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Development of semliki forest virus in mouse brain: An electron microscopic study
ExPERmENTAL.
AND
moLEmlLAR
Development
PATHOLOGY
12, 1-13 (1970)
of Semliki Forest Virus in Mouse Brain: An Electron
Microscopic
Study
PHILIP M. GRIMLEY AND ROBERT M. FRIEDMAN Pathologic
Anatomy
Bmnch
and Lubomtory Bethesda, Received
of Pathology,
Maryland June
National
Cancer
Institute,
20014 25, 1969
Electron microscopic studies have suggested characteristic differences in the morphogenesis of several arbovirus subgroups (Murphy et al., 1968). In cells infected by the Group A arboviruses, cytoplasmic accumulation of “precursor particles” or virus cores is a commonly reported feature (Morgan et al., 1961; Mussgay and Weibel, 1962; Chain et al., 1966; Grimley et al., 1968). The signiflcance of core particles, their relationship to the arbovirus growth cycle, and the major pathways of virion maturation have been variously interpreted, Some investigators have emphasized the formation of virions within cytoplasmic vacuoles and explained the release of infectious particles by exocytosis or cytolysis (Morgan et al., 1961; Erlandson et al., 1967). Others have proposed that budding of virus cores through the plasmalemma provides the main route for extracellular virion formation (Mussgay and Weibel, 1962; Acheson and Tamm, 1967; Higashi et al., 1967). While it is generally assumed that group A arboviruses acquire their outer lipoprotein envelope from host cell membrane elements (Pfefferkorn and Hunter, 1963; Friedman and Pa&m, 19691, a recent electron microscopic study of Semliki Forest virus (SFVl in mouse neurons led to the novel suggestion that virions might mature. without direct passage through cell membranes (McGee-Russell and Gosztonyi, 1967). In comparative studies of cell cultures infected by group A arboviruses, we observed the formation of two major types of cytopathic vacuoles (CPV) and related these to phases of virus growth (Grimley et al., 1968; Grimley and Friedman, 1969). Early and late forms of cytopathic vacuoles (CPV-I and CPVII) were observed in both Sindbis and SFV infections of chick or mouse cells (Grimley and Friedman, 1969). The present ultrastructural investigation was undertaken in order to re-evaluate the morphogenesis of SFV in brain cells and to compare the development of cytopathic vacuoles with events in vitro. The results provide new evidence of a general similarity in the sequence of arbovirus maturation. Transitional steps in the envelopment of intracellular virions are elucidated. MATERIALS AND METHODS Experimental infection. Suckling BALB/c mice were injected intracerebrally with 30-100 pfu of Semliki Forest virus which had been passaged in primary chick embryo cultures. Virus pools were prepared as described previously 1 Copyright
0 1970 by Academic
Pteas, Inc.
2
GRIMLEY
AND
FRIEDMAN
(Grimley et al., 1968; Friedman and Pastan, 1969). The injection vehicle was a balanced tissue culture medium (#1640 RPMI, Grand Island Biologicals, Grand Island, N.Y.) with 10% fetal calf serum added. Approximately 0.05-0.1 cc. of fluid with virus was injected. In two sets of experiments, litters of mice were injected 24 or 48 hours after birth and sacrificed when moribund (approximately 40 hours). In another set of experiments, parallel litters were injected at 8-hour intervals, and mice were sacrificed at various times after the death of some mice in the first injected litter. This procedure provided a spectrum of infections from 32-48 hours. Virus assay. Animals anesthetized with ether were sacrificed by decapitation at 6-48 hours after virus infection. Whole blood was collected in volumetric tubes and immediately frozen to -7OOC. Brains were weighed and frozen whole. After thawing, brain samples were ground with sterile sand, centrifuged at lOOOg, and prepared as a 10% suspension in the tissue culture medium with 10% serum. Beginning with 0.2 cc. of suspension, serial dilutions were prepared in the same medium. Aliquots of blood serum were similarly diluted. Plaque assay was performed on chick embryo cell monolayers as described by Friedman and Sonnabend (1965). Electron microscopy. Brains were fixed for electron microscopy by perfusion of 4% glutaraldehyde (Chambers et al., 1968) through the left ventricle of the heart or by direct immersion and slicing of the rapidly dissected brain. Thin slices from representative regions of brain were embedded in paraffin and stained with hematoxylin and eosin (Chambers et al., 1968). Small blocks of cortex or cerebellum were post-fixed with 1% osmium tetroxide and embedded for electron microscopy in Luft’s epon formula according to reported methods (Grimley et al., 1968). Thin sections were prepared with an automatic ultratome, stained with 10% uranyl acetate in methanol and with lead citrate, coated with carbon and examined at original magnifications of 9,000x to 37,000~ in an Hitachi-Perkin Elmer HU-11E microscope. RESULTS
Light microscopic examination of representative sections from infected suckling brains disclosed scant evidence of virus effect. Inflammatory reaction was absent or minimal and a few zones of eosinophilic necrosis or cellular edema were scattered in various regions, primarily the cortex as described by McGeeRussell and Gosztonyi (1967). Cell inclusion bodies were not observed. The virus growth curve is illustrated in Fig. 1. Virus titers in the brain suspensions increased progressively until 40 hours after innoculation and began to plateau shortly before death of the animals. Peak virus production at 48 hours was l,OOO,OOO-fold greater than infectivity at 8 hours, but represented only a lOO-fold increase over that detected at 24 hours. Viremia developed after 12 hours of infection. Rise in blood virus titers paralleled the growth of virus in the brain up to 32 hours, but the maximum blood titer was only 1% of maximum brain titers. In brains examined by electron microscopy at 32-36 hours after SFV injection, search for infected cells in selected samples was generally unrewarding. At
SEMLIKI
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8
FOREST
16 HOURS
24 AFTER
3
VIRUS
32
40
48
INFECTION
FIG. 1. Growth curve for Semliki Forest Virus (SFV) in suckling mouse brain and blood. Each point represents pooled brain suspension or whole blood from 2 or more animals.
36-38 hours the first signs of virus development could occasionally be detected. Mature 50 rnp diameter virions were clustered in the interstitial spaces, often in long 6les between apposed glial and neuronal bodies or processes (Fig. 2). Thorough examination of several brain samples taken at 36-40 hours after initiation of infection disclosed a number of vacuoles (Figs. 2, 3) resembling the CPV-I previously described in SFV infected tissue cultures (Grimley et al., 1968). These cytopathic vacuoles often occurred in planes of section where CPV-II and virus core accumulations were absent or minimal. The CPV-I consisted of multiple membrane vesicles averaging 65-70 rnF in external diameter and arranged in circular profiles. Each vesicle contained an irregular central density (Fig. 3). The limiting membrane at the periphery of neuronal CPV-I was less distinct than that observed in tissue culture cells, and the component vesicles were larger. Some CPV-I appeared to be disrupted or degenerated and contained intermingled dense particles resembling virus cores. Multiple CPV-I were only rarely detected in a single plane of section (Fig. 2). Budding of mature virions from the plasmalemma of neural processes was apparent in focal regions (Fig. 4). The neural processes could be identified by the presence of abundant, aligned neurotubules, clusters of synaptic vesicles, or synaptolemmal thickening at regions of synaptic junction. Unattached 28-30 rnp diameter virus cores could occasionally be identified in the axoplasm. These cores had a distinctive “knobby” surface at high magnification. (Faulkner and McGee-Russell, 1968). Budding of virus cores into channels of the endoplasmic reticulum was not infrequent and in rare planes of section simultaneous budding of serially aligned virus cores was observed (Fig. 5). Some of the channels evidently derived from
4
GRIMLEY
AND FRIEDMAN
FIG. 2. Periphery of neuron in region of early infection. Mature virions are sandwiched in the interstitial space between cell membranes. Cytoplasmic vacuoles of O.5-O.7-r diameter contain circumferentially arranged vesicles with an irregular central density. X40,000. FIG. 3. Structure similar to type I cytopathic vacuole (CPV-I) in perinuclear cytoplasm. Lining vesicles of spherical form average 65 rnti diameter and contain irregular central densities. ~47,500.
SEMLIKI
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VIRUS
5
FIG. 4. Evidence of mature virus budding from neuronal processes in mouse brain. (A) Longitudinal view of neuraxon with aligned neurotubules. ~60,000. (B) Cross sections of cell processes including neuraxon with neurotubules (arrow). X60,000. (C) Multiple virus buds and accumulation of virions in interstitial space. X47,500. (D) Cell process with budding virion and cytoplasmic &NCture bearing virus cores (arrow). ~60,000.
6
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cistenae of the Golgi complex (Fig. 6) while others may have originated as lamellae of granular endoplasmic reticulum which had lost their ribosomal complement. Some micrographs suggested progressive detachment of ribosomes and replacement by virus cores (Fig. 7). One micrograph demonstrated budding of a virus core through clearly identifiable granular endoplasmic reticulum (Fig. 8). Budding of virus intracellularly was accompanied by a demonstrable thickening of the overlying cytomembrane which was destined to form the virus envelope (Fig. 9). By 40 hours after injection of SFV, large numbers of neurons were infected, and most samples examined by electron microscopy exhibited advanced intracellular phases of virus development. Virus cores accumulated as infection progressed and surrounded vacuolar membranes classified as CPV-II (Fig. 7, 9, 10). Some of the vacuoles contained long straight tubular invaginations which measured up to 50 ml in external diameter. The latter possessed a 22 rnr lucent core and resembled forms described in tissue culture by Erlandson, et al. (1967). On cross sections (Fig. ll), the invaginated tubules and vacuolar membrane surrounded by virus cores resembled a rosette, similar to the “assembly areas” emphasized by McGee-Russell and Gosztonyi (1967). Another type of membrane structure which often accompanied advanced intracellular virus infection is illustrated in Figs. 7 and 10. This consisted of an array of intermeshed tubular membranes averaging 25-30 rnw in diameter.The tubules appeared angulated and in some planes of section were organized in orderly patterns. This structure may be described as a “microtubular reticulum.” It was characteristically associated with converging cisternae of the endoplasmic reticulum but was not surrounded by virus cores. Similar structures have been described in Group B arbovirus infection (Murphy et al., 1968) and lymphoma cells (Chandra, 1968). Coated tubules in crystalloid array were frequently present in the cytoplasm of infected cells (Fig. 121, occasionally in close proximity to regions of microtubular reticulum. The coated tubules averaged 30 rnp in outer diameter and demonstrated an 80-90-A electron lucent core in cross section. DISCUSSION
Biochemical and immunologic studies of arbovirus replication are most conveniently and safely performed with cell cultures, but the significance of in vitro experiments might be uncertain if the modes of viral morphogenesis in vitro and in uiuo differed to the extent previously postulated by McGee-Russell and Gosztonyi (1967). Present observations offer new evidence of ultrastructural similarities of SFV development in mouse neurons and mammalian or avian cell cultures. Two morphologic pathways of virion maturation have been defined: surface budding virions which are easily detected in the early phases of infection in vitro were noted in regions of brain where ultrastructural evidence of intracellular virus maturation was minimal. Accumulation of intracytoplasmic virus cores and virions within cellular vacuoles marked the advanced stages of neuronal infection and also paralleled events in vitro. In contrast to some previous tissue culture studies of Group A arboviruses (Mussgay and Weibel, 1962;
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5. Sites of virus budding within cytoplasmic membrane channels. Note presence of single cores which are apparently free in the hyaloplasm (arrow). X 60,00& FIG. 6. Budding of virus into reticular channels of Golgi region. Some mature virions reside within Golgi cistemae. X32,000. FIG.
dense
ViNS
FIG. 7. Field demonstrating cytopathic changes in advanced infection. Structure near center (B resembles degenerating CPV-I. Channels of endoplasmic reticulum (ER) appear to acquire an outer layer of 28-30 mp virus cores. Formation of straight tubular imaginations singly or in groups (arrows) appears to be a secondary modification of the core associated vacuoles (CPV-II). Note re. gion of angulated tubules within dilated channel of endoplasmic reticulum (lower left). x40,ooO. Fm. 8. Budding of virus core into dilated chmel of granular endoplasmic reticulum. Membrane in this region appears thickened by a fuzzy electron-dense coat. ~47,500.
8
SEMLIKI
FIG. 9. Type II cytopathic vacuoles (CPV-II) membranous channel (arrow) is accompanied body within several of the cores. X112,000.
FOREST
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with outer rim of virus cores. by local membrane thickening.
Budding of core into Note dense central
Acheson and Tamm, 1967), transitional stages in the intracellular budding of virus particles could be clearly identified in mouse neurons, and the juxtaposed cytomembranes appeared locally modified at the budding sites (Figs. 8, 9). Correlation of the findings in brain and tissue cultures suggests that some apparent discrepancies in the arbovirus maturation sequence may have been due to differences in the stage of infection at the time of sampling for electron microscopy. Reports emphasizing the surface budding of Group .A arboviruses have often focused attention upon samples prepared during the rapid growth phase (Mussgay and Weibel, 1962; Chain et al., 1966; Ach’eson and Tamm 1967), while reports emphasizing intracellular virion development have been based upon the examination of relatively late samples (Morgan et al., 1961; Eklandson et al., 1967; Higashi et al., 1967). It is nevertheless possible that the host cell environment may preferentially modify the pathways of virus maturation or limit appropriate budding sites. Such an effect has not been clearly demonstrated in systems thus far reported (Grimley and Friedman, 1969). Identification of CPV-I in SFV-infected brain was of particular interest. These structures form during the logarithmic phase of tissue culture infections with SFV and Sindbis virus (Grimley et al., 1966; Grimley and Friedman, 1969) and
FIG. tubular
10. Accumulation of mature virus within cytopathic vacuoles (arrows). Zone of microreticulum at left. X 23,403. FIG. 11. High magnification of 50-ma tubular structures inside of vacuoles with virus cores. In cross section, these complexes appear as rosettes, and secondary thickening of the membranes is apparent. Note the channels of granular endoplasmic reticulum and virus cores in close proximity. X~,~. 10
SEMLIKI
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11
FIG. 12. Closely packed aggregate of coated tubules observed in advanced infection. ~57,ooO.
have been associated with replication of early viral RNA (Friedman and Berezesky, 1967; Grimley et al., 1966). In neurons they also appear to precede CPV-II. Yasuzumi and Tsubo (1965) described abundant “glohoid bodies” in Japanese B encephalitis infections of mouse brain, and some of the micrographs published by Murphy et al. (1968), particularly Fig. 5 and 15, suggest that round “membranous vesicles” described by them may represent analogous structures in St. Louis encephalitis infection. Tissue culture studies have shown that CPV-I may degenerate as infection progresses, so that their detection depends upon appropriate chronologic sampling. This is obviously difficult in asynchronous central nervous system infections (McGee-Russell and Gosztonyi, 1967) and further studies of early stages in tissue culture arbovirus infections appear necessary to ascertain the incidence of CPV-I. Accumulation of noninfectious virus cores around cytopathic vacuoles designated CPV-II appears to be a characteristic in late Group A arbovirus infections (Rabin and Jenson, 1967; Murphy et al., 1966). We conclude that many or all of these vacuoles derive from the granular endoplasmic reticulum. Evidence for this is circumstantial, since direct connections of CPV-II to the endoplasmic reticulum are difficult to trace, even in serial sections (Morgan et al., 1961). Nevertheless, occasional micrographs have suggested transitions of endoplasmic reticulum to CPV-II (Chain et al., 1966; Grimley et al., 1966), and the synthesis of SFV core proteins is associated with membrane bound ribosomes (Fried-
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GRIMLEY
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FRIEDMAN
man and Berezesky, 1967; Friedman, 1968). Secondary modification of CPV-II membranes appears to occur in late infection and may be preparative to intravacuolar budding of the virus cores (Erlandson et al., 1967; Higashi et al., 1967). The significance of straight tubular structures within CPV-II is uncertain, but they are a relatively late phenomenon and may represent formation of aberrant virus or excessive viral lipoproteins (Higashi et al., 1967), rather than a significant pathway of virus production (McGee-Russell and Gosztonyi, 1967). Recent studies of cycloheximide treated cells have shown that CPV-II fail to form when viral protein synthesis is inhibited after several hours of infection, but that surface-budding of virus continues in the absence of protein synthesis for up to 2 hours (Friedman and Grimley, 1969). These results indicate the existence of a large reservoir of completed virus cores after 6 hours of infection. Present observations of virus budding from neural processes provide a dramatic demonstration of virus core migration in long cell processes distal to the protein synthesizing and assembly sites. A tendency of virus to bud from pseudopodia has been noted in tissue culture (Acheson and Tamm, 1967; Chain et al., 1966), and the findings suggest passive transport of virus cores in hyaloplasmic or axoplasmic streams (Lubinska, 1964). The latter could be important in local or distant dissemination of the neurotropic arboviruses (Sabin and Olitsky, 1937). Differences in the morphogenesis of arbovirus subgroups have been considered by Murphy et al. (1968) and can now be more clearly defined: the major ultrastructural distinction in Group A infections is accumulation of excessive virus cores in the late stages of growth. The occurrence of CPV-I in various arbovirus subgroups remains to be fully evaluated. Intracellular virus budding is apparent in Group A, Group B, Bunyamwera, and California group arboviruses, but surface budding virus has been described primarily in Group A infections. Microtubular reticulum is a host cell response to several types of virus infection, including Groups A and B arboviruses. SUMMARY Electron microscopic examination of mouse brains infected with Semliki Forest virus (SFV) disclosed two major pathways of virion maturation not previously demonstrated in neurons: (1) budding of 28-30 mp virus cores through the cell surface (plasmalemma); (2) budding of virus cores into cytoplasmic vacuoles and cistemae of the Golgi membrane complex or endoplasmic reticulum. In regions of advanced neuronal infection, virus cores accumulated in large numbers and attached to the limiting membranes of type II cytopathic vacuoles (CPV-II), some of which appeared to derive from the endoplasmic reticulum. Transmigration of virus cores from cytomembrane assembly areas to sites of envelopment at the plasmalemma was evidenced by the presence of virus cores and surface-budding virions in neuronal processes. Axoplasmic transport of virus cores may thus be a factor in spread of arbovirus infections. Intracytoplasmic vacuoles enclosing membranous vesicles of tX-70-mp diameter were identified in regions of early infection; they corresponded to the type I cytopathic vacuoles (CPV-I) recently observed in tissue cultures and associated with viral RNA replication. Unusual cytomembrane proliferations, described as a “microtubular reticulum” developed regularly in the advanced stages of intracellular SFV infection and aggregates of 30 ma straight tubules with an electron-dense matrix occasionally accumulated in close proximity. The ultrastructural findings in SFV-infected neurons correlated closely with electron microscopic and biochemical studies of mesenchymal cell cultures, and indicate broad similarities in the maturation sequence of Group A arbovimses. ACKNOWLEDGMENTS We thank
Mrs.
Dorothy
Hughes
and
Mr.
Herman
Michelitch
for valuable
technical
assistance.
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REFERENCES N. H., and TAMM, I. (1967). Replication of Semliki Forest Virus: An electron microscopic study. Virology 32, 126143. CHAIN, M. M. T., DOANE, F. W., and McLE~, D. M. (1966). Morphological development of Chikungunya virus. Can. J. Microbial. 12, 895-900. CHAMBERS, R. W., BOWLMG, M. C., and GRIMLEY, P. M. (1966). Glutaraldehyde fixation in routine hi&pathology. Arch. Pathol. 85,16-30. CHANDRA, S. (1966). Undulating tubules associated with endoplasmic reticulum in pathologic tissues. Lab. Invest. 18, 422-428. ERLANDSON,R. A., BABCOCK, V. I., SOUTHAM, C. M., BAILEY, R. B., and SHIPKEY, F. H. (1967). Semliki Forest Virus in Hep-2 cell cultures. J. Viral. 1, 966-1969. FAULKNER, P., and MCGEE-RUSSELL, S. M. (1966). Purification and structure of Semliki Forest Virus isolated from mouse brain. Can. J. Microbial. 14, 153-160. FRIEDMAN, R. M. (1966). Protein synthesis directed by an arbovirus. J. Viral. 2, 26-32. FRIEDMAN, R. M., and BEREZESKY,I. K. (1967). Cytoplasmic fractions associated with Semliki Forest Virus ribonucleic acid replication. J. Viral. 1, 374-383. FRIEDMAN, R. M., and GRIMLEY, P. M. (1969). Inhibition of arbovirus assembly by cycloheximide. ACHESON,
J. Viral.
(Kyoto).
4, 292-299.
FRIEDMAN, R. M., and PASTAN, I. (1969). an arbovirus. J. Mol. Biol. 40, 107-115. FRIEDMAN, R. M., and SONNABEND, J. A. J. Immunol.
Nature and function of the structural phospholipids of (1965).
Inhibition
of interferon action by puromycin.
95, 696-703.
GRIMLEY, P. M., BEREZESKY,I. K., and FRIEDMAN,R. M. (1966). Cytoplasmic structures associated with an arbovirus infection: Loci of viral ribonucleic acid synthesis. J. Viral. 2, 1326-1338. GRIMLEY, P. M., and FRIEDMAN, R. M. (1969). Development of cytopathic vacuoles in cells infected by Group A arboviruses. Bact. Proc. 185(V231). HIGASHI, N., MATSUMOTO, A., TABATA, K., and NAGATOMO, Y. (1967). Electron microscope study of development of Chikungunya virus in green monkey kidney stable (Vera) cells. Virology 3.3, 55-69. LUBINSKA, L. (1964). Axoplasmic streaming in regenerating and in normal nerve fibers. In “Mechanisms of Neural Regeneration Progress in Brain Research” (M. Singer and J. P. Schade, eds.), Vol. 13, pp. 1-216. Elsevier, New York. MCGEE-RUSSELL, S. M., and GOSZTONYI,G. G. (1967). Assembly of Semliki Forest Virus in brain. Nature
214, 1204-1206.
MORGAN, C., HOWE, C., and ROSE, H. M. (1961). Structure and development of viruses as observed in the electron microscope V. Western Equine encephalomyelitis virus. J. Erptl. Med. 113, 219-234.
MURPHY, F. A., HARRISON,A. K., and T~IANABOS, T. (1966). Electron microscopic observations of mouse brain infected with Bunyamwera group arboviruses. J. Viral. 2, 1315-1325. MURPHY, F. A., HARRISON,A. K., GARY, G. W. JR., WHITFIELD, M. S.. and FORRESTER,F. T. (1966). St. Louis encephalitis virus infection of mice. Electron microscopic studies of central nervous system. Lab. Invest. 19, 652-662. MUSSGAY, M. and WEIBEL, J. (1962). Electron microscopic and biological studies on the growth of Venezuelan Equine encephalitis virus in KB cells. Virology 16, 52-62. PFEFFERKORN,E. R., and HUNTER, H. S. (1963). The source of the ribonucleic acid and phospholipid of Sindbis virus. Virology 20, 446-456. RABIN, E. R., and JENSON,A. B. (1967). Electron microscopic studies of animal viruses with emphasis on in vivo infections. hogr. Med. Viral. 9,392-450. SABIN, A. B., and OLIT~KY, P. K. (1937). Influence of host factors on the neuroinvasiveness of Vesicular Stomatitis virus. J. Exptl. Med. 66, 35-57. YASUZUMI, G., and TSUBO, I. (1965). Analysis of the development of Japanese B encephalitis virus II. Electron microscope studies of neurons infected with Japanese B encephalitis virus. J. Ultrastruct.
Res. 12. 304-316.
AND
moLEmlLAR
Development
PATHOLOGY
12, 1-13 (1970)
of Semliki Forest Virus in Mouse Brain: An Electron
Microscopic
Study
PHILIP M. GRIMLEY AND ROBERT M. FRIEDMAN Pathologic
Anatomy
Bmnch
and Lubomtory Bethesda, Received
of Pathology,
Maryland June
National
Cancer
Institute,
20014 25, 1969
Electron microscopic studies have suggested characteristic differences in the morphogenesis of several arbovirus subgroups (Murphy et al., 1968). In cells infected by the Group A arboviruses, cytoplasmic accumulation of “precursor particles” or virus cores is a commonly reported feature (Morgan et al., 1961; Mussgay and Weibel, 1962; Chain et al., 1966; Grimley et al., 1968). The signiflcance of core particles, their relationship to the arbovirus growth cycle, and the major pathways of virion maturation have been variously interpreted, Some investigators have emphasized the formation of virions within cytoplasmic vacuoles and explained the release of infectious particles by exocytosis or cytolysis (Morgan et al., 1961; Erlandson et al., 1967). Others have proposed that budding of virus cores through the plasmalemma provides the main route for extracellular virion formation (Mussgay and Weibel, 1962; Acheson and Tamm, 1967; Higashi et al., 1967). While it is generally assumed that group A arboviruses acquire their outer lipoprotein envelope from host cell membrane elements (Pfefferkorn and Hunter, 1963; Friedman and Pa&m, 19691, a recent electron microscopic study of Semliki Forest virus (SFVl in mouse neurons led to the novel suggestion that virions might mature. without direct passage through cell membranes (McGee-Russell and Gosztonyi, 1967). In comparative studies of cell cultures infected by group A arboviruses, we observed the formation of two major types of cytopathic vacuoles (CPV) and related these to phases of virus growth (Grimley et al., 1968; Grimley and Friedman, 1969). Early and late forms of cytopathic vacuoles (CPV-I and CPVII) were observed in both Sindbis and SFV infections of chick or mouse cells (Grimley and Friedman, 1969). The present ultrastructural investigation was undertaken in order to re-evaluate the morphogenesis of SFV in brain cells and to compare the development of cytopathic vacuoles with events in vitro. The results provide new evidence of a general similarity in the sequence of arbovirus maturation. Transitional steps in the envelopment of intracellular virions are elucidated. MATERIALS AND METHODS Experimental infection. Suckling BALB/c mice were injected intracerebrally with 30-100 pfu of Semliki Forest virus which had been passaged in primary chick embryo cultures. Virus pools were prepared as described previously 1 Copyright
0 1970 by Academic
Pteas, Inc.
2
GRIMLEY
AND
FRIEDMAN
(Grimley et al., 1968; Friedman and Pastan, 1969). The injection vehicle was a balanced tissue culture medium (#1640 RPMI, Grand Island Biologicals, Grand Island, N.Y.) with 10% fetal calf serum added. Approximately 0.05-0.1 cc. of fluid with virus was injected. In two sets of experiments, litters of mice were injected 24 or 48 hours after birth and sacrificed when moribund (approximately 40 hours). In another set of experiments, parallel litters were injected at 8-hour intervals, and mice were sacrificed at various times after the death of some mice in the first injected litter. This procedure provided a spectrum of infections from 32-48 hours. Virus assay. Animals anesthetized with ether were sacrificed by decapitation at 6-48 hours after virus infection. Whole blood was collected in volumetric tubes and immediately frozen to -7OOC. Brains were weighed and frozen whole. After thawing, brain samples were ground with sterile sand, centrifuged at lOOOg, and prepared as a 10% suspension in the tissue culture medium with 10% serum. Beginning with 0.2 cc. of suspension, serial dilutions were prepared in the same medium. Aliquots of blood serum were similarly diluted. Plaque assay was performed on chick embryo cell monolayers as described by Friedman and Sonnabend (1965). Electron microscopy. Brains were fixed for electron microscopy by perfusion of 4% glutaraldehyde (Chambers et al., 1968) through the left ventricle of the heart or by direct immersion and slicing of the rapidly dissected brain. Thin slices from representative regions of brain were embedded in paraffin and stained with hematoxylin and eosin (Chambers et al., 1968). Small blocks of cortex or cerebellum were post-fixed with 1% osmium tetroxide and embedded for electron microscopy in Luft’s epon formula according to reported methods (Grimley et al., 1968). Thin sections were prepared with an automatic ultratome, stained with 10% uranyl acetate in methanol and with lead citrate, coated with carbon and examined at original magnifications of 9,000x to 37,000~ in an Hitachi-Perkin Elmer HU-11E microscope. RESULTS
Light microscopic examination of representative sections from infected suckling brains disclosed scant evidence of virus effect. Inflammatory reaction was absent or minimal and a few zones of eosinophilic necrosis or cellular edema were scattered in various regions, primarily the cortex as described by McGeeRussell and Gosztonyi (1967). Cell inclusion bodies were not observed. The virus growth curve is illustrated in Fig. 1. Virus titers in the brain suspensions increased progressively until 40 hours after innoculation and began to plateau shortly before death of the animals. Peak virus production at 48 hours was l,OOO,OOO-fold greater than infectivity at 8 hours, but represented only a lOO-fold increase over that detected at 24 hours. Viremia developed after 12 hours of infection. Rise in blood virus titers paralleled the growth of virus in the brain up to 32 hours, but the maximum blood titer was only 1% of maximum brain titers. In brains examined by electron microscopy at 32-36 hours after SFV injection, search for infected cells in selected samples was generally unrewarding. At
SEMLIKI
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16 HOURS
24 AFTER
3
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32
40
48
INFECTION
FIG. 1. Growth curve for Semliki Forest Virus (SFV) in suckling mouse brain and blood. Each point represents pooled brain suspension or whole blood from 2 or more animals.
36-38 hours the first signs of virus development could occasionally be detected. Mature 50 rnp diameter virions were clustered in the interstitial spaces, often in long 6les between apposed glial and neuronal bodies or processes (Fig. 2). Thorough examination of several brain samples taken at 36-40 hours after initiation of infection disclosed a number of vacuoles (Figs. 2, 3) resembling the CPV-I previously described in SFV infected tissue cultures (Grimley et al., 1968). These cytopathic vacuoles often occurred in planes of section where CPV-II and virus core accumulations were absent or minimal. The CPV-I consisted of multiple membrane vesicles averaging 65-70 rnF in external diameter and arranged in circular profiles. Each vesicle contained an irregular central density (Fig. 3). The limiting membrane at the periphery of neuronal CPV-I was less distinct than that observed in tissue culture cells, and the component vesicles were larger. Some CPV-I appeared to be disrupted or degenerated and contained intermingled dense particles resembling virus cores. Multiple CPV-I were only rarely detected in a single plane of section (Fig. 2). Budding of mature virions from the plasmalemma of neural processes was apparent in focal regions (Fig. 4). The neural processes could be identified by the presence of abundant, aligned neurotubules, clusters of synaptic vesicles, or synaptolemmal thickening at regions of synaptic junction. Unattached 28-30 rnp diameter virus cores could occasionally be identified in the axoplasm. These cores had a distinctive “knobby” surface at high magnification. (Faulkner and McGee-Russell, 1968). Budding of virus cores into channels of the endoplasmic reticulum was not infrequent and in rare planes of section simultaneous budding of serially aligned virus cores was observed (Fig. 5). Some of the channels evidently derived from
4
GRIMLEY
AND FRIEDMAN
FIG. 2. Periphery of neuron in region of early infection. Mature virions are sandwiched in the interstitial space between cell membranes. Cytoplasmic vacuoles of O.5-O.7-r diameter contain circumferentially arranged vesicles with an irregular central density. X40,000. FIG. 3. Structure similar to type I cytopathic vacuole (CPV-I) in perinuclear cytoplasm. Lining vesicles of spherical form average 65 rnti diameter and contain irregular central densities. ~47,500.
SEMLIKI
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5
FIG. 4. Evidence of mature virus budding from neuronal processes in mouse brain. (A) Longitudinal view of neuraxon with aligned neurotubules. ~60,000. (B) Cross sections of cell processes including neuraxon with neurotubules (arrow). X60,000. (C) Multiple virus buds and accumulation of virions in interstitial space. X47,500. (D) Cell process with budding virion and cytoplasmic &NCture bearing virus cores (arrow). ~60,000.
6
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FRIEDMAN
cistenae of the Golgi complex (Fig. 6) while others may have originated as lamellae of granular endoplasmic reticulum which had lost their ribosomal complement. Some micrographs suggested progressive detachment of ribosomes and replacement by virus cores (Fig. 7). One micrograph demonstrated budding of a virus core through clearly identifiable granular endoplasmic reticulum (Fig. 8). Budding of virus intracellularly was accompanied by a demonstrable thickening of the overlying cytomembrane which was destined to form the virus envelope (Fig. 9). By 40 hours after injection of SFV, large numbers of neurons were infected, and most samples examined by electron microscopy exhibited advanced intracellular phases of virus development. Virus cores accumulated as infection progressed and surrounded vacuolar membranes classified as CPV-II (Fig. 7, 9, 10). Some of the vacuoles contained long straight tubular invaginations which measured up to 50 ml in external diameter. The latter possessed a 22 rnr lucent core and resembled forms described in tissue culture by Erlandson, et al. (1967). On cross sections (Fig. ll), the invaginated tubules and vacuolar membrane surrounded by virus cores resembled a rosette, similar to the “assembly areas” emphasized by McGee-Russell and Gosztonyi (1967). Another type of membrane structure which often accompanied advanced intracellular virus infection is illustrated in Figs. 7 and 10. This consisted of an array of intermeshed tubular membranes averaging 25-30 rnw in diameter.The tubules appeared angulated and in some planes of section were organized in orderly patterns. This structure may be described as a “microtubular reticulum.” It was characteristically associated with converging cisternae of the endoplasmic reticulum but was not surrounded by virus cores. Similar structures have been described in Group B arbovirus infection (Murphy et al., 1968) and lymphoma cells (Chandra, 1968). Coated tubules in crystalloid array were frequently present in the cytoplasm of infected cells (Fig. 121, occasionally in close proximity to regions of microtubular reticulum. The coated tubules averaged 30 rnp in outer diameter and demonstrated an 80-90-A electron lucent core in cross section. DISCUSSION
Biochemical and immunologic studies of arbovirus replication are most conveniently and safely performed with cell cultures, but the significance of in vitro experiments might be uncertain if the modes of viral morphogenesis in vitro and in uiuo differed to the extent previously postulated by McGee-Russell and Gosztonyi (1967). Present observations offer new evidence of ultrastructural similarities of SFV development in mouse neurons and mammalian or avian cell cultures. Two morphologic pathways of virion maturation have been defined: surface budding virions which are easily detected in the early phases of infection in vitro were noted in regions of brain where ultrastructural evidence of intracellular virus maturation was minimal. Accumulation of intracytoplasmic virus cores and virions within cellular vacuoles marked the advanced stages of neuronal infection and also paralleled events in vitro. In contrast to some previous tissue culture studies of Group A arboviruses (Mussgay and Weibel, 1962;
SEMLIKI
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5. Sites of virus budding within cytoplasmic membrane channels. Note presence of single cores which are apparently free in the hyaloplasm (arrow). X 60,00& FIG. 6. Budding of virus into reticular channels of Golgi region. Some mature virions reside within Golgi cistemae. X32,000. FIG.
dense
ViNS
FIG. 7. Field demonstrating cytopathic changes in advanced infection. Structure near center (B resembles degenerating CPV-I. Channels of endoplasmic reticulum (ER) appear to acquire an outer layer of 28-30 mp virus cores. Formation of straight tubular imaginations singly or in groups (arrows) appears to be a secondary modification of the core associated vacuoles (CPV-II). Note re. gion of angulated tubules within dilated channel of endoplasmic reticulum (lower left). x40,ooO. Fm. 8. Budding of virus core into dilated chmel of granular endoplasmic reticulum. Membrane in this region appears thickened by a fuzzy electron-dense coat. ~47,500.
8
SEMLIKI
FIG. 9. Type II cytopathic vacuoles (CPV-II) membranous channel (arrow) is accompanied body within several of the cores. X112,000.
FOREST
9
VIRUS
with outer rim of virus cores. by local membrane thickening.
Budding of core into Note dense central
Acheson and Tamm, 1967), transitional stages in the intracellular budding of virus particles could be clearly identified in mouse neurons, and the juxtaposed cytomembranes appeared locally modified at the budding sites (Figs. 8, 9). Correlation of the findings in brain and tissue cultures suggests that some apparent discrepancies in the arbovirus maturation sequence may have been due to differences in the stage of infection at the time of sampling for electron microscopy. Reports emphasizing the surface budding of Group .A arboviruses have often focused attention upon samples prepared during the rapid growth phase (Mussgay and Weibel, 1962; Chain et al., 1966; Ach’eson and Tamm 1967), while reports emphasizing intracellular virion development have been based upon the examination of relatively late samples (Morgan et al., 1961; Eklandson et al., 1967; Higashi et al., 1967). It is nevertheless possible that the host cell environment may preferentially modify the pathways of virus maturation or limit appropriate budding sites. Such an effect has not been clearly demonstrated in systems thus far reported (Grimley and Friedman, 1969). Identification of CPV-I in SFV-infected brain was of particular interest. These structures form during the logarithmic phase of tissue culture infections with SFV and Sindbis virus (Grimley et al., 1966; Grimley and Friedman, 1969) and
FIG. tubular
10. Accumulation of mature virus within cytopathic vacuoles (arrows). Zone of microreticulum at left. X 23,403. FIG. 11. High magnification of 50-ma tubular structures inside of vacuoles with virus cores. In cross section, these complexes appear as rosettes, and secondary thickening of the membranes is apparent. Note the channels of granular endoplasmic reticulum and virus cores in close proximity. X~,~. 10
SEMLIKI
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11
FIG. 12. Closely packed aggregate of coated tubules observed in advanced infection. ~57,ooO.
have been associated with replication of early viral RNA (Friedman and Berezesky, 1967; Grimley et al., 1966). In neurons they also appear to precede CPV-II. Yasuzumi and Tsubo (1965) described abundant “glohoid bodies” in Japanese B encephalitis infections of mouse brain, and some of the micrographs published by Murphy et al. (1968), particularly Fig. 5 and 15, suggest that round “membranous vesicles” described by them may represent analogous structures in St. Louis encephalitis infection. Tissue culture studies have shown that CPV-I may degenerate as infection progresses, so that their detection depends upon appropriate chronologic sampling. This is obviously difficult in asynchronous central nervous system infections (McGee-Russell and Gosztonyi, 1967) and further studies of early stages in tissue culture arbovirus infections appear necessary to ascertain the incidence of CPV-I. Accumulation of noninfectious virus cores around cytopathic vacuoles designated CPV-II appears to be a characteristic in late Group A arbovirus infections (Rabin and Jenson, 1967; Murphy et al., 1966). We conclude that many or all of these vacuoles derive from the granular endoplasmic reticulum. Evidence for this is circumstantial, since direct connections of CPV-II to the endoplasmic reticulum are difficult to trace, even in serial sections (Morgan et al., 1961). Nevertheless, occasional micrographs have suggested transitions of endoplasmic reticulum to CPV-II (Chain et al., 1966; Grimley et al., 1966), and the synthesis of SFV core proteins is associated with membrane bound ribosomes (Fried-
12
GRIMLEY
AND
FRIEDMAN
man and Berezesky, 1967; Friedman, 1968). Secondary modification of CPV-II membranes appears to occur in late infection and may be preparative to intravacuolar budding of the virus cores (Erlandson et al., 1967; Higashi et al., 1967). The significance of straight tubular structures within CPV-II is uncertain, but they are a relatively late phenomenon and may represent formation of aberrant virus or excessive viral lipoproteins (Higashi et al., 1967), rather than a significant pathway of virus production (McGee-Russell and Gosztonyi, 1967). Recent studies of cycloheximide treated cells have shown that CPV-II fail to form when viral protein synthesis is inhibited after several hours of infection, but that surface-budding of virus continues in the absence of protein synthesis for up to 2 hours (Friedman and Grimley, 1969). These results indicate the existence of a large reservoir of completed virus cores after 6 hours of infection. Present observations of virus budding from neural processes provide a dramatic demonstration of virus core migration in long cell processes distal to the protein synthesizing and assembly sites. A tendency of virus to bud from pseudopodia has been noted in tissue culture (Acheson and Tamm, 1967; Chain et al., 1966), and the findings suggest passive transport of virus cores in hyaloplasmic or axoplasmic streams (Lubinska, 1964). The latter could be important in local or distant dissemination of the neurotropic arboviruses (Sabin and Olitsky, 1937). Differences in the morphogenesis of arbovirus subgroups have been considered by Murphy et al. (1968) and can now be more clearly defined: the major ultrastructural distinction in Group A infections is accumulation of excessive virus cores in the late stages of growth. The occurrence of CPV-I in various arbovirus subgroups remains to be fully evaluated. Intracellular virus budding is apparent in Group A, Group B, Bunyamwera, and California group arboviruses, but surface budding virus has been described primarily in Group A infections. Microtubular reticulum is a host cell response to several types of virus infection, including Groups A and B arboviruses. SUMMARY Electron microscopic examination of mouse brains infected with Semliki Forest virus (SFV) disclosed two major pathways of virion maturation not previously demonstrated in neurons: (1) budding of 28-30 mp virus cores through the cell surface (plasmalemma); (2) budding of virus cores into cytoplasmic vacuoles and cistemae of the Golgi membrane complex or endoplasmic reticulum. In regions of advanced neuronal infection, virus cores accumulated in large numbers and attached to the limiting membranes of type II cytopathic vacuoles (CPV-II), some of which appeared to derive from the endoplasmic reticulum. Transmigration of virus cores from cytomembrane assembly areas to sites of envelopment at the plasmalemma was evidenced by the presence of virus cores and surface-budding virions in neuronal processes. Axoplasmic transport of virus cores may thus be a factor in spread of arbovirus infections. Intracytoplasmic vacuoles enclosing membranous vesicles of tX-70-mp diameter were identified in regions of early infection; they corresponded to the type I cytopathic vacuoles (CPV-I) recently observed in tissue cultures and associated with viral RNA replication. Unusual cytomembrane proliferations, described as a “microtubular reticulum” developed regularly in the advanced stages of intracellular SFV infection and aggregates of 30 ma straight tubules with an electron-dense matrix occasionally accumulated in close proximity. The ultrastructural findings in SFV-infected neurons correlated closely with electron microscopic and biochemical studies of mesenchymal cell cultures, and indicate broad similarities in the maturation sequence of Group A arbovimses. ACKNOWLEDGMENTS We thank
Mrs.
Dorothy
Hughes
and
Mr.
Herman
Michelitch
for valuable
technical
assistance.
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