- Email: [email protected]
Terrapene heart (TH-1), a continuous cell line from the heart of the box turtle Terrapene carolina
Q 1967 by Academic Press Inc. Experimental
263
Celi Research 48, 263-268 (1967)
TERRAPENE CELL LINE
HEART
(TH-l),
FROM THE HEART
A CONTINUOUS OF THE BOX TURTLE
TERRAPENECAROLINA" HF.
CLARK
and D. T. KARZON3
Department of Pediatrics and Department of Bacteriology School of Medicine, State University of New York at Buffalo, Received
January
and Immunology, Buffalo, N.Y., U.S.A.
16, 196F
THEdemonstration
by Wolf et al. [22, 253 and Griitzner [la] that cells from poikilothermic vertebrates could be readily cultivated using methods and media developed for mammalian cells led to the rapid development of continuous cell lines from fish [5, 9, 11, 251 and amphibia [8, 15, 241. The preparation of primary cell cultures from turtle [7, 171, lizard [18-201, and snake [13, 14, 19, 201 has also been described. Recently a continuously propagated line of cells from the marine turtle (Chelonia my&s) was described in a brief report [21]. The present paper describes the development of a serially propagated cell line (TH-1) derived from the terrestrial Eastern box turtle Terrapene Carolina which has been under continuous cultivation for four and one-half years.
MATERIALS
AND
METHODS
The heart of an adult box turtle was minced and trypsinized for 19 hr at 4°C in 30 ml of 0.25 per cent trypsin in phosphate buffered saline (PBS), pH 7.3. The resultant cell suspension was sedimented at 100 g for IO min at 23°C resuspended in 5 ml of Eagle’s basal medium (BME) containing 0.15 per cent NaHCO, and 10 per cent calf serum (BME-CSIO) and plated in a 30 ml plastic flask (Falcon) at room temperature (ca 23°C). The medium was replaced whenever the culture became acid (approximately weekly). Cells were passed by decanting the old medium and rinsing the culture briefly with 0.25 per cent trypsin in PBS. When a majority of cells were detached (ca 5-10 min; longer on plastic than on glass), the remaining 1 These studies were initiated in the Special Projects Unit, Viral and Rickettsial Section, National Communicable Disease Center, with the encouragement of Dr C. C. Shepard, Director. 2 Supported in part by research grant CA-08737 from the National Cancer Institute and training grant AI-098 from the National Institute of Allergy and Infectious Diseases. 3 Recipient of Research Career Award AI-1136 from the National Institute of Allergy and Infectious Diseases. 4 Revised version received April 24, 1967. Experimental
Cell Research 48
264
H F. Clark and D. T. Karzon
cells were suspended by vigorous pipetting with media and replated. At the fifth passage, cells were transferred onto glass bottles. Two-fold divisions were routinely employed. After 27 months (passages 14 to 27), fetal calf serum (non-heat-inactivated) was substituted for calf serum in the medium. Media contained 100 units/ml of penicillin and 100 pg/ml of streptomycin. RESULTS
By 28 days, the primary culture of Terrapene heart cells consisted of mixed frbroblasts and epithelial cells which covered 75 per cent of the surface. The second passage achieved 95 per cent cover of mixed cell type at 18 days. Subsequent passages achieved confluent growth in from 7 to 14 days. By the sixth passage, 76 days after primary seeding, fibroblasts had disappeared and cells were entirely of an epithelial type. TH-1 cells have an epithelial morphology ranging from cuboidal to slightly fusiform. Minimum and maximum cell diameters are approximately 20 ,u to 30 ,U and 50 ,u to 60 ,u, respectively. Nuclei are of uniform size, approximately 10 ,U in diameter, with one or two nucleoli. Eosinophilic cytoplasmic inclusions are frequently present adjacent to the nucleus in normal cells. The cells exhibit contact inhibition and may occasionally be oriented in whorl-like configurations. The effect of the standard growth medium BME-CSlO, Eagle’s minimal essential medium with 10 per cent calf serum (MEM-CSlO), and two special media isotonic for frog cells described by Auclair [l j and Freed [S] on TH-1 cell growth was studied. Growth in the frog media was satisfactory, but not superior to that in BME-CSlO. Growth in MEM-CSlO was the poorest observed in any of the media tested. Alkaline conditions (pH >8.0) retarded the growth of TH-1 cells. Therefore, medium used for growth was acidified with 0.1 N HCl to reduce the pH below 7.2 before use. Cell stocks were routinely passaged using approximately 1 x lo5 cells/ml. To determine the minimum cell concentration capable of yielding confluent growth, serial two-fold dilutions of 19th passage cells were plated in 4 oz prescription bottles. Growth was retarded at low plating concentrations but confluent growth was eventually attained in cultures inoculated with as few as 316 cells/ml, the lowest concentration tested. However, confluent growth in cultures with this cell input was obtained only after 468 days of incubation. Passaged cells mere typically in small clumps. Hence, colony forming efficiency could not be readily determined. TH-1 cells were routinely grown at room temperature (approximately 23’C). Cell doubling time at this temperature was 3 to 5 days. In early Experimental
Cell Research 45
Cell line from the box turtle
T. Carolina
265
passages, attempts to grow TH-1 cells at 36°C were uniformly unsuccessful. However, cell cultures grown out at 23’C could be subsequently maintained at 36°C for up to 3 weeks in media containing 20 to 50 per cent calf serum. TH-1 cells grew successfully for a few passages at 30°C but then degenerated. The emergence of cell sublines with increased temperature tolerance is described in a subsequent report [4]. Assessment of methods of freeze-storage of TH-1 cells was undertaken. Dimethylsulfoxide (DMSO) was a superior additive compared to glycerol. Optimal recovery was obtained when cells were frozen in medium containing 7.5 to 15 per cent DMSO and 10 to 40 per cent heat-inactivated (30 min at 56%) calf serum. Near 100 per cent recovery of frozen cells was never attained, but viable TH-1 cells were recovered after 6 months’ storage at - 70°C. Cultures have been successfully stored for 12 months at room temperature with monthly changes of media or for periods of 16 months at 14°C without media changes. Cultures deteriorated rapidly at 4°C. The susceptibility of TH-1 cells to viruses was determined by inoculating 2 or 3 tubes of cells with 0.1 ml of virus in the form of undiluted cell culture or 10 per cent suspensions of egg or animal tissue. Cells inoculated with mammalian or avian viruses were incubated at 36°C. Cells inoculated with viruses from poikilothermic vertebrates were maintained at 23°C. All virusinoculated cultures were maintained in BME with 10 or 20 CS. Cultures were observed for cytopathic effect (CPE) for at least 14 days. Cultures showing cytopathic or toxic change were harvested by freezing and thawing and re-inoculated into TH-1 cells without dilution. Four general types of results were obtained: (1) Vaccinia, herpes simplex, vesicular stomatitis and pseudorabies viruses were serially propagated with CPE in TH-1 cells at 36°C. Vesicular stomatitis virus showed evidence suggestive of auto-interference. Passage of undiluted VSV caused a minimum degree of CPE from which the cell culture often completely recovered. Passage of VSV at a dilution of 1O-2 caused severe CPE, completely destroying the cell sheet. Each of nine amphibian viruses, LTl-LT4, L4-L5 (isolated in this laboratory) and FVl-FV3 (isolated by Dr A. Granoff), were all rapidly cytopathic in TH-1 cells maintained at room temperature. Cytopathic effects caused by each of these amphibian agents were similar. (2) Two strains of cell culture-adapted canine distemper virus (CDV) (strain Onderstepoort AV3, and Buffalo 4856 ferret kidney) and a strain of chorioallantoic membrane-adapted CDV (Onderstepoort) caused syncytial Experimental
Cell Research 48
266
H F. Clark
and D. T. Karzon
CPE during several passages in TH-1 cells. However, each was lost after less than eight passages. No CPE was obtained with the virulent dog brain passaged Snyder Hill strain of CDV. (3) Sindbis virus, Sendai virus, adenoviruses 1 through 7, Rous sarcoma virus (Bryan’s high titer strain) and certain stocks of the Kansas strain of Newcastle disease virus on original inoculation into TH-1 cells caused rapid cytopathic or toxic changes that were not passageable. (4) Many viruses demonstrated no CPE (cultures were not tested for inapparent replication). These are listed below. Reoviruses
Reovirus 1, Lang Picornaviruses
Poliovirus 1, Mahoney Poliovirus 2, MEF-1 Poliovirus 3, Saukett Echovirus 4, Buffalo Shropshire/56 Echovirus 4, Pesacek Echovirus 6, Buffalo Collopy/55 Coxsackie B4, JVB Bovine enterovirus, M-6 Avian encephalomyelitis, 1143
Influenza A2/Jap 305157 Influenza A2/Buffalo C45/63 Influenza B/GL 1739/54 Influenza C 1413, 5/11/63 1233 Parainfluenza I, C35 Parainfluenza 2, Green Respiratory syncytial virus, Long Mumps, Enders Mumps, Buffalo C143/64 Measles, Edmonston, virulent, AV3 Measles, Philadelphia-26 Papovavirus
Shope papilloma, tumor Human papilloma, tumor
Adenoviruses
Canine hepatitis, Cornell 1
Fish virus
Herpesviruses
Equine rhinopneumonitis, Cornell 6540 Canine herpesvirus, F205V Varicella, Buffalo C260/63 Infectious bovine rhinotracheitis, Colorado 1 Avian laryngotracheitis, Cornell CAM Myxoviruses
Influenza A, PR8
Infectious pancreatic necrosis, Wolf Unclassified
viruses
Rabies, CVS, mouse brain Virus diarrhea of cattle, N.Y. 1 Hog cholera, PAV-1, Baker Suckling mouse cataract agent (SMCA) Clark, allantoic fluid GT-48, Clark, allantoic fluid
DISCUSSION
TH-1 cells grow well in mammalian media or in media isotonic for frog cells. For convenience, the mammalian medium BME was routinely employed. Fish cell cultures grow well in mammalian media [9, 11, 251, but Experimental
Cell Research 48
Cell line from the box turtle
T. Carolina
267
amphibian cell cultures appear to require medium of reduced tonicity [l, 2, 8, 15, 16, 241. Growth of reptilian cell cultures has been reported in media of both mammalian [13, 14, 17-201 and reptilian [7] tonicity. In this laboratory, primary cell cultures and continuous cell lines from several species of reptiles have been successfully propagated in mammalian medium. Reptiles, fish and amphibians all have plasma tonicity well below that of mammals, but it may be significant that amphibian tonicity is slightly lower than that of most mammals and fish. TH-1 supports the growth of amphibian viruses FVl-FV3, LTl-LT4, and L4-L5. These viruses have a very wide host range in cells incubated at 23°C or 30°C [3, lo]. TH-1 supports the replication with CPE of herpes simplex, pseudorabies, vaccinia, and vesicular stomatitis viruses at 36°C. Vaccinia replication has been reported in primary cell cultures derived from lizard [18] and turtle [6, 171. Herpes simplex replication in primary turtle cells has also been reported [S]. The growth of pseudorabies and vesicular stomatitis viruses in poikilothermic cell cultures has not been previously reported. Poikilothermic cell lines such as TH-1 may provide rewarding information on the effect of widely divergent host cell species on virus growth and attenuation. Such cell lines may also provide a useful tool for the study of the effect of temperature on cell and virus synthetic processes. SUMMARY
A serially propagated epithelial cell line (TH-1) was established from the heart of a box turtle (Terrapene Carolina). TH-1 cells grew in mammalian media at well as in media of reduced toncity designed for amphibian cells. TH-1 cells routinely were grown and maintained at room temperature (23°C). An incubation temperature of 30°C was tolerated for only a few passages, while a temperature of 36°C supported no cell growth. TH-1 cells could be maintained for brief periods at 36°C. At this temperature replication of vaccinia, herpes simplex, pseudorabies and vesicular stomatitis viruses was obtained. TH-1 cells supported the replication of frog viruses at 23°C. It is suggested that poikilothermic cell lines may offer unusual opportunities to study the growth of homothermic vertebrate viruses in host cells phylogenetically far removed from the normal host. REFERENCES 1. AUCLAIR, W., Nature 192, 467 (1961). 2. BALLS, M. and RUBEN, L. N., Ezpfl Cell Res. 43, 694 (1966). 3. CLARK, H F. and KARZON, D. T., Fed. Proc. 24, 319 (1965).
Experimentul
Cell Research 48
268
H F. Clark and D. T. Karzon
4. CLARK, H F. and KARZON, D. T., Expfl Cell Res. 48, 269 (1967). 5. CLEM, L. W., MOEWUS, L. and SIGEL, M. M., Proc. SOC. Expfl Biol. Med. 108, 762 (1961). ~ I 6. FAUC~NNIER, B., Ann. Inst. Pasteur 105, 439 (1963). 7. FAUCONNIER, B. and PACHOPOS, M., ibid. 102, 661 (1962). 8. FREED, J. J., ExpfZ Cell Res. 26, 327 (1962). 9. FRYER. J. L.. YUSHA. A. and PILCHER. K. S., Ann. N.Y. Acad. Sci. 126. 566 (1965). 10. GRANO~F, A.,‘CAME, P. E. and BREEZE; D. C.,’ Virology 29, 133 (1966). \ ’ 11. GRAVELL, M. and MALSBERGER, R. G., Ann. N. Y. Acad. Sci. 126, 555 (1965). 12. GR~TZNER, L., Zenf. Bakf. Parasif. Znfekf. Hyg. 173, 195 (1958). 13. KAZAR, J., REHACEK, J. and BREZINA, R., J. Hyg. Epidemiol. Microbial. Zmmunol. 10, 240 (1966). 14. MELENDEZ, L., SPALATIN, J. and HANSON, R. P., Am. J. Vet. Res. 26, 1451 (1965). 15. RAFFERTY, K. A., JR., Ann. N. Y. Acad. Sci. U.S. 126, 3 (1965). 16. SHAH, C. q., Experientia 18, 239 (1962). 17. SHINDAROV. L.. Compi. Rend. Acad. B&are Sci. 15, 539 (1962). 18. SHINDAROV; L.‘and TONEV, E., Acfa Vi&. 9, 459 (i965). ~ ’ 19. SOMOGYIOVA, J., Biologia 19, 257 (1964). 20. SOMOGYIOVA, J. and REHACEK, J., Acfa Virol. 9, 286 (1965). 21. WADDELL, G. H. and SIGEL, M. M., Bacf. Proc., p. 99 (1965). 22. WOLF, K. and DUNBAR, C. E., Proc. Sot. Expfl Biof. Med. 95, 455 (1957). 23. WOLF, K. and QUIMBY, M. C., Science 144, 1578 (1964).
24. 25.
ibid. 135, 1065 (1962).
WOLF,
Experimental
K.,
QUIMBY,
M. C., PYLE,
Cell Research 48
E. A. and
DEXTER,
R. P., Science
132, 1890 (1960).
263
Celi Research 48, 263-268 (1967)
TERRAPENE CELL LINE
HEART
(TH-l),
FROM THE HEART
A CONTINUOUS OF THE BOX TURTLE
TERRAPENECAROLINA" HF.
CLARK
and D. T. KARZON3
Department of Pediatrics and Department of Bacteriology School of Medicine, State University of New York at Buffalo, Received
January
and Immunology, Buffalo, N.Y., U.S.A.
16, 196F
THEdemonstration
by Wolf et al. [22, 253 and Griitzner [la] that cells from poikilothermic vertebrates could be readily cultivated using methods and media developed for mammalian cells led to the rapid development of continuous cell lines from fish [5, 9, 11, 251 and amphibia [8, 15, 241. The preparation of primary cell cultures from turtle [7, 171, lizard [18-201, and snake [13, 14, 19, 201 has also been described. Recently a continuously propagated line of cells from the marine turtle (Chelonia my&s) was described in a brief report [21]. The present paper describes the development of a serially propagated cell line (TH-1) derived from the terrestrial Eastern box turtle Terrapene Carolina which has been under continuous cultivation for four and one-half years.
MATERIALS
AND
METHODS
The heart of an adult box turtle was minced and trypsinized for 19 hr at 4°C in 30 ml of 0.25 per cent trypsin in phosphate buffered saline (PBS), pH 7.3. The resultant cell suspension was sedimented at 100 g for IO min at 23°C resuspended in 5 ml of Eagle’s basal medium (BME) containing 0.15 per cent NaHCO, and 10 per cent calf serum (BME-CSIO) and plated in a 30 ml plastic flask (Falcon) at room temperature (ca 23°C). The medium was replaced whenever the culture became acid (approximately weekly). Cells were passed by decanting the old medium and rinsing the culture briefly with 0.25 per cent trypsin in PBS. When a majority of cells were detached (ca 5-10 min; longer on plastic than on glass), the remaining 1 These studies were initiated in the Special Projects Unit, Viral and Rickettsial Section, National Communicable Disease Center, with the encouragement of Dr C. C. Shepard, Director. 2 Supported in part by research grant CA-08737 from the National Cancer Institute and training grant AI-098 from the National Institute of Allergy and Infectious Diseases. 3 Recipient of Research Career Award AI-1136 from the National Institute of Allergy and Infectious Diseases. 4 Revised version received April 24, 1967. Experimental
Cell Research 48
264
H F. Clark and D. T. Karzon
cells were suspended by vigorous pipetting with media and replated. At the fifth passage, cells were transferred onto glass bottles. Two-fold divisions were routinely employed. After 27 months (passages 14 to 27), fetal calf serum (non-heat-inactivated) was substituted for calf serum in the medium. Media contained 100 units/ml of penicillin and 100 pg/ml of streptomycin. RESULTS
By 28 days, the primary culture of Terrapene heart cells consisted of mixed frbroblasts and epithelial cells which covered 75 per cent of the surface. The second passage achieved 95 per cent cover of mixed cell type at 18 days. Subsequent passages achieved confluent growth in from 7 to 14 days. By the sixth passage, 76 days after primary seeding, fibroblasts had disappeared and cells were entirely of an epithelial type. TH-1 cells have an epithelial morphology ranging from cuboidal to slightly fusiform. Minimum and maximum cell diameters are approximately 20 ,u to 30 ,U and 50 ,u to 60 ,u, respectively. Nuclei are of uniform size, approximately 10 ,U in diameter, with one or two nucleoli. Eosinophilic cytoplasmic inclusions are frequently present adjacent to the nucleus in normal cells. The cells exhibit contact inhibition and may occasionally be oriented in whorl-like configurations. The effect of the standard growth medium BME-CSlO, Eagle’s minimal essential medium with 10 per cent calf serum (MEM-CSlO), and two special media isotonic for frog cells described by Auclair [l j and Freed [S] on TH-1 cell growth was studied. Growth in the frog media was satisfactory, but not superior to that in BME-CSlO. Growth in MEM-CSlO was the poorest observed in any of the media tested. Alkaline conditions (pH >8.0) retarded the growth of TH-1 cells. Therefore, medium used for growth was acidified with 0.1 N HCl to reduce the pH below 7.2 before use. Cell stocks were routinely passaged using approximately 1 x lo5 cells/ml. To determine the minimum cell concentration capable of yielding confluent growth, serial two-fold dilutions of 19th passage cells were plated in 4 oz prescription bottles. Growth was retarded at low plating concentrations but confluent growth was eventually attained in cultures inoculated with as few as 316 cells/ml, the lowest concentration tested. However, confluent growth in cultures with this cell input was obtained only after 468 days of incubation. Passaged cells mere typically in small clumps. Hence, colony forming efficiency could not be readily determined. TH-1 cells were routinely grown at room temperature (approximately 23’C). Cell doubling time at this temperature was 3 to 5 days. In early Experimental
Cell Research 45
Cell line from the box turtle
T. Carolina
265
passages, attempts to grow TH-1 cells at 36°C were uniformly unsuccessful. However, cell cultures grown out at 23’C could be subsequently maintained at 36°C for up to 3 weeks in media containing 20 to 50 per cent calf serum. TH-1 cells grew successfully for a few passages at 30°C but then degenerated. The emergence of cell sublines with increased temperature tolerance is described in a subsequent report [4]. Assessment of methods of freeze-storage of TH-1 cells was undertaken. Dimethylsulfoxide (DMSO) was a superior additive compared to glycerol. Optimal recovery was obtained when cells were frozen in medium containing 7.5 to 15 per cent DMSO and 10 to 40 per cent heat-inactivated (30 min at 56%) calf serum. Near 100 per cent recovery of frozen cells was never attained, but viable TH-1 cells were recovered after 6 months’ storage at - 70°C. Cultures have been successfully stored for 12 months at room temperature with monthly changes of media or for periods of 16 months at 14°C without media changes. Cultures deteriorated rapidly at 4°C. The susceptibility of TH-1 cells to viruses was determined by inoculating 2 or 3 tubes of cells with 0.1 ml of virus in the form of undiluted cell culture or 10 per cent suspensions of egg or animal tissue. Cells inoculated with mammalian or avian viruses were incubated at 36°C. Cells inoculated with viruses from poikilothermic vertebrates were maintained at 23°C. All virusinoculated cultures were maintained in BME with 10 or 20 CS. Cultures were observed for cytopathic effect (CPE) for at least 14 days. Cultures showing cytopathic or toxic change were harvested by freezing and thawing and re-inoculated into TH-1 cells without dilution. Four general types of results were obtained: (1) Vaccinia, herpes simplex, vesicular stomatitis and pseudorabies viruses were serially propagated with CPE in TH-1 cells at 36°C. Vesicular stomatitis virus showed evidence suggestive of auto-interference. Passage of undiluted VSV caused a minimum degree of CPE from which the cell culture often completely recovered. Passage of VSV at a dilution of 1O-2 caused severe CPE, completely destroying the cell sheet. Each of nine amphibian viruses, LTl-LT4, L4-L5 (isolated in this laboratory) and FVl-FV3 (isolated by Dr A. Granoff), were all rapidly cytopathic in TH-1 cells maintained at room temperature. Cytopathic effects caused by each of these amphibian agents were similar. (2) Two strains of cell culture-adapted canine distemper virus (CDV) (strain Onderstepoort AV3, and Buffalo 4856 ferret kidney) and a strain of chorioallantoic membrane-adapted CDV (Onderstepoort) caused syncytial Experimental
Cell Research 48
266
H F. Clark
and D. T. Karzon
CPE during several passages in TH-1 cells. However, each was lost after less than eight passages. No CPE was obtained with the virulent dog brain passaged Snyder Hill strain of CDV. (3) Sindbis virus, Sendai virus, adenoviruses 1 through 7, Rous sarcoma virus (Bryan’s high titer strain) and certain stocks of the Kansas strain of Newcastle disease virus on original inoculation into TH-1 cells caused rapid cytopathic or toxic changes that were not passageable. (4) Many viruses demonstrated no CPE (cultures were not tested for inapparent replication). These are listed below. Reoviruses
Reovirus 1, Lang Picornaviruses
Poliovirus 1, Mahoney Poliovirus 2, MEF-1 Poliovirus 3, Saukett Echovirus 4, Buffalo Shropshire/56 Echovirus 4, Pesacek Echovirus 6, Buffalo Collopy/55 Coxsackie B4, JVB Bovine enterovirus, M-6 Avian encephalomyelitis, 1143
Influenza A2/Jap 305157 Influenza A2/Buffalo C45/63 Influenza B/GL 1739/54 Influenza C 1413, 5/11/63 1233 Parainfluenza I, C35 Parainfluenza 2, Green Respiratory syncytial virus, Long Mumps, Enders Mumps, Buffalo C143/64 Measles, Edmonston, virulent, AV3 Measles, Philadelphia-26 Papovavirus
Shope papilloma, tumor Human papilloma, tumor
Adenoviruses
Canine hepatitis, Cornell 1
Fish virus
Herpesviruses
Equine rhinopneumonitis, Cornell 6540 Canine herpesvirus, F205V Varicella, Buffalo C260/63 Infectious bovine rhinotracheitis, Colorado 1 Avian laryngotracheitis, Cornell CAM Myxoviruses
Influenza A, PR8
Infectious pancreatic necrosis, Wolf Unclassified
viruses
Rabies, CVS, mouse brain Virus diarrhea of cattle, N.Y. 1 Hog cholera, PAV-1, Baker Suckling mouse cataract agent (SMCA) Clark, allantoic fluid GT-48, Clark, allantoic fluid
DISCUSSION
TH-1 cells grow well in mammalian media or in media isotonic for frog cells. For convenience, the mammalian medium BME was routinely employed. Fish cell cultures grow well in mammalian media [9, 11, 251, but Experimental
Cell Research 48
Cell line from the box turtle
T. Carolina
267
amphibian cell cultures appear to require medium of reduced tonicity [l, 2, 8, 15, 16, 241. Growth of reptilian cell cultures has been reported in media of both mammalian [13, 14, 17-201 and reptilian [7] tonicity. In this laboratory, primary cell cultures and continuous cell lines from several species of reptiles have been successfully propagated in mammalian medium. Reptiles, fish and amphibians all have plasma tonicity well below that of mammals, but it may be significant that amphibian tonicity is slightly lower than that of most mammals and fish. TH-1 supports the growth of amphibian viruses FVl-FV3, LTl-LT4, and L4-L5. These viruses have a very wide host range in cells incubated at 23°C or 30°C [3, lo]. TH-1 supports the replication with CPE of herpes simplex, pseudorabies, vaccinia, and vesicular stomatitis viruses at 36°C. Vaccinia replication has been reported in primary cell cultures derived from lizard [18] and turtle [6, 171. Herpes simplex replication in primary turtle cells has also been reported [S]. The growth of pseudorabies and vesicular stomatitis viruses in poikilothermic cell cultures has not been previously reported. Poikilothermic cell lines such as TH-1 may provide rewarding information on the effect of widely divergent host cell species on virus growth and attenuation. Such cell lines may also provide a useful tool for the study of the effect of temperature on cell and virus synthetic processes. SUMMARY
A serially propagated epithelial cell line (TH-1) was established from the heart of a box turtle (Terrapene Carolina). TH-1 cells grew in mammalian media at well as in media of reduced toncity designed for amphibian cells. TH-1 cells routinely were grown and maintained at room temperature (23°C). An incubation temperature of 30°C was tolerated for only a few passages, while a temperature of 36°C supported no cell growth. TH-1 cells could be maintained for brief periods at 36°C. At this temperature replication of vaccinia, herpes simplex, pseudorabies and vesicular stomatitis viruses was obtained. TH-1 cells supported the replication of frog viruses at 23°C. It is suggested that poikilothermic cell lines may offer unusual opportunities to study the growth of homothermic vertebrate viruses in host cells phylogenetically far removed from the normal host. REFERENCES 1. AUCLAIR, W., Nature 192, 467 (1961). 2. BALLS, M. and RUBEN, L. N., Ezpfl Cell Res. 43, 694 (1966). 3. CLARK, H F. and KARZON, D. T., Fed. Proc. 24, 319 (1965).
Experimentul
Cell Research 48
268
H F. Clark and D. T. Karzon
4. CLARK, H F. and KARZON, D. T., Expfl Cell Res. 48, 269 (1967). 5. CLEM, L. W., MOEWUS, L. and SIGEL, M. M., Proc. SOC. Expfl Biol. Med. 108, 762 (1961). ~ I 6. FAUC~NNIER, B., Ann. Inst. Pasteur 105, 439 (1963). 7. FAUCONNIER, B. and PACHOPOS, M., ibid. 102, 661 (1962). 8. FREED, J. J., ExpfZ Cell Res. 26, 327 (1962). 9. FRYER. J. L.. YUSHA. A. and PILCHER. K. S., Ann. N.Y. Acad. Sci. 126. 566 (1965). 10. GRANO~F, A.,‘CAME, P. E. and BREEZE; D. C.,’ Virology 29, 133 (1966). \ ’ 11. GRAVELL, M. and MALSBERGER, R. G., Ann. N. Y. Acad. Sci. 126, 555 (1965). 12. GR~TZNER, L., Zenf. Bakf. Parasif. Znfekf. Hyg. 173, 195 (1958). 13. KAZAR, J., REHACEK, J. and BREZINA, R., J. Hyg. Epidemiol. Microbial. Zmmunol. 10, 240 (1966). 14. MELENDEZ, L., SPALATIN, J. and HANSON, R. P., Am. J. Vet. Res. 26, 1451 (1965). 15. RAFFERTY, K. A., JR., Ann. N. Y. Acad. Sci. U.S. 126, 3 (1965). 16. SHAH, C. q., Experientia 18, 239 (1962). 17. SHINDAROV. L.. Compi. Rend. Acad. B&are Sci. 15, 539 (1962). 18. SHINDAROV; L.‘and TONEV, E., Acfa Vi&. 9, 459 (i965). ~ ’ 19. SOMOGYIOVA, J., Biologia 19, 257 (1964). 20. SOMOGYIOVA, J. and REHACEK, J., Acfa Virol. 9, 286 (1965). 21. WADDELL, G. H. and SIGEL, M. M., Bacf. Proc., p. 99 (1965). 22. WOLF, K. and DUNBAR, C. E., Proc. Sot. Expfl Biof. Med. 95, 455 (1957). 23. WOLF, K. and QUIMBY, M. C., Science 144, 1578 (1964).
24. 25.
ibid. 135, 1065 (1962).
WOLF,
Experimental
K.,
QUIMBY,
M. C., PYLE,
Cell Research 48
E. A. and
DEXTER,
R. P., Science
132, 1890 (1960).