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Cell infection by a nuclear polyhedrosis virus
VIROLOGY
42, 311-318
Cell
(1970)
Infection
by
a Nuclear
Polyhedrosis
Virus
K. A. HARRAP Insect
Pathology
Unit,
Department Accepted
of Forestry, June
University 3,
of Oxford,
England
1970
Infection through the microvilli of the gut columnar cells is suggested as the initial stage of infection by a nuclear polyhedrosis virus. The virus envelope, or outer membrane, appears to have an important function in such cell infection as virus particles outside the microvilli are enveloped whereas those within the microvilli are not. Assembly of the virus appears to occur in t,he columnar cell nucleus and many enveloped virus particles can be found in the cytoplasm. Such virus particles can also be found in close association with the underlying tracheal epithelium cells. Nuclear polyhedrosis virus replication subsequently becomes established in these cells. The importance of the virus envelope in the infection of the cell is discussed.
medium (Trager, 1935), and pieces of gut and fat body were fixed in 5% (v/v) glutaraldehyde for 1 hour followed by two washes of 15 min each and a subsequent fixation in 2% (w/v) osmium tetroxide. Trager’s B medium was used both as a diluent for the fixatives and for the intermediate washes. The fixed tissue was washed again and dehydrated through a graded ethanol series prior to embedding in Epikote. Thin sections were made on a LKB ultramicrotome and stained in 2% (w/v) uranyl acetate followed by lead citrate (Reynolds, 1963). The stained sections were examined on an AEI EMGB electron microscope at 60 kV.
INTRODUCTION
A pathway of infection by nuclear polyhedrosis viruses has been proposed previously (Harrap and Robertson, 1968)) based on observations of a restricted virus development in gut epithelium cells. The process of initial infection of such cells was not observed at that time, but further work has now revealed a mechanism by which cell infection may occur. Some of the results of this work have been reported in a preliminary form (Harrap, 1969), and a complete account is presented in this communication. MATERIALS
AND
METHODS
Larvae of the small tortoiseshell butterfly, Aglais urticae L. (Lepidoptera: Nymphalidae) were fed on leaves of stinging nettle (Urtica dioica L.) previously coated with a suspension of nuclear polyhedra. This inoculum was prepared according to the methods described by Longworth and Cunningham (1968). The original virus stock was obtained from a population of A. urticae larvae occurring naturally in Oxfordshire, EQgland, which was found to be infected with nuclear polyhedrosis virus. The larvae were removed from the food plant and killed at predetermined intervals. The cadavers were dissected under Trager’s B
RESULTS
Virus particles were observed in the nuclei of some gut columnar cells in dissections made from larvae collected 24 hours after infection feeding. The time intervals are of limited significance, however, as it was difficult to determine the exact time at which any individual larva commenced feeding on the virus-treated food plant. However, it is possible to deduce the overall progression of the virus infection from the time intervals. Evidence of virus multiplication in tracheal epithelium and fat body cells was found at progressively later stages. 311
312
HARRAP
Examination of areas of microvilli at the apex of some columnar cells showed that occasional enveloped virus particles were present. The virus envelope and the plasma membrane of the microvillus were often found in close apposition (Figs. 1 and 2). The envelope appeared to surround the virus particle very loosely, and the densely staining virus particle within it was frequently curved or twisted. This condition was also observed in fat body and tracheal epithelium cells in which nuclear polyhedrosis virus was developing, and there is no reason to regard it as atypical. Complete fusion of the virus envelope with the plasma membrane of the microvillus was not observed, but microvilli were often seen bearing balloon-like projections from the plasma membrane (Fig. 3). Such projections could be residual virus envelopes seen after the virus particles had passed into the cell cytoplasm, though it is also possible that they were produced from the plasma membrane of the microvilli. Virus particles were found within the microvilli of columnar cells, and in this situation no surrounding virus envelope was visible (Figs. 4 and 5). Occasionally, a virus particle was observed within the cell cytoplasm at the base of the microvilli, where it appeared to be disintegrating (Fig. 6). Columnar cells with virus particles in the microvilli and many of the adjacent columnar cells were often found with evidence of virus replication in the nuclei. Harrap and Robertson (1968) reported that such nuclei were not characterized by any gross reorganization of the nuclear chromatin though many virus particles could be seen within them (Fig. 7). The length of the virus particles was found to be somewhat variable, especially in regions where they appeared to be aligned with a small stroma of regular repeating units (Fig. 8). The infected nuclei also contained many membranous vesicles which may have a function in the envelopment of the virus particles. Small masses of material were present in the infected nuclei examined, which were structurally identical with polyhedron protein as recognized by the presence of a cubic lattice pattern with the usual 4 nm spacing (Fig. 9). Enveloped virus particles were frequently observed on the periphery of such
masses but no virus particles were embedded within the polyhedron protein as occurs in the formation of typical polyhedra (Harrap and Robertson, 1968). In columnar cells with infected nuclei the area of cytoplasm between the nucleus and the basal lamina usually contained many enveloped virus particles (Figs. 10 and 11). It is quite possible that these virus particles originated in the nucleus because parts of the nuclear membrane were no longer evident in infected nuclei and the nuclear content and the cell cytoplasm were continuous in these regions. The envelope itself was not acquired as a result of a budding process at the nuclear membrane as enveloped virus particles were also found in the nucleus. The envelope surrounded the virus particle loosely and was usually spherical as would be expected if it were semipermeable and both the lluid contained by it and the cytoplasmic fluid of the cell were in equilibrium. The virus particle within this spherical envelope was often bent so that in certain planes of section two virus particles appeared to be enclosed by a single virus envelope. It was very difficult to determine whether the enveloped virus particles in the basal cytoplasm of the columnar cell were within or external to the deeply infolded plasma membrane. It is cIear, however, that the enveloped virus particles can traverse the plasma membrane as they could be observed outside the cell cytoplasm in the substance of the basal lamina. Whether or not the virus envelope acquires some components of the plasma membrane as a result of this process is not known because neither the site of the traverse of the enveloped virus particles across the plasma membrane nor the manner of its accomplishment have been ascertained. Tr:.cheal epithelium cells were frequently found adjacent to the basal lamina and evidence of early stages of virus replication could be seen in these cells from 48 hours after infection feeding (Fig. 12). Virus replication in fat body cells was not observed until 72 hours or more after infection feeding. DISCUSSION
It is logical to arrange the process of virus infection observed in the midgut tissue of A.
CELL
FIG. FIG. FIG. FIG.
INFECTION
BY AN INSECT
313
VIRUS
1. Enveloped virus particle (evp) among microvilli at the apex of a columnar 2. Enveloped virus particle (evp) in contact with cell microvilli. 3. Balloon-like projections (arrowed) on columnar cell microvilli. 4. Virus particles (arrowed) within columnar cell microvilli.
cell.
FIG. 5. A virus particle FIG. 6. A virus particle FIG. 7. Virus replication
within a columnar cell microvillus. in the cell cytoplasm at the base of the microyilli. in a columnar cell nucleus vp, virus particles; 314
pp, polyhedron
protein.
FIG. 8. \.irus part.icles aligned with FIG. 9. Small masses of polyhedron is shown in the inset. Note how the FIG. 10. Enveloped virus particles FIG. 11. Enveloped virus part,icles
a stroma of repeat.ing unit,s (arrowed) in a columnar cell nuelel IS. protein in a virus-infected columnar cell nucleus. The area mark ed nuclear membrane (arrowed) is ruptured. grouped in the cytoplasm at the base of an infected columnar CC:ll. in the cytoplasm at the base of an infected columnar cell. 315
316
HARRA4P
FIG. 12. A tracheal epithelium cell at an early stage of virus (arrowed) in the enlarged cell nucleus ((ecn). Enveloped virus (cc) cytoplasm and basal lamina @I). t, tracheole.
urticae larvae as a sequenceof events in the early stages of nuclear polyhedrosis virus infection. If the virus particles in and adjacent to the microvilli of the columnar cells were in stages of release from the cell
infection. particles
A few virus are present
particles are visible in the columnar cell
then it is difficult to postulate an initia1 source of virus infection, other than possibly the tracheal epithelium cells, as virus development was not observed in any other tissue at this stage. However, the extent of
CELL
INFECTION
BY
virus infection and replication in the tracheal epithelium cells progressed with time and even if these cells could act as sources of infection for the gut columnar cells it is difficult to explain how they could become infected as a result of feeding polyhedra to the larva. Clearly, it is more reasonable to suggest that the observed virus assembly in the columnar cells constitutes the primary focus of virus infection and the following pathway of infection is therefore proposed. When nuclear polyhedra are ingested the polyhedron protein is dissolved in the alkaline gut secretions possibly assisted by proteolytic enzymes. This may be a rather slow process so that by the time the enveloped virus particles are eventually released by the dissolution of the polyhedron protein they lrave been carried into the midgut of the larva by the peristaltic movements of the gut wall. The peritrophic membrane surrounding the contents of the gut lumen consists of a network of strands of chitin and protein forming a mesh with holes 150-200 nm in diameter (Alercer and Day, 1952; Waterhouse, 1953). Some of the enveloped virus particles could pass through the peritrophic membrane and come into contact with the microvilli of the columnar cells as a result of movements of the microvilli characteristic of absorptive cells. The envelope of the virus particle may then become attached to the plasma membrane of a microvillus, and a resulting fusion of the plasma membrane and the virus envelope could allow the nucleocapsid to enter the microvillus. The nucleocapsid could then be carried into the cell as a result of some form of absorptive gradient. Similar observations have recently been made in the early stages of granulosis virus infection of Trichoplusia //.i (Summers, 1969). As nuclear polyhedrosis and granulosis virus particles are structurally very similar, the possibility of a comparable cell infection system is encouraging. Enveloped virus particles which do not come into contact with columnar cell microvilli in this way are probably destroyed fairly quickly in the alkaline environment of the gut lumen. This could account for the difficulty in locating infection in columnar cells, as only a small number of localized cells may become infected. It is significant
AN
INSECT
VIRUS
317
that although the regions of infected columnar cells in A. urticae larvae were not extensive, every cell in such regions was infected. Virus assembly apparently occurs in the nucleus of the cell as virus particles were observed here in different structural states. However, the most significant feature of the development of nuclear polyhedrosis virus in the nuclei of columnar cells is that although enveloped virus particles are formed together with some polyhedron protein the polyhedron protein is produced only as very small masses and never surrounds the enveloped virus particles. The production of polyhedron protein may be a cellular responseto virus infection in which caseeither columnar cells respond poorly or the virus is capable of overcoming the reaction. If, as is more likely, production of polyhedron protein results from a virus-directed synthesis then the mechanism of incorporation of virus particles is either effectively blocked or columnar cells are incapable of synthesizing the large amounts of protein necessary. It is also possible that the deposition of polyhedron protein around the enveloped virus particles is dependent solely on the numbers of these particles. Such enveloped particles appear to pass into the cytoplasm of the columnar cell as a result of the rupture or dissolution of the nuclear membrane which is presumably a reflection of the virus development. Thus there may never be sufficient numbers of enveloped virus particles present for normal polyhedron formation to occur. In the cytoplasm, the enveloped virus particles between the nucleus and the basal membrane may be drawn along some form of gradient in the cell which prevents their general distribution throughout the cell cytoplasm. The virus particles become grouped at the base of the cell, traverse the plasma membrane and are carried through the basal lamina where they come into contact with tracheal epithelium cells. These cells are then infected probably as a result of an attachment of the virus envelope and cell membrane in a similar manner to the mechanism postulated for the infection of columnar cells. The importance of the virus envelope in the infection process is again apparent and it is likely that virus particles lacking the envelope are incapable
318
HARRAP
of infecting cells. The extent of virus infection in the gut therefore influences the spread of virus infection into the tracheal epithelium cells and these provide an effective portal to other susceptible tissues, such as hemocytes, fat body, and hypodermis. Infection in all these tissues is characterized by the formation of typical polyhedra containing virus particles. In these tissues a large proportion of the virus particles produced in any one cell have no further infective function in that host as the virus particles in the polyhedra will not be released until the polyhedra are ingested by another larva. The spread of the virus infection must therefore be dependent on those enveloped virus particles not incorporated into polyhedra. Such virus particles are released when the nucleus ruptures as a result of the large number of mature polyhedra which have developed. The advantage of virus infection occurring initially in a tissue where this occlusion process does not take place is obvious and columnar cells infected with nuclear polyhedrosis virus can be regarded as “virus factories” whose entire output is available for the successful establishment of virus infection in the host. Such a mechanism of cell infection supports the view that nuclear polyhedrosis and granulosis virus particles are enveloped viruses, the nucleocapsid being equivalent to the so-called intimate membrane (Bergold, 1953) and its contents. It seems likely that possession of the virus envelope is a prerequisite for cell infection, and the enveloped virus particle would therefore be the virion. It is significant that during the formation of normal polyhedra only enveloped virus particles are occluded by crystallizing polyhedron protein even though unenveloped
virus particles are also present in the nucleus. The structure of the virus envelope has been investigated in some detail as it apparently has such a significant function in the initiation of cell infection. The results of this work will be reported elsewhere. ACKNOWLEDGMENTS The work reported here formed part of a thesis submitted for the Degree of Doctor of Philosophy, University of Oxford. The electron microscope and its ancillary equipment were purchased with a grant from the Natural Environment Research Council. REFERENCES BERGOLD, G. H. (1953). Insect viruses. Advan. Virus Res. 1, 91-139. HARHAP, K. A. (1969). Proc. Znl. Congr. Viral. Is1 1968. Znt. Viral. 1, 281. HARKAP, K. A., and ROIIEHTSON, J. S. (19G8). A possible infection pathway in the development of a nuclear polyhedrosis virus. J. Gen. Viral. 3, 221-225. LONGIVORTH, J. F., and CUNNINGHAM, J. C. (1968). The activation of occult nuclear-polyhedrosis viruses by foreign nuclear polyhedra. J. Znverle. Palhol. 10, 361-367. MINCER, E. H., and DAY, M. F. (1952). The fine structure of the peritrophic membrane in Dirippus morosus. Rio/. Bull. 103, 384-394. KEYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Hiol. 17, 208-212. SUMMERS, M. 11. (1969). Apparent in vivo pathway of granulosis virus invasion and infection. J. Viral. 4, 188-190. TRAGEH, W. (1935). Cultivation of the virus of grasserie in silkworm tissue cultures. J. Exp. Med. 61, 501-513. WATEIIHOUSE, II. F. (1953). The occurence and significance of the peritrophic membrane, with special reference to adult Lepidoptera and Diptera. Bust. J. Zool. 1, 299-318.
42, 311-318
Cell
(1970)
Infection
by
a Nuclear
Polyhedrosis
Virus
K. A. HARRAP Insect
Pathology
Unit,
Department Accepted
of Forestry, June
University 3,
of Oxford,
England
1970
Infection through the microvilli of the gut columnar cells is suggested as the initial stage of infection by a nuclear polyhedrosis virus. The virus envelope, or outer membrane, appears to have an important function in such cell infection as virus particles outside the microvilli are enveloped whereas those within the microvilli are not. Assembly of the virus appears to occur in t,he columnar cell nucleus and many enveloped virus particles can be found in the cytoplasm. Such virus particles can also be found in close association with the underlying tracheal epithelium cells. Nuclear polyhedrosis virus replication subsequently becomes established in these cells. The importance of the virus envelope in the infection of the cell is discussed.
medium (Trager, 1935), and pieces of gut and fat body were fixed in 5% (v/v) glutaraldehyde for 1 hour followed by two washes of 15 min each and a subsequent fixation in 2% (w/v) osmium tetroxide. Trager’s B medium was used both as a diluent for the fixatives and for the intermediate washes. The fixed tissue was washed again and dehydrated through a graded ethanol series prior to embedding in Epikote. Thin sections were made on a LKB ultramicrotome and stained in 2% (w/v) uranyl acetate followed by lead citrate (Reynolds, 1963). The stained sections were examined on an AEI EMGB electron microscope at 60 kV.
INTRODUCTION
A pathway of infection by nuclear polyhedrosis viruses has been proposed previously (Harrap and Robertson, 1968)) based on observations of a restricted virus development in gut epithelium cells. The process of initial infection of such cells was not observed at that time, but further work has now revealed a mechanism by which cell infection may occur. Some of the results of this work have been reported in a preliminary form (Harrap, 1969), and a complete account is presented in this communication. MATERIALS
AND
METHODS
Larvae of the small tortoiseshell butterfly, Aglais urticae L. (Lepidoptera: Nymphalidae) were fed on leaves of stinging nettle (Urtica dioica L.) previously coated with a suspension of nuclear polyhedra. This inoculum was prepared according to the methods described by Longworth and Cunningham (1968). The original virus stock was obtained from a population of A. urticae larvae occurring naturally in Oxfordshire, EQgland, which was found to be infected with nuclear polyhedrosis virus. The larvae were removed from the food plant and killed at predetermined intervals. The cadavers were dissected under Trager’s B
RESULTS
Virus particles were observed in the nuclei of some gut columnar cells in dissections made from larvae collected 24 hours after infection feeding. The time intervals are of limited significance, however, as it was difficult to determine the exact time at which any individual larva commenced feeding on the virus-treated food plant. However, it is possible to deduce the overall progression of the virus infection from the time intervals. Evidence of virus multiplication in tracheal epithelium and fat body cells was found at progressively later stages. 311
312
HARRAP
Examination of areas of microvilli at the apex of some columnar cells showed that occasional enveloped virus particles were present. The virus envelope and the plasma membrane of the microvillus were often found in close apposition (Figs. 1 and 2). The envelope appeared to surround the virus particle very loosely, and the densely staining virus particle within it was frequently curved or twisted. This condition was also observed in fat body and tracheal epithelium cells in which nuclear polyhedrosis virus was developing, and there is no reason to regard it as atypical. Complete fusion of the virus envelope with the plasma membrane of the microvillus was not observed, but microvilli were often seen bearing balloon-like projections from the plasma membrane (Fig. 3). Such projections could be residual virus envelopes seen after the virus particles had passed into the cell cytoplasm, though it is also possible that they were produced from the plasma membrane of the microvilli. Virus particles were found within the microvilli of columnar cells, and in this situation no surrounding virus envelope was visible (Figs. 4 and 5). Occasionally, a virus particle was observed within the cell cytoplasm at the base of the microvilli, where it appeared to be disintegrating (Fig. 6). Columnar cells with virus particles in the microvilli and many of the adjacent columnar cells were often found with evidence of virus replication in the nuclei. Harrap and Robertson (1968) reported that such nuclei were not characterized by any gross reorganization of the nuclear chromatin though many virus particles could be seen within them (Fig. 7). The length of the virus particles was found to be somewhat variable, especially in regions where they appeared to be aligned with a small stroma of regular repeating units (Fig. 8). The infected nuclei also contained many membranous vesicles which may have a function in the envelopment of the virus particles. Small masses of material were present in the infected nuclei examined, which were structurally identical with polyhedron protein as recognized by the presence of a cubic lattice pattern with the usual 4 nm spacing (Fig. 9). Enveloped virus particles were frequently observed on the periphery of such
masses but no virus particles were embedded within the polyhedron protein as occurs in the formation of typical polyhedra (Harrap and Robertson, 1968). In columnar cells with infected nuclei the area of cytoplasm between the nucleus and the basal lamina usually contained many enveloped virus particles (Figs. 10 and 11). It is quite possible that these virus particles originated in the nucleus because parts of the nuclear membrane were no longer evident in infected nuclei and the nuclear content and the cell cytoplasm were continuous in these regions. The envelope itself was not acquired as a result of a budding process at the nuclear membrane as enveloped virus particles were also found in the nucleus. The envelope surrounded the virus particle loosely and was usually spherical as would be expected if it were semipermeable and both the lluid contained by it and the cytoplasmic fluid of the cell were in equilibrium. The virus particle within this spherical envelope was often bent so that in certain planes of section two virus particles appeared to be enclosed by a single virus envelope. It was very difficult to determine whether the enveloped virus particles in the basal cytoplasm of the columnar cell were within or external to the deeply infolded plasma membrane. It is cIear, however, that the enveloped virus particles can traverse the plasma membrane as they could be observed outside the cell cytoplasm in the substance of the basal lamina. Whether or not the virus envelope acquires some components of the plasma membrane as a result of this process is not known because neither the site of the traverse of the enveloped virus particles across the plasma membrane nor the manner of its accomplishment have been ascertained. Tr:.cheal epithelium cells were frequently found adjacent to the basal lamina and evidence of early stages of virus replication could be seen in these cells from 48 hours after infection feeding (Fig. 12). Virus replication in fat body cells was not observed until 72 hours or more after infection feeding. DISCUSSION
It is logical to arrange the process of virus infection observed in the midgut tissue of A.
CELL
FIG. FIG. FIG. FIG.
INFECTION
BY AN INSECT
313
VIRUS
1. Enveloped virus particle (evp) among microvilli at the apex of a columnar 2. Enveloped virus particle (evp) in contact with cell microvilli. 3. Balloon-like projections (arrowed) on columnar cell microvilli. 4. Virus particles (arrowed) within columnar cell microvilli.
cell.
FIG. 5. A virus particle FIG. 6. A virus particle FIG. 7. Virus replication
within a columnar cell microvillus. in the cell cytoplasm at the base of the microyilli. in a columnar cell nucleus vp, virus particles; 314
pp, polyhedron
protein.
FIG. 8. \.irus part.icles aligned with FIG. 9. Small masses of polyhedron is shown in the inset. Note how the FIG. 10. Enveloped virus particles FIG. 11. Enveloped virus part,icles
a stroma of repeat.ing unit,s (arrowed) in a columnar cell nuelel IS. protein in a virus-infected columnar cell nucleus. The area mark ed nuclear membrane (arrowed) is ruptured. grouped in the cytoplasm at the base of an infected columnar CC:ll. in the cytoplasm at the base of an infected columnar cell. 315
316
HARRA4P
FIG. 12. A tracheal epithelium cell at an early stage of virus (arrowed) in the enlarged cell nucleus ((ecn). Enveloped virus (cc) cytoplasm and basal lamina @I). t, tracheole.
urticae larvae as a sequenceof events in the early stages of nuclear polyhedrosis virus infection. If the virus particles in and adjacent to the microvilli of the columnar cells were in stages of release from the cell
infection. particles
A few virus are present
particles are visible in the columnar cell
then it is difficult to postulate an initia1 source of virus infection, other than possibly the tracheal epithelium cells, as virus development was not observed in any other tissue at this stage. However, the extent of
CELL
INFECTION
BY
virus infection and replication in the tracheal epithelium cells progressed with time and even if these cells could act as sources of infection for the gut columnar cells it is difficult to explain how they could become infected as a result of feeding polyhedra to the larva. Clearly, it is more reasonable to suggest that the observed virus assembly in the columnar cells constitutes the primary focus of virus infection and the following pathway of infection is therefore proposed. When nuclear polyhedra are ingested the polyhedron protein is dissolved in the alkaline gut secretions possibly assisted by proteolytic enzymes. This may be a rather slow process so that by the time the enveloped virus particles are eventually released by the dissolution of the polyhedron protein they lrave been carried into the midgut of the larva by the peristaltic movements of the gut wall. The peritrophic membrane surrounding the contents of the gut lumen consists of a network of strands of chitin and protein forming a mesh with holes 150-200 nm in diameter (Alercer and Day, 1952; Waterhouse, 1953). Some of the enveloped virus particles could pass through the peritrophic membrane and come into contact with the microvilli of the columnar cells as a result of movements of the microvilli characteristic of absorptive cells. The envelope of the virus particle may then become attached to the plasma membrane of a microvillus, and a resulting fusion of the plasma membrane and the virus envelope could allow the nucleocapsid to enter the microvillus. The nucleocapsid could then be carried into the cell as a result of some form of absorptive gradient. Similar observations have recently been made in the early stages of granulosis virus infection of Trichoplusia //.i (Summers, 1969). As nuclear polyhedrosis and granulosis virus particles are structurally very similar, the possibility of a comparable cell infection system is encouraging. Enveloped virus particles which do not come into contact with columnar cell microvilli in this way are probably destroyed fairly quickly in the alkaline environment of the gut lumen. This could account for the difficulty in locating infection in columnar cells, as only a small number of localized cells may become infected. It is significant
AN
INSECT
VIRUS
317
that although the regions of infected columnar cells in A. urticae larvae were not extensive, every cell in such regions was infected. Virus assembly apparently occurs in the nucleus of the cell as virus particles were observed here in different structural states. However, the most significant feature of the development of nuclear polyhedrosis virus in the nuclei of columnar cells is that although enveloped virus particles are formed together with some polyhedron protein the polyhedron protein is produced only as very small masses and never surrounds the enveloped virus particles. The production of polyhedron protein may be a cellular responseto virus infection in which caseeither columnar cells respond poorly or the virus is capable of overcoming the reaction. If, as is more likely, production of polyhedron protein results from a virus-directed synthesis then the mechanism of incorporation of virus particles is either effectively blocked or columnar cells are incapable of synthesizing the large amounts of protein necessary. It is also possible that the deposition of polyhedron protein around the enveloped virus particles is dependent solely on the numbers of these particles. Such enveloped particles appear to pass into the cytoplasm of the columnar cell as a result of the rupture or dissolution of the nuclear membrane which is presumably a reflection of the virus development. Thus there may never be sufficient numbers of enveloped virus particles present for normal polyhedron formation to occur. In the cytoplasm, the enveloped virus particles between the nucleus and the basal membrane may be drawn along some form of gradient in the cell which prevents their general distribution throughout the cell cytoplasm. The virus particles become grouped at the base of the cell, traverse the plasma membrane and are carried through the basal lamina where they come into contact with tracheal epithelium cells. These cells are then infected probably as a result of an attachment of the virus envelope and cell membrane in a similar manner to the mechanism postulated for the infection of columnar cells. The importance of the virus envelope in the infection process is again apparent and it is likely that virus particles lacking the envelope are incapable
318
HARRAP
of infecting cells. The extent of virus infection in the gut therefore influences the spread of virus infection into the tracheal epithelium cells and these provide an effective portal to other susceptible tissues, such as hemocytes, fat body, and hypodermis. Infection in all these tissues is characterized by the formation of typical polyhedra containing virus particles. In these tissues a large proportion of the virus particles produced in any one cell have no further infective function in that host as the virus particles in the polyhedra will not be released until the polyhedra are ingested by another larva. The spread of the virus infection must therefore be dependent on those enveloped virus particles not incorporated into polyhedra. Such virus particles are released when the nucleus ruptures as a result of the large number of mature polyhedra which have developed. The advantage of virus infection occurring initially in a tissue where this occlusion process does not take place is obvious and columnar cells infected with nuclear polyhedrosis virus can be regarded as “virus factories” whose entire output is available for the successful establishment of virus infection in the host. Such a mechanism of cell infection supports the view that nuclear polyhedrosis and granulosis virus particles are enveloped viruses, the nucleocapsid being equivalent to the so-called intimate membrane (Bergold, 1953) and its contents. It seems likely that possession of the virus envelope is a prerequisite for cell infection, and the enveloped virus particle would therefore be the virion. It is significant that during the formation of normal polyhedra only enveloped virus particles are occluded by crystallizing polyhedron protein even though unenveloped
virus particles are also present in the nucleus. The structure of the virus envelope has been investigated in some detail as it apparently has such a significant function in the initiation of cell infection. The results of this work will be reported elsewhere. ACKNOWLEDGMENTS The work reported here formed part of a thesis submitted for the Degree of Doctor of Philosophy, University of Oxford. The electron microscope and its ancillary equipment were purchased with a grant from the Natural Environment Research Council. REFERENCES BERGOLD, G. H. (1953). Insect viruses. Advan. Virus Res. 1, 91-139. HARHAP, K. A. (1969). Proc. Znl. Congr. Viral. Is1 1968. Znt. Viral. 1, 281. HARKAP, K. A., and ROIIEHTSON, J. S. (19G8). A possible infection pathway in the development of a nuclear polyhedrosis virus. J. Gen. Viral. 3, 221-225. LONGIVORTH, J. F., and CUNNINGHAM, J. C. (1968). The activation of occult nuclear-polyhedrosis viruses by foreign nuclear polyhedra. J. Znverle. Palhol. 10, 361-367. MINCER, E. H., and DAY, M. F. (1952). The fine structure of the peritrophic membrane in Dirippus morosus. Rio/. Bull. 103, 384-394. KEYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Hiol. 17, 208-212. SUMMERS, M. 11. (1969). Apparent in vivo pathway of granulosis virus invasion and infection. J. Viral. 4, 188-190. TRAGEH, W. (1935). Cultivation of the virus of grasserie in silkworm tissue cultures. J. Exp. Med. 61, 501-513. WATEIIHOUSE, II. F. (1953). The occurence and significance of the peritrophic membrane, with special reference to adult Lepidoptera and Diptera. Bust. J. Zool. 1, 299-318.