&OURNAL OF ULTRASTRUCTURE RESEARCH 5 3 , 1 9 3 - 2 0 3

(1975)

Electron Microscopy Studies on Banzi Virus Particle and Its Development in the Suckling Mice Brains I C.-M. CALBERG-BAcQ, F. RENTIER-DELRUE, P. M. OSTERRIETHAND P. Y. DUCHESNE Universit~ de Liege, Laboratoire de Microbiologie G~n~rale. 32 boulevard de la Constitution, 4000 Liege (Belgium) and Universit~ de Liege, Institute d'Anatomie, rue des Pitteurs, 4000 Liege (Belgium). Received December 30, 1974; and in revised form May 28, I975 Banzi virus [Flavivirus (Togaviridae)] was purified° from infected mice brains. Negatively stained, the particle appears roughly spherical (525 A in diameter), its envelope bears thin projections, its central core (270 ,~ in diameter) has a polygonal outline suggesting a cubic symmetry. Hexagonal structures (120 ~ in diameter) might represent the capsomeres. In thin sections, the sizes of the total particle and of the core are 440 and 285 ,~, respectively. The viral development in suckling mice brains is cytoplasmic. An enlargement of the endoplasmic reticulum cysternae is observed, which is followed by an extensive hypertrophy of the intracytoplasmic membranous material. This latter material consists of numerous vesicles located inside the cysternae and a peculiar assembly of concentric lamellae. Mature virus particles appear in the extracytoplasmic spaces delimited by these membranes. They are also found outside the cells, either in groups or isolated and fixed to a plasma membrane.

The arthropod-born Banzi virus (H 336), originally isolated from a 9-yr old boy in Tongaland (22), is closely related to Uganda S and Yellow Fever viruses and belongs to the serological group B of Casals (7). According to the international nomenclature of Wildy (26), it should be classified into the flavivirus genus of the Togaviridae family. The occurrence of neutralizing antibodies against Banzi virus in human sera from Angola, Botswana, Mozambique, Nyassaland, and Tongaland was demonstrated. The virus was isolated from cattle, sheep, sentinel hamsters, and several species of culex in the same areas. Few hosts, including mice, were found susceptible to experimental infections and a cytopathic effect was observed on different types of cell cultures (Catalogue of Arthropod-born Viruses, 1971). At present, there are no published data on the biology of the Banzi virus. This paper deals with electron microscopy studies made on the purified

virions and on the development of the virus in suckling mice brains.

A preliminary account of this work was presented at the Joint Meeting of the Belgian, Dutch and German Societies of Electron Microscopy in Liege (Belgium), 1973.

Purification of the Virus

MATERIALS AND METHODS

Virus The H 336 Banzi virus, lyophilized after its eighth intracerebral passage in mice brains, was kindly supplied by Dr. J. Casals (Yale Arbovirus Research Unit, New Haven, Conn.). For the ninth passage this material was used after rehydration in 0.25 ml phosphate buffered saline, pH 7.0 (BS:NaC1, 0.13 M; Na2HPO4, 8 mM; KH~PO~, 1.5 mM; KC1, 2.7 raM; CaC12, 10 mM; MgCI~, 0.5 mM). The virus stock preparations consisted in homogenized brain suspensions in BS (0.8 ml/brain) containing penicillin (100 U/ml) and streptomycin (100 ttg/ml), clarified by centrifugation (3.000g for 15 rain) and stored at -80°C.

Experimental Infection One-day old "Swiss albino" mice were infected by intracerebral inoculation of 0.02 ml of the stock preparation. The mice developed signs of disease about 24 hr later. The brains were harvested when 5% of the animals were dead, i.e., about 40 hr after inoculation.

The starting material consisted of 100 infected brains homogenized in 100 ml of BS. The virus was 193

Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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CALBERG-BACQ ET AL.

purified by selective precipitation of nonviral mate- particles the r a c k e t - s h a p e d aspect frerials with protamine sulfate followingthe technique of quently observed with togaviruses (Fig. 2). Cheng (8) as modified by Osterrieth and CalbergSome particles show a net polygonal outBacq (18). line, either hexagonal or p e n t a g o n a l (Fig. 3). S o m e t i m e s the central viral structure is Electron Microscopy Negative staining. The viral suspensions were seen through the broken envelope (Fig. 4, mixed with an equal volume of 2% phosphotungstate, arrow) revealing structures t h a t look like pH 6.5 (6), and deposited as droplets on Formar-car- large capsomeres (Fig. 5, arrow). M a n y bon coated grids. hexagons 100-120 A in d i a m e t e r are presThin sections. Specimens of midhrain, cerebral, ent in the p r e p a r a t i o n s (Fig. 6); t h e y m i g h t and cerebellar cortex were cut into blocks and fixed at 4°C for 1 hr in phosphate buffer, pH 7.4, containing represent the isolated capsomeres. In addition to p r o t a m i n e precipitation, 2.5% glutaraldehyde. Blocks were postfixed with 1% OsO4 in SCrensen phosphate buffer, pH 7.4 rinsed a t t e m p t s to purify the Banzi virus were and dehydrated through an ethanol series. Propylene m a d e by extracting the c o n t a m i n a n t s with oxide was used for the final dehydration and the fluorocarbon, a technique used with anmaterial was embedded in Epon or araldite. Thin other flavivirus (the Dengue virus, 23). No sections were stained with uranyl acetate (24) and lead citrate (20). Control sections of noninfected intact Banzi particle was recovered even if brains were made. The preparations were observed the p r e p a r a t i o n s were fixed with glutaralwith a Siemens Elmiskop 101 and a Philips EM 201 dehyde (1% final) or f o r m a l d e h y d e (1% microscopes. final) before extraction with fluorocarbon. T h e resulting p r e p a r a t i o n s showed viral RESULTS debris and some largely extended enveThe Banzi Virion lopes. As observed with the electron microViral Replication in the Mouse Brain scope, the purified virus p r e p a r a t i o n s are Usually suckling mice were infected with homogeneous and contain a high concen0.02 ml of the brain suspension o b t a i n e d tration of complete, roughly spherical virions. The external envelope bears very from the preceding passage. Table II thin projections giving the particle its (Expts. 1, 2, a n d 3) gives the infectious characteristic "fuzzy" a p p e a r a n c e (Fig. yields o b t a i n e d 40 hr after inoculation. T h e 1). T a b l e I gives the m e a n dimensions titer a p p e a r s to increase as the n u m b e r of passages increases. T a b l e II (Expts. 4 and measured. On the basis of these data, the length of the projections is around 75 J~. 5) also shows t h a t by using a dilute inocuThis is certainly a m i n i m u m value since lure, the infection, which can be followed the projections are not extended around up to 72 hr, gives rise to a very high the particle. The distance between two infectious titer. T h i n sections in the midbrain, cerebral, projections is 65 ± 2 ~t. T h e p r e p a r a t i o n s always contain m a n y and cerebellar cortex of infected mice were disrupted envelopes and this aspect is not observed. Essentially, the replication of the modified by freezing and thawing. In some Banzi virus proceeds in the s a m e way in virions, the loose envelope is collapsed on the various tissues examined. In all cases, the central structure, hence giving the the cell nuclei r e m a i n unaffected, the mito-

FIGs. 1-6. Negative staining of purified Banzi virus preparations. × 200 000. Fro. 1. General appearance of the preparation. FIG. 2. Racket-shaped particles. Fro. 3. Particles with polygonal outline. FIGs. 4, 5. The arrows point to particles with a disrupted envelope showing the central core. FIc. 6. The arrows point to isolated hexagonal structures.

STRUCTURE AND D E V E L O P M E N T OF BANZI VIRUS

195

196

CALBERG-BACQ E T AL. TABLE1 Mean dimensions (A)

Virus

After negative staining

Banzi (flavivirus) Semliki forest (alphavirus)

In thin sections

Total

Without Projections

Core

Total

Core

525 ~ 8 680 (17)

380 ± 5 530 (17)

270 ± 30 378 (17)

440 ± 7 ±500 (11)

284 ± 8 280 (11)

TABLE I1 VIRAL MULTIPLICATION IN THE MOUSE BRAIN Number of passages

Experiment

Inoculum (LDso/brain)

Duration of infection (hr) at brain harvest

Yield (LD~o/brain)

11 17 20

1 2 3 4 5

1 × 104

40

2.5 x i0 ~

2 x 106

40

• 5 × 108

2x

40

1.9x

40

2.5 × I0 ~ 2.5 x 101°

106

2 x 101 2 X

chondria are well-preserved and the mature virus particles are recognizable thanks to their typical morphology and their specific location in the infected tissues. They have a central core often polygonal in outline surrounded by a thin electron dense layer on which projections can sometimes be distinguished (Fig 7; cf. Table II for the mean dimensions measured). When located in the extracellular spaces, the mature particles occur either alone or in groups, often in a more or less close association with the plasma membrane (Fig. 8). When observed inside the cell, they are always extracytoplasmic and occur in spaces that are delimited by membranes derived from the endoplasmic reticulum (Figs. 12-14). Eventual viral nucleoids, i.e., intracytoplasmic forms of the virus still de-

101

72

i0 ~

prived of the external envelope, are difficult to localize. Even in areas where many mature viruses are present, there are no structures which can be differentiated from ribosomes. In early stage of infection (Table I, Expt. 4) the cytoplasm is still well organized and contains ribosomes in rosettes. In the perikaryon of numerous nerve cells, however, both the Golgi apparatus and the endoplasmic reticulum cysternae have undergone considerable enlargement (Fig. 9). Small vesicles also appear developing from the endoplasmic reticulum membrane inside the enlarged cysternae (Figs. 10 and 11). In the limited areas where they are present, the mature viruses are found either in the cysternae or budding through the membrane. Essentially, brains, harvested from mice

Fins. 7, 8. Banzi virions in thin sections; parts of brain cells 40 hr after infection with a heavy inoculum (Expt. 3, Table II). Insert, particle with distinct projections. In Fig. 8, the arrow points to a particle in connection with a plasma membrane, x 100 000. Fias. 9-11. Early stage of infection; parts of the perikaryon of cells 40 hr after infection with a dilute inoculum (Expt. 4, Table II). x 50 000. FIG. 9. Enlargement of the endoplasmic reticulum cysternae. FIGS. 10, 11. Budding of vesicles inside the cysternae.

STRUCTURE AND DEVELOPMENT OF BANZI VIRUS

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CALBERG-BACQ E T AL.

STRUCTURE AND DEVELOPMENT OF BANZI VIRUS

199

either 40 hr after infection with a heavy T h e tissue is modified too much, however, inoculum or 72 hr after infection with a to claim whether or not the glial cells play a dilute inoculum, exhibit the same cellular role in the infectious process. alterations. However, in the latter case, to M a n y virus particles are found in the which the following description is limited, extracellular spaces. In an a t t e m p t to vetthe alterations are more frequent and more ify whether the images of virions fixed to profound; the viral multiplication is also the marginal cell m e m b r a n e were repremore extensive (Table II). Strikingly, the sentative of the first step of infection, brain infected cytoplasm exhibits an enormously sections m a d e during the viral eclipse hypertrophied m e m b r a n o u s s y s t e m . Nu- phase were examined. T h e mice were inocmerous vesicles (600-1000 A), similar to ulated with 1.5 × 108 LDso/brain and the those observed in the earlier stage of infec- brains were harvested 2 hr later. T h e tion, are r a n d o m l y distributed inside the recovered infectivity was 3.7 × 103 LDs0/ enlarged cysternae (Fig. 12). Sometimes brain but no viral particle could be dethey are aligned along a double m e m b r a n e tected. figure formed by two cysternae close toDISCUSSION gether (Fig. 13). T h e cells also contain very T h e Banzi virus presents features of a characteristic assemblies of concentric lamellae (Figs. 15 and 16), which may arise typical togavirus (13, 14). It is a roughly from the fusion of flattened Golgi vesicles spherical enveloped virion; its envelope (Fig. 18, arrow), they frequently include does not tightly fit the central core and mitochondria. Both vesicles and lamellae bears characteristic thin projections; the m a y derive from the same m e m b r a n o u s polygonal outline of the core suggests a m a t e r i a l since, s o m e t i m e s , t h e y form cubic s y m m e t r y ; and viral replication in mixed figures (Fig. 17). Finally, cytoplas- the mouse brain occurs only in the cytomic dense regions m a d e of imbricated plasm, where it induces an i m p o r t a n t extubules are seen in the vicinity of a vesicu- tension of the m e m b r a n o u s structures. In thin sections, the Banzi virion exlar area (Fig. 12, T) or in close connection with the concentric lamellae (Figs. 15 and hibits dimensions consistent with those 16, T). These structures might be involved observed after negative staining. These are in the synthesis of the viral central core. c h a r a c t e r i s t i c of the genus Flavivirus M a n y m a t u r e viruses are present inside which is thought to comprise the smallest the cysternae either a t t a c h e d to the mere- e n v e l o p e d a n i m a l viruses. If the albrane or located at the external side of the phaviruses appear to be larger after negavesicles. Budding images can be observed tive staining, their m e a n d i a m e t e r in thin (Fig. 12). Viruses are also found isolated in sections, however, is not significantly difsmaller vacuoles (Fig. 14); in the lamellar ferent from t h a t of the flaviviruses. T a b l e I areas, they appear between two lamellae or gives as an example the comparative diin a central vesicle (Fig. 19). T h e virions mensions of the Semliki Forest virion. This are produced by the nerve cells both in discrepancy reflects the flexibility of the their perikaryons and in their extensions. Semliki Forest nucleocapsid (3) which, in FIGs. 12 14. Intracellular localization of mature particles in a late stage of infection. × 50 000. FIG. 12. Parts of brain cells 72 hr after infection with a dilute inoculum (Expt. 5, Table II); a vesicuJar region containing numerous mature particles. The arrow points to a budding figure. T, a cytoplasmic dense region made of imbricated tubules. FIG. 13. Parts of brain cells 40 hr after infection with a heavy inoculum (Expt. 3, Table II). Vesicles are aligned along a double membrane figure. FIG. 14. Parts of brain cells 40 hr after infection with a heavy inoculum (Expt. 3, Table II). Mature particles are isolated in small vacuoles.

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CALBERG-BACQ E T AL.

FIGS. 15, 16. Figures of concentric lamellae; parts of the cytoplasm of brain cells 72 hr after infection with a dilute inoculum (Expt. 5, Table II). T, tubular regions, x 50 000.

Ftas. 17, 18. Details of concentric lamellae figures; parts of the cytoplasm of brain cells 72 hr after infection with dilute inoculum (Expt. 5, Table II). × 50 000. FtG. 17. Mixed figure of concentric lamellae and aligned vesicles. Fro. 18. The arrow points to incomplete formation of lamellae. Flo. 19. Localization of mature virus particles inside the concentric lamellae; part of the cytoplasm of a brain cell 40 hr after infection with a heavy inoculum (Expt. 3, Table II). × 44 000. 201

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CALBERG-BACQ ET AL.

thin section, is observed under its contracted form. Thus, it follows that the core of the alphaviruses and the nonflexible nucleocapsid of the flaviviruses might be built according to the same pattern. Also note that isolated cores from an alphavirus (Sindbis) and from a flavivirus (Kunjin) were found to have the same sedimentation constant (4). The preparations of purified Banzi virions contain isolated hexagonal structures, 120 ~ in diameter. The same structures are seen on particles that have a disrupted envelope. Identical subunits were assigned to be the capsomeres of Semliki Forest virus (17) and of Sindbis virus (15). Maturation of the Banzi virus in the mouse brain cells clearly involves a specialized intracytoplasmic membranous material, which probably originates from the endoplasmic reticulum and the Golgi apparatus. This proliferation of the internal membranes and the appearance of mature particles within the external spaces of vesicular regions is a constant feature of flavivirus replication both in cell cultures and in brains (1, 2, 5, 10, 19, 21, 27, 28). Note, however, that the occurrence of the concentric membranes so frequently encountered in Banzi infected cells was only reported during infection with the Powassan virus (25), with the Omsk Hemorrhagic Fever virus (2•); they are seldom observed in Central European Encephalitis and Zimmern infections (2). Hence, this extreme cytoplasmic alteration appears as a characteristic feature of flavivirus infection although it is not always observed or occurs with a much lower frequency. It might be related to the development of a very acute disease (i.e., a very rapid infection) in young animals. The frequent occurrence of groups of Banzi viruses in the extracellular spaces supports the idea that the release of the mature particles proceeds through the fusion of a virus producing vesicle with the external cell membrane. The only clear image of such a process, however, has been

reported by Dalton (9) with the Langat virus. A budding process of the Banzi viruses through the plasma membrane could not be demonstrated. If it occurs, it is not very frequent and the figures of the flavivirus maturation, even in the intracytoplasmic cysternae, are not as distinct budding images as those given by alphaviruses. The alphaviruses (11) and the non-arthropod-born togavirus, Rubella virus (12) exhibit both external and intraeytoplasmic maturation with a preferential site at the marginal cell membrane. A second important morphological difference is the synthesis of a very great amount of precursors of alphavirus particles. These nucleoids either appear as aggregates in the form of crystalline areas or they occur around vesicles in a characteristic way, which allows their identification. Such a situation does not occur during Banzi virus or other flavivirus multiplication and it has not been possible to identify precursor particles with certainty. Inclusions of tubular material identical to those found in Banzi infected cells (Figs. 12 and 15) have been described during both alphavirus (16) and flavivirus (2) replication. They might be involved in viral RNA synthesis (12). From most of the morphological data available, it appears that the nuclei are not involved in the replication of flaviviruses. While it is certain that the nerve cells do support this multiplication, the glial cells may also be involved in this phenomenon (9). We are very grateful to Dr. L. Simar (Service d'Anatomo-Pathologie, Universit~ de Liege) for the thin sectioning and to Prof. R. Bronchart (Servicede Recherches Ultrastructurales, Universit~ de Liege) for the use of the electron microscope.We thank also Mr. F. G~rard for technical assistance. This work has been supported in part by grants (to P.M.O.) from the "Fonds de la Recherche ScientifiqueM~dicale" (No. 20.110) and (to P.Y.D.) from the "Fonds de la Recherche Scientifique." REFERENCES 1. BELL, T. M., FIELD, E. J., AND NARANG, H. K., Arch. Ges. Virusforseh. 35, 183 (1971).

STRUCTURE AND DEVELOPMENT OF BANZI VIRUS 2. BLINZINGER, K., Ann. Inst. Pasteur 123, 497 (1972). 3. BONSDORFF, VON C. H., Comment. Biol. 74, 53 (1973). 4. BOULTON,R. W., AND WESTAWAY,E. G., Virology 49, 283 (1971). 5. BOULTON, S. P., AND WEBB, H. E., Brain 94, 411 (1971). 6. BRENNER, S., AND HORNE, R. W., Biochim. Biophys. Acta 34, 103 (1959). 7. CASALS,J., Trans. N. Y. Acad. Sci. 19 (Ser. 2), 219 (1957). 8. CHENa, P. Y., Virology 14, 124 (1961). 9. DALTON,S., Ann. Inst. Pasteur 123, 489 (1972). 10. DAVID-WEsT,S., LABZOFFSKY,N. A., ANDHAMVAS, J. J., Arch. Ges. Virusforsch 36, 372 (1972). 11. GRIMLEY, V. M., AND FRIEDMAN, R. M., Exptl. Molec. Pathol. 12, 1 (1970). 12. HIGASHI,N., Progr. Med. Virol 15, 331 (1973). 13. HORZINEK,M. C., J. Gen. Virol. 20, 87 (1973). 14. HORZINEK, M. C. Progr. Med. Virol. 16, 109 (1973). 15. HORZINEK, M. C., AND MUSSGAY, M., J. Virol. 4, 514 (1969}. 16. LASCANO,E. F., BERR1A, M. I., ANDBARRERA-ORo, J. G., J. Virol. 4, 271 (1969).

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17. OSTERRIETH, P. M., Mem. Soc. Roy. Sci. Liege 5 (Ser. 16), 125 (1968). 18 OSTERRIETH, P. M,, AND CALBERG-BACQ, C.-M., J. Gen. Microbiol. 43, 19 (1968). 19. PEAT, A., AND BELL, T. i . , Arch. Ges. Virusforsch. 31, 230 (1970). 20. REYNOLDS,E. S., g. Cell. Biol. 17, 208 (1963). 21. SHESTOPALOVA,N. M., REINGOLD, V. N., GAGARINA, A. V., KORNILOVA,E. A., PoPov, G. V., AND CHUMAKOV,M. P., J. Ultrastruct. Res 40, 458 (1972). 22. SM1THBURN,K. C., PATERSON,H. E., HEYMAN,C. S., AND WINTER, P. A. D., S. African Med. J. 33, 959 (1959). 23. STEVENS,W. M. ANDSCHLESINGER~R. W., Virology 27, 103 (1965). 24. WATSON, M. L., g. Biophys. Biochem. Cytol. 4, 475 (1958). 25. WHITNEY,M., Internat. Virol., 2, 163 (1972). 26. WILDY, P., Classification and Nomenclature of Viruses, Monographs in Virology, Vol. V. Karger, Basel, 1971. 27. YASUZUMI,G., AND TSUEO, I., J. Ultrastruct. Res. 12, 304 (1965). 28. YASUZUMI, G., TSUBO, I., SUGIHASA, R., AND NAKAI, Y., J. Ultrastruct. Res. II, 213 (1964).