384

Printed in Sweden Copyright ~ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved

3. ULTRASTRUCTURE RESEARCH 47, 384-399 (1974)

Studies on a Nuc lear Polyhedrosis Virus in

Bombyx mori Cells in Vitro

1. Multiplication Kinetics and Ultrastructural Studies

RAJENDRA RAGHOW 1 and T. D. C. GRACE

Division of Entomology, CSIRO, P.O. Box 1700, Canberra City, A. C. T. 2601

Received June 12, 1973 and in revised form December 4, 1973

The analysis of the multiplication kinetics of Bombyx mori nuclear polyhedrosis virus (BMNPV) in Grace's B. mori cell line reveals three phases of viral develop- ment: (a)a latent phase (0-12 hours), (b) an exponential phase (16-48 hours), and (c) a stationary phase (48 hours onwards). Ultrastructural observations of infected cells at various times during these three phases reveal a sequence of events of virus replication as follows: (i) Membrane-enclosed virus particles enter the cell by phagocytosis and viropexis (in the early part of the latent phase). Naked NPV rods are seen attached to the nuclear membrane in the nuclear pore area. (ii) The precursor of the virogenic stroma (PVS) appears as electron-dense patches in the nucleus 16 hours after infection (beginning of the exponential phase). It changes into an electron-dense network associated with large numbers of naked viral rods 32 hours after infection. (iii) About 90% of the virions become membrane-enclosed (36 hours after infection) followed by their occlusion into polyhedra (40 hours after infection). (iv) The maximum titre is attained 48 hours after infection. Paracrystalline arrays of tubular aber- rant forms, probably of viral origin, and proliferation of membrane profiles were observed in the infected cells.

Most of the developmental studies on nuclear polyhedrosis viruses (NPV) have used whole insects (4, 12-14, 16, 28, 31, 37, 38, 39; for review see 6, 10, 27). However, in such in vivo studies there are two major disadvantages: (a) the difficulty of synchroniz- ing the infection process, and (b) the difficulty of following quantitatively the subse-

quent multiplication and growth of the virus within the cells. In attempts to overcome these disadvantages there have been several investigations on primary cell lines in vitro (2, 8, 15, 18, 21-24, 33, 35, 36). Cultured amoebocytes (20) and hemocytes (29)

1 Present address: Biochemistry Department. School of General Studies, Australian National University, Canberra, A.C.T., Australia.

NPV IN Bombyx rnori CELLS in vitro 385

have also been used for deve lopmenta l studies of an NPV. G o o d w i n et al. (7) r epor ted

repl ica t ion of Spodoptera frugiperda N V P in an S. frugiperda cell line es tabl ished f rom

pupa l ovar ian tissue. In the above reports , no direct quant i t a t ive studies have been

made concurren t ly wi th observat ions on the u l t ras t ruc ture of the virus-infected cells

at var ious stages of development .

In the present s tudy we have corre la ted the viral deve lopmen t as seen under the

electron microscope , s ta r t ing f rom virus pene t ra t ion fo l lowed b y repl ica t ion and oc-

clusion into po lyhed ra of Bombyx mori N P V in Grace ' s B. mori cell line, wi th direct

es t imat ion of virus t i tre (LDs0 units/106 cells) a t var ious stages of the growth cycle.

The ma in emphasis i s on the early events, especially virus entry, and on the subse-

quent changes in the cell nucleus after infection.

MATERIALS AND METHODS

Bombyx mori cells from an established line (9) were grown in prescription bottles and petri dishes in medium containing 1% bovine plasma albumin and 1% heat-treated haemo- lymph from Antheraea pernyi (Guer.); 106 cells in 2.5 ml of medium were seeded into 60-ram petri dishes.

The stock virus was concentrated under sterile conditions by centrifugation (10 000 g, 10 minutes) of the medium from 3-day-old infected B. mori cultures. The stock virus consisted of only nonoccluded virions and a very small proport ion of naked viral rods (T. D. C. Grace, unpublished observations). Stock virus was stored frozen at - 2 0 ° until use. I t was estimated that the stock contained about 8.9 × 105 LD~0 units per ml.

To initiate infection 0.1 ml of the solution stock virus was added to each culture (108 cells per petri dish). Adsorption of virus continued for 1 hour at 28 °. The controls were mock- infected with 0.1 ml of medium from 3-day-old uninfected cultures. Beginning at the end of adsorption period (zero time) samples were taken at regular intervals during the 70 hours of infection period.

At each time, the cells were gently agitated, suspended in the growth medium, and 1.25 ml (5 × 105 cells) were frozen at - 2 0 ° for virus assay. The other 1.25 ml were centrifuged (270 g for 5 minutes) and the cell pellet was processed for electron microscopy as described before (25). The sections were examined in a Siemens Elmiskop 1 operating at 60 kV.

Samples for virus assay were thawed and sonicated for 1 minute at 15 kHz, with the tube in crushed ice. The cell debris was centrifuged at 10 000 g for 10 minutes. The supernatant was decanted and the pellet was dispersed in 0.1 ml of 0.004 M NaHCOs (pH 10.0) for 15 minutes with occasional shaking. The suspension was spun at 10 000 g and the combined supernatants made to a volume of 5 ml with growth medium without haemolymph, so diluting the NaHCO3 by a fac to r of 50. The same treatment was given to the control cells. The treatment with NaHCO~ was essential after the appearance of polyhedra, i.e., 40 hr onwards, but for the sake of uniformity all the samples received this treatment. Serial 10-fold dilutions were made in medium without hemolymph, and at each dilution 10 ~1 of virus suspension were injected through a proleg into each of 10 fifth-instar larvae of B. mori. At least 4 dilutions were used at each time. The LDs0 was determined by the method of Reed and Muench (26).

386 RAGHOW AND GRACE

RESULTS

GROWTH KINETICS

The LDs0 time curve of infected cells is shown in Fig. 1. It is evident that there is art initial decrease in the titre which stays at a low level for 12 hours. After the latent period of about 12 hours there is a rapid rise in the infectious titre, and within the next 30 hours the maximum titre (2.2 x 10 ~ LDs0/106 cells) is attained. There is little change in the titre in the 48-72-hour period. More than 90 % cells showed polyhedra in their nuclei in these later stages of infection.

ULTRASTRUCTURE STUDIES Uninfected cells

The uninfected cell of B. mori has the typical ultrastructural morphology of a cell in tissue culture (Fig. 2). There is a large nucleus surrounded by a nuclear membrane with a prominent nucleolus. The appearance of the chrolnatin material is greatly de- pendent on the physiological state of the cell. In most of the cells not undergoing mitosis, the chromatin material is evenly diffused throughout the nucleus. In the cytoplasm, most of the well-characterised organelles, i.e., mitochondria, rough and smooth endoplasmic reticulum, ribosomes, vacuoles, and lysosomes etc., are seen.

Infected cells

Virus entry and uneoating. Steps in the entry and uncoating of the virus particles in these cells are illustrated in Figs. 3-11. These stages took place from zero time (see Materials and Methods) to 8 hours. It is evident from the figures that the pathway of viral entry is through attachment of the viral particle to the plasma membrane (Fig. 3) followed by an active invagination of the plasma membrane by phagocytosis (Figs. 4-7). The immediate lining of the plasma membrane, near the tip of the entering NPV particle clearly shows some spherical structures (arrows, Fig. 5; see also Discus- sion), which are absent where the apparent phagocytosis has progressed further (Figs. 6 and 7). It is notable that all the entering particles have their membranes intact, and at no stage were naked viral rods seen penetrating the plasma membranes. However, most of the particles observed in the cell cytoplasm 4-8 hours after infection showed only part of the membrane (Fig. 8).

The next stages in the entry pathway are visible in Figs. 9-11. The naked rods can be seen attached to the nuclear membrane. Some of these viral rods are only partially electron opaque (Figs. 10, 11).

The first change in the nucleus of the infected ceils was seen 16 hours after infection. Discrete patches of electron-dense material appeared, interspersed with diffused elec-

NPV!N B o m b y x rnori CELLS in vitro 387

_m

t D o 5

t ~

£21 --.1

o 4

--.I

10 20 3O 40 50 60 Time in hours

FIG. 1. Growth curve of BMNPV in Bombyx mori cells. For details see Materials and Methods.

tron opacity in the nuclei (Fig. 12). This is probably the precursor of the virogenic stroma (PVS). Examination of the PVS at higher magnification (Fig. 13) did not reveal any virus particles associated with it at this time; neither were any virus particles seen elsewhere in the nucleus. The nucleolus did not have any obvious alteration in its ultrastructure. A nucleus at 24 hours after infection showed an extensive network of electron opaque material, the virogenic stroma (VS, 38), with which were associated many viral rods. The characteristic feature of the virogenic stroma at this stage was the complete lack of membrane-bound particles associated with it. Naked viral rods

Fta. 2. A mock-infected cell of B. mori at zero time. N, Nucleus; Nu, nucleolus; M, mitochondria; V, vacuole, x 6 300. Fins. 3-11. Various stages in the entry of virus. FIG. 3. Virus particle with expanded membrane is seen attached to the plasma membrane of the cell. x 60 000.

Fins. 4 and 5. Virus particles seen in the initial stages of penetration. Arrow pointing to the circular structures near the tip of the entering particle, x 60 000. FIGs. 6 and 7. Apparently advanced stages in the entry process. The membrane of the virion is still intact, x 60 000.

388 R A G H O W A N D G R A C E

NPV IN Bombyx mori CELLS in vitro 3 8 9

390 RAGHOW AND GRACE

of uniform diameter but varying lengths are seen close to the electron opaque VS (Fig. 14). The number of viral rods associated with the VS had increased greatly 32 hours after infection (Fig. 15) and all were invariably naked.

By 36 hours after infection (Fig. 16) most of the viral rods were encapsulated in membranes and the VS appeared in only 5-10 % of the infected cell nuclei. At this time, however, there were no indications of occlusion of the viral particles in poly- hedra.

At 40 hours after infection, the majority of the cells contained very small developing polyhedra. A section of a representative cell at this time is shown in Fig. 17. Many particles can be seen being occluded (arrows) along the peripheral regions of the pro- tein crystal. Often more than one particle is encapsulated within a common membrane and occluded in a polyhedron (Figs. 17 and 18).

Figure 18 represents a section across a typical cell at 72 hours after infection. The majority of the virus particles have been occluded into the polyhedra. The notable feature of this late stage of infection is that most of the membrane-enclosed particles are close to the polyhedra whereas the nonenveloped virus rods are randomly located within the nucleus. At that time, the last in the growth curve, the nuclear membrane is intact. There was no alteration in the cytoplasmic organization of the infected cell at any stage after infection. However, there appeared to be inhibition of cell division as judged by the total number of cells in infected and control cultures (R. Raghow, unpublished observations).

Occasionally, in some of the infected nuclei there was an extensive proliferation of the membranous structures (Fig. 19). A few infected nuclei exhibited, along with membrane proliferation, a large number of tubular structures the diameter of which were the same as the viral rods (Fig. 20). These structures were generally many times longer than the viral rods but were "empty" or electron lucent throughout most of their lengths.

DISCUSSION

The experiments described above reveal three distinct phases in virus growth in B. mori cells in vitro. There is (i) latent phase (0-12 hours), (ii) a rapid increase or exponential phase (16-48 hours) followed by (iii) a stationary phase (48 hours on- wards). Aizawa (1) and Vaughn and Faulkner (34) noted similar phases during the development of BMNPV in vivo. However, a direct quantitative comparison with

FIG. 8. A virus particle seen in the cytoplasm 2 br after infection. The membranes are only partially intact (arrow). x 60 000. F~. 9. A naked viral rod apparently attached to the nuclear pore. The particle is electron-dense throughout. N, Nucleus; NM, nuclear membrane, x 60 000. Fits. 10 and 11. Partially electron dense, naked viral rods attached to the nuclear membrane, x 80 000.

NPV IN Bombyx mori CELLS in vitro 391

392 R A G H O W AND GRACE

?,

NPV IN Bombyx mori CELLS #l vitro 393

FIG. 14. A section of VS 24 hr after infection showing network of granular electron-dense material and few naked particles closely associated with it. × 60 0(I0.

their in vivo observations cannot be made because, in both of the above-mentioned studies, growth kinetics were based mainly on the determination of infectious titre

in the haemolymph of the infected insects. The electron microscopic observations at various times during these three phases of growth show a sequence of events starting from the entry of the virus to the final occlusion of the virions in the polyhedral bo- dies.

The entry of tlae intact virus particles by phagocytosis occurs in the initial 2 hours of the latent phase. These observations support the mechanism of viral entry by viropexis (5). The role of the circular structures just below the plasma membrane, in contact with the entering virus parti~le is possibly to assist the process of penetration

by phagocytosis. The NPV rods attached to the nuclear membrane (an event seen during 4-6 hr of the latent period) are naked. The exact mechanism by which the

FIGS. 12-20. The infected nuclei of Bombyx mori cells showing a sequence of events of viral develop- ment during a single-cycle growth experiment. FIG. 12. The distinct areas of electron-dense material (PVS) in the nucleus 16 hr after infection. Mingled with these patches there is some diffused electron dense material. Nu, Nucleolus. x 15 000. FIG. 13. Magnified view of the PVS revealing the granular appearance, x 60 000.

394 RAGHOW AND GRACE

membrane around the virus is removed is not clear f rom our studies. The mechanisms of entry in vivo have been investigated in a variety of insect viruses (11, 12, 16, 17, 19, 30, 40). Some of these studies suggest that viral entry is by fusion of the viral envelope with the plasma membrane of the susceptible cell, followed by the entry of the naked nucleocapsid (11, 12, 16, 30). On the other hand, entry of viral particles by

phagocytosis and viropexis is evident in some insect viruses (11, 17). Recent observa- tions on a poxvirus of Amsacta moorei have revealed that this virus can enter the host tissues either by fusion or viropexis depending on the conditions of infection (11). Such a dual mode of entry was also reported for vesicular stomatitis virus infect-

ing L cells (see 5 for review). The at tachment of naked viral rods (some of which are only partially electron

dense) to the nuclear pores is very significant, but clear-cut evidence for the entry of the whole nucleocapsid or only its contents is lacking. Summers (30) has provided

some evidence that with the granulous virus of Trichoplusia ni only the contents of the viral rods enter the nucleus. Such a mechanism would very likely operate in NPV of B. mori.

The first well-defined change in the nucleus, is the appearance of PVS at 16 hours after infection, which coincides with the beginning of the exponential phase of multi- plication kinetics. Dur ing most of the exponential phase the nuclei of the infected

cells show all impor tant stages of viral synthesis and assembly. In the next 32 hours the PVS becomes more network-like with the viral rods associated with it. I t finally almost disappears when the polyhedra appear in the infected nuclei. Dur ing this period

of rapid viral synthesis as judged by electron microscopy, the infectious titre rises by about 4 log units. Apar t f rom the studies of Xeros (38) and Benz (3), very little is known about the biochemical nature of the VS. However, the VS has been implicated in viral assembly (3, 13, 39).

The impor tant facts about viral occlusion are as follows: (a) Examinat ion of large numbers of sections of infected nuclei showed that occlusion does not start until mos t of the viral rods are enveloped. (b) Our studies indicate that the same polyhedron

Fro. 15. VS 32 hr after infection appears more condensed. The number of naked viral rods has in- creased considerably, x 60 000. FIG. 16. Part of a nucleus 36 hr after infection. Most of the particles are membrane-bound although at this stage occlusion is positively absent. × 60 000. FIG. 17. A nucleus 36 hr after infection showing the beginning of occlusion. Some enveloped viruses (arrows) can be seen being occluded in the protein matrix (P). x 60 000. FIG. 18. A late stage in the infection cycle represented by a section of a nucleus 72 hr after infection. Majority of the enveloped viruses (filled arrow) have either been occluded or are in close proximity to the polyhedra (P); the naked particles (hollow arrow) are randomly spread in the nucleus. × 10 000. FIG. 19. Aberrant membranous structures in the nuclei of B. mori 48 hr after infection. Naked virus particles can be seen (arrow). x 30 000. FI6. 20. Another aberration in the infected nucleus in the form of a paracrystalline array of rods (* and a variety of membrane profiles, x 24 000.

NPV IN Bombyx mori CZLLS in vitro 3 9 5

2 6 - 741839 a r. Ultrastructare Research

396 R A G H O W AND G R A C E

NPV IN Bombyx mori CELLS in vitro 397

398 R A G H O W A N D G R A C E

can contain both kind of particles, i.e., many nucleocapsids, common to a single envelope and a single nucleocapsid within an envelope. This is in contradiction to Goodwin (personal communication) who described the so called "multiple-embedded' and "single embedded" viruses and assigned some kind of specificity in the occlusion of the two types.

From 48 hours onwards nonoccluded virions can be seen in the cytoplasm of the infected cells, although as late as 72 hours, when most of the nuclei are packed with polyhedra, the nuclear membranes in the majority of the cells are still intact. It is obvious that some of the particles find their way out of the nucleus long before the general disorganization of the cellular structures begins to take place in the late stages of infection. These observations are significant in relation to the pathway of in vivo infections. Harrap and Robertson (14) noted that although there was multi- plication of NPV in the columnar cells of Aglais urticae midgut there was no occlusion of virions into the polyhedra. They also detected some particles in the cytoplasm of the infected cells, and their role in the virus spread f rom columnar cells to the other sus- ceptible tissues was indicated. These observations on spread of virus in vivo have been extended to the study of some other insect viruses (17, 19, 32). Thus, it appears that our observations are reminiscent of viral spread as it occurs in vivo. Recently Dales (5) has concluded that "viropexis appears to be a predominant, but not exclusive mechanism for internalizing many animal agents." Our present observations on the entry of BMNPV into B. mori cells lend support to this conclusion.

The extensive proliferation of membranes and the appearance of paracrystalline arrays of rod-shaped structures similar in diameter to the naked viral rods seen in some infected nuclei were possibly nothing but aberrations caused during viral as- sembly; that they play a direct role in the biosynthesis of virus is unlikely. Such aber- rant forms during the replication of insect viruses have been described before (27, 30).

It is a pleasure to thank Dr B. K. Filshie for his help in electron microscopy and Hilary Mende and Rhonda Christian for expert technical assistance.

REFERENCES

1. AIZAWA, K., J. Insect Pathol. 1, 67 (1959). 2. AIZAWA, K., and VAaO, C., Ann. Inst. Pasteur 96, 455 (1959). 3. BENZ, G., J. Insect Pathol. 2, 215 (1963). 4. BIRD, F. To, Can. J. Microbiol. 10, 49 (1964). 5. DALES, S., Bacteriol. Rev. 37, 103 (1973). 6. DALGARNO, L., and DAVEY, M. W., in GIBBS, A. J. (Ed.), Viruses and Invertebrates, p.

245. North-Holland PUN., Amsterdam, 1973. 7. GOODWIN, R. H., VAUGHN, J. L., ADAMS, J. R. and LOULODES, S. J., J. Invert. PathoL

16, 284 (1970).

NPV IN Bombyx mori CELLS in vitro 399

8. GRACE, T. D. C., Science 129, 249 (1958). 9. - - Nature (London) 216, 613 (1967).

10. - - Advan. Virus Res. 14, 201 (1969). 11. GRANADOS, R. R., Virology 52, 305 (1973). 12. HARRAP, K. A., ibid. 42, 311 (1970). 13. - - ibid. 50, 133 (1972)." 14. HARRAP, K. A. and ROBERTSON, J. S., J. Gen. Virol. 3, 221 (1968). 15. HE1TER, F., Ann. Epiphyt. 14, 213 (1963). 16. KAWANISHI, C. Y., SUMMERS, M. D., STOLTZ, D. B., and ARNOTT, H. J., J. Invert.

Pathol. 20, 104 (1972). 17. KISLEV, N., HARPAZ, I. and ZELCER, A., J. Invert. Pathol. 14, 245 (1969). 18. KRYW~ENCZYK, J. and Sore, S. S., J. Invert. Pathol. 9, 568 (1967). 19. LEUTENEGGER, R., Virology. 32, 109 (1967). 20. MART~GNONL M. E. and SCALLION, R. J., Nature (London) 190, 1133 (1961). 2l. MEDVEDEVA, N. B., Vopr. Virusol. (in Russian) 4, 449 (1959). 22. MILOSERDOVA, V. D., ibid. 10, 417 (1965). 23. QUIOT, J. M. and LUOANL J., lnt. Colloq. Invertebr. Tissue Culture 2nd, p. 233 (1968). 24. QUIOT, J. M., VAGO, C. and PARADIS, S., Entomophaga 15, 437 (1970). 25. RA~HOW, R. S., GRACE, T. D. C., FILSHIE, B. K., BARTLEY, W. and DALGARN0, L. J.

Gen. Virol. 21, 109 (1973). 26. REED, L. J. and MUENCH, H. A., Amer. J. Hyg. 27, 493 (1938). 27. SMtTI~, K. M., in MARAMOROSCH, K. and KURSTAK, E. (Eds.), Comparative Virology,

p. 479. Academic Press, New York, 1971. 28. SMITH, K. M. and XEROS, N., Parasitology 43, 178 q953). 29. Sore, S. S. and CUNNINGHAM, J. C., J. Invert. Pathol. 19, 51 (1972). 30. SUMMERS, M. D., J. Ultrastruct. Res. 35, 606 (1971). 3l. SUMMERS, M. D. and ARNOTT, H. J., ibid. 28, 462 (1969). 32. TANADA, Y. and LEUTENEGGER, R., ibid. 30, 589 (1970). 33. VAUGHN, J. L. and STANLEY, M. S. M., J. Invert. Pathol. 16, 357 (1970). 34. VAtJGHN, J. L. and FAULKNER, P., Virology 20, 484 (1963). 35. VAGO, C., QUIOT, J. M., and ARMAGIER, A., C. g. Acad. Sci. Set. D 269, 978 (1969). 36. VAGO, C., and BERGOIN, M., Advan. Virus Res. 13, 247 (1968). 37. XEROS, N., Nature (London) 172, 548 (1953). 38. - - ibid. 175, 588 (1955). 39. - - ibid. 178, 412 (1956). 40. - - - - J. Insect Pathol. 6, 225 (1964).