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Studies of the morphology of Murray Valley encephalitis and Japanese encephalitis viruses growing in cultured mosquito cells
34, 435-443
VIROLOGY
Studies Japanese
(1968)
of the
Morphology
Encephalitis
of Murray
Viruses
Growing
B. K. FILSHIE Division
of Entomology,
Valley
AND
in Cultured
Mosquito
and Cells’
J. REHACEK2
Commonwealth Scientific and Industrial Canberra, Australia Accepted
Encephalitis
October
Research
Organization,
30, 1967
The development of Murray Valley and Japanese encephalitis viruses within cultured mosquito cells has been followed by electron microscopy. Although cytopathic effects were observed in cells infected with JE virus, no such changes were detected in MVE-infected cells. Large clusters of virus particles appear to be taken into cells by phagocytosis. During the replication process, particles are always found within the endoplasmic reticulum, which sometimes takes on a characteristic form in heavily infected cells. Incomplete particles and nucleoids were not observed.
Marshall, in press) and JE virus, Nakayama strain, in 45th mouse brain passage were used in the experiments. Both strains were obtained from Dr. I. D. ZIarshall, *John Curtin School of &Zedical Research, Australian National University, Canberra. Cells. The stable cell line derived from larvae of Aedes aegypti (Grace, 1966) was employed in the experiments. ,4t the beginning of the experiments the cells had undergone 150-200 passages. Persistently infected cells were grown in 2-ounce medicine bottles. In experiments involving acute infections, cells were grown in Jena glass test tubes. The medium developed by Grace (1962) was used, and cuItures were incubat.ed at 30”. Meth.ods of infectim and titration. Test tubes containing about 5.5 X lo5 mosquito cells were inoculated with about 108 MVE or JE plaque-forming units (PFU). Ever!, 3 days the medium was changed, and from day 6 t,o day 27 after inoculation cells were taken for electron microscopy. From da? 6 onward the number of cells varied between lo6 and 106.“, the titer of RiVE virus varied bet,ween 104.6 and 106.” PE‘U/ml and t,he
INTRODUCTION
Surprisingly little use has been made of arthropod cells for investigations of arboviruses at. the fine structural level. Bergold and Weibel (1962) demonstrated yellow fever virus in salivary glands of Aedes aegypti, and Bowne and Jones (1966) detected bluet,ongue virus particles in Culicoides variipennis. Now that a line of mosquito cells can be sucessfully maintained in tissue culture (Grace, 1966), new perspectives of a number of problems are revealed, including those concerning the developmental morphology of arboviruses. In the present investigations electron micrographs of Muray Valley encephalitis (MVE) and Japanese encephalitis (JE) virus particles within these cells are reported and are compared with arboviruses propagated in vertebrate cells. MATERIALS
AND
METHODS
Viruses. NVE virus, strain 96961/,53 in 7th mouse brain passage (Hawkes and 1 This work was supported in part by the U.S. Public Health Service, Grant No. Al-02462-07. 2 Visiting fellow from the Institute of Virology, Czechoslovak Academy of Sciences, Bratislava, Czechoslovakia (present address).
titer
of
JE
104.g PFUiml. 435
virus
varied
bet.ween
l@
anti
436
FILSHIE
AND
Persistently infected cells growing in the 3rd to 5th passages (Rehacek, in preparation) were selected for morphological examination if the virus titers exceeded lo4 PFU/ml. For virus titration, cells and media were twice frozen and thawed, and pipetted to disrupt the cells. After centrifugation at 1500 rpm for 3 minutes, the supernatant was prepared for virus assay in tenfold serial dilutions in gelatin saline. Virus titers were determined using the plaque technique on chick embryo fibroblasts. Preparation for electron microscopy. Cells were removed from the tubes or bottles by gently pipetting the media. After centrifugation at 600 rpm for 3 minutes, the cells were resuspended in a small volume of the supernatant, transferred to “Spinco” polyethylene microcentrifuge tubes, and again sedimented at 600 rpm for 3 minutes. The resulting pellet of cells was fixed with 2.5 % glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 (Millonig, 1962) for 224 hours at room temperature, washed in several changes of buffer for a total of 2-4 hours, postfixed with 1% osmium textroxide in 0.1 M phosphate buffer at pH 7.2 for 2 hours at room temperature, dehydrated through an acetone series and embedded in Araldite. Sections approximately 500 A thick were cut, with a Porter-Blum MT-2 uItramictotome, mounted on carbon films, and stained with 5% aqueous uranyl acetate for 1 hour at room temperature, followed by lead citrate (Reynolds, 1963) for lo-20 minutes at room temperature. The sections were examined in a Siemens Elmiskop I electron microscope operated at 80 kV and electron-optical magnifications up to 40,000 times. RESULTS
Virus Particles External profiles of both MVE and JE particles in thin sections were found to be either circular or elliptical (Fig. 2), but the latter appearance could have resulted from compression introduced during sectioning. The fine structure and dimensions of RXVE and JE particles appear identical in thin sections. We have not detected any morphological differences between extracellular
REHACEK
particles and particles found within the endoplasmic reticulum. Particles are approximately 409 A in diameter. The outer layer of each particle consists of a single electron-dense membrane about 25 A in thickness separated by a less dense layer about 25 A in thickness from a central, dense core approximately 280 A in diameter (Fig. 2). In some images the outer covering of the core appears to form a distinct membrane 10-20 A in thickness (Fig. 2, inset). We infer from these observations that MVE and JE virus particles consist of a nucleoid approximately 250 A in diameter enveloped by a triple-layered unit membrane approximately 75 A in thickness. Infected Cells In the initial experiments cells were grown in medicine bottles. However, neither extranor intracellular particles were observed by electron microscopy during the first 24 days after inoculation. We suspected that the multiplicity and hence the probability of detecting particles in thin sections was low. In all further experiments involving acute infection, cells were cultivated in test tubes, where higher multiplicities were achieved. MVE Virus. Particles were first observed in acutely infected cells 6 days after inoculation and in persistently infected cultures, in the 5th passage, on day 6 after seeding. In infected cells, mature particles are always contained within vacuoles or elements of the endoplasmic reticulum, never free in the cytoplasm. From day 6 onward large vacuoles frequently contained single particles and crystalline groups of particles, in addition to other debris apparently ingested from the culture medium by phagocytosis (Figs. 1 and 2). On day 9, about 5 % of the cells (observed as thin sections) contained virus particles, which were now confined mainly to the endoplasmic reticulum. In some of these cells the reticulum was arranged in characteristic patterns, the presence of which seemed to be correlated with the presence of relatively large numbers of particles within the reticulum. Figure 3 is a section of a cell where this pattern is particularly well devel-
FIG. 1. Section uf a cell from a culture 9 days after present in this part of the cytoplasm, and most contain of the micrograph is not completely enclosed by the cell with uranyl acetate and lead citrate. X30,CQO. FIG. 2. MVE virus particles packed closely together in a cell, 9 days after infection. Endoplasmic reticulum, stained with many1 acetat,e and lead citrate. X120,900.
infection with virus particles. at this stage.
MVE. Phagocytic vacuoles I’, are The vacuole at the extreme right Mitochondrion, &f. Section stained
in a crystalline array within ER; microtubule, MT; Inset is a single particle,
a phagocytic vacuole ribosomes, R. Section X250,000.
old.
It consists esseriti:rlly of tmxs of inter-
comlecting smooth-membrancd vesicles and c%twnae, from \vhich radiate cist,ernne studdrd with rihosomes. Sumerous virus part iclw :w found lvithin the rihowme-covered rcticalum and to a lesser extent, \vithin the cwtr:~!I~~ placed regions of smooth memhran~ (I:igs:. 3 and 4). To\vnrd the plasma menltwanw of infected cells, vesicles containing viriw particles are devoid of a covering of rihosomes (Fig. 5). Occasionall>- the limiting mCmhranc of these vesicles is seen to hc joined to the plasma membrane (Fig. 3). Infwted cells ~~suall~~ carrv clusters of virus p:lrticlcs :tdherirg to their surfaces (I’&. 5).
.JI:’ 17ims. I’uticlw \\-erc~fimt SWII irr tlw entloplasmic reticulum of the mosquito ~11s ;3 d:r~~ after inoculation. Within the cells the viruses are aln-:tys rncloscd caithw in largc~ vacuoles or in thr reticulum (I’@. S), hut the patterns of smooth and put iclc-rovcwcl memhr:mes sew in A I\‘I<-irifec~c~ci (*(‘I Is uwc’ not detected in cells corlt:Gr\g .TIq: virus particles. In the, qJE-inforted culturrs grc:ttel numbers of cells undergoing :tutol~& were observed th:tn in either the ;\IVIC-iufwted crlltures or in thr contrtk. .\I:in>. of the morihuntl cells contained p:wticles bit bin elements
7).
of the
rridophmir
rr~ticlllllrn
(I:ig.
FIG. 4. A serial section to that of Wig. 3, at higher magnifcatiun. A few virus particles are located within the smooth membrar~~us regimw (SM), brat. the majority lie within ribosome-covered cisterrtae which radiate from these regions. Section stained with umnyl aeet,ate and lead cit,ratc. X60,000. FIG. 5. Near the plasma membrane (P&f) of infected cells, vesicles containing MT’E virus part,icles (right) are devoid of a covering of ribosomcs. Grot~ps of virus part,icles are freqllent,ly observed adhering to the surface of infected cells (upper left). Cellular projections, PR. Section stained with uranyl acetate and lead citrate. X60,OOO. 439
FIO. 6. JE virus particles adhering to the plasma membrane UC a mosquit,o cell. ‘l’hr virun is irk its fifth passage t,hrollgh the cells, which wcrc fixed 2 days after illfection. Section sc:ii~~r~l wi( tl r~r:r~l) 1 acetate and lead cit,rate. X60,000. FIG. 7. A moribund cell showing .JFZ virus particles wit.hin sacs of the cndoplasmic tnt iruhlm (arrows) (fifth passage, 2 days after infection). Degenerating mitochondrion, M; ribosomes, I<. Swtio~l stailjetl with uranyl acetate and lead citrate. X60,000. FIG. 8. .JE virus particles (arrows) localed within thr rough endoplasmic ret ir.lllllrrl of ;I rnosql~itc) cell (first pussagc, 12 days aft,er infection). Section stained wit 11 ~II.HII~~ acelate RIIC~ lc~r~l VII r:ttc XliO,OOO. 440
FIG. 9. Section of a PK cell wiLh JE particles (arrows) conlained in the Golgi apparatus (G) and also in-the rough endoplasmic reticulum (ER). X60,000. Flo. 10. Another Golgi region of the same cell at higher magnification. The virus particles are contained within the cistcrnae and vesicles of the Golgi apparatus. Some of the particles appear to be attached to t.he inner surfaces of the membranes (arrows). Microtuhule, MT; nucleus, N. Section stained with uranyl acetate and lead cit,rate. X120,000. 441
1-22
FILSHIE
AND
F’or purposes of comparison of t,he development of JE virus between invertebrate nnd vertebrate cells, the experiments of Ota (1965) were repeated using pig kidney (PK) cells and the Xakayama strain of JE. This virus was not adapted to PK cells, but nevertheless, normal particles were found in the cells 20 hours after inoculation, in the endoplasmic reticulum (Fig. 9) and the Golgi complex (Figs. 9 and 10). We have observed close contacts and pedicellar attachments between vesicular membranes and virus particules similar to those found by Ot,a (196.5) (arrows, Fig. 10). However, we cannot. confirm t’hat any COIItinuit,y exists between the capsid membrane of t,he virion and the unit membrane structure of t,he vesicle (cf. Ota, 196.5). DISCUSSION
Alosquito cells infected with MVE virus show no cytopathic effects of any significance in our experiments. In contrast, in pig kidney cells infected wit,h JE virus, particles appear only in cells showing degenerative changes. The methods of fixation and embedding employed in our investigation have resulted in improved preservation of cellular detail. In part,icular, the int,ernal virus-containing membrane systems of infect,ed cells can now be classified’into phagoq%ic vacuoles, particle-covered and smooth endoplasmic reticulum, Golgi complex, et’c. It, is now possible with t,his additional informntion to envisage certain stages of t,he process of replication of JIVE virus wit,hin cultuled insect cells. However, it should be pointjed out that throughout our investigations, repeated dificulty \vas encountered in obtaining a high multiplicity of virus. Even h cult,ures lvhere the heaviest, infections were achieved, the ratio of l’l+YJ:cell was never much greater t)han 2: 1. Consequently, very fc\v cells in individual cultures contained large numbers of virus part,icles, and our proposals are based on the examinat.ion of :L relatively small sample of infected cells irl a very large number of thin sections. Infect,ion appears to result from the ingestion of part,icles, geneAl\. in large clust,ers, h3 1)h:tgocytoais. The appearawe of single parti-
REHACEK
cles lvithin t)he cisternae of th(l endoplasmic ret’iculum suggests that. this is their sit’e of assembly; in individual cells containing large numbers of particles, t,he ret,iculum is ap parently organized into a charact,eristic pat,tern for this purpose. On the basis of our results, it has not been possible t,o distinguish, during t’he later stages of infect,ion, between inoculated and progeny virus part,icles lying within cyt80plasmic vacuoles. In a recent study of Semliki Forest, virus, a group A arbovirus, Acheson and Tumm (1967) demonstrated clearly the presence of viral nucleoids in the cytoplasmic matrix of chick embryo cells and that the final stage in the assembly of virus particles probabl> occurs at, t’he plasma membrane. \vhere :I portion of t#he membrane is incorporated into the outer coat of the virus part,icle. From our result)s and those of Ota (196:i), it, seems likely that final maturation of group 13 arboviruses takes place at, the internal men)branes of the ccl! as distinct from mat,ur:1tion at the plasma membrane. for group .I arboviruses. However, in infrcted cell:: w have not observed incomplete particles 01 nuclroids which might throw further light, on the assembly mechanism of the group H arboviruses. ACKNOWLEI
)( ;JII+xTS
Wr are indebted to I)r. I. I). Marshall, Johll Ctutill School of Medical Research, ,4nst raliall National Ultiversity, for providiug the virlts atltl t,hr facilities ilr his laboratory :III~ to Kay ~IvCaskill and Rosemary Miller for tcrhlliral assist XIICP. REFEREKCISS ACHESON, N. I-1., and TAMM, I. (l!Nii). Replication of Semliki Forest virlls: all electrorl microscopic stktdy. Virology 32, 12X-l-G. BERGOLU. (;. II., and WEIHEL, J. (19ti2). I)c)tnollstration of yellow fever virus with thr rlp~~t ran microscope. I-iro/og!/ 17, 551~5fii. BOWNE, J. Ct., and JOKES, It. II. (19Ni). Ohsrrv;ttions on bllletongl~e virlls ilr the salivary glands of a11 insect vec’tor, C'u/icoides NIVipennis. i’irology 30, 12i-1X3. GR.ZVE. T. 1). c. (1962). 15st:rblishmelit of follr strains uf cells frclrn illsec-l 1 isslles grc,wt, in Vi/rYJ. .Ya/cr,.f? 196, 78%78%
ARBOVIRUSES
GROWING
IN CULTURED
GRACE, T. D. C. (1966). Establishment of a line of mosquito (Aedes aegvpti L.) cells grown in vitro. Nature 211, 366-367. HAWKES, R. A., and MARSHALL, I. D. (1967). Studies of arboviruses by agar gel diffusion. Am. J. Epidemiol. 86, 28-44. MILLONIG, G. (1962). Further observations on a phosphate buffer for osmium solutions in fixation. Proc. 6th Intern. Congr. Electron
MOSQUITO
CELLS
443
Microscopy, Philadelphia, 1968, Vol. 2, P8. Academic Press, New York. OTA, Z. (1965). Electron microscope study of the development of Japanese B encephalitis virus in porcine kidney stable (PS) cells. Virology 26, 372-378. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain for electron microscopy. J. Cell Biol. 17, 208-212.
VIROLOGY
Studies Japanese
(1968)
of the
Morphology
Encephalitis
of Murray
Viruses
Growing
B. K. FILSHIE Division
of Entomology,
Valley
AND
in Cultured
Mosquito
and Cells’
J. REHACEK2
Commonwealth Scientific and Industrial Canberra, Australia Accepted
Encephalitis
October
Research
Organization,
30, 1967
The development of Murray Valley and Japanese encephalitis viruses within cultured mosquito cells has been followed by electron microscopy. Although cytopathic effects were observed in cells infected with JE virus, no such changes were detected in MVE-infected cells. Large clusters of virus particles appear to be taken into cells by phagocytosis. During the replication process, particles are always found within the endoplasmic reticulum, which sometimes takes on a characteristic form in heavily infected cells. Incomplete particles and nucleoids were not observed.
Marshall, in press) and JE virus, Nakayama strain, in 45th mouse brain passage were used in the experiments. Both strains were obtained from Dr. I. D. ZIarshall, *John Curtin School of &Zedical Research, Australian National University, Canberra. Cells. The stable cell line derived from larvae of Aedes aegypti (Grace, 1966) was employed in the experiments. ,4t the beginning of the experiments the cells had undergone 150-200 passages. Persistently infected cells were grown in 2-ounce medicine bottles. In experiments involving acute infections, cells were grown in Jena glass test tubes. The medium developed by Grace (1962) was used, and cuItures were incubat.ed at 30”. Meth.ods of infectim and titration. Test tubes containing about 5.5 X lo5 mosquito cells were inoculated with about 108 MVE or JE plaque-forming units (PFU). Ever!, 3 days the medium was changed, and from day 6 t,o day 27 after inoculation cells were taken for electron microscopy. From da? 6 onward the number of cells varied between lo6 and 106.“, the titer of RiVE virus varied bet,ween 104.6 and 106.” PE‘U/ml and t,he
INTRODUCTION
Surprisingly little use has been made of arthropod cells for investigations of arboviruses at. the fine structural level. Bergold and Weibel (1962) demonstrated yellow fever virus in salivary glands of Aedes aegypti, and Bowne and Jones (1966) detected bluet,ongue virus particles in Culicoides variipennis. Now that a line of mosquito cells can be sucessfully maintained in tissue culture (Grace, 1966), new perspectives of a number of problems are revealed, including those concerning the developmental morphology of arboviruses. In the present investigations electron micrographs of Muray Valley encephalitis (MVE) and Japanese encephalitis (JE) virus particles within these cells are reported and are compared with arboviruses propagated in vertebrate cells. MATERIALS
AND
METHODS
Viruses. NVE virus, strain 96961/,53 in 7th mouse brain passage (Hawkes and 1 This work was supported in part by the U.S. Public Health Service, Grant No. Al-02462-07. 2 Visiting fellow from the Institute of Virology, Czechoslovak Academy of Sciences, Bratislava, Czechoslovakia (present address).
titer
of
JE
104.g PFUiml. 435
virus
varied
bet.ween
l@
anti
436
FILSHIE
AND
Persistently infected cells growing in the 3rd to 5th passages (Rehacek, in preparation) were selected for morphological examination if the virus titers exceeded lo4 PFU/ml. For virus titration, cells and media were twice frozen and thawed, and pipetted to disrupt the cells. After centrifugation at 1500 rpm for 3 minutes, the supernatant was prepared for virus assay in tenfold serial dilutions in gelatin saline. Virus titers were determined using the plaque technique on chick embryo fibroblasts. Preparation for electron microscopy. Cells were removed from the tubes or bottles by gently pipetting the media. After centrifugation at 600 rpm for 3 minutes, the cells were resuspended in a small volume of the supernatant, transferred to “Spinco” polyethylene microcentrifuge tubes, and again sedimented at 600 rpm for 3 minutes. The resulting pellet of cells was fixed with 2.5 % glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 (Millonig, 1962) for 224 hours at room temperature, washed in several changes of buffer for a total of 2-4 hours, postfixed with 1% osmium textroxide in 0.1 M phosphate buffer at pH 7.2 for 2 hours at room temperature, dehydrated through an acetone series and embedded in Araldite. Sections approximately 500 A thick were cut, with a Porter-Blum MT-2 uItramictotome, mounted on carbon films, and stained with 5% aqueous uranyl acetate for 1 hour at room temperature, followed by lead citrate (Reynolds, 1963) for lo-20 minutes at room temperature. The sections were examined in a Siemens Elmiskop I electron microscope operated at 80 kV and electron-optical magnifications up to 40,000 times. RESULTS
Virus Particles External profiles of both MVE and JE particles in thin sections were found to be either circular or elliptical (Fig. 2), but the latter appearance could have resulted from compression introduced during sectioning. The fine structure and dimensions of RXVE and JE particles appear identical in thin sections. We have not detected any morphological differences between extracellular
REHACEK
particles and particles found within the endoplasmic reticulum. Particles are approximately 409 A in diameter. The outer layer of each particle consists of a single electron-dense membrane about 25 A in thickness separated by a less dense layer about 25 A in thickness from a central, dense core approximately 280 A in diameter (Fig. 2). In some images the outer covering of the core appears to form a distinct membrane 10-20 A in thickness (Fig. 2, inset). We infer from these observations that MVE and JE virus particles consist of a nucleoid approximately 250 A in diameter enveloped by a triple-layered unit membrane approximately 75 A in thickness. Infected Cells In the initial experiments cells were grown in medicine bottles. However, neither extranor intracellular particles were observed by electron microscopy during the first 24 days after inoculation. We suspected that the multiplicity and hence the probability of detecting particles in thin sections was low. In all further experiments involving acute infection, cells were cultivated in test tubes, where higher multiplicities were achieved. MVE Virus. Particles were first observed in acutely infected cells 6 days after inoculation and in persistently infected cultures, in the 5th passage, on day 6 after seeding. In infected cells, mature particles are always contained within vacuoles or elements of the endoplasmic reticulum, never free in the cytoplasm. From day 6 onward large vacuoles frequently contained single particles and crystalline groups of particles, in addition to other debris apparently ingested from the culture medium by phagocytosis (Figs. 1 and 2). On day 9, about 5 % of the cells (observed as thin sections) contained virus particles, which were now confined mainly to the endoplasmic reticulum. In some of these cells the reticulum was arranged in characteristic patterns, the presence of which seemed to be correlated with the presence of relatively large numbers of particles within the reticulum. Figure 3 is a section of a cell where this pattern is particularly well devel-
FIG. 1. Section uf a cell from a culture 9 days after present in this part of the cytoplasm, and most contain of the micrograph is not completely enclosed by the cell with uranyl acetate and lead citrate. X30,CQO. FIG. 2. MVE virus particles packed closely together in a cell, 9 days after infection. Endoplasmic reticulum, stained with many1 acetat,e and lead citrate. X120,900.
infection with virus particles. at this stage.
MVE. Phagocytic vacuoles I’, are The vacuole at the extreme right Mitochondrion, &f. Section stained
in a crystalline array within ER; microtubule, MT; Inset is a single particle,
a phagocytic vacuole ribosomes, R. Section X250,000.
old.
It consists esseriti:rlly of tmxs of inter-
comlecting smooth-membrancd vesicles and c%twnae, from \vhich radiate cist,ernne studdrd with rihosomes. Sumerous virus part iclw :w found lvithin the rihowme-covered rcticalum and to a lesser extent, \vithin the cwtr:~!I~~ placed regions of smooth memhran~ (I:igs:. 3 and 4). To\vnrd the plasma menltwanw of infected cells, vesicles containing viriw particles are devoid of a covering of rihosomes (Fig. 5). Occasionall>- the limiting mCmhranc of these vesicles is seen to hc joined to the plasma membrane (Fig. 3). Infwted cells ~~suall~~ carrv clusters of virus p:lrticlcs :tdherirg to their surfaces (I’&. 5).
.JI:’ 17ims. I’uticlw \\-erc~fimt SWII irr tlw entloplasmic reticulum of the mosquito ~11s ;3 d:r~~ after inoculation. Within the cells the viruses are aln-:tys rncloscd caithw in largc~ vacuoles or in thr reticulum (I’@. S), hut the patterns of smooth and put iclc-rovcwcl memhr:mes sew in A I\‘I<-irifec~c~ci (*(‘I Is uwc’ not detected in cells corlt:Gr\g .TIq: virus particles. In the, qJE-inforted culturrs grc:ttel numbers of cells undergoing :tutol~& were observed th:tn in either the ;\IVIC-iufwted crlltures or in thr contrtk. .\I:in>. of the morihuntl cells contained p:wticles bit bin elements
7).
of the
rridophmir
rr~ticlllllrn
(I:ig.
FIG. 4. A serial section to that of Wig. 3, at higher magnifcatiun. A few virus particles are located within the smooth membrar~~us regimw (SM), brat. the majority lie within ribosome-covered cisterrtae which radiate from these regions. Section stained with umnyl aeet,ate and lead cit,ratc. X60,000. FIG. 5. Near the plasma membrane (P&f) of infected cells, vesicles containing MT’E virus part,icles (right) are devoid of a covering of ribosomcs. Grot~ps of virus part,icles are freqllent,ly observed adhering to the surface of infected cells (upper left). Cellular projections, PR. Section stained with uranyl acetate and lead citrate. X60,OOO. 439
FIO. 6. JE virus particles adhering to the plasma membrane UC a mosquit,o cell. ‘l’hr virun is irk its fifth passage t,hrollgh the cells, which wcrc fixed 2 days after illfection. Section sc:ii~~r~l wi( tl r~r:r~l) 1 acetate and lead cit,rate. X60,000. FIG. 7. A moribund cell showing .JFZ virus particles wit.hin sacs of the cndoplasmic tnt iruhlm (arrows) (fifth passage, 2 days after infection). Degenerating mitochondrion, M; ribosomes, I<. Swtio~l stailjetl with uranyl acetate and lead citrate. X60,000. FIG. 8. .JE virus particles (arrows) localed within thr rough endoplasmic ret ir.lllllrrl of ;I rnosql~itc) cell (first pussagc, 12 days aft,er infection). Section stained wit 11 ~II.HII~~ acelate RIIC~ lc~r~l VII r:ttc XliO,OOO. 440
FIG. 9. Section of a PK cell wiLh JE particles (arrows) conlained in the Golgi apparatus (G) and also in-the rough endoplasmic reticulum (ER). X60,000. Flo. 10. Another Golgi region of the same cell at higher magnification. The virus particles are contained within the cistcrnae and vesicles of the Golgi apparatus. Some of the particles appear to be attached to t.he inner surfaces of the membranes (arrows). Microtuhule, MT; nucleus, N. Section stained with uranyl acetate and lead cit,rate. X120,000. 441
1-22
FILSHIE
AND
F’or purposes of comparison of t,he development of JE virus between invertebrate nnd vertebrate cells, the experiments of Ota (1965) were repeated using pig kidney (PK) cells and the Xakayama strain of JE. This virus was not adapted to PK cells, but nevertheless, normal particles were found in the cells 20 hours after inoculation, in the endoplasmic reticulum (Fig. 9) and the Golgi complex (Figs. 9 and 10). We have observed close contacts and pedicellar attachments between vesicular membranes and virus particules similar to those found by Ot,a (196.5) (arrows, Fig. 10). However, we cannot. confirm t’hat any COIItinuit,y exists between the capsid membrane of t,he virion and the unit membrane structure of t,he vesicle (cf. Ota, 196.5). DISCUSSION
Alosquito cells infected with MVE virus show no cytopathic effects of any significance in our experiments. In contrast, in pig kidney cells infected wit,h JE virus, particles appear only in cells showing degenerative changes. The methods of fixation and embedding employed in our investigation have resulted in improved preservation of cellular detail. In part,icular, the int,ernal virus-containing membrane systems of infect,ed cells can now be classified’into phagoq%ic vacuoles, particle-covered and smooth endoplasmic reticulum, Golgi complex, et’c. It, is now possible with t,his additional informntion to envisage certain stages of t,he process of replication of JIVE virus wit,hin cultuled insect cells. However, it should be pointjed out that throughout our investigations, repeated dificulty \vas encountered in obtaining a high multiplicity of virus. Even h cult,ures lvhere the heaviest, infections were achieved, the ratio of l’l+YJ:cell was never much greater t)han 2: 1. Consequently, very fc\v cells in individual cultures contained large numbers of virus part,icles, and our proposals are based on the examinat.ion of :L relatively small sample of infected cells irl a very large number of thin sections. Infect,ion appears to result from the ingestion of part,icles, geneAl\. in large clust,ers, h3 1)h:tgocytoais. The appearawe of single parti-
REHACEK
cles lvithin t)he cisternae of th(l endoplasmic ret’iculum suggests that. this is their sit’e of assembly; in individual cells containing large numbers of particles, t,he ret,iculum is ap parently organized into a charact,eristic pat,tern for this purpose. On the basis of our results, it has not been possible t,o distinguish, during t’he later stages of infect,ion, between inoculated and progeny virus part,icles lying within cyt80plasmic vacuoles. In a recent study of Semliki Forest, virus, a group A arbovirus, Acheson and Tumm (1967) demonstrated clearly the presence of viral nucleoids in the cytoplasmic matrix of chick embryo cells and that the final stage in the assembly of virus particles probabl> occurs at, t’he plasma membrane. \vhere :I portion of t#he membrane is incorporated into the outer coat of the virus part,icle. From our result)s and those of Ota (196:i), it, seems likely that final maturation of group 13 arboviruses takes place at, the internal men)branes of the ccl! as distinct from mat,ur:1tion at the plasma membrane. for group .I arboviruses. However, in infrcted cell:: w have not observed incomplete particles 01 nuclroids which might throw further light, on the assembly mechanism of the group H arboviruses. ACKNOWLEI
)( ;JII+xTS
Wr are indebted to I)r. I. I). Marshall, Johll Ctutill School of Medical Research, ,4nst raliall National Ultiversity, for providiug the virlts atltl t,hr facilities ilr his laboratory :III~ to Kay ~IvCaskill and Rosemary Miller for tcrhlliral assist XIICP. REFEREKCISS ACHESON, N. I-1., and TAMM, I. (l!Nii). Replication of Semliki Forest virlls: all electrorl microscopic stktdy. Virology 32, 12X-l-G. BERGOLU. (;. II., and WEIHEL, J. (19ti2). I)c)tnollstration of yellow fever virus with thr rlp~~t ran microscope. I-iro/og!/ 17, 551~5fii. BOWNE, J. Ct., and JOKES, It. II. (19Ni). Ohsrrv;ttions on bllletongl~e virlls ilr the salivary glands of a11 insect vec’tor, C'u/icoides NIVipennis. i’irology 30, 12i-1X3. GR.ZVE. T. 1). c. (1962). 15st:rblishmelit of follr strains uf cells frclrn illsec-l 1 isslles grc,wt, in Vi/rYJ. .Ya/cr,.f? 196, 78%78%
ARBOVIRUSES
GROWING
IN CULTURED
GRACE, T. D. C. (1966). Establishment of a line of mosquito (Aedes aegvpti L.) cells grown in vitro. Nature 211, 366-367. HAWKES, R. A., and MARSHALL, I. D. (1967). Studies of arboviruses by agar gel diffusion. Am. J. Epidemiol. 86, 28-44. MILLONIG, G. (1962). Further observations on a phosphate buffer for osmium solutions in fixation. Proc. 6th Intern. Congr. Electron
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CELLS
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Microscopy, Philadelphia, 1968, Vol. 2, P8. Academic Press, New York. OTA, Z. (1965). Electron microscope study of the development of Japanese B encephalitis virus in porcine kidney stable (PS) cells. Virology 26, 372-378. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain for electron microscopy. J. Cell Biol. 17, 208-212.