Journalof General Virology(1991), 72, 2537-2539. Printedin Great Britain
2537
Spontaneously productive C-type retrovirus infection of fish cell lines G. N. Frerichs, z* Diana Morgan, 2 D. Hart, 2 Christine Skerrow, ~ R. J. Roberts ~ and D. E. Onions 2 l Institute of Aquaculture, University of Stirling, Stifling FK9 4LA and 2Department of Veterinary Pathology, University of Glasgow Veterinary School, Bearsden, Glasgow G61 1QH, U.K.
The spontaneous production and release of morphologically typical, 85 to 90 nm diameter C-type retrovirus particles from four cell lines derived from three species of warmwater fish have been identified. Virus pellets from cell culture supernatants showed high levels of
MnZ+-dependent reverse transcriptase activity at 24 °C. Peak enzyme activity was associated with a 1-16 g/ml sucrose gradient fraction. All four isolates induced a cytolytic infection of a bluegill fry cell line within 6 to 10 days.
The spontaneous or chemically induced release of C-type retroviruses from a wide variety of low-passage cell cultures and high-passage established cell lines derived from non-neoplastic mammalian tissues has been well documented and reviewed by Teich (1982). Similar viruses have not hitherto been reported in fish cell cultures, but C-type particles have been observed in lesions from epidermal hyperplasia of northern pike (Esox lucius) (Winqvist et al., 1968; Yamamoto et al., 1983), epidermal hyperplasia and dermal sarcoma of walleye perch (Stizostedion vitreum) (Walker, 1969; Yamamoto et al., 1985), swimbladder fibrosarcoma of Atlantic salmon (Salmo salar) (Duncan, 1978) and fin papillomatosis of European smelt (Osmerus eperlanus) (Anders, 1989). C-type particles and reverse transcriptase activity associated with 1.15 to 1.16 g/ml sucrose gradient fractions have also been identified in tissue from northern pike lymphosarcoma (Papas et al., 1976) and white sucker (Catostomus commersoni) papillomatosis (Sonstegard, 1977). Cell-free transmission studies have suggested a viral aetiology for northern pike lymphosarcoma (Mulcahy & O'Leary, 1970; Brown et al., 1975), but none of the above C-type viruses has been isolated in cell culture and the pathogenic significance of these agents has yet to be established. As part of the procedure to characterize a number of newly developed cell lines derived from southeast Asian freshwater fish species, we have now identified piscine C-type retroviruses in the form of spontaneously productive infections of four cell lines derived from three unrelated species of fish. Two of the infected cell lines originated from striped snakehead fish (Ophicephalus striatus) captured at different times and locations in Thailand, the third line was derived from a climbing perch (Anabas testudineus) in
Thailand and the fourth from a snakeskin gourami (Trichogaster pectoralis) imported into the U.K. from Singapore. One snakehead line (SSN-1) was initiated from whole fry tissue and the second (SSN-3) from caudal peduncle tissue of clinically healthy juvenile fish. The climbing perch line (CP) and the gourami line (SGP) were also developed from juvenile caudal peduncle tissue. Primary monolayer cultures were initiated and cell lines subsequently established by standard fish tissue and cell culture trypsinization and transfer procedures (Wolf, 1979) using Leibovitz L-15 medium supplemented with 10~ foetal calf serum for cell growth. All cultures were established and routinely maintained at 25 °C. Electron microscopy of uranyl acetate-lead citratestained ultrathin sections of seemingly normal monolayer cultures of each cell line at approximate passage levels 80 (SSN-1), 50 (SSN-3), 30 (CP) and 20 (SGP) showed all four lines to be infected with morphologically similar C-type virus particles. Progeny virions formed at the plasma membrane and finally budded off as roughly spherical 85 to 90 nm diameter particles, with a centrally located nucleoid separated by an electron-lucent zone from an outer envelope carrying surfac e projections (Fig. 1). Extracellular virus pellets were obtained from clarified supernatant culture fluids by ultracentrifugation at 150000 g for 60 min and were assayed for reverse transcriptase (RT) activity at room temperature (24 °C) essentially according to the method of Klement & Nicolson (1977). Pellets were resuspended in freshly prepared disruption buffer and 25 lal samples were added to an equal volume of reaction mix containing either 40 l.tg/ml poly(rA).oligo(dT) or 40 ~tg/ml poly(dA).oligo(dT) and 2 mM*MnC12 or 20 mM-MgClz. Supernatants from all four cell lines showed high levels of Mn :÷dependent RT activity, with poly(rA).oligo(dT):poly-
0001-0428 © 1991 SGM
2538
Short communication
Fig. 1. C-type retrovirus particle formation and release in snakehead fish cells (SSN-1) Bar represents 100 nm.
Fig. 2. BF-2cellcytopathology. (a) Normal monolayer; (b) comparable monolayer 8 days after infection with extracellular gourami cell line (SGP) retrovirus.
Table 1. Cell culture supernatant R T activity at 24 °C Synthetic template c.p.m. Cell culture
Cation added
poly(rA)
poly(dA)
Ratio rA :dA
SSN-1
Mn z+ Mg 2÷ Mn 2÷ Mg 2+ Mn 2+ Mg 2+ Mn ~+ Mg 2+ Mn 2+ Mg 2÷
304218 6732 360 286 174356 859 278608 31636 194215 5706
9626 6236 443 417 1522 856 22906 10178 10736 7614
31.6 1"1 0-8 0.7 114.5 1-0 I2-2 3-1 180-5 0-7
SSN-2 SSN-3 SGP CP
(dA). oligo(dT) ratios of [3H]TTP incorporation of 31.6 (SSN-1), 114.5 (SSN-3), 180.5 (CP) and 12.2 (SGP) (Table 1). Peak enzyme activity was associated with a 1.16 g/ml sucrose density gradient fraction, corresponding to the buoyant density of C-type retroviruses. In
contrast to these findings, a third striped snakehead fish cell line (SSN-2), similarly derived from caudal peduncle tissue of wild juvenile fish in Thailand, showed no electron microscopic or RT assay evidence of retrovirus infection either before or immediately after treatment of the cells with 50 ~tg/ml 5-iododeoxyuridine for 48 h. All four retrovirus isolates were found to induce a cytolytic infection in monolayer cultures of the BF-2 cell line (ATCC no. CCL91) derived from bluegill fry (Lepomis machrochirus). Cytopathic effects were first observed as a generalized depletion of cells, giving the monolayer a mesh-like appearance 6 to 10 days after inoculation with infective supernatant fluids (Fig. 2). Cell degeneration and depletion normally continued until the monolayer was completely destroyed 10 to 14 days layer. Productive retrovirus infection of the BF-2 cells was identified by electron microscopy. The susceptibility of BF-2 cells to exogenous retrovirus infection was confirmed by inoculating 25 cm z flasks of subconfluent cells with 1 ml culture medium or RT-positive medium
Short communication
Table 2. Triplicate BF-2 cell culture supernatant R T activity at 22 °C following infection with SSN-1 cell culture fluid or normal medium
Infective medium
Days postinfection
Normal
0
SSN-I
0
Normal
8
SSN-I
8
Normal
15
SSN-1
15
Normal
22
SSN-I
22
Synthetic template (c.p.m.) poly(rA)
poly(dA)
Ratio rA :dA
648 658 572 595 546 582 291 285 335 2615 6235 3635 762 963 826 17187 25 328 30093 1105 2106 2507 97 045 16294 27050
932 1082 904 970 826 880 369 387 317 763 810 947 1098 1465 1137 1584 853 1578 1198 2090 2251 2482 697 979
0.7 0-6 0.6 0.6 0-7 0.7 0.8 0-7 1.1 3.4 7.7 3.9 0.7 0.7 0.7 10-9 29.7 19.1 0.9 1.0 1.1 39.1 23.4 27.6
from SSN-1 cells. Polybrene was added to a final concentration of 4 Ixg/ml and the inoculum left for 1-5 h before the addition of further medium without polybrene. The cells were maintained at 22 °C and passaged when confluent, which was usually once a week. Medium was removed 2 days after subculture, clarified at 10000g for 10 min and stored at - 7 0 ° C until assayed. RT activity in the supernatant medium from SSN-l-infected BF-2 cells increased progressively with each subculture, whereas mock-infected cells showed no comparable degree of enzyme activity (Table 2). The retroviruses described in the present report are firmly believed to be of piscine origin, as no other type of cell culture was ever handled within the laboratory prior to the first identification of a virus-infected fish cell line. Further studies are required to determine whether proviral sequences related to the released retroviruses are present in the DNA of tissues of the corresponding fish species to resolve whether the cell culture isolates are genetically acquired (endogenous) or transmissible (ex-
2539
ogenous) infections. The relatedness of this group of cell culture viruses to the C-type viruses isolated from other animal classes, as well as the C-type viruses which have been observed in association with neoplastic conditions of teleost fish, has yet to be established. Apart from these considerations however, the availability of a cultureadapted piscine C-type retrovirus affords the opportunity for fundamental research into the nature of retrovirus infections in the phylogenetically most simple and primitive of vertebrate animals. We thank Stuart Millar and Mary Alexander for technical assistance. This work was supported by the U.K. Overseas Development Administration.
References ANDERS, K. (1989). A herpes virus associated with an epizootic epidermal papillomatosis in European smolt (Osmerus eperlanus). In Viruses of Lower Vertebrates, pp. 184 197. Edited by W. Ahne & E. Kurstak. Heidelberg: Springer-Verlag. BROWN, E. R., KEITH, L., HAZDRA,J. J. & ARNDT, T. (1975). Tumors in fish caught in polluted waters: possible explanations. In Comparative Leukemia Research 1973, pp. 47-57. Edited by Y. Ito & R. M. Dutcher. Basel: S. Karger. DUNCAN, 1. B. (1978). Evidence for an oncovirus in swimbladder fibrosarcoma of Atlantic salmon Salmo salar L. Journal of Fish Diseases l, 127-131. KLEMENT, V. & NICOLSON, M. O. (1977). Methods for assays of RNA tumor viruses. Methods in Virology 6, 6~80. MULCAHY, M. F. & O'LEARY, A. (1970). Cell-free transmission of lymphasarcoma in the northern pike Esox lucius L. (Pisces; Esocidae). Experientia 26, 891. PAPAS, T. S., DAnLaERt~, J. E. & SONSTEGARD, R. A. (1976). Type C virus in lymphosarcoma in northern pike (Esox lucius). Nature, London 261, 506-508. SONST~GARD, R. A. (1977). Environmental carcinogenesis studies in fishes of the Great Lakes of North America. Annals of the New York Academy of Sciences 298, 261-269. TEICH, N. (1982). Taxonomy of retroviruses. In Molecular Biology of Tumor Viruses, pp. 25-207. Edited by R. Weiss, N. Teich, H. Varmus & J. Coffin. New York: Cold Spring Harbor Laboratory. WALKER, R. (1969). Virus associated with epidermal hyperplasia in fish. National Cancer Institute Monograph 31, 195-207. WINQVIST, G., LJUNGBERG,O. & HELLSTROEM,B. (1968). Skin turnouts of northern pike (Esox lucius L.) II. Viral particles in epidermal proliferations. Bulletin de l'Office International des Epizooties 69, 1023-1031. WOLF, K. (1979). Cold-blooded vertebrate cell and tissue culture. Methods in Enzymology 58, 466-477. YAMAMOTO, T., KELLY, R. K. & NIELSON, O. (1983). Epidermal hyperplasias of northern pike (Esox lucius) associated with herpesvirus and C-type particles. Archives of Virology 79, 255-272. YAMAMOTO, T., KELLY, R. K. & NIELSON, O. (1985). Morphological differentiation of virus-associated skin tumors of walleye (Stizostedion vitreum vitreum). Fish Pathology 20, 361-372.
(Received 13 June 1991 ," Accepted 17 June 1991)
2537
Spontaneously productive C-type retrovirus infection of fish cell lines G. N. Frerichs, z* Diana Morgan, 2 D. Hart, 2 Christine Skerrow, ~ R. J. Roberts ~ and D. E. Onions 2 l Institute of Aquaculture, University of Stirling, Stifling FK9 4LA and 2Department of Veterinary Pathology, University of Glasgow Veterinary School, Bearsden, Glasgow G61 1QH, U.K.
The spontaneous production and release of morphologically typical, 85 to 90 nm diameter C-type retrovirus particles from four cell lines derived from three species of warmwater fish have been identified. Virus pellets from cell culture supernatants showed high levels of
MnZ+-dependent reverse transcriptase activity at 24 °C. Peak enzyme activity was associated with a 1-16 g/ml sucrose gradient fraction. All four isolates induced a cytolytic infection of a bluegill fry cell line within 6 to 10 days.
The spontaneous or chemically induced release of C-type retroviruses from a wide variety of low-passage cell cultures and high-passage established cell lines derived from non-neoplastic mammalian tissues has been well documented and reviewed by Teich (1982). Similar viruses have not hitherto been reported in fish cell cultures, but C-type particles have been observed in lesions from epidermal hyperplasia of northern pike (Esox lucius) (Winqvist et al., 1968; Yamamoto et al., 1983), epidermal hyperplasia and dermal sarcoma of walleye perch (Stizostedion vitreum) (Walker, 1969; Yamamoto et al., 1985), swimbladder fibrosarcoma of Atlantic salmon (Salmo salar) (Duncan, 1978) and fin papillomatosis of European smelt (Osmerus eperlanus) (Anders, 1989). C-type particles and reverse transcriptase activity associated with 1.15 to 1.16 g/ml sucrose gradient fractions have also been identified in tissue from northern pike lymphosarcoma (Papas et al., 1976) and white sucker (Catostomus commersoni) papillomatosis (Sonstegard, 1977). Cell-free transmission studies have suggested a viral aetiology for northern pike lymphosarcoma (Mulcahy & O'Leary, 1970; Brown et al., 1975), but none of the above C-type viruses has been isolated in cell culture and the pathogenic significance of these agents has yet to be established. As part of the procedure to characterize a number of newly developed cell lines derived from southeast Asian freshwater fish species, we have now identified piscine C-type retroviruses in the form of spontaneously productive infections of four cell lines derived from three unrelated species of fish. Two of the infected cell lines originated from striped snakehead fish (Ophicephalus striatus) captured at different times and locations in Thailand, the third line was derived from a climbing perch (Anabas testudineus) in
Thailand and the fourth from a snakeskin gourami (Trichogaster pectoralis) imported into the U.K. from Singapore. One snakehead line (SSN-1) was initiated from whole fry tissue and the second (SSN-3) from caudal peduncle tissue of clinically healthy juvenile fish. The climbing perch line (CP) and the gourami line (SGP) were also developed from juvenile caudal peduncle tissue. Primary monolayer cultures were initiated and cell lines subsequently established by standard fish tissue and cell culture trypsinization and transfer procedures (Wolf, 1979) using Leibovitz L-15 medium supplemented with 10~ foetal calf serum for cell growth. All cultures were established and routinely maintained at 25 °C. Electron microscopy of uranyl acetate-lead citratestained ultrathin sections of seemingly normal monolayer cultures of each cell line at approximate passage levels 80 (SSN-1), 50 (SSN-3), 30 (CP) and 20 (SGP) showed all four lines to be infected with morphologically similar C-type virus particles. Progeny virions formed at the plasma membrane and finally budded off as roughly spherical 85 to 90 nm diameter particles, with a centrally located nucleoid separated by an electron-lucent zone from an outer envelope carrying surfac e projections (Fig. 1). Extracellular virus pellets were obtained from clarified supernatant culture fluids by ultracentrifugation at 150000 g for 60 min and were assayed for reverse transcriptase (RT) activity at room temperature (24 °C) essentially according to the method of Klement & Nicolson (1977). Pellets were resuspended in freshly prepared disruption buffer and 25 lal samples were added to an equal volume of reaction mix containing either 40 l.tg/ml poly(rA).oligo(dT) or 40 ~tg/ml poly(dA).oligo(dT) and 2 mM*MnC12 or 20 mM-MgClz. Supernatants from all four cell lines showed high levels of Mn :÷dependent RT activity, with poly(rA).oligo(dT):poly-
0001-0428 © 1991 SGM
2538
Short communication
Fig. 1. C-type retrovirus particle formation and release in snakehead fish cells (SSN-1) Bar represents 100 nm.
Fig. 2. BF-2cellcytopathology. (a) Normal monolayer; (b) comparable monolayer 8 days after infection with extracellular gourami cell line (SGP) retrovirus.
Table 1. Cell culture supernatant R T activity at 24 °C Synthetic template c.p.m. Cell culture
Cation added
poly(rA)
poly(dA)
Ratio rA :dA
SSN-1
Mn z+ Mg 2÷ Mn 2÷ Mg 2+ Mn 2+ Mg 2+ Mn ~+ Mg 2+ Mn 2+ Mg 2÷
304218 6732 360 286 174356 859 278608 31636 194215 5706
9626 6236 443 417 1522 856 22906 10178 10736 7614
31.6 1"1 0-8 0.7 114.5 1-0 I2-2 3-1 180-5 0-7
SSN-2 SSN-3 SGP CP
(dA). oligo(dT) ratios of [3H]TTP incorporation of 31.6 (SSN-1), 114.5 (SSN-3), 180.5 (CP) and 12.2 (SGP) (Table 1). Peak enzyme activity was associated with a 1.16 g/ml sucrose density gradient fraction, corresponding to the buoyant density of C-type retroviruses. In
contrast to these findings, a third striped snakehead fish cell line (SSN-2), similarly derived from caudal peduncle tissue of wild juvenile fish in Thailand, showed no electron microscopic or RT assay evidence of retrovirus infection either before or immediately after treatment of the cells with 50 ~tg/ml 5-iododeoxyuridine for 48 h. All four retrovirus isolates were found to induce a cytolytic infection in monolayer cultures of the BF-2 cell line (ATCC no. CCL91) derived from bluegill fry (Lepomis machrochirus). Cytopathic effects were first observed as a generalized depletion of cells, giving the monolayer a mesh-like appearance 6 to 10 days after inoculation with infective supernatant fluids (Fig. 2). Cell degeneration and depletion normally continued until the monolayer was completely destroyed 10 to 14 days layer. Productive retrovirus infection of the BF-2 cells was identified by electron microscopy. The susceptibility of BF-2 cells to exogenous retrovirus infection was confirmed by inoculating 25 cm z flasks of subconfluent cells with 1 ml culture medium or RT-positive medium
Short communication
Table 2. Triplicate BF-2 cell culture supernatant R T activity at 22 °C following infection with SSN-1 cell culture fluid or normal medium
Infective medium
Days postinfection
Normal
0
SSN-I
0
Normal
8
SSN-I
8
Normal
15
SSN-1
15
Normal
22
SSN-I
22
Synthetic template (c.p.m.) poly(rA)
poly(dA)
Ratio rA :dA
648 658 572 595 546 582 291 285 335 2615 6235 3635 762 963 826 17187 25 328 30093 1105 2106 2507 97 045 16294 27050
932 1082 904 970 826 880 369 387 317 763 810 947 1098 1465 1137 1584 853 1578 1198 2090 2251 2482 697 979
0.7 0-6 0.6 0.6 0-7 0.7 0.8 0-7 1.1 3.4 7.7 3.9 0.7 0.7 0.7 10-9 29.7 19.1 0.9 1.0 1.1 39.1 23.4 27.6
from SSN-1 cells. Polybrene was added to a final concentration of 4 Ixg/ml and the inoculum left for 1-5 h before the addition of further medium without polybrene. The cells were maintained at 22 °C and passaged when confluent, which was usually once a week. Medium was removed 2 days after subculture, clarified at 10000g for 10 min and stored at - 7 0 ° C until assayed. RT activity in the supernatant medium from SSN-l-infected BF-2 cells increased progressively with each subculture, whereas mock-infected cells showed no comparable degree of enzyme activity (Table 2). The retroviruses described in the present report are firmly believed to be of piscine origin, as no other type of cell culture was ever handled within the laboratory prior to the first identification of a virus-infected fish cell line. Further studies are required to determine whether proviral sequences related to the released retroviruses are present in the DNA of tissues of the corresponding fish species to resolve whether the cell culture isolates are genetically acquired (endogenous) or transmissible (ex-
2539
ogenous) infections. The relatedness of this group of cell culture viruses to the C-type viruses isolated from other animal classes, as well as the C-type viruses which have been observed in association with neoplastic conditions of teleost fish, has yet to be established. Apart from these considerations however, the availability of a cultureadapted piscine C-type retrovirus affords the opportunity for fundamental research into the nature of retrovirus infections in the phylogenetically most simple and primitive of vertebrate animals. We thank Stuart Millar and Mary Alexander for technical assistance. This work was supported by the U.K. Overseas Development Administration.
References ANDERS, K. (1989). A herpes virus associated with an epizootic epidermal papillomatosis in European smolt (Osmerus eperlanus). In Viruses of Lower Vertebrates, pp. 184 197. Edited by W. Ahne & E. Kurstak. Heidelberg: Springer-Verlag. BROWN, E. R., KEITH, L., HAZDRA,J. J. & ARNDT, T. (1975). Tumors in fish caught in polluted waters: possible explanations. In Comparative Leukemia Research 1973, pp. 47-57. Edited by Y. Ito & R. M. Dutcher. Basel: S. Karger. DUNCAN, 1. B. (1978). Evidence for an oncovirus in swimbladder fibrosarcoma of Atlantic salmon Salmo salar L. Journal of Fish Diseases l, 127-131. KLEMENT, V. & NICOLSON, M. O. (1977). Methods for assays of RNA tumor viruses. Methods in Virology 6, 6~80. MULCAHY, M. F. & O'LEARY, A. (1970). Cell-free transmission of lymphasarcoma in the northern pike Esox lucius L. (Pisces; Esocidae). Experientia 26, 891. PAPAS, T. S., DAnLaERt~, J. E. & SONSTEGARD, R. A. (1976). Type C virus in lymphosarcoma in northern pike (Esox lucius). Nature, London 261, 506-508. SONST~GARD, R. A. (1977). Environmental carcinogenesis studies in fishes of the Great Lakes of North America. Annals of the New York Academy of Sciences 298, 261-269. TEICH, N. (1982). Taxonomy of retroviruses. In Molecular Biology of Tumor Viruses, pp. 25-207. Edited by R. Weiss, N. Teich, H. Varmus & J. Coffin. New York: Cold Spring Harbor Laboratory. WALKER, R. (1969). Virus associated with epidermal hyperplasia in fish. National Cancer Institute Monograph 31, 195-207. WINQVIST, G., LJUNGBERG,O. & HELLSTROEM,B. (1968). Skin turnouts of northern pike (Esox lucius L.) II. Viral particles in epidermal proliferations. Bulletin de l'Office International des Epizooties 69, 1023-1031. WOLF, K. (1979). Cold-blooded vertebrate cell and tissue culture. Methods in Enzymology 58, 466-477. YAMAMOTO, T., KELLY, R. K. & NIELSON, O. (1983). Epidermal hyperplasias of northern pike (Esox lucius) associated with herpesvirus and C-type particles. Archives of Virology 79, 255-272. YAMAMOTO, T., KELLY, R. K. & NIELSON, O. (1985). Morphological differentiation of virus-associated skin tumors of walleye (Stizostedion vitreum vitreum). Fish Pathology 20, 361-372.
(Received 13 June 1991 ," Accepted 17 June 1991)
