- Email: [email protected]
Development of an icosahedral virus in hemocytes of Galleria mellonella (L.)
VIROLOGY
24,
%o-u)h
(1964)
Development
of an Icosahedral
Virus
in Wemocytes
of
Ga~~~~ia ~e~/o~e~~a (I..) RUTH Division of Invertebrate
Pathology,
LEUTENEGGER University
of California,
Berkeley,
California
Accepted June 16, 1964
The proliferation of an icosahedral virus in insect hemoeytes was studied by means of electron mieroscoDv . 1 of ultrathin sections. Evidence is presented that virus development occurs in the cytoplasm. INTRODUCTION
Several viruses are known to proliferate in insect hemocytes, in particular many nucleopolyhedrosis viruses, granulosis viruses, and Pa~l~otellavir~s (Wittig, 1962). Electron n~icroscopi~ techniques were used by Bergold (1952) to demonstrate virus rods and developmental forms of nucleopolyhedrosis virus in silkworrn hemocytes. Xeros and Smith (1956) demonstrated the development of polyhedra in blood cells of Tipula paludosa Meigen with the aid of electron microscopy. Martignoni and Scallion (1961) observed the proliferation of nucleopolyhedrosis virus in insect hemocyte monolayers, in zl~~ro. Developmental forms of virus were seen in electron micrographs of blood cells from larvae of Christoneura fumijerana (Clemens) and Pieris rapae (Linnaeus) suffering from granulosis (Bird, 1958). Bird concluded that viral proliferation might have occurred in these blood cells. Wittig (1960) suggested that the presence of capsules in blood cells of ~hr~~~o~~~ra ?~u?~~~ana(Hiibner) larvae infected with granulosis virus might be attributed to phagocytosis. In this note I present evidence concerning the development of an icosahedral virus (Steinhaus and Leutenegger, 1963) in blood cells from larvae of GalEel-ia n~ellon~lla (L.). MATERIALS
AND METHODS
The virus was originally isolated from a scarab, Sericesthis pruinosa (Dalman) (Steinhaus and Leutenegger, 1963). In this paper
it will be referred to as Sericesthis iridescent virus (SIV). The virus suspension was prepared as follows. Last-instar larvae of Galleria mellonella (L.) were inoculated intrahemocoelically with SIV suspensions filtered through ~Iillipore membranes (HA). After approxin~ately 3 weeks at room temperature (20--25°C) the larvae died of frank SIV infection. They were triturated in distilled water and the resulting suspension was centrifuged in a clinical centrifuge at 1400 g. The supernatant was further centrifuged for 30 minutes at 14,337 g (at R,,) in a model L Beckman ultracentrifuge (rotor 40.2). The res~tillg pellet was resuspended in distilled water and passed through a Millipore filter (HA). Nine microliters of this suspension were injected into the hemocoel of last-instar larvae. At 6 hours, 48 hours, and 5 days following inoculation, the blood was collected by means of fire-polished capillaries (~~artignoni and Milstead, 1964) and centrifuged for 10 minutes at 1400 g in a clinical ce~ltrifuge. The sedimented cells were fixed at 0-5°C for 20-50 minutes in 1% OsO* buffered with Verona1 acetate at pH 7.2 according to Zetterqvist (quoted by Pease, 1960). Following fixation the pellet was dehydrated in increasing concentrations of ethyl alcohol, then stained with 2% phosphotungstic acid in absolute alcohol and enlb~ded in Maraglas. Ultrathin sections were cut with glass knives, mounted in a Porter-Blum microtome. The sections were placed on Formvar-coated copper grids and
200
DEVELOPMENT
FIG. 1. Blood cell from a larva
OF ICOSAHEDRAL
VIRUS
201
of Galleria mellonella 6 hours after inoculation, showing only a few particles. N, nucleus. FIG. 2. Blood cell from a larva of Gulleria mellonella 48 hours after inoculation. The number of virus particles has increased, and areas of dense granulated material are present (arrow). M, mitochondria; N, nucleus.
virus
202
~EUTENEGG~~
FIG. 3. Various viral stages and typical dense areas (arrow), possibly sites of virus multiplication, are visible in the cytoplasm of a blood cell from a Galleria nzeUoneZlularva 48 hours after inoculation.
DEVELOPMENT
OF ICOSAHEDRAL
VIRUS
203
FIG. 4. Formation of membranous structures (arrow) in the cytoplasm of a blood cell from Galleria mellonella larva 48 hours after inoculation. M, mitochondria; AV, nucleus.
examined in an RCA EMU-3 electron microscope. RESULTS
The presence of viral developmental stages, similar to those described by Smith and Hills (1962) and Bird (1962) for the Tipula iridescent virus (TIV) can be demonstrated in ultrathin sections in the blood cells of G. mellonella during the course of the disease. Six hours after inoculation a few immature viral particles occur in the cytoplasm, predominantly enclosed by vesicles (Fig. 1). The region normally occupied by the core appears in most instances to be occupied by a less electron-dense material, except for a few particles which seem to cont’ain a central mass. At 48 hours postinoculation the number of virus particles in the cytoplasm has increased (Fig. 2). Mature particles along with various immature forms are present. The infection leads to striking cytoplasmic changes as evidenced by the
formation of dense areas and membranous structures not found in normal cells (Figs. 3 and 4). The dense, granulated areas are probably sites within which virus replication occurs (Fig. 3). In the later phases of the disease (5 days after inoculation) the cytoplasm is nearly completely replaced by virus. The nucleus remains unaffected, as in the case of virus growth in fat-body cells (Smith, 1963). CONCLUSIONS
The icosahedral SIV particles develop in the cytoplasm of blood cells of G. mellonella larvae. As immature particles can be found as early as 6 hours following inoculation, it is unlikely that the blood cells are phagocytozing particles produced in other sites of virus multiplication since no cytolysis occurs at this early stage. The evidence is inconclusive regarding the sequence of virus maturation. Smith (1963) proposed that the outer shell of TIV develops first, followed by the
204
LEUTENEGGER
formation of a primary threadlike body which eventually fills the particle at maturation. Bird (1962), working with TIV, claims that a central core forms first and later an outer shell. The empty shells seen by Smith were thought by Bird to represent sections that missed the central core. The developmental sequence of the icosahedral virus shown in the illustrations of this paper seems to support Smith’s hypothesis, since the empty shells are often observed in clusters, while the “pulling” of cores observed by Bird (1962) cannot be other than a random event (see his Fig. 4). Sections made above and below the core would provide shells with smaller diameters (Thomas and Williams, 1961) than those observed here. Further investigations are necessary to study this phenomenon more thoroughly. It should be emphasized that, at the time of this writing, it is not known whether or not SIV is identical with or related to TIV, except as described previously by Steinhaus and Leutenegger (1963). The formation of vesicles in the cytoplasm during the early phase of viral development suggests that the uptake of the infective stage might occur by pinocytosis. This most interesting aspect of the viral cycle will be the object of further investigations. ACKNOWLEDGMENTS The author wishes to express deep appreciation to Dr. M. E. Msrtignoni for his criticism of the manuscript’. REFERENCES BERGOLD, G. H. (1952). Demonstration of the polyhedral virus in blood cells of silkworms. Biochim. Biophys. Acta 8, 397-400.
BIRD, F. T. (1958). Histopathology of granulosis viruses in insects. Can. J. Microbial. 4, 267-272. BIRD, F. T. (1962). On the development of the Tip& iridescent virus particle. Can. J. Microbiol. 8, 533-535. MARTIGNONI, M. E., and MILSTEAD, J. E. (1964). Hypoproteinemia in a noctuid larva during the course of nucleopolyhedrosis. J. Insect Pathol. (in press). MARTIGNONI, M. E., and SCALLION, R. J. (1961). Multiplication in vitro of a nuclear polyhedrosis virus in insect amoebocytes. Nature 190, 11331134. PEASE, D. C. (1960). “Histological Techniques for Electron Microscopy.” Academic Press, New York. SMITH, K. M. (1963). Cytoplasmic virus diseases. In “Insect Pathology, An Advanced Treatise” (E. A. Steinhaus, ed.), Vol. l., pp. 457-497. Academic Press, New York. SMITH, K. M., and HILLS, G. J. (1962). Multiplication and ultrastructure of insect viruses. Proc. Intern. Congr. Entomol. llth, Vienna 1960 Vol. 2, pp. 823-827. STEINHACS, E. A., and LEUTENEGGER, R. (1963). Icosahedral virus from a scarab (Sericesthis). J. Insect Pathol. 5, 266-270. THOMAS, R. S., and WILLIAMS, R. C. (1961). Localization of DNA and protein in Tip& iridescent virus by enzymatic digestion and electron microscopy. J. Biophys. Biochem. cytoz. 11, 15-29. WJTTIG, G. (1960). Untersuchungen am Blut gesunder und granulosekranker Raupen von murinana (HB.). 2. Angezu. Choristoneura Entomol. 46, 385-400. WITTIG, G. (1962). The pathology of insect blood cells: a review. Am. Zoologist 2, 257-273. XEROS, N., and SMITH, K. M. (1956). Further studies on the development of viruses in cells of insects. Proc. Intern. Cons. Electron Microscopy 3rd London 1964 pp. 259-262. Royal Microscopical Society, London.
24,
%o-u)h
(1964)
Development
of an Icosahedral
Virus
in Wemocytes
of
Ga~~~~ia ~e~/o~e~~a (I..) RUTH Division of Invertebrate
Pathology,
LEUTENEGGER University
of California,
Berkeley,
California
Accepted June 16, 1964
The proliferation of an icosahedral virus in insect hemoeytes was studied by means of electron mieroscoDv . 1 of ultrathin sections. Evidence is presented that virus development occurs in the cytoplasm. INTRODUCTION
Several viruses are known to proliferate in insect hemocytes, in particular many nucleopolyhedrosis viruses, granulosis viruses, and Pa~l~otellavir~s (Wittig, 1962). Electron n~icroscopi~ techniques were used by Bergold (1952) to demonstrate virus rods and developmental forms of nucleopolyhedrosis virus in silkworrn hemocytes. Xeros and Smith (1956) demonstrated the development of polyhedra in blood cells of Tipula paludosa Meigen with the aid of electron microscopy. Martignoni and Scallion (1961) observed the proliferation of nucleopolyhedrosis virus in insect hemocyte monolayers, in zl~~ro. Developmental forms of virus were seen in electron micrographs of blood cells from larvae of Christoneura fumijerana (Clemens) and Pieris rapae (Linnaeus) suffering from granulosis (Bird, 1958). Bird concluded that viral proliferation might have occurred in these blood cells. Wittig (1960) suggested that the presence of capsules in blood cells of ~hr~~~o~~~ra ?~u?~~~ana(Hiibner) larvae infected with granulosis virus might be attributed to phagocytosis. In this note I present evidence concerning the development of an icosahedral virus (Steinhaus and Leutenegger, 1963) in blood cells from larvae of GalEel-ia n~ellon~lla (L.). MATERIALS
AND METHODS
The virus was originally isolated from a scarab, Sericesthis pruinosa (Dalman) (Steinhaus and Leutenegger, 1963). In this paper
it will be referred to as Sericesthis iridescent virus (SIV). The virus suspension was prepared as follows. Last-instar larvae of Galleria mellonella (L.) were inoculated intrahemocoelically with SIV suspensions filtered through ~Iillipore membranes (HA). After approxin~ately 3 weeks at room temperature (20--25°C) the larvae died of frank SIV infection. They were triturated in distilled water and the resulting suspension was centrifuged in a clinical centrifuge at 1400 g. The supernatant was further centrifuged for 30 minutes at 14,337 g (at R,,) in a model L Beckman ultracentrifuge (rotor 40.2). The res~tillg pellet was resuspended in distilled water and passed through a Millipore filter (HA). Nine microliters of this suspension were injected into the hemocoel of last-instar larvae. At 6 hours, 48 hours, and 5 days following inoculation, the blood was collected by means of fire-polished capillaries (~~artignoni and Milstead, 1964) and centrifuged for 10 minutes at 1400 g in a clinical ce~ltrifuge. The sedimented cells were fixed at 0-5°C for 20-50 minutes in 1% OsO* buffered with Verona1 acetate at pH 7.2 according to Zetterqvist (quoted by Pease, 1960). Following fixation the pellet was dehydrated in increasing concentrations of ethyl alcohol, then stained with 2% phosphotungstic acid in absolute alcohol and enlb~ded in Maraglas. Ultrathin sections were cut with glass knives, mounted in a Porter-Blum microtome. The sections were placed on Formvar-coated copper grids and
200
DEVELOPMENT
FIG. 1. Blood cell from a larva
OF ICOSAHEDRAL
VIRUS
201
of Galleria mellonella 6 hours after inoculation, showing only a few particles. N, nucleus. FIG. 2. Blood cell from a larva of Gulleria mellonella 48 hours after inoculation. The number of virus particles has increased, and areas of dense granulated material are present (arrow). M, mitochondria; N, nucleus.
virus
202
~EUTENEGG~~
FIG. 3. Various viral stages and typical dense areas (arrow), possibly sites of virus multiplication, are visible in the cytoplasm of a blood cell from a Galleria nzeUoneZlularva 48 hours after inoculation.
DEVELOPMENT
OF ICOSAHEDRAL
VIRUS
203
FIG. 4. Formation of membranous structures (arrow) in the cytoplasm of a blood cell from Galleria mellonella larva 48 hours after inoculation. M, mitochondria; AV, nucleus.
examined in an RCA EMU-3 electron microscope. RESULTS
The presence of viral developmental stages, similar to those described by Smith and Hills (1962) and Bird (1962) for the Tipula iridescent virus (TIV) can be demonstrated in ultrathin sections in the blood cells of G. mellonella during the course of the disease. Six hours after inoculation a few immature viral particles occur in the cytoplasm, predominantly enclosed by vesicles (Fig. 1). The region normally occupied by the core appears in most instances to be occupied by a less electron-dense material, except for a few particles which seem to cont’ain a central mass. At 48 hours postinoculation the number of virus particles in the cytoplasm has increased (Fig. 2). Mature particles along with various immature forms are present. The infection leads to striking cytoplasmic changes as evidenced by the
formation of dense areas and membranous structures not found in normal cells (Figs. 3 and 4). The dense, granulated areas are probably sites within which virus replication occurs (Fig. 3). In the later phases of the disease (5 days after inoculation) the cytoplasm is nearly completely replaced by virus. The nucleus remains unaffected, as in the case of virus growth in fat-body cells (Smith, 1963). CONCLUSIONS
The icosahedral SIV particles develop in the cytoplasm of blood cells of G. mellonella larvae. As immature particles can be found as early as 6 hours following inoculation, it is unlikely that the blood cells are phagocytozing particles produced in other sites of virus multiplication since no cytolysis occurs at this early stage. The evidence is inconclusive regarding the sequence of virus maturation. Smith (1963) proposed that the outer shell of TIV develops first, followed by the
204
LEUTENEGGER
formation of a primary threadlike body which eventually fills the particle at maturation. Bird (1962), working with TIV, claims that a central core forms first and later an outer shell. The empty shells seen by Smith were thought by Bird to represent sections that missed the central core. The developmental sequence of the icosahedral virus shown in the illustrations of this paper seems to support Smith’s hypothesis, since the empty shells are often observed in clusters, while the “pulling” of cores observed by Bird (1962) cannot be other than a random event (see his Fig. 4). Sections made above and below the core would provide shells with smaller diameters (Thomas and Williams, 1961) than those observed here. Further investigations are necessary to study this phenomenon more thoroughly. It should be emphasized that, at the time of this writing, it is not known whether or not SIV is identical with or related to TIV, except as described previously by Steinhaus and Leutenegger (1963). The formation of vesicles in the cytoplasm during the early phase of viral development suggests that the uptake of the infective stage might occur by pinocytosis. This most interesting aspect of the viral cycle will be the object of further investigations. ACKNOWLEDGMENTS The author wishes to express deep appreciation to Dr. M. E. Msrtignoni for his criticism of the manuscript’. REFERENCES BERGOLD, G. H. (1952). Demonstration of the polyhedral virus in blood cells of silkworms. Biochim. Biophys. Acta 8, 397-400.
BIRD, F. T. (1958). Histopathology of granulosis viruses in insects. Can. J. Microbial. 4, 267-272. BIRD, F. T. (1962). On the development of the Tip& iridescent virus particle. Can. J. Microbiol. 8, 533-535. MARTIGNONI, M. E., and MILSTEAD, J. E. (1964). Hypoproteinemia in a noctuid larva during the course of nucleopolyhedrosis. J. Insect Pathol. (in press). MARTIGNONI, M. E., and SCALLION, R. J. (1961). Multiplication in vitro of a nuclear polyhedrosis virus in insect amoebocytes. Nature 190, 11331134. PEASE, D. C. (1960). “Histological Techniques for Electron Microscopy.” Academic Press, New York. SMITH, K. M. (1963). Cytoplasmic virus diseases. In “Insect Pathology, An Advanced Treatise” (E. A. Steinhaus, ed.), Vol. l., pp. 457-497. Academic Press, New York. SMITH, K. M., and HILLS, G. J. (1962). Multiplication and ultrastructure of insect viruses. Proc. Intern. Congr. Entomol. llth, Vienna 1960 Vol. 2, pp. 823-827. STEINHACS, E. A., and LEUTENEGGER, R. (1963). Icosahedral virus from a scarab (Sericesthis). J. Insect Pathol. 5, 266-270. THOMAS, R. S., and WILLIAMS, R. C. (1961). Localization of DNA and protein in Tip& iridescent virus by enzymatic digestion and electron microscopy. J. Biophys. Biochem. cytoz. 11, 15-29. WJTTIG, G. (1960). Untersuchungen am Blut gesunder und granulosekranker Raupen von murinana (HB.). 2. Angezu. Choristoneura Entomol. 46, 385-400. WITTIG, G. (1962). The pathology of insect blood cells: a review. Am. Zoologist 2, 257-273. XEROS, N., and SMITH, K. M. (1956). Further studies on the development of viruses in cells of insects. Proc. Intern. Cons. Electron Microscopy 3rd London 1964 pp. 259-262. Royal Microscopical Society, London.