 310 was established from a single ring-necked pheasant embryo obtained from fertile eggs from Hanaford Valley Game Farm, Centralia, Washington. A primary culture was prepared by established methods (I). The cells were grown at 37 or 35” in Ham’s FlO supplemented with 10% tryptose phosphate broth, 5% calf serum, and once established, 1% dimethyl sulfoxide (DMSO), although 1% DMSO was not required for growth. Cells were passaged initially whenever confluency was reached. Presently, Ph 310 cells are plated at approximately 2-3 x 106/100-mm dish and passaged every 5-7 days. The medium is not changed between passages, and in fact, frequently changing the medium appears to be detrimental to cell growth. A portion of the original culture was frozen at the second passage (PII) enabling comparison of late passage Ph 310 cells with early passage cells. Cells from some ring-necked pheasant embryos have been shown to contain an endogenous oncornavirus virus belonging 548 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and Japanese quail (Q) cells, but not on C/ BE or Ph cells preinfected with RAV-61 (21, indicating a virus of subgroup F host range (2, 3). After cloning three times through quail cells, RSV (RAV-F) from single foci grew to high titer (> 5 x lo5 FFU/ml) in both Q and C/BE cells. These results showed that an infectious subgroup F virus had been induced in Ph 310 cells which could grow in cells lacking endogenous subgroup F information. Conclusive evidence for the induction of a leukosis virus was furnished by end-point dilution of the RSV (RAV-F) which yielded a nontransforming virus of F subgroup host range and interference pattern, called here RAV-310. Ph 310 cells appeared by several criteria to be normal ring-necked pheasant cells. The cells have retained a fibroblastic mor-
549
phology (Fig. 1A and B) which is in contrast to that of the Ph 310 cells transformed by RSV (RAV-0) (Fig. 1C) which are enlarged and multinucleate. When 2.5 x lo5 cells were plated in soft agar (8) no colonies of transformed cells were obtained. Under the same conditions the effrciency of agar colony formation by RSVtransformed chicken cells was 3%. Karyotype analysis was performed in collaboration with Dr. R. Shoffner. Chromosome spreads from Ph 310 cells were indistinguishable from those of normal ringnecked pheasant fibroblasts. There were no recognizable changes in morphology among the six largest chromosome pairs and no aneuploidy of these chromosomes was seen. The growth characteristics of Ph 310 are presented in Fig. 2. Cell growth was ob-
FIG. 1. Photomicrographs of Ph 310 cells. (A) Cells after 11 months of culture (subconfluent). (B) Cells after 5 months of culture (confluent). (C) Cells grown from a focus of Ph 310 transformed by RSV (RAV-0). Wright’s stain, approximately 100 x. (D) Clones of Ph 310 grown in CM, 12 days after plating 100 cells, stained with Giemsa. (E) Clone from (D), magnified about 40 X.
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FIG. 2. Cultures of Ph 310 growing in Ham’s FlO supplemented with 10% tryptose phosphate broth, 5% calf serum, were trypsinized and plated on 60mm dishes in the same medium. Cell densities were determined by cell counts in hemacytometer chambers. For determination of cell number at 8 and 10 days after seeding the medium was changed at 5 or 6 days. Initial number of cells plated: Cl, 5 x 10”; 0, 1 x loj; A, 3 x W; n, 5 x 105; 0, 1 x 10”; 0, 1.8 x 10”.
served at initial plating densities of greater than about 3.5 x lo3 cells/cm2. The greatest saturation density observed in confluent monolayers was about 2 x 105/ cm2 which was similar to the density obtained with normal chick fibroblasts, but less than that of transformed cells. The cells grow slowly at 37” with a generation time of about 3 days. Cells grow at low density when plated in cloning medium (CM, Ham’s FlO containing 10% calf serum, 12% tryptose phosphate broth, 4% chick serum, 1% 100x vitamins (Gibco), 8 mglml of folic acid, and 0.5% DMSO). When 25-100 single cells (estimated from hemacytometer counts) were plated in 60mm dishes in CM, cell clones grew (Fig. 1D). The efficiency of plating of Ph 310
cells in CM is between 20 and 50%. The cell clones derived from single (or small numbers of cells), appear to be normal fibroblasts (Fig. 1E). If cover slips on which single or small numbers of clones were growing, were transferred to new dishes, secondary clones were found on the dish surface after about 1 week, indicating that the Ph 310 cells could detach from the plastic surface, reattach, and divide. Studies are currently in progress to characterize clones derived from cells plated at low density. It will be of interest to determine whether low density growth of these cells induces or selects for RAV-310 viral or subviral expression. The susceptibility of Ph 310 to different subgroups of avian oncornaviruses has been determined (Table 1). The original Ph 310 at low passage number (IV) were susceptible to subgroups A, C, E, and F. After 9 months of continuous culture, the cells became much less sensitive to transformation by subgroups A and C, although the resistance was not absolute. The sensitivity of these cells to transformation by subgroup E, the subgroup of chicken endogenous virus (61, remained approximately the same as that of Japanese quail cells or C/O chicken cells. Ph 310 was also sensitive to subgroup F virus induced from the same line by infection with RSV(-), further indicating that Ph 310 was not producing interfering endogenous F subgroup virus. Ph 310 cells were also sensitive to infection by subgroup E RAV’s, RAV-0 (4 1 and RAVSO, a recombinant between RAV-0 and exogenous virus with E subgroup host-range (9). Some surprising results were obtained when Ph 310 cells were infected by various subgroup RAV’s (Table 2). First, it was found that preinfection with RAV-0, RAV60 (not shown), or RAV-61 increased sensitivity of Ph 310 to transformation by subgroups A and C RSV. Several examples of avian oncornavirus enhancement have been previously reported in which preinfection by subgroup a leukosis virus increased focus formation by sarcoma viruses of subgroups B (10-12) or E (13). The effect in Ph 310 cells was the reverse; preinfection by subgroups E or F enhanced
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EFFICIENCY OF Focus Cell type
TABLE 1 FORMATION OF SUBGROUPS A, C, E, AND F Rous SARCOMA VIRUS ON Ph 310 AND OTHER AVIAN CELL@ Infecting Virus PR-RSV-A
Q" Ph 310 Ph 310 PIV’ C/E6 C/BE” CIOb
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1.0 0.001-0.002 0.9 1.0 1.2 N?M
RSV (RAV-1)
PR-RSV-C
1.0 0.003 NT NV 0.46 N!P
1.0 0.2-0.8 9.6 160 110 NF
PR-RSV-E
RSV (RAV-0)
1.0 0.91 2.7 0 0 NTJ
1.0 0.8 0.5 0 0 1.0
RSV (RAV-F) 1.0 1.0 1.0 NF 1.0 NF
” The efficiency of focus formation is expressed relative to that on Japanese quail cells (Q). Quail cells were from embryonated eggs obtained from Life Sciences, Inc., St. Petersburg, Florida. All embryonated chicken eggs were from H & N Incorporated, Redmond, Wash. The titers on permissive host cells were lo”10” FFU/ml (subgroups A, E, and F on quail; subgroup C on chick). * Q, C/E, C/BE, and C/O cells were tested at second passage. ’ Cells from the same embryo as Ph 310 tested at fourth passage. ’ NT = not tested.
TABLE
2
EFFECT OF PREINFECTION WITH RAV ON Focus FORMATION BY RSV” Preinfected by Sarcoma virus RAV-Ob A. Ph 310 PR-A PR-C PR-E RSV(RAV-F) B. Ph 310 PVIH PR-E
RAV-O(Ph)’
RAV-61
None
80-300 12-50 0.02-0.38 1.0
120 10 0.06 NTd
30 15 0.01-0.018 0.001
1.0 1.0 1.0 1.0
1.0
N?M
1.0
1.0
a Ph cells, 8 x 109, were infected with 0.05 ml of RAV stock. After 4 days at 37”, cells were transferred and challenged with dilutions of different sarcoma viruses. The efficiency of focus formation by sarcoma viruses relative to that on nonpreinfected pheasant cells is reported. In some cases a range of values from separate experiments is reported. * RAV-0 spontaneously released from L7 V+ C/A cells. ’ RAV-0 passaged three times in Ph 310 cells. d NT = not tested. c Cells from the original pheasant embryo at eighth transfer.
focus formation by subgroup A. The enhancement seen here was probably not a simple multiplicity dependence phenomenon since increasing the multiplicity of infecting PR-A virus did not lead to a better efficiency of transformation. Even at high m.o.i., Ph 310 transformed poorly after PR-A infection compared to a portion of the same culture at low cell passage number (data not shown). Second, it was found that only one passage of RAV-0 infected Ph 310 cells was sufficient to estab-
lish interference with subgroup E sarcoma viruses, a phenomenon not observed in the progenitor Ph 310 cells. It, therefore, appeared that RAV-0 replicated more efficiently in Ph 310 cells than the original low passage culture, indicating that Ph 310 should prove useful for the assay of RAV-0 (14). Finally, it was observed that RAV-61 (subgroup F) interferes with infection by PR-E in established Ph 310 cells but not in low passage Ph 310 cells. It had been previously reported that F virus in-
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terferes with E sarcoma virus in quail and pheasant cells (2, 3). These results indicate there have been changes in Ph 310 cells after long-term culture which have altered susceptibility to exogenous subgroups of RSV and have led to new oncornavirus interactions. It has not yet been determined whether these new viral interactions are affected intracellularly or t.hrough changes at the cell surface. Further studies are being conducted to analyze the interactions of different viruses in Ph 310 cells. ACKNOWLEDGMENTS I am grateful to Dr. Bill Mason for performing the gs tests and to Dr. R. Shoffner for the karyotype analysis. I thank Nancy Hopt and Paul Smith for helpful technical assistance, and Mr. and Mrs. R. Cramer of the Hanaford Valley Game Farm for supplying pheasant eggs. This study was supported by U.S. Public Health Service Grants No. CA 12895, No. CA 15784, and No. CA 10895 from the National Cancer Institute, and an American Cancer Society Institutional Research Grant.
REFERENCES 1. VOGT, P., In “Fundamental Technique in Virology (K. Habel and N. P. Salzman, eds.), pp. 198-211. Academic Press, New York, 1969. 2. HANAFUSA, T., and HANAFUSA, H., Virology 51, 247-251 (1973). 3. FUJITA, D., CHEN, Y., FRIIS, R., and VOGT, P., Virology 60, 558-571 (1974). 4. PAYNE, L., and CHUBB, R., J. Gen. Virol. 3,379391 (1968). 5. HALPERN, M., BOLOGNESI, D., FRIIS, R., and MASON, W., J. Virol. 15, 1131-1140 (1975). 6. VOGT, P., and FRIIS, R., Virology 43, 223-234 (1971). 7. HANAFUSA, H., MIYAMOTO, T., and HANAFUSA, T., Virology 40, 55-64 (1970). 8. WYKE, J., and LINIAL, M., Virology 53, 152-161 (1973). 9. HANAFUSA, H., HAYWARD, W., CHEN, J., and HANAFUSA, T., Cold Spring Harbor Syrnp. Quant. Biol. 39 (Part 2), 1139-1144 (1974). 10. VOGT, P., Virology 25, 237-247 (1965). 11. HANAFUSA, H., Virology 25, 248-255 (1965). 12. TOYOSHIMA, K., and VOGT, P., Virology 38,414426 (1969). 13. ISHIZAKI, R., and SHIMUZO, T., Virology 40,415417 (1970). 14. LINIAL, M., and NEIMAN, P., Virology, in press.
73, 548-552 (1976)
A Line of Ring-Necked
Pheasant Cells Susceptible Avian Oncornaviruses MAXINE
Department of Medicine, Division of Oncology, Washington 98195, and the Fred Hutchinson
to Infection
by
LINIAL
University of Washington School of Medicine, Cancer Research Center, Seattle, Washington
Seattle, 98104
Accepted May 4,1976 Ring-necked pheasant cells from a single embryo have been continuously cultured in this laboratory since March, 1975, and have been designated Ph 310. The established cell line grows as fibroblastic cells and has no properties associated with cell transformation. No viral-associated proteins were detected in these Ph 310 cells, although a subgroup F virus could be induced after infection with Bryan high titer Rous sarcoma virus. Ph 310 cells are highly susceptible to avian oncornavirus subgroups E and F but not A or C.
to subgroup F, called RAV-61 (2) or RSV(RPV) (3). Therefore, the Ph 310 cells were tested for the presence of viral proteins such as avian oncornavirus groupspecific (gs) antigens (4) and helper activity associated with envelop protein gp 85 (5). Complement fixation tests for gs antigen (6) were negative for this antigen in Ph 310 cells. Infection of Ph 310 with RSV (RAV-0) did not rescue a virus with subgroup F properties able to form foci on C/E or C/BE chicken cells, as would be expected if subgroup F ring-necked pheasant endogenous virus envelop proteins were present (2, 3). Further, no RNAdependent DNA polymerase activity was detected in Ph 310 supernatants. Therefore, Ph 310 cells appeared to be free of all subgroup F viral gene products by these measurements. However, Ph 310 cells do contain the genetic information encoding for subgroup F leukosis virus. A portion of the Ph 310 culture was infected with envelop defective Bryan high titer Rous sarcoma virus, BH RSV or RSV(-), using Sendai virusmediated fusion (7). One month after infection, RSV(-) Ph 310 cells were not producing infectious virus. After 3 months of culture, an infectious virus was found in the cell supernatant. This virus formed foci with equal efficiency on C/BE, Ph 310,
The study of avian oncornaviruses has been limited by the lack of nontransformed avian cell lines. This report describes the properties of a line of ringnecked pheasant cells (Phasimus colchicus torquatus) which has been in continuous culture since March, 1975. Ph 310 was established from a single ring-necked pheasant embryo obtained from fertile eggs from Hanaford Valley Game Farm, Centralia, Washington. A primary culture was prepared by established methods (I). The cells were grown at 37 or 35” in Ham’s FlO supplemented with 10% tryptose phosphate broth, 5% calf serum, and once established, 1% dimethyl sulfoxide (DMSO), although 1% DMSO was not required for growth. Cells were passaged initially whenever confluency was reached. Presently, Ph 310 cells are plated at approximately 2-3 x 106/100-mm dish and passaged every 5-7 days. The medium is not changed between passages, and in fact, frequently changing the medium appears to be detrimental to cell growth. A portion of the original culture was frozen at the second passage (PII) enabling comparison of late passage Ph 310 cells with early passage cells. Cells from some ring-necked pheasant embryos have been shown to contain an endogenous oncornavirus virus belonging 548 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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COMMUNICATIONS
and Japanese quail (Q) cells, but not on C/ BE or Ph cells preinfected with RAV-61 (21, indicating a virus of subgroup F host range (2, 3). After cloning three times through quail cells, RSV (RAV-F) from single foci grew to high titer (> 5 x lo5 FFU/ml) in both Q and C/BE cells. These results showed that an infectious subgroup F virus had been induced in Ph 310 cells which could grow in cells lacking endogenous subgroup F information. Conclusive evidence for the induction of a leukosis virus was furnished by end-point dilution of the RSV (RAV-F) which yielded a nontransforming virus of F subgroup host range and interference pattern, called here RAV-310. Ph 310 cells appeared by several criteria to be normal ring-necked pheasant cells. The cells have retained a fibroblastic mor-
549
phology (Fig. 1A and B) which is in contrast to that of the Ph 310 cells transformed by RSV (RAV-0) (Fig. 1C) which are enlarged and multinucleate. When 2.5 x lo5 cells were plated in soft agar (8) no colonies of transformed cells were obtained. Under the same conditions the effrciency of agar colony formation by RSVtransformed chicken cells was 3%. Karyotype analysis was performed in collaboration with Dr. R. Shoffner. Chromosome spreads from Ph 310 cells were indistinguishable from those of normal ringnecked pheasant fibroblasts. There were no recognizable changes in morphology among the six largest chromosome pairs and no aneuploidy of these chromosomes was seen. The growth characteristics of Ph 310 are presented in Fig. 2. Cell growth was ob-
FIG. 1. Photomicrographs of Ph 310 cells. (A) Cells after 11 months of culture (subconfluent). (B) Cells after 5 months of culture (confluent). (C) Cells grown from a focus of Ph 310 transformed by RSV (RAV-0). Wright’s stain, approximately 100 x. (D) Clones of Ph 310 grown in CM, 12 days after plating 100 cells, stained with Giemsa. (E) Clone from (D), magnified about 40 X.
550
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6 1
8
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FIG. 2. Cultures of Ph 310 growing in Ham’s FlO supplemented with 10% tryptose phosphate broth, 5% calf serum, were trypsinized and plated on 60mm dishes in the same medium. Cell densities were determined by cell counts in hemacytometer chambers. For determination of cell number at 8 and 10 days after seeding the medium was changed at 5 or 6 days. Initial number of cells plated: Cl, 5 x 10”; 0, 1 x loj; A, 3 x W; n, 5 x 105; 0, 1 x 10”; 0, 1.8 x 10”.
served at initial plating densities of greater than about 3.5 x lo3 cells/cm2. The greatest saturation density observed in confluent monolayers was about 2 x 105/ cm2 which was similar to the density obtained with normal chick fibroblasts, but less than that of transformed cells. The cells grow slowly at 37” with a generation time of about 3 days. Cells grow at low density when plated in cloning medium (CM, Ham’s FlO containing 10% calf serum, 12% tryptose phosphate broth, 4% chick serum, 1% 100x vitamins (Gibco), 8 mglml of folic acid, and 0.5% DMSO). When 25-100 single cells (estimated from hemacytometer counts) were plated in 60mm dishes in CM, cell clones grew (Fig. 1D). The efficiency of plating of Ph 310
cells in CM is between 20 and 50%. The cell clones derived from single (or small numbers of cells), appear to be normal fibroblasts (Fig. 1E). If cover slips on which single or small numbers of clones were growing, were transferred to new dishes, secondary clones were found on the dish surface after about 1 week, indicating that the Ph 310 cells could detach from the plastic surface, reattach, and divide. Studies are currently in progress to characterize clones derived from cells plated at low density. It will be of interest to determine whether low density growth of these cells induces or selects for RAV-310 viral or subviral expression. The susceptibility of Ph 310 to different subgroups of avian oncornaviruses has been determined (Table 1). The original Ph 310 at low passage number (IV) were susceptible to subgroups A, C, E, and F. After 9 months of continuous culture, the cells became much less sensitive to transformation by subgroups A and C, although the resistance was not absolute. The sensitivity of these cells to transformation by subgroup E, the subgroup of chicken endogenous virus (61, remained approximately the same as that of Japanese quail cells or C/O chicken cells. Ph 310 was also sensitive to subgroup F virus induced from the same line by infection with RSV(-), further indicating that Ph 310 was not producing interfering endogenous F subgroup virus. Ph 310 cells were also sensitive to infection by subgroup E RAV’s, RAV-0 (4 1 and RAVSO, a recombinant between RAV-0 and exogenous virus with E subgroup host-range (9). Some surprising results were obtained when Ph 310 cells were infected by various subgroup RAV’s (Table 2). First, it was found that preinfection with RAV-0, RAV60 (not shown), or RAV-61 increased sensitivity of Ph 310 to transformation by subgroups A and C RSV. Several examples of avian oncornavirus enhancement have been previously reported in which preinfection by subgroup a leukosis virus increased focus formation by sarcoma viruses of subgroups B (10-12) or E (13). The effect in Ph 310 cells was the reverse; preinfection by subgroups E or F enhanced
SHORT
EFFICIENCY OF Focus Cell type
TABLE 1 FORMATION OF SUBGROUPS A, C, E, AND F Rous SARCOMA VIRUS ON Ph 310 AND OTHER AVIAN CELL@ Infecting Virus PR-RSV-A
Q" Ph 310 Ph 310 PIV’ C/E6 C/BE” CIOb
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1.0 0.001-0.002 0.9 1.0 1.2 N?M
RSV (RAV-1)
PR-RSV-C
1.0 0.003 NT NV 0.46 N!P
1.0 0.2-0.8 9.6 160 110 NF
PR-RSV-E
RSV (RAV-0)
1.0 0.91 2.7 0 0 NTJ
1.0 0.8 0.5 0 0 1.0
RSV (RAV-F) 1.0 1.0 1.0 NF 1.0 NF
” The efficiency of focus formation is expressed relative to that on Japanese quail cells (Q). Quail cells were from embryonated eggs obtained from Life Sciences, Inc., St. Petersburg, Florida. All embryonated chicken eggs were from H & N Incorporated, Redmond, Wash. The titers on permissive host cells were lo”10” FFU/ml (subgroups A, E, and F on quail; subgroup C on chick). * Q, C/E, C/BE, and C/O cells were tested at second passage. ’ Cells from the same embryo as Ph 310 tested at fourth passage. ’ NT = not tested.
TABLE
2
EFFECT OF PREINFECTION WITH RAV ON Focus FORMATION BY RSV” Preinfected by Sarcoma virus RAV-Ob A. Ph 310 PR-A PR-C PR-E RSV(RAV-F) B. Ph 310 PVIH PR-E
RAV-O(Ph)’
RAV-61
None
80-300 12-50 0.02-0.38 1.0
120 10 0.06 NTd
30 15 0.01-0.018 0.001
1.0 1.0 1.0 1.0
1.0
N?M
1.0
1.0
a Ph cells, 8 x 109, were infected with 0.05 ml of RAV stock. After 4 days at 37”, cells were transferred and challenged with dilutions of different sarcoma viruses. The efficiency of focus formation by sarcoma viruses relative to that on nonpreinfected pheasant cells is reported. In some cases a range of values from separate experiments is reported. * RAV-0 spontaneously released from L7 V+ C/A cells. ’ RAV-0 passaged three times in Ph 310 cells. d NT = not tested. c Cells from the original pheasant embryo at eighth transfer.
focus formation by subgroup A. The enhancement seen here was probably not a simple multiplicity dependence phenomenon since increasing the multiplicity of infecting PR-A virus did not lead to a better efficiency of transformation. Even at high m.o.i., Ph 310 transformed poorly after PR-A infection compared to a portion of the same culture at low cell passage number (data not shown). Second, it was found that only one passage of RAV-0 infected Ph 310 cells was sufficient to estab-
lish interference with subgroup E sarcoma viruses, a phenomenon not observed in the progenitor Ph 310 cells. It, therefore, appeared that RAV-0 replicated more efficiently in Ph 310 cells than the original low passage culture, indicating that Ph 310 should prove useful for the assay of RAV-0 (14). Finally, it was observed that RAV-61 (subgroup F) interferes with infection by PR-E in established Ph 310 cells but not in low passage Ph 310 cells. It had been previously reported that F virus in-
552
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terferes with E sarcoma virus in quail and pheasant cells (2, 3). These results indicate there have been changes in Ph 310 cells after long-term culture which have altered susceptibility to exogenous subgroups of RSV and have led to new oncornavirus interactions. It has not yet been determined whether these new viral interactions are affected intracellularly or t.hrough changes at the cell surface. Further studies are being conducted to analyze the interactions of different viruses in Ph 310 cells. ACKNOWLEDGMENTS I am grateful to Dr. Bill Mason for performing the gs tests and to Dr. R. Shoffner for the karyotype analysis. I thank Nancy Hopt and Paul Smith for helpful technical assistance, and Mr. and Mrs. R. Cramer of the Hanaford Valley Game Farm for supplying pheasant eggs. This study was supported by U.S. Public Health Service Grants No. CA 12895, No. CA 15784, and No. CA 10895 from the National Cancer Institute, and an American Cancer Society Institutional Research Grant.
REFERENCES 1. VOGT, P., In “Fundamental Technique in Virology (K. Habel and N. P. Salzman, eds.), pp. 198-211. Academic Press, New York, 1969. 2. HANAFUSA, T., and HANAFUSA, H., Virology 51, 247-251 (1973). 3. FUJITA, D., CHEN, Y., FRIIS, R., and VOGT, P., Virology 60, 558-571 (1974). 4. PAYNE, L., and CHUBB, R., J. Gen. Virol. 3,379391 (1968). 5. HALPERN, M., BOLOGNESI, D., FRIIS, R., and MASON, W., J. Virol. 15, 1131-1140 (1975). 6. VOGT, P., and FRIIS, R., Virology 43, 223-234 (1971). 7. HANAFUSA, H., MIYAMOTO, T., and HANAFUSA, T., Virology 40, 55-64 (1970). 8. WYKE, J., and LINIAL, M., Virology 53, 152-161 (1973). 9. HANAFUSA, H., HAYWARD, W., CHEN, J., and HANAFUSA, T., Cold Spring Harbor Syrnp. Quant. Biol. 39 (Part 2), 1139-1144 (1974). 10. VOGT, P., Virology 25, 237-247 (1965). 11. HANAFUSA, H., Virology 25, 248-255 (1965). 12. TOYOSHIMA, K., and VOGT, P., Virology 38,414426 (1969). 13. ISHIZAKI, R., and SHIMUZO, T., Virology 40,415417 (1970). 14. LINIAL, M., and NEIMAN, P., Virology, in press.