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Scanning and freeze-fracture electron microscopy of rabies virus infection in murine neuroblastoma cells
Comp. lmmun. Microbiol. in[ect. Dis., Vol. 5, Nos. 1 3, pp. I 8, 1982. Printed in Great Britain.
[)147-9571/82,'010001 07 $03.00!0 ~ 1982 Pergamon Press Ltd.
SCANNING AND F R E E Z E - F R A C T U R E ELECTRON MICROSCOPY OF RABIES VIRUS INFECTION IN M U R I N E NEUROBLASTOMA CELLS
Y u z o IWASAKI* a n d NOBUYUKI MINAMOTO'~ * National Center for Nervous, Mental and Muscular Disorders, 2620 Ogawahigashi, Kodaira, Tokyo 187, and t Department of Veterinary Public Health, Faculty of Agriculture, Gifu University, Gifu 504, Japan A~tract--A persistent infection of CVS strain of rabies virus was established in murine neuroblastoma cells, C-1300, by a serial passage of the cells infected with a low input multiplicity. Little cytopathic effects were seen in the infected cultures and the cell growth was not interfered, although 90 100°~,of the cells were bearing intracytoplasmic inclusions and the infectious virus was constantly recovered from the supernatant. Scanning electron microscopy disclosed preferential budding of the virus from fine cellular processes with a relative sparing of the cell body. Freezefracture of the infected cells revealed that the intracytoplasmic inclusion (matrix) was composed of an aggregate of fine particles with a diameter of approximately 200 A. Intramembrane particles were distributed sparsely and randomly in the viral envelope. Key words: Persistent infection, rabies virus, neuroblastoma cell, freeze-fracture, scanning electron microscopy MICROSCOPIE
ELECTRONIQUE
PAR CONGELATION RABIQUE
A BALAYAGE
D'UNE
DE CELLULES
INFECTION
ET FRACTURE
PAR LE VIRUS
DE NEUROBLASTOME
MURIN
R~ume--L'infection persistante de cellules de neuroblastome murin C-1300 par la souche CVS du virus rabique a ~te rralisee grfice '~ des passage en srrie de cellules infectees par une faible quantite de virus. I1 a et~ constat/~ peu d'effet cytopathog~ne dans les cultures infect~es et la croissance cellulaire n'a pas ~t~ modifi~e, bien que 90 100°~i des cellules aient d~veloppe des inclusions intracytoplasmiques et que les mrmes infections aient toujours bt~ retrouv~es dans le surnageant de la culture. L'examen en microscopie 61ectronique fi balayage a r~vrle que le bourgeonnement du virus se faisait de prrf~rence ~ partir des fins prolongements cellulaires, ~pargnant relativement le corps de la cellule. La fracture par cong~lation des cellules infectees a rbv~le que les inclusions intracytoplasmiques (matrice) ~taient compos~es d'un agr~gat de fines particules d'un diamrtre approximatif de 200/~. Les particules intramembranaires etaient eparpillres au hasard dans l'enveloppe virale. Mots-clefs: Infection persistante, virus rabique, cellules de neuroblastome, fracture par cong~lation, microscopie electronique ~, balayage
INTRODUCTION In the p a s t t w o d e c a d e s , a v a r i e t y o f tissue c u l t u r e cells w e r e u s e d in the s t u d y o f rabies v i r u s i n f e c t i o n . A m o n g t h o s e d i v e r s e tissue c u l t u r e systems, c e r t a i n lines o f n e u r o b l a s t o m a cells are u n i q u e in the sense t h a t the p r o p a g a t i o n o f rabies v i r u s e s in t h o s e cells r e s t o r e s the v i r u l e n c e for a n i m a l s t h a t was o n c e lost o r s u b s t a n t i a l l y r e d u c e d d u r i n g the p a s s a g e s in o t h e r tissue c u l t u r e s y s t e m s [1]. T h u s , it c a n be a s s u m e d t h a t t h o s e n e u r o b l a s t o m a cells s h a r e s o m e c o m m o n p r o p e r t i e s w i t h n e u r o n a l cells, the m a i n sites o f rabies v i r u s 1
2
Y. IWASAKIand N. MINAMOTO
replication in the central nervous system [2], with reference to the regulation of the virulence of rabies virus. A murine neuroblastoma cell line, C-1300, clone NA, is one of those cell lines with such a unique property and it could be a suitable in vitro model for the study of the cellular events in rabies virus infected neuronal cells. We have previously reported an electron microscopic study of NA cells acutely infected with rabies virus [3]. In this communication we describe scanning and freeze-fracture electron microscopic observations of NA cells persistently infected with CVS strain of rabies virus. MATERIALS AND METHODS A murine neuroblastoma cell line, C-1300, clone NA [4], was originally supplied by Dr. F. A. McMorris of the Wistar Institute and maintained in our laboratory. For the establishment of persistently infected cells, NA cells were seeded in Falcon T-25 plastic flasks at a density of 104 cells/cm 2, infected with CVS strain of rabies virus propagated in mouse brain tissue, at a multiplicity of 1 TCIDso per cell and were maintained in Eagle's minimum essential medium supplemented with 10'~0 fetal bovine serum at 37 C. The cells were split every 8 10 days at a ratio of I : 5 and, at the time of cell transfer, a small portion of cells were seeded on glass cover slips for the detection of immunofluorescent viral antigens and the cytological study. For electron microscopy, the cells were seeded on plastic cover slips (Lux Scientific Corp., U.S.A.) at the 25th cell transfer level and the cells on cover slips were fixed at 24 and 96 hr after seeding with 2.5°~, glutaraldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.3, 285 mOsM. The cells for scanning electron microscopy was post-fixed with 1'!00sO ~ in PBS, dehydrated with a graded series of acetone and then dried in a Hitachi HCP-2 critical point dryer. The specimens were coated with platinum-palladium at a thickness of 100 A and examined with a Hitachi S-700 scanning electron microscope operating at 10 or 15 kV. For freeze-fracture of the cells in monolayers on plastic cover slips, the method of Pauli et al. [5] was adapted to a freeze-etch device developed by Eiko Engineering Co. (Ibaraki, Japan). The cover slips with the cells were cut to the size of 2 x 2 mm and immersed in 25°~i glycerol in PBS for 1 hr and then a small piece of cover slip with the cell face down was mounted on a copper disk with a drop of mounting medium, containing 30'!~; polyvinyl alcohol, 30~'0 glycerol and 2°i, DMSO. The specimens were quenched in liquid nitrogen and fractured at a stage temperature of - 100 C under a vacuum of 1 x 10-7 Torr. The fracture surface was immediately shadowed with platinum-carbon. The replicas were retrieved after digestion with commercial bleaching solution. After rinsing with several changes of distilled water, the replicas were mounted on 300 mesh copper grids and examined in a Hitachi H-600 electron microscope operating at 100 kV. RESULTS AND DISCUSSION The most striking feature of the neuroblastoma cells persistently infected with rabies virus was the absence of any cytopathic effects detectable under a light microscope. Plating efficiency and growth curve of the cells were also identical in both cozqrol and infected cultures (data not shown).
Fig. 2. Control N A cells, 24 hr after splitting, x 440. Fig. 3. Rabies virus infected N A cells at 25th passage, 24 hr after splitting, x 440. Fig. 4. A high power view of the area indicated by a rectangle in Fig. 3. Note the virus budding from fine cellular processes (arrows), x 20,000.
Fig. 5. A freeze-fracture profile of a rabies virus infected NA cell. M: matrix, G: golgi apparatus, Np: nucleoplasm Ne: nuclear membrane, x 10,000. Fig. 6. A high power view of the matrix shown in Fig. 5. An arrow indicates a chain of particles, × 40,000.
Fig. 7. Two virions
(arrows)
in the vicinity
Fig. 8. Virus budding Fig. 9. A small cluster of virions showing
of a matrix
from a cell process,
P face (fine arrows)
(M),
x 150,000.
x 150,000.
and E face (bald arrows)
of the envelope,
x 120,000.
SEM and freeze-fracture study of rabies in culture
7
-/
to
IOO
t.)
+-_ Q_
50 '~ Lt-
%
g
A -CVS
I
I0
[
20
J
50
No of cell transfer
]
40
I
50
o
g_
Fig. 1. Virus yield and development of viral antigens, Closed circle: virus titre; open circle: °,i of FA positive cells.
As early as the 5th cell transfer, small eosinophilic intracytoplasmic inclusions developed in 90-100°o of the cells in infected cultures and the size and location of these inclusions well corresponded to discrete immunofluorescent granules stained with anti-rabies virus antibodies. Infectious virus was constantly recovered from the supernatant of the infected cultures as late as the 52nd cell transfer (Fig. 1). By scanning electron microscopy, the virus infected and control cultures were hardly distinguishable at low power views (Figs. 2 and 3). In both cultures, the cells with a moderately villous surface were interconnected with long cell processes. Mitotic figures were not uncommon. At a higher power view, however, virus budding from fine cellular processes was readily detectable in the virus infected cultures (Fig. 4). Virions were being released from the tips and sides of these fine processes (Fig, 4, arrows). Unlike acutely infected neuroblastoma cells with a high input multiplicity [3], the virus budding from the cell body was rarely observed in this persistently infected culture. Figure 5 is a representative freeze-fracture profile of a rabies virus infected NA cell. A homogeneous granular region (Np) is a cut surface of nucleoplasm and a small portion of nuclear membrane (Ne) is also exposed (mostly P face). A tubulovesicular structure (G) in a juxtanuclear region is a Golgi apparatus. A large inclusion, which locates the outside of the Golgi apparatus, is a matrix (M) composed of nucleocapsids of rabies virus. Figure 6 is a high power view of the matrix shown in Fig. 5. The matrix is composed of an aggregate of fine particles, approximately 200 A in diameter. The size of these particles is much larger than that of intramembrane particles, approximately 80 100 A in this preparation, and occasionally they form short chains of a single row (Fig. 6, arrow). These matrices are often seen in the cells of infected cultures but not of control cultures. On rare occasions, virions are seen in the vicinity of the matrix (Fig. 7, arrows). Figure 8 shows P and E faces (p and e) of such a fine cellular process as illustrated in Fig. 4. The diameter of this process is approximately 65 tam and a bullet-shaped tip (v) of this process could be a virus particle being released at the point indicated by arrows. Figure 9 shows a small aggregate of virus particles found in extracellular space. Fine and bald arrows indicate P and E faces of the virions, respectively. As illustrated in these plates, membrane associated particles of 80 100 A diameter distributed sparsely and randomly in the viral envelope and the density of these particles were much lower than that of intramembrane particles in the plasma membrane of the cell.
8
Y. |WASAK1and N. MINAMOTO
A relative a b u n d a n c e o f virus b u d d i n g from fine cellular processes o f the cells with grossly n o r m a l a p p e a r a n c e was impressive. A l t h o u g h scanning m i c r o s c o p y alone is n o t sufficient to identify rabies virus particles, the b u d d i n g o f rabies virus from these cellular processes has a l r e a d y been c o n f i r m e d by scanning a n d t r a n s m i s s i o n electron m i c r o s c o p i c o b s e r v a t i o n s o f whole m o u n t cells [6]. These o b s e r v a t i o n s m a y share some a n a l o g y with the cellular events o f rabies virus infection in the central nervous system where the infection often progresses in the obsence o f h i s t o p a t h o l o g i c a l changes a n d the virus b u d d i n g from the p e r i k a r y o n is f o u n d only u n d e r certain e x p e r i m e n t a l c o n d i t i o n s [3, 7, 8]. S p a r i n g o f the cell b o d y in virus b u d d i n g can be significant in the survival o f host cells since the virus b u d d i n g from the p l a s m a m e m b r a n e results in the d i s t u r b a n c e o f the m e m b r a n e structure a n d c o u l d interfere with the vital activity o f the cell m e m b r a n e as t r a n s m e m b r a n e t r a n s p o r t o f substances. Thus, the preferential release o f p r o g e n y virus from the cellular process m a y play a beneficial role in the e s t a b l i s h m e n t o f chronic persistent infection.
REFERENCES 1. Clark, H. F., Rabies virus increase in virulence when propagated in neuroblastoma cell culture, Science 199, 1072-1075 (1978). 2. Matsumoto, S., Electron microscope studies of rabies virus in mouse brain, J. Cell Biol. 19, 565-591 (1963). 3. Iwasaki, Y. and Clark, H.F., Rabies virus infection in mouse neuroblastoma cells, Lab. Invest. 36, 578-584 (1977). 4. McMorris, F. A. and Ruddle, F. H., Expression of neuronal phenotypes in neuroblastoma cell hybrids, Devl. Biol. 39, 226 231 (1974). 5. Pauli, B. U., Weinstein, R. S., Soble, L. W., Freeze-fracture of monolayer cultures, J. Cell Biol. 72, 763 769 (1977). 6. Iwasaki, Y., Application of the critical point dried whole cell technique to the study of animal rhabdoviruses, Intervirology 9, 214 225 (1978). 7. lwasaki, Y. and Clark, H. F_ Cell to transmission of virus in the central nervous system II. Experimental rabies in mouse, Lab. Invest. 33, 391--399 (1975). 8. Perl, D. P., The pathology of rabies in the central nervous system, in The Natural Histoo ~o f Rabies (Ed. Baer, G. M.), Vol. 1, pp. 235-272. Academic Press, New York (1975).
[)147-9571/82,'010001 07 $03.00!0 ~ 1982 Pergamon Press Ltd.
SCANNING AND F R E E Z E - F R A C T U R E ELECTRON MICROSCOPY OF RABIES VIRUS INFECTION IN M U R I N E NEUROBLASTOMA CELLS
Y u z o IWASAKI* a n d NOBUYUKI MINAMOTO'~ * National Center for Nervous, Mental and Muscular Disorders, 2620 Ogawahigashi, Kodaira, Tokyo 187, and t Department of Veterinary Public Health, Faculty of Agriculture, Gifu University, Gifu 504, Japan A~tract--A persistent infection of CVS strain of rabies virus was established in murine neuroblastoma cells, C-1300, by a serial passage of the cells infected with a low input multiplicity. Little cytopathic effects were seen in the infected cultures and the cell growth was not interfered, although 90 100°~,of the cells were bearing intracytoplasmic inclusions and the infectious virus was constantly recovered from the supernatant. Scanning electron microscopy disclosed preferential budding of the virus from fine cellular processes with a relative sparing of the cell body. Freezefracture of the infected cells revealed that the intracytoplasmic inclusion (matrix) was composed of an aggregate of fine particles with a diameter of approximately 200 A. Intramembrane particles were distributed sparsely and randomly in the viral envelope. Key words: Persistent infection, rabies virus, neuroblastoma cell, freeze-fracture, scanning electron microscopy MICROSCOPIE
ELECTRONIQUE
PAR CONGELATION RABIQUE
A BALAYAGE
D'UNE
DE CELLULES
INFECTION
ET FRACTURE
PAR LE VIRUS
DE NEUROBLASTOME
MURIN
R~ume--L'infection persistante de cellules de neuroblastome murin C-1300 par la souche CVS du virus rabique a ~te rralisee grfice '~ des passage en srrie de cellules infectees par une faible quantite de virus. I1 a et~ constat/~ peu d'effet cytopathog~ne dans les cultures infect~es et la croissance cellulaire n'a pas ~t~ modifi~e, bien que 90 100°~i des cellules aient d~veloppe des inclusions intracytoplasmiques et que les mrmes infections aient toujours bt~ retrouv~es dans le surnageant de la culture. L'examen en microscopie 61ectronique fi balayage a r~vrle que le bourgeonnement du virus se faisait de prrf~rence ~ partir des fins prolongements cellulaires, ~pargnant relativement le corps de la cellule. La fracture par cong~lation des cellules infectees a rbv~le que les inclusions intracytoplasmiques (matrice) ~taient compos~es d'un agr~gat de fines particules d'un diamrtre approximatif de 200/~. Les particules intramembranaires etaient eparpillres au hasard dans l'enveloppe virale. Mots-clefs: Infection persistante, virus rabique, cellules de neuroblastome, fracture par cong~lation, microscopie electronique ~, balayage
INTRODUCTION In the p a s t t w o d e c a d e s , a v a r i e t y o f tissue c u l t u r e cells w e r e u s e d in the s t u d y o f rabies v i r u s i n f e c t i o n . A m o n g t h o s e d i v e r s e tissue c u l t u r e systems, c e r t a i n lines o f n e u r o b l a s t o m a cells are u n i q u e in the sense t h a t the p r o p a g a t i o n o f rabies v i r u s e s in t h o s e cells r e s t o r e s the v i r u l e n c e for a n i m a l s t h a t was o n c e lost o r s u b s t a n t i a l l y r e d u c e d d u r i n g the p a s s a g e s in o t h e r tissue c u l t u r e s y s t e m s [1]. T h u s , it c a n be a s s u m e d t h a t t h o s e n e u r o b l a s t o m a cells s h a r e s o m e c o m m o n p r o p e r t i e s w i t h n e u r o n a l cells, the m a i n sites o f rabies v i r u s 1
2
Y. IWASAKIand N. MINAMOTO
replication in the central nervous system [2], with reference to the regulation of the virulence of rabies virus. A murine neuroblastoma cell line, C-1300, clone NA, is one of those cell lines with such a unique property and it could be a suitable in vitro model for the study of the cellular events in rabies virus infected neuronal cells. We have previously reported an electron microscopic study of NA cells acutely infected with rabies virus [3]. In this communication we describe scanning and freeze-fracture electron microscopic observations of NA cells persistently infected with CVS strain of rabies virus. MATERIALS AND METHODS A murine neuroblastoma cell line, C-1300, clone NA [4], was originally supplied by Dr. F. A. McMorris of the Wistar Institute and maintained in our laboratory. For the establishment of persistently infected cells, NA cells were seeded in Falcon T-25 plastic flasks at a density of 104 cells/cm 2, infected with CVS strain of rabies virus propagated in mouse brain tissue, at a multiplicity of 1 TCIDso per cell and were maintained in Eagle's minimum essential medium supplemented with 10'~0 fetal bovine serum at 37 C. The cells were split every 8 10 days at a ratio of I : 5 and, at the time of cell transfer, a small portion of cells were seeded on glass cover slips for the detection of immunofluorescent viral antigens and the cytological study. For electron microscopy, the cells were seeded on plastic cover slips (Lux Scientific Corp., U.S.A.) at the 25th cell transfer level and the cells on cover slips were fixed at 24 and 96 hr after seeding with 2.5°~, glutaraldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.3, 285 mOsM. The cells for scanning electron microscopy was post-fixed with 1'!00sO ~ in PBS, dehydrated with a graded series of acetone and then dried in a Hitachi HCP-2 critical point dryer. The specimens were coated with platinum-palladium at a thickness of 100 A and examined with a Hitachi S-700 scanning electron microscope operating at 10 or 15 kV. For freeze-fracture of the cells in monolayers on plastic cover slips, the method of Pauli et al. [5] was adapted to a freeze-etch device developed by Eiko Engineering Co. (Ibaraki, Japan). The cover slips with the cells were cut to the size of 2 x 2 mm and immersed in 25°~i glycerol in PBS for 1 hr and then a small piece of cover slip with the cell face down was mounted on a copper disk with a drop of mounting medium, containing 30'!~; polyvinyl alcohol, 30~'0 glycerol and 2°i, DMSO. The specimens were quenched in liquid nitrogen and fractured at a stage temperature of - 100 C under a vacuum of 1 x 10-7 Torr. The fracture surface was immediately shadowed with platinum-carbon. The replicas were retrieved after digestion with commercial bleaching solution. After rinsing with several changes of distilled water, the replicas were mounted on 300 mesh copper grids and examined in a Hitachi H-600 electron microscope operating at 100 kV. RESULTS AND DISCUSSION The most striking feature of the neuroblastoma cells persistently infected with rabies virus was the absence of any cytopathic effects detectable under a light microscope. Plating efficiency and growth curve of the cells were also identical in both cozqrol and infected cultures (data not shown).
Fig. 2. Control N A cells, 24 hr after splitting, x 440. Fig. 3. Rabies virus infected N A cells at 25th passage, 24 hr after splitting, x 440. Fig. 4. A high power view of the area indicated by a rectangle in Fig. 3. Note the virus budding from fine cellular processes (arrows), x 20,000.
Fig. 5. A freeze-fracture profile of a rabies virus infected NA cell. M: matrix, G: golgi apparatus, Np: nucleoplasm Ne: nuclear membrane, x 10,000. Fig. 6. A high power view of the matrix shown in Fig. 5. An arrow indicates a chain of particles, × 40,000.
Fig. 7. Two virions
(arrows)
in the vicinity
Fig. 8. Virus budding Fig. 9. A small cluster of virions showing
of a matrix
from a cell process,
P face (fine arrows)
(M),
x 150,000.
x 150,000.
and E face (bald arrows)
of the envelope,
x 120,000.
SEM and freeze-fracture study of rabies in culture
7
-/
to
IOO
t.)
+-_ Q_
50 '~ Lt-
%
g
A -CVS
I
I0
[
20
J
50
No of cell transfer
]
40
I
50
o
g_
Fig. 1. Virus yield and development of viral antigens, Closed circle: virus titre; open circle: °,i of FA positive cells.
As early as the 5th cell transfer, small eosinophilic intracytoplasmic inclusions developed in 90-100°o of the cells in infected cultures and the size and location of these inclusions well corresponded to discrete immunofluorescent granules stained with anti-rabies virus antibodies. Infectious virus was constantly recovered from the supernatant of the infected cultures as late as the 52nd cell transfer (Fig. 1). By scanning electron microscopy, the virus infected and control cultures were hardly distinguishable at low power views (Figs. 2 and 3). In both cultures, the cells with a moderately villous surface were interconnected with long cell processes. Mitotic figures were not uncommon. At a higher power view, however, virus budding from fine cellular processes was readily detectable in the virus infected cultures (Fig. 4). Virions were being released from the tips and sides of these fine processes (Fig, 4, arrows). Unlike acutely infected neuroblastoma cells with a high input multiplicity [3], the virus budding from the cell body was rarely observed in this persistently infected culture. Figure 5 is a representative freeze-fracture profile of a rabies virus infected NA cell. A homogeneous granular region (Np) is a cut surface of nucleoplasm and a small portion of nuclear membrane (Ne) is also exposed (mostly P face). A tubulovesicular structure (G) in a juxtanuclear region is a Golgi apparatus. A large inclusion, which locates the outside of the Golgi apparatus, is a matrix (M) composed of nucleocapsids of rabies virus. Figure 6 is a high power view of the matrix shown in Fig. 5. The matrix is composed of an aggregate of fine particles, approximately 200 A in diameter. The size of these particles is much larger than that of intramembrane particles, approximately 80 100 A in this preparation, and occasionally they form short chains of a single row (Fig. 6, arrow). These matrices are often seen in the cells of infected cultures but not of control cultures. On rare occasions, virions are seen in the vicinity of the matrix (Fig. 7, arrows). Figure 8 shows P and E faces (p and e) of such a fine cellular process as illustrated in Fig. 4. The diameter of this process is approximately 65 tam and a bullet-shaped tip (v) of this process could be a virus particle being released at the point indicated by arrows. Figure 9 shows a small aggregate of virus particles found in extracellular space. Fine and bald arrows indicate P and E faces of the virions, respectively. As illustrated in these plates, membrane associated particles of 80 100 A diameter distributed sparsely and randomly in the viral envelope and the density of these particles were much lower than that of intramembrane particles in the plasma membrane of the cell.
8
Y. |WASAK1and N. MINAMOTO
A relative a b u n d a n c e o f virus b u d d i n g from fine cellular processes o f the cells with grossly n o r m a l a p p e a r a n c e was impressive. A l t h o u g h scanning m i c r o s c o p y alone is n o t sufficient to identify rabies virus particles, the b u d d i n g o f rabies virus from these cellular processes has a l r e a d y been c o n f i r m e d by scanning a n d t r a n s m i s s i o n electron m i c r o s c o p i c o b s e r v a t i o n s o f whole m o u n t cells [6]. These o b s e r v a t i o n s m a y share some a n a l o g y with the cellular events o f rabies virus infection in the central nervous system where the infection often progresses in the obsence o f h i s t o p a t h o l o g i c a l changes a n d the virus b u d d i n g from the p e r i k a r y o n is f o u n d only u n d e r certain e x p e r i m e n t a l c o n d i t i o n s [3, 7, 8]. S p a r i n g o f the cell b o d y in virus b u d d i n g can be significant in the survival o f host cells since the virus b u d d i n g from the p l a s m a m e m b r a n e results in the d i s t u r b a n c e o f the m e m b r a n e structure a n d c o u l d interfere with the vital activity o f the cell m e m b r a n e as t r a n s m e m b r a n e t r a n s p o r t o f substances. Thus, the preferential release o f p r o g e n y virus from the cellular process m a y play a beneficial role in the e s t a b l i s h m e n t o f chronic persistent infection.
REFERENCES 1. Clark, H. F., Rabies virus increase in virulence when propagated in neuroblastoma cell culture, Science 199, 1072-1075 (1978). 2. Matsumoto, S., Electron microscope studies of rabies virus in mouse brain, J. Cell Biol. 19, 565-591 (1963). 3. Iwasaki, Y. and Clark, H.F., Rabies virus infection in mouse neuroblastoma cells, Lab. Invest. 36, 578-584 (1977). 4. McMorris, F. A. and Ruddle, F. H., Expression of neuronal phenotypes in neuroblastoma cell hybrids, Devl. Biol. 39, 226 231 (1974). 5. Pauli, B. U., Weinstein, R. S., Soble, L. W., Freeze-fracture of monolayer cultures, J. Cell Biol. 72, 763 769 (1977). 6. Iwasaki, Y., Application of the critical point dried whole cell technique to the study of animal rhabdoviruses, Intervirology 9, 214 225 (1978). 7. lwasaki, Y. and Clark, H. F_ Cell to transmission of virus in the central nervous system II. Experimental rabies in mouse, Lab. Invest. 33, 391--399 (1975). 8. Perl, D. P., The pathology of rabies in the central nervous system, in The Natural Histoo ~o f Rabies (Ed. Baer, G. M.), Vol. 1, pp. 235-272. Academic Press, New York (1975).