Research in Veterinary Science 1992, 53, 399-401
Occurrence of retrovirus-like particles in various cellular and intercellular compartments of the venom glands from Bothropsjararacussu S. M. CARNEIRO, Laborat6rio de Imunoquimica, Instituto Butantan, Av. Vital Brasil, 1500, $6o Paulo, SP05504, Brazil, H. T A N A K A , J. J. KISIELIUS, Sef~o de Microscopia Eletrdnica, Instituto Adolfo Lutz, A. SESSO, Laborat6rio de Patologia Molecular, Faeuldade de Medicina da Universidade de S6o Paulo, Brazil
Retrovirus-like particles were detected in venom glands from Bothropsjararacussuduring electron microscopy. Type C-like particles were found inter- and intracellularly in gland and vessel lumina and scattered in the connective tissue. They were about 100 nm in diameter, with an electron dense core and bilaminar external membrane. Shapes suggestive of a budding process from the plasma membrane were also observed. Less frequently, type A-like particles, about 80 nm diameter with an electronlucent core, appeared in association with the membranes of the endoplasmic reticulum of the secretory cells.
RETROVIRUSES were first identified in poikilothermic animals by Zeigel and Clark (1969) during electron microscopic examination of a spleen cell line from a Russell's viper (Vipera russelli) bearing a sarcoma. Since then, C-type particles have been isolated and characterised from two tumour-free V russelli heart cell lines and from one embryonic rhabdomyosarcoma of a corn snake (Elaphe guttata) (Lunger et al 1974, Clark et al 1979). The particles described were morphologically and biochemically similar to mammalian and avian retroviruses. They possessed an RNA genome, and an RNA-dependent DNA polymerase activity and presented typical stages of maturation by budding from plasma membranes. The particles were about 110 nm in diameter and possessed a dense core about 50 nm in diameter. They were antigenically distinct from avian and murine leukaemia viruses (Clark and Lunger 1981). Juxtanuclear aggregates of type A particles were reported by Lunger and Clark (1977) in the cytoplasmic matrix of cultured viper spleen cells. As in some cases of homoeothermic animals, these particles were associated with type-C virus-producing cells and their general morphological features were similar to type-A particles found in chicken embryo cells infected by Rous sarcoma virus (De Giuli et al 1975, Maul and Lewandowski 1975). They measured 60 to 70 nm in diameter, consisted of an electronlucent central core (26 to 29 nm) surrounded by an outer electron dense shell, and they lacked an enveloping membrane. Spike-like projections were seen radiating 399
from the outer electron dense component (Lunger and Clark 1977). The present authors have detected two kinds of retrovirus-like particles while examining venom gland samples prepared for conventional electron microscopy and, to their knowledge, this is the first report on the finding of such types of retrovirus-like particles in glands from apparently healthy snakes. Venom gland fragments from 15 Bothrops jararacussu two-monthold littermates, after decapitation, were processed and embedded by conventional techniques of transmission electron microscopy. Ultrathin sections were stained for 10 minutes with 2 per cent uranyl acetate aqueous solution and l0 'minutes with lead stain (Reynolds 1963). Seven of the 15 snakes had retrovirus-like particles in some glandular areas. Type C-like mature particles (Figs 1a, b) were found inter- and intracellularly in gland and vessel lumina, and scattered in the connective tissue, mainly close to
FIG 1: Type C-like particles in the intercellular spaces of secretory cells (a). Bar = 1-0 ~,m. Type C particles with electron dense cores and distinct bilaminar external membranes (b). Bar = 0.1 Bm
400
S. M. Carneiro, H. Tanaka, J. J. Kisielius, A. Sesso
FIG 2: Sequential stages of budding of type C-like particles from the surface of a fibroblast (a) and secretory cell basolateral regions (b and c). Mature detached particles are seen in the space between two secretory cells (d). Bar = 0.1 gm
collagen fibrils. In fibroblasts, pericytes and endothelial cells they appeared inside uncoated endocytic vesicles. In the secretory cells, they were usually seen inside secretory and other vesicles. They were about 100 nm in diameter and had a central or slightly eccentric electron dense core, a moderately electronlucent halo and a distinct bilaminar external membrane (Fig lb). Immature particles differed from the mature ones in that their core was electronlucent. In all these cell types the particles were seen in sequential stages of the budding process from plasmalemma as is described for mammalian type-C virus particles. In an initial budding stage, electron dense material in a crescent shape accumulated below the slightly bowed cell membrane (Fig 2a). The budding particles close to the detachment stage were progressively more encircled by the unit membrane derived from the host cell and had a circular or an almost circular electron dense ring surrounding a moderately electron dense core (Figs 2b, c). When the particle detached from the host membrane, the core became more electron dense and frequently eccentric (Fig 2d). Another type of particle was seen mainly associated with endoplasmic reticulum membranes. Occasionally, it was found in the secretory cell cytoplasm, the membranous areas appearing as predominantly smooth reticulum membranes, with numerous particles about 70 to 80 nm in diameter with a central electronlucent core 28 to 30 nm in diameter surrounded by an electron dense ring and by a moderately electron dense layer (Fig 3a). In some transections, a bilaminar membrane was observed circumscribing the particle almost entire-
FIG 3: Type A-like particles associated with the membranous area of the secretory cell cytoplasm. A bilaminar membrane partially surrounding each particle can be distinguished. Arrows indicate images suggestive of continuity between the endoplasmic reticulum membrane and the membrane surrounding the particle (a). Bar = 0-1 gm. Type A-like particles inside rough endoplasmic cisternae (b) and presenting ribosome-like granules apposed to them (arrow) (c). Bar = 0.1 gm
ly. Images suggestive of continuity between the bilayer membrane surrounding the particle and the endoplasmic reticular membrane were observed (Fig 3a). Similar particles were seen occupying the intracisternal space of granular endoplasmic reticulum, some of them presenting ribosome-like granules closely apposed to their surface (Figs 3b, c). The supposition of the first particles described in this paper being type-C was based on their morphology, clear cut bilayer membrane envelope, size range, extracellular or intravacuolar localisation and on the typical budding process from plasmalemma membranes. The characterisation of the particles found only in the secretory cell cytoplasm was not so clear. They displayed some features in common with A-type particles described in homoeothermic vertebrates (Dalton 1972) or in snakes (Lunger and Clark 1977): similar size, electronlucent core, apposition to ribosome-like granules, intracytoplasmic or intracisternal localisation and co-residence with C-type particles. They were never seen budding from plasmalemma or endomembranes; however, in the present study, they were always seen closely associated with endoplasmic reticulum membranes suggesting, at least, a kind of incomplete membrane envelope.
Retrovirus-like particles in venom glandsfrom Bothrops jararacussu The authors were not able to collect and examine other organs or tissues from the snakes or from their mother, because by the time this finding was made they were already dead. Attempts to test the oncogenic potential of snake retroviruses by injecting viper spleen virus into adult geckos have been unsuccessful (Clark and Lunger 1981). The presence of viruses in snake venom has not been searched for systematically. The small dense particles observed in thin sections of a centrifuged venom pellet from the tumour-bearing V ~usselli, were interpreted as representing a type of secretory granule (Zeigel and Clark 1971). However, encapsulated herpesviruses were detected by electron microscopy in reconstituted lyophilised venom from an Indian cobra (Naja naja) and from a banded krait (Bungarusfasciatus) (Monroe et al 1968). This discovery and the finding of mature retrovirus-like particles in the gland lumina of the present snakes indicate that at least part of the particles had their morphology reasonably preserved not only from a putative lytic action of the venom but also from the deleterious effects of lyophilisation. These results lead one to conjecture that the possibility of virus transmission by snake bite into susceptible hosts cannot be ruled out. Acknowledgements The authors thank Dr L. A. B. M. Lula for providing the snakes; Mr Helio Correa for technical assistance and Dr Wilson Fernandes for laboratory facilities.
401
References CLARK, H. F. & LUNGER, P. D. (1981) Viruses. In: Diseases of the Reptilia. Eds J. E. Cooper and O. F. Jackson. London, New York, Academic Press. pp135-163 CLARK, H. F., ANDERSEN, P. R. & LUNGER, P. D. (1979) Propagation and characterisation of a C-type virus from a rhabdomyosarcoma of a corn snake. Journal of General Virology 43, 673-683 DALTON, A. J. (1972) Rr~Atumor viruses-terminology and ultrastructural aspects of virion morphology and replication. Journal of the National Cancer Institute 49, 323-327 DE GIULI, C., HANAFUSA, H., KAWAI, S., DALES, S., CHEN, J. H. & HSU, K. C. (1975) Relationship between A-type and Ctype particles in cells infected by Rons sarcoma virus. Proceedings of the National Academy of Sciences USA 72, 3706-3710 LUNGER, P. D. & CLAR~, H. F. (I977) Intracytoplasmic type A particles in'viper spleen cells. Journal of the National CancerInstitute 58, 809-811 LUNGER, P. D.,'HARDY, W. D. & CLARK, H.F. (1974) C-type virus particles in a reptilian tumor. Journal of the National Cancer Institute 52, 1231-1235 MAUL, G. G. & LEWANDOWSKI, L. J. (1975) Detection of typeA oncornavirus-like structures in chicken embryo cells after infection with Rous sarcoma virus. Journal of Virology 16, 1071-1074 MONROE, J. H., SHIBLEY, G.¢P., SCHIDLOVSKY, G., NAKAI, T., HOWATSON, A. F., WIVEL, N. W. & O'CONNOR, T. E. (1968) Action of snake venom on a Rauscher virus. Journal of the National Cancer Institute 40, 135-145 REYNOLDS, E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 17, 208-212 ZEIGEL, R. F. & CLARK, H. F. (1969) Electron microscopic observations on a C-type virus in cell cultures derived from a tumorbearing viper. Journal of the National Cancer Institute 43, 1097-1102 ZEIGEL, R. F. & CLARK, H. F. (I971) Histologic and electron microscopic observations on a tumor-bearing viper: establishment of a C-type virus-producing ceil line. Journal of the National Cancer Institute 46, 309-321
Received December 31, 1991 Accepted August 7, 1992
Occurrence of retrovirus-like particles in various cellular and intercellular compartments of the venom glands from Bothropsjararacussu S. M. CARNEIRO, Laborat6rio de Imunoquimica, Instituto Butantan, Av. Vital Brasil, 1500, $6o Paulo, SP05504, Brazil, H. T A N A K A , J. J. KISIELIUS, Sef~o de Microscopia Eletrdnica, Instituto Adolfo Lutz, A. SESSO, Laborat6rio de Patologia Molecular, Faeuldade de Medicina da Universidade de S6o Paulo, Brazil
Retrovirus-like particles were detected in venom glands from Bothropsjararacussuduring electron microscopy. Type C-like particles were found inter- and intracellularly in gland and vessel lumina and scattered in the connective tissue. They were about 100 nm in diameter, with an electron dense core and bilaminar external membrane. Shapes suggestive of a budding process from the plasma membrane were also observed. Less frequently, type A-like particles, about 80 nm diameter with an electronlucent core, appeared in association with the membranes of the endoplasmic reticulum of the secretory cells.
RETROVIRUSES were first identified in poikilothermic animals by Zeigel and Clark (1969) during electron microscopic examination of a spleen cell line from a Russell's viper (Vipera russelli) bearing a sarcoma. Since then, C-type particles have been isolated and characterised from two tumour-free V russelli heart cell lines and from one embryonic rhabdomyosarcoma of a corn snake (Elaphe guttata) (Lunger et al 1974, Clark et al 1979). The particles described were morphologically and biochemically similar to mammalian and avian retroviruses. They possessed an RNA genome, and an RNA-dependent DNA polymerase activity and presented typical stages of maturation by budding from plasma membranes. The particles were about 110 nm in diameter and possessed a dense core about 50 nm in diameter. They were antigenically distinct from avian and murine leukaemia viruses (Clark and Lunger 1981). Juxtanuclear aggregates of type A particles were reported by Lunger and Clark (1977) in the cytoplasmic matrix of cultured viper spleen cells. As in some cases of homoeothermic animals, these particles were associated with type-C virus-producing cells and their general morphological features were similar to type-A particles found in chicken embryo cells infected by Rous sarcoma virus (De Giuli et al 1975, Maul and Lewandowski 1975). They measured 60 to 70 nm in diameter, consisted of an electronlucent central core (26 to 29 nm) surrounded by an outer electron dense shell, and they lacked an enveloping membrane. Spike-like projections were seen radiating 399
from the outer electron dense component (Lunger and Clark 1977). The present authors have detected two kinds of retrovirus-like particles while examining venom gland samples prepared for conventional electron microscopy and, to their knowledge, this is the first report on the finding of such types of retrovirus-like particles in glands from apparently healthy snakes. Venom gland fragments from 15 Bothrops jararacussu two-monthold littermates, after decapitation, were processed and embedded by conventional techniques of transmission electron microscopy. Ultrathin sections were stained for 10 minutes with 2 per cent uranyl acetate aqueous solution and l0 'minutes with lead stain (Reynolds 1963). Seven of the 15 snakes had retrovirus-like particles in some glandular areas. Type C-like mature particles (Figs 1a, b) were found inter- and intracellularly in gland and vessel lumina, and scattered in the connective tissue, mainly close to
FIG 1: Type C-like particles in the intercellular spaces of secretory cells (a). Bar = 1-0 ~,m. Type C particles with electron dense cores and distinct bilaminar external membranes (b). Bar = 0.1 Bm
400
S. M. Carneiro, H. Tanaka, J. J. Kisielius, A. Sesso
FIG 2: Sequential stages of budding of type C-like particles from the surface of a fibroblast (a) and secretory cell basolateral regions (b and c). Mature detached particles are seen in the space between two secretory cells (d). Bar = 0.1 gm
collagen fibrils. In fibroblasts, pericytes and endothelial cells they appeared inside uncoated endocytic vesicles. In the secretory cells, they were usually seen inside secretory and other vesicles. They were about 100 nm in diameter and had a central or slightly eccentric electron dense core, a moderately electronlucent halo and a distinct bilaminar external membrane (Fig lb). Immature particles differed from the mature ones in that their core was electronlucent. In all these cell types the particles were seen in sequential stages of the budding process from plasmalemma as is described for mammalian type-C virus particles. In an initial budding stage, electron dense material in a crescent shape accumulated below the slightly bowed cell membrane (Fig 2a). The budding particles close to the detachment stage were progressively more encircled by the unit membrane derived from the host cell and had a circular or an almost circular electron dense ring surrounding a moderately electron dense core (Figs 2b, c). When the particle detached from the host membrane, the core became more electron dense and frequently eccentric (Fig 2d). Another type of particle was seen mainly associated with endoplasmic reticulum membranes. Occasionally, it was found in the secretory cell cytoplasm, the membranous areas appearing as predominantly smooth reticulum membranes, with numerous particles about 70 to 80 nm in diameter with a central electronlucent core 28 to 30 nm in diameter surrounded by an electron dense ring and by a moderately electron dense layer (Fig 3a). In some transections, a bilaminar membrane was observed circumscribing the particle almost entire-
FIG 3: Type A-like particles associated with the membranous area of the secretory cell cytoplasm. A bilaminar membrane partially surrounding each particle can be distinguished. Arrows indicate images suggestive of continuity between the endoplasmic reticulum membrane and the membrane surrounding the particle (a). Bar = 0-1 gm. Type A-like particles inside rough endoplasmic cisternae (b) and presenting ribosome-like granules apposed to them (arrow) (c). Bar = 0.1 gm
ly. Images suggestive of continuity between the bilayer membrane surrounding the particle and the endoplasmic reticular membrane were observed (Fig 3a). Similar particles were seen occupying the intracisternal space of granular endoplasmic reticulum, some of them presenting ribosome-like granules closely apposed to their surface (Figs 3b, c). The supposition of the first particles described in this paper being type-C was based on their morphology, clear cut bilayer membrane envelope, size range, extracellular or intravacuolar localisation and on the typical budding process from plasmalemma membranes. The characterisation of the particles found only in the secretory cell cytoplasm was not so clear. They displayed some features in common with A-type particles described in homoeothermic vertebrates (Dalton 1972) or in snakes (Lunger and Clark 1977): similar size, electronlucent core, apposition to ribosome-like granules, intracytoplasmic or intracisternal localisation and co-residence with C-type particles. They were never seen budding from plasmalemma or endomembranes; however, in the present study, they were always seen closely associated with endoplasmic reticulum membranes suggesting, at least, a kind of incomplete membrane envelope.
Retrovirus-like particles in venom glandsfrom Bothrops jararacussu The authors were not able to collect and examine other organs or tissues from the snakes or from their mother, because by the time this finding was made they were already dead. Attempts to test the oncogenic potential of snake retroviruses by injecting viper spleen virus into adult geckos have been unsuccessful (Clark and Lunger 1981). The presence of viruses in snake venom has not been searched for systematically. The small dense particles observed in thin sections of a centrifuged venom pellet from the tumour-bearing V ~usselli, were interpreted as representing a type of secretory granule (Zeigel and Clark 1971). However, encapsulated herpesviruses were detected by electron microscopy in reconstituted lyophilised venom from an Indian cobra (Naja naja) and from a banded krait (Bungarusfasciatus) (Monroe et al 1968). This discovery and the finding of mature retrovirus-like particles in the gland lumina of the present snakes indicate that at least part of the particles had their morphology reasonably preserved not only from a putative lytic action of the venom but also from the deleterious effects of lyophilisation. These results lead one to conjecture that the possibility of virus transmission by snake bite into susceptible hosts cannot be ruled out. Acknowledgements The authors thank Dr L. A. B. M. Lula for providing the snakes; Mr Helio Correa for technical assistance and Dr Wilson Fernandes for laboratory facilities.
401
References CLARK, H. F. & LUNGER, P. D. (1981) Viruses. In: Diseases of the Reptilia. Eds J. E. Cooper and O. F. Jackson. London, New York, Academic Press. pp135-163 CLARK, H. F., ANDERSEN, P. R. & LUNGER, P. D. (1979) Propagation and characterisation of a C-type virus from a rhabdomyosarcoma of a corn snake. Journal of General Virology 43, 673-683 DALTON, A. J. (1972) Rr~Atumor viruses-terminology and ultrastructural aspects of virion morphology and replication. Journal of the National Cancer Institute 49, 323-327 DE GIULI, C., HANAFUSA, H., KAWAI, S., DALES, S., CHEN, J. H. & HSU, K. C. (1975) Relationship between A-type and Ctype particles in cells infected by Rons sarcoma virus. Proceedings of the National Academy of Sciences USA 72, 3706-3710 LUNGER, P. D. & CLAR~, H. F. (I977) Intracytoplasmic type A particles in'viper spleen cells. Journal of the National CancerInstitute 58, 809-811 LUNGER, P. D.,'HARDY, W. D. & CLARK, H.F. (1974) C-type virus particles in a reptilian tumor. Journal of the National Cancer Institute 52, 1231-1235 MAUL, G. G. & LEWANDOWSKI, L. J. (1975) Detection of typeA oncornavirus-like structures in chicken embryo cells after infection with Rous sarcoma virus. Journal of Virology 16, 1071-1074 MONROE, J. H., SHIBLEY, G.¢P., SCHIDLOVSKY, G., NAKAI, T., HOWATSON, A. F., WIVEL, N. W. & O'CONNOR, T. E. (1968) Action of snake venom on a Rauscher virus. Journal of the National Cancer Institute 40, 135-145 REYNOLDS, E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 17, 208-212 ZEIGEL, R. F. & CLARK, H. F. (1969) Electron microscopic observations on a C-type virus in cell cultures derived from a tumorbearing viper. Journal of the National Cancer Institute 43, 1097-1102 ZEIGEL, R. F. & CLARK, H. F. (I971) Histologic and electron microscopic observations on a tumor-bearing viper: establishment of a C-type virus-producing ceil line. Journal of the National Cancer Institute 46, 309-321
Received December 31, 1991 Accepted August 7, 1992
