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Formation of poliovirus in monkey kidney tissue culture cells
VIROLOGY
16, 325-333 (1962)
Formation
of Poliovirus HEATHER
Department
in Monkey
DONALD MAYOR
of Virology
Kidney AND
Cells’
LIANE E. JORDAN
and Epidemiology, Baylor Medicine, Houston, Texas Accepted
Tissue Culture
University
College
of
November SO, 1961
Cytochemical and fluorescent antibody techniques have been utilized to study the release of poliovirus. Electron microscopic studies of ultrathin sections of epoxyembedded monkey kidney cells infected with poliovirus (types 1 and 2 virulent and attenuated) have revealed cytoplasmic arrays of spherical particles each approximately 25 rnp in diameter. Similar particles were found occasionally in “blebs” at the plasma membrane. Particles were detected only when infected cultures were incubated at 30°C. At this temperature fully infective poliovirus is formed but its release is inhibited. Particles were not encountered in infected cultures incubated at 37” or in control preparations. INTRODUCTION
Poliovirus particles have been demonstrated in ultrathin sections of cultured human cells infected with the virulent type 1 Brunhilde strain, occurring in the cytoplasm either as crystalline arrays (Stuart and Fogh, 1959,196l; Fogh and Stuart, 1960) or inlaid within filamentous protein material (Fogh, 1961). Characteristic virus particles were found only after inoculation with this highly virulent strain. Other members of the enterovirus group, for example, ECHO type 19 (Nuiiez-Montiel and Weibel, 1960), ECHO type 9 (Rifkind et al., 1961)) and ECHO type 4 (It. Jamison, unpublished) have been detected in infected monkey kidney tissue cultures by thin-sectioning techniques, yet poliovirus particles have not been previously demonstrated in monkey kidney (MK) tissue culture even though this is the host cell of choice for routine propagation of the virus. In all reported attempts to locate poliovirus particles in MK tissue cultures using the electron microscope, procedures utilizing methacrylate resin have been employed, and the vagaries of explosion artifact and ‘Aided tion.
by a grant from the National
polymerization damage with this material are well known. We decided to use an epoxy resin and a very brief dehydration and embedding schedule in an attempt to protect the tissue culture cells from undue extraction of fine structural components and to subject them to a minimum of polymerization damage. Howes and Melnick (1957) first showed that poliovirus multiplication is characterized by an accumulation of intracellular virus which is soon released. Roisman (1959) demonstrated that when poliovirus is grown at 30” the release is inhibited. Cytochemical and fluorescent antibody techniques have proved to be useful tools for demonstrating the release phenomenon and studying its control (Mayor, 1961). It seemed worth while, therefore, to integrate these results with studies at the fine structural level ; to this end the infected tissue cultures were incubated at 30” before searching for intracellular poliovirus particles with the electron microscope. The results of these experiments are reported in this paper. MATERIALS
Tissue
Founda-
epithelial 325
AND METHODS
Trypsin-dispersed MK cells were grown in 16-ounce
culture.
326
MAYOR
AND JORDAN
bottles using methods and the lactalbumin hydrolyzate medium described by Melnick (1956j. Virus. The following poliovirus strains were used: type 1, Brunhilde (virulent), LSc (attenuated) ; type 2, MEF-1 (virulent), YSK (attenuated). When the cell monolayers formed a confluent sheet, usually within a week, the cells were washed twice with fresh medium and inoculated with 1 ml of a virus suspension containing approximately lo7 PFU/ml. As there are approximately 10’ cells per monolayer in a 16-ounce bottle, these inoculating conditions corresponded to an approximate input multiplicity of one. After an adsorption time of approximately 1% hours at 37” the virus inoculum was decanted and 10 ml of growth medium containing cysteine (Melnick et al., 1957) was added. The infected cultures were incubated at 37” for an additional 2 hours and were then transferred to a 30” incubator for an additional 15 hours (overnight). Although most of the cells in the monolayer were still attached to the glass, an advanced cytopathic effect was observed in at least 75% of them. Cultures were usually chilled at 4” for 1 or 2 hours prior to fixation for electron microscopy in order to aid possible aggregation of virus particles to form crystalline arrays and were processed for ultrathin sectioning as a monolayer according to the following schedule. Fixdon: 10 minutes in 1% buffered osmium tetroxide pH 7.8 in the cold. Dehydration: 35% ethyl alcohol, 5 minutes; 50% ethyl alcohol, 3 minutes; 70% ethyl alcohol, 3 minutes; 95% ethyl alcohol, 3 minutes; absolute ethyl alcohol, 5 minutes; 0.1% phosphotungstic acid in absolute ethyl alcohol, 10 minutes; 2 changes of propylene oxide, each 10 minutes ; 50: 50 mixture propylene oxide and CIBA 502 araldite resin, 11/2-2 hours. At this stage, the cells were harvested with a rubber policeman and centrifuged at low speed for 5-10 minutes; the resulting pellets were then embedded in the epoxy resin with DMPs02 as accelerator according to Luft’s procedure (1961). Ultrathin sec’ 2 , 4 , 6 - Tri (dimethylaminomethyl) phenol Rohm and Haas Co., Philadelphia, Pemuylvania.
tions were cut with a SIROFLEX ultramicrotome and examined in an RCA EMU 3D and a Siemens Elmiskop 1. Cytochemical and immunofluorescent techniques. Tissue cultures were grown on
11 x 22 mm coverslips in Leighton tubes and infected with poliovirus in an identical manner to that described (Mayor, 1961). Processing techniques for acridine orange and fluorescent antibody staining and for ultraviolet microscopy and photography are also given in full in this paper. Incubation times and temperatures were the same as for the tissue cultures destined for electron microscopy described above. RESULTS
Cytochemistry
and Immunofluorescence
The correlated cytochemical and immunofluorescent studies indicated typical release of poliovirus at 37”. With the acridine orange staining technique numerous cytoplasmic outpouchings brilliantly stained for RNA were observed at the walls of infected cells after incubation overnight (Fig. 1). Similar blebs filled with antigen were detected at this stage by the fluorescent antibody technique (Fig. 2). Release was observed in at least 50% of the cells still attached to the glass in the infected monolayers. By contrast, after overnight incubation at 30” both viral antigen and nucleic acid appeared to be maintained within the cytoplasm of infected cells (Figs. 3 and 4) and most of these cells were still attached to the glass. Electron
Microscopy
The cell shown in ultrathin section in Fig. 5 is a typical monkey kidney cell from an uninfected confluent tissue culture monolayer. In the cytoplasm mitochondria are well defined and the endoplasmic reticulum sparse. The nucleolus is visible. Figure 6 shows a portion of the cytoplasm of a cell from a typical culture 18 hours after infection with poliovirus. Numerous dense masses (DM) with diameters ranging from 0.2 p to as large as 4 p are shown. Some thin sections of cells revealed as many as 50 cross sections of these massesand, although
POLIOVIRUS
FORMATION
327
FIG. 1. Monkey kidney cells 8 hours after inoculation with Brunhilde (type 1) strain virulent poliovirus. Acridine orange technique. Release of RNA material at. the cell walls is evident. Black and white reproduction from color original. Magnification: X 2000. FIG. 2. Monkey kidney cells 8 hours after inoculation with MEF-1 (type 2) virulent poliovirus. Fluorescent antibody technique. Cells are rounded and full of ant,igen. Kel~~asc~ of antigen in “blebs” at the cell walls can be observed. Magnification: X 1000. FIG. 3. Monkey kidney cells 21 hours after inoculation with LSc (type 1) attenaalrcl poliovirus. Culture incubated at 30”. Fluorescent ant,ibodp technique. Antigen is maintained within the cells and unstained nuclei are clear. Magnificat,ion: X 1000. FIG. 4. Monkey kidney cells 21 hours after inoculation with YSK (type 2) atjtenuwtc~d poliovirus. Culture incubated at 30”. Acridine orange technique. Note intense RNA staining at, the cell prriphery and absence of virus rel<~asc~.Black and white reproduction fronr color original. Magnification: X 2000.
they were never encountered in cells which contained typical crystalline arrays of virus particles, they were often observed in the same electron microscopic field and appeared to be related to the infection. Their presence served as a useful indicator in de-
tiding whether a particular preparation warranted an exhaustive search for virus particles. Similar cytoplasmic masses were observed by Ntiiiee-Montiel and Weibel (1960) in monkey kidney cells infected with ECHO 19 virus, but these often oc-
328
MAYOR
FIG. 5. Ultrathin
JORDAN
section of normal monkey kidney cell. In the nucleus (N) a nuclec ,lus Double nuclear membrane (NIV), mitochondria (Mi), and plasma mc:mare clearly defined. Endoplasmic reticulum (ER) is sparse. Magnificatil 0Il:
(Aic) is evident. br: ane (PM) X 20,000.
AND
POLIOVlRUS
FORMATION
FIG. 6. Ultrathin section of portion of the cytoplasm of a monkey kidney cell from n culture 18 hours after inoculation with Brunhilde (type 1) strain of poliovirus. Numerous dense masses (DM) and vacuoles (Vn) are evident, ;?lo virus particles c-an he olw~~vt~l. Magnification : X 33,000.
::2. 9
MAYOR
FIG. culture crystals particle chondria
AND
JORDAN
7. Ultrathin section of portion of the cytoplasm of a monkey kidney cell from a 18 hours after inoculation with LSc (type 1) attenuated poliovirus. Two large virus (VC) with a clear crystal interface (CZ) are clearly discerned. The center-to-center spacing is approximately 25 mN. Numerous vacuoles (Vu) and well-preserved mito(Mi) are present. Magnification: X 44,000.
curred in close proximity to arrays of virus particles. We have occasionally observed a few similar masses in cells from supposedly uninfected cultures, but in very low concentration. Figure 7 shows a portion of the cytoplasm from an infected cell containing a large
well-developed viral inclusion. It appears to consist of two crystalline arrays in close juxtaposition and a crystal interface (CI) is cleary visible. The upper crystal (VC) exhibits a hexagonal profile. The center-tocenter particle spacing is 22-25 rnp, in good agreement with the figure obtained by
POLIOVIRUS
Stuart and Fogh (1959) for poliovirus in FL cells. Numerous cross sections of mitochondria are present and these appear to be very electron dense. An association between viral arrays and endoplasmic reticulum is shown in Fig. 8, where the virus particles appear to follow closely the contours of a cytoplasmic vacuole (T’a) . This was quite a common observation in both virulent and attenuated strains. Although release of poliovirus is definitely inhibited by growth poliovirus particles at. 30”, extracellular were occasionally observed in a few specimens. The virus particles were usually associated with a membrane component (M), possibly a portion of the endoplasmic reticulum, or were contained within a limiting membrane to form a “bleb” (VI?) (Figs. 9 and 10). Numerous mitochondria with welldefined cristae were observed in extracellular space whenever virus blebs were encountered (Figs. 9 and 10). Cytoplasmic arrays of poliovirus particles have been detected in monkey kidney cells infected with both virulent and attenuated strains of the virus. Characteristic particles were observed solely in the cytoplasm or in extracellular space of specimens incubated at 30”. never within the nucleus of these cells or in control preparations. Virus particles were not located in specimens incubated at 37” throughout the whole growth cycle. The crystalline arrays appear similar to those observed by Stuart and Fogh (1959: 1961) and the particle diameters are in good agreement with their figures, and with t,he known diameter of poliovirus (Taylor and McCormick, 1956). Stuart and Fogh observed that mitochondria became fewer with time after infection and could not identify any mitochondria in polioinfected FL cells at the stage when virus particles appeared. We found, however, numerous well-preserved mitoehondria in cells containing virus particle arrays (Fig. 7) and many in extracellular space whenever virus release was observed (Figs. 9 and 10). It is possible that the use of epoxy resin plays some part in this finding, as it is generally
FORMATIOS
:i:s1
agreed that epoxies are superior to acrylic resins for preserving fine structural detail. The possible association of virus particles with the endoplasmic reticulum, both intracellularly (Fig. 8) and extracellularly (Figs. 9 and lo), may give a clue as to their origin and is in agreement with the findings of Stuart and Fogh (1961). Rifkind et al. (1961) in a study of the structure and development of ECHO 9 virus observed numerous cytoplasmic protrusions containing virus particles shed into the culture medium. Our results are in accordance with their speculations that virus release occurs through a weakening of the plasma membrane and subsequent discharge of cytoplasmic organelles and virus particles. Our studies indicate that the control of virus release is a very important factor in being able to locate intracellular poliovirus particles in monkey kidney cells, Release can be controlled to a certain extent kinetically during the growth cycle by growing the virus at lower temperatures, thus inhibiting release while maintaining a high intracellular titer of virus. In addition, at 30” a greater proportion of infected cells remain attached to the glass than in cultures maintaincd at 37”, making it possible with our embedding techniques to deal with a more uniform population and greater numbers of infected cells, known from cytochomical and immunofluoresccnt techniques to contain virus particles. Stuart and Fogh (personal communication) worked solely with ~11s free in tile medium, a fact that could account’ for their failure to find virus particles in monkey kidney cells as most, of these cells had probably released their intracellular virus by &is time. Virus rclcasc may also be controlled during t’he preparation of specimens for the electron microscope by using gentle embedding and polymerizing procedures such as are possible with the epoxy resins. The continued appearance of numerous well-preserved mitochondria in our preparations indicate a reasonable level of preservation of fine structural details. These studies suggest that in such viruscell systems whcrc the detection of characteristic virus particles has so far cludcd investigators, that the release phenomenon be
332
MAYOR
AND JORDAN
POLIOVIRUS
studied first, perhaps with the aid of cytochemical and immunofluorescent techniques, and that when this can be controlled preference be given to epoxy resins over the acrylic variety as embedding media. ACKNOWLEDGMENT We are grateful to Dr. Joseph L. Melnick for his continued interest and encouragement. We wish to thank Mr. Craig Wallis for tissue culture work and Mr. Richard Jamison for help in preparing the plates. Electron microscope facilities were generously made available by Dr. Martin Catoni at the University of Texas Dental Branch. REFERENCES FOGH, J. (1961). Filamentous organization of poliovirus particles. Virology 14, 495-497. FOGH, J., and STUART, D. C., JR. (1960). Intracellular crystals of polioviruses in HeLa cells. Virology 11, 308-311. HOWES, D. W., and MELNICK, J. L. (1957). The growth cycle of poliovirus in monkey kidney cells. I. Maturation and release of virus in monolayer cultures. Virology 4, 97-108. LUFT, L. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol.
9, 409414. MAYOR, H. D. (1961). Cytochemical and fluorescent antibody studies on the growth of poliovirus in tissue culture. Texas Repts. Biol. and Med. 19, 106-122. MELSICK, J. L. (1956). Tissue culture methods for
FORMATION
3x1
the cultivation of poliomyelitis and other viruses. In “Diagnostic Procedures for Virus and Rickettsial Diseases,” 2nd ed., pp. 97-152. American Public Health Association. MELNICK, J. L., HSIUNG, G. D., RAPPAPORT, C., HOWES, D., and REISSIG, M. (1957). Factors influencing the proliferation of viruses. Texas Repts. Biol. and Med. 15, 496-533. NELSOX, E., HAGER, H., and Kovacs, E. (1969). Intracellular virus crystals in the central nervous system of mice following rodent polioencephalitis (MM strain) infection. J. Biophys. Biochem. Cytol. 8, 825-828. NGREz-MONTIEL, and WEIBEL, J. (1960). Electron microscope study of ECHO 19 virus infection in monkey kidney cells. J. Biophys. Biochem. Cytol. 8, 29-295. RIFKIND, R. A., GODMAN, G. C., HOWE, C., MORGAX, C., and ROSE, H. M. (1961). Structure and development of viruses as observed in the electron microscope. VI. ECHO viruses, type 9. J. Exptl. Med. 114, 1-11. ROIZMAN, B. (1959). Preparation of high potenq poliovirus in FL cell cultures at room temperat,ure. Proc. Sot. Exptl. Biol. Med. 101, 41&411. STUART, D. C., JR., and FOGH, J. (1959). Electron microscopic demonstration of intracellular poliovirus crystals. Exptl. Cell Research 18, 378-381. STUART, D. C., JR., and FOGH, J. (1961). Micromorphology of FL cells infected with polio and 13, 177-190. Coxsackie viruses. Virology TAYLOR, A. R. and MCCORDIICK, M. J. (1956). Electron microscopy of poliomyelitis virus. I’nle J Biol. and Med. 28, 589-597.
FIG. 8. Ultrathin section showing portion of a cytoplasmic array of poliovirus particles (VC) grouped around a vacuole (Vu). Magnification: X 55,999. FIG. 9. Ultrathin section showing virus particles and cell organelles in extracellular space. Numerous virus particles (VP) are observed either contained within a “bleb” (VB) or associated with a membrane component (M). Mitochondria (Mi) and vacuoles (Vu) are present outside the plasma membrane (PM). Magnification: X 35,009. FIG. 10. Ultrathin section again showing virus particles (VP), membrane components (M), mitochondria (Mi), and vacuoles (Vu), in extracellular space. A large bleb (VB) containing many virus particles is clearly evident. Magnification: X 55.000.
16, 325-333 (1962)
Formation
of Poliovirus HEATHER
Department
in Monkey
DONALD MAYOR
of Virology
Kidney AND
Cells’
LIANE E. JORDAN
and Epidemiology, Baylor Medicine, Houston, Texas Accepted
Tissue Culture
University
College
of
November SO, 1961
Cytochemical and fluorescent antibody techniques have been utilized to study the release of poliovirus. Electron microscopic studies of ultrathin sections of epoxyembedded monkey kidney cells infected with poliovirus (types 1 and 2 virulent and attenuated) have revealed cytoplasmic arrays of spherical particles each approximately 25 rnp in diameter. Similar particles were found occasionally in “blebs” at the plasma membrane. Particles were detected only when infected cultures were incubated at 30°C. At this temperature fully infective poliovirus is formed but its release is inhibited. Particles were not encountered in infected cultures incubated at 37” or in control preparations. INTRODUCTION
Poliovirus particles have been demonstrated in ultrathin sections of cultured human cells infected with the virulent type 1 Brunhilde strain, occurring in the cytoplasm either as crystalline arrays (Stuart and Fogh, 1959,196l; Fogh and Stuart, 1960) or inlaid within filamentous protein material (Fogh, 1961). Characteristic virus particles were found only after inoculation with this highly virulent strain. Other members of the enterovirus group, for example, ECHO type 19 (Nuiiez-Montiel and Weibel, 1960), ECHO type 9 (Rifkind et al., 1961)) and ECHO type 4 (It. Jamison, unpublished) have been detected in infected monkey kidney tissue cultures by thin-sectioning techniques, yet poliovirus particles have not been previously demonstrated in monkey kidney (MK) tissue culture even though this is the host cell of choice for routine propagation of the virus. In all reported attempts to locate poliovirus particles in MK tissue cultures using the electron microscope, procedures utilizing methacrylate resin have been employed, and the vagaries of explosion artifact and ‘Aided tion.
by a grant from the National
polymerization damage with this material are well known. We decided to use an epoxy resin and a very brief dehydration and embedding schedule in an attempt to protect the tissue culture cells from undue extraction of fine structural components and to subject them to a minimum of polymerization damage. Howes and Melnick (1957) first showed that poliovirus multiplication is characterized by an accumulation of intracellular virus which is soon released. Roisman (1959) demonstrated that when poliovirus is grown at 30” the release is inhibited. Cytochemical and fluorescent antibody techniques have proved to be useful tools for demonstrating the release phenomenon and studying its control (Mayor, 1961). It seemed worth while, therefore, to integrate these results with studies at the fine structural level ; to this end the infected tissue cultures were incubated at 30” before searching for intracellular poliovirus particles with the electron microscope. The results of these experiments are reported in this paper. MATERIALS
Tissue
Founda-
epithelial 325
AND METHODS
Trypsin-dispersed MK cells were grown in 16-ounce
culture.
326
MAYOR
AND JORDAN
bottles using methods and the lactalbumin hydrolyzate medium described by Melnick (1956j. Virus. The following poliovirus strains were used: type 1, Brunhilde (virulent), LSc (attenuated) ; type 2, MEF-1 (virulent), YSK (attenuated). When the cell monolayers formed a confluent sheet, usually within a week, the cells were washed twice with fresh medium and inoculated with 1 ml of a virus suspension containing approximately lo7 PFU/ml. As there are approximately 10’ cells per monolayer in a 16-ounce bottle, these inoculating conditions corresponded to an approximate input multiplicity of one. After an adsorption time of approximately 1% hours at 37” the virus inoculum was decanted and 10 ml of growth medium containing cysteine (Melnick et al., 1957) was added. The infected cultures were incubated at 37” for an additional 2 hours and were then transferred to a 30” incubator for an additional 15 hours (overnight). Although most of the cells in the monolayer were still attached to the glass, an advanced cytopathic effect was observed in at least 75% of them. Cultures were usually chilled at 4” for 1 or 2 hours prior to fixation for electron microscopy in order to aid possible aggregation of virus particles to form crystalline arrays and were processed for ultrathin sectioning as a monolayer according to the following schedule. Fixdon: 10 minutes in 1% buffered osmium tetroxide pH 7.8 in the cold. Dehydration: 35% ethyl alcohol, 5 minutes; 50% ethyl alcohol, 3 minutes; 70% ethyl alcohol, 3 minutes; 95% ethyl alcohol, 3 minutes; absolute ethyl alcohol, 5 minutes; 0.1% phosphotungstic acid in absolute ethyl alcohol, 10 minutes; 2 changes of propylene oxide, each 10 minutes ; 50: 50 mixture propylene oxide and CIBA 502 araldite resin, 11/2-2 hours. At this stage, the cells were harvested with a rubber policeman and centrifuged at low speed for 5-10 minutes; the resulting pellets were then embedded in the epoxy resin with DMPs02 as accelerator according to Luft’s procedure (1961). Ultrathin sec’ 2 , 4 , 6 - Tri (dimethylaminomethyl) phenol Rohm and Haas Co., Philadelphia, Pemuylvania.
tions were cut with a SIROFLEX ultramicrotome and examined in an RCA EMU 3D and a Siemens Elmiskop 1. Cytochemical and immunofluorescent techniques. Tissue cultures were grown on
11 x 22 mm coverslips in Leighton tubes and infected with poliovirus in an identical manner to that described (Mayor, 1961). Processing techniques for acridine orange and fluorescent antibody staining and for ultraviolet microscopy and photography are also given in full in this paper. Incubation times and temperatures were the same as for the tissue cultures destined for electron microscopy described above. RESULTS
Cytochemistry
and Immunofluorescence
The correlated cytochemical and immunofluorescent studies indicated typical release of poliovirus at 37”. With the acridine orange staining technique numerous cytoplasmic outpouchings brilliantly stained for RNA were observed at the walls of infected cells after incubation overnight (Fig. 1). Similar blebs filled with antigen were detected at this stage by the fluorescent antibody technique (Fig. 2). Release was observed in at least 50% of the cells still attached to the glass in the infected monolayers. By contrast, after overnight incubation at 30” both viral antigen and nucleic acid appeared to be maintained within the cytoplasm of infected cells (Figs. 3 and 4) and most of these cells were still attached to the glass. Electron
Microscopy
The cell shown in ultrathin section in Fig. 5 is a typical monkey kidney cell from an uninfected confluent tissue culture monolayer. In the cytoplasm mitochondria are well defined and the endoplasmic reticulum sparse. The nucleolus is visible. Figure 6 shows a portion of the cytoplasm of a cell from a typical culture 18 hours after infection with poliovirus. Numerous dense masses (DM) with diameters ranging from 0.2 p to as large as 4 p are shown. Some thin sections of cells revealed as many as 50 cross sections of these massesand, although
POLIOVIRUS
FORMATION
327
FIG. 1. Monkey kidney cells 8 hours after inoculation with Brunhilde (type 1) strain virulent poliovirus. Acridine orange technique. Release of RNA material at. the cell walls is evident. Black and white reproduction from color original. Magnification: X 2000. FIG. 2. Monkey kidney cells 8 hours after inoculation with MEF-1 (type 2) virulent poliovirus. Fluorescent antibody technique. Cells are rounded and full of ant,igen. Kel~~asc~ of antigen in “blebs” at the cell walls can be observed. Magnification: X 1000. FIG. 3. Monkey kidney cells 21 hours after inoculation with LSc (type 1) attenaalrcl poliovirus. Culture incubated at 30”. Fluorescent ant,ibodp technique. Antigen is maintained within the cells and unstained nuclei are clear. Magnificat,ion: X 1000. FIG. 4. Monkey kidney cells 21 hours after inoculation with YSK (type 2) atjtenuwtc~d poliovirus. Culture incubated at 30”. Acridine orange technique. Note intense RNA staining at, the cell prriphery and absence of virus rel<~asc~.Black and white reproduction fronr color original. Magnification: X 2000.
they were never encountered in cells which contained typical crystalline arrays of virus particles, they were often observed in the same electron microscopic field and appeared to be related to the infection. Their presence served as a useful indicator in de-
tiding whether a particular preparation warranted an exhaustive search for virus particles. Similar cytoplasmic masses were observed by Ntiiiee-Montiel and Weibel (1960) in monkey kidney cells infected with ECHO 19 virus, but these often oc-
328
MAYOR
FIG. 5. Ultrathin
JORDAN
section of normal monkey kidney cell. In the nucleus (N) a nuclec ,lus Double nuclear membrane (NIV), mitochondria (Mi), and plasma mc:mare clearly defined. Endoplasmic reticulum (ER) is sparse. Magnificatil 0Il:
(Aic) is evident. br: ane (PM) X 20,000.
AND
POLIOVlRUS
FORMATION
FIG. 6. Ultrathin section of portion of the cytoplasm of a monkey kidney cell from n culture 18 hours after inoculation with Brunhilde (type 1) strain of poliovirus. Numerous dense masses (DM) and vacuoles (Vn) are evident, ;?lo virus particles c-an he olw~~vt~l. Magnification : X 33,000.
::2. 9
MAYOR
FIG. culture crystals particle chondria
AND
JORDAN
7. Ultrathin section of portion of the cytoplasm of a monkey kidney cell from a 18 hours after inoculation with LSc (type 1) attenuated poliovirus. Two large virus (VC) with a clear crystal interface (CZ) are clearly discerned. The center-to-center spacing is approximately 25 mN. Numerous vacuoles (Vu) and well-preserved mito(Mi) are present. Magnification: X 44,000.
curred in close proximity to arrays of virus particles. We have occasionally observed a few similar masses in cells from supposedly uninfected cultures, but in very low concentration. Figure 7 shows a portion of the cytoplasm from an infected cell containing a large
well-developed viral inclusion. It appears to consist of two crystalline arrays in close juxtaposition and a crystal interface (CI) is cleary visible. The upper crystal (VC) exhibits a hexagonal profile. The center-tocenter particle spacing is 22-25 rnp, in good agreement with the figure obtained by
POLIOVIRUS
Stuart and Fogh (1959) for poliovirus in FL cells. Numerous cross sections of mitochondria are present and these appear to be very electron dense. An association between viral arrays and endoplasmic reticulum is shown in Fig. 8, where the virus particles appear to follow closely the contours of a cytoplasmic vacuole (T’a) . This was quite a common observation in both virulent and attenuated strains. Although release of poliovirus is definitely inhibited by growth poliovirus particles at. 30”, extracellular were occasionally observed in a few specimens. The virus particles were usually associated with a membrane component (M), possibly a portion of the endoplasmic reticulum, or were contained within a limiting membrane to form a “bleb” (VI?) (Figs. 9 and 10). Numerous mitochondria with welldefined cristae were observed in extracellular space whenever virus blebs were encountered (Figs. 9 and 10). Cytoplasmic arrays of poliovirus particles have been detected in monkey kidney cells infected with both virulent and attenuated strains of the virus. Characteristic particles were observed solely in the cytoplasm or in extracellular space of specimens incubated at 30”. never within the nucleus of these cells or in control preparations. Virus particles were not located in specimens incubated at 37” throughout the whole growth cycle. The crystalline arrays appear similar to those observed by Stuart and Fogh (1959: 1961) and the particle diameters are in good agreement with their figures, and with t,he known diameter of poliovirus (Taylor and McCormick, 1956). Stuart and Fogh observed that mitochondria became fewer with time after infection and could not identify any mitochondria in polioinfected FL cells at the stage when virus particles appeared. We found, however, numerous well-preserved mitoehondria in cells containing virus particle arrays (Fig. 7) and many in extracellular space whenever virus release was observed (Figs. 9 and 10). It is possible that the use of epoxy resin plays some part in this finding, as it is generally
FORMATIOS
:i:s1
agreed that epoxies are superior to acrylic resins for preserving fine structural detail. The possible association of virus particles with the endoplasmic reticulum, both intracellularly (Fig. 8) and extracellularly (Figs. 9 and lo), may give a clue as to their origin and is in agreement with the findings of Stuart and Fogh (1961). Rifkind et al. (1961) in a study of the structure and development of ECHO 9 virus observed numerous cytoplasmic protrusions containing virus particles shed into the culture medium. Our results are in accordance with their speculations that virus release occurs through a weakening of the plasma membrane and subsequent discharge of cytoplasmic organelles and virus particles. Our studies indicate that the control of virus release is a very important factor in being able to locate intracellular poliovirus particles in monkey kidney cells, Release can be controlled to a certain extent kinetically during the growth cycle by growing the virus at lower temperatures, thus inhibiting release while maintaining a high intracellular titer of virus. In addition, at 30” a greater proportion of infected cells remain attached to the glass than in cultures maintaincd at 37”, making it possible with our embedding techniques to deal with a more uniform population and greater numbers of infected cells, known from cytochomical and immunofluoresccnt techniques to contain virus particles. Stuart and Fogh (personal communication) worked solely with ~11s free in tile medium, a fact that could account’ for their failure to find virus particles in monkey kidney cells as most, of these cells had probably released their intracellular virus by &is time. Virus rclcasc may also be controlled during t’he preparation of specimens for the electron microscope by using gentle embedding and polymerizing procedures such as are possible with the epoxy resins. The continued appearance of numerous well-preserved mitochondria in our preparations indicate a reasonable level of preservation of fine structural details. These studies suggest that in such viruscell systems whcrc the detection of characteristic virus particles has so far cludcd investigators, that the release phenomenon be
332
MAYOR
AND JORDAN
POLIOVIRUS
studied first, perhaps with the aid of cytochemical and immunofluorescent techniques, and that when this can be controlled preference be given to epoxy resins over the acrylic variety as embedding media. ACKNOWLEDGMENT We are grateful to Dr. Joseph L. Melnick for his continued interest and encouragement. We wish to thank Mr. Craig Wallis for tissue culture work and Mr. Richard Jamison for help in preparing the plates. Electron microscope facilities were generously made available by Dr. Martin Catoni at the University of Texas Dental Branch. REFERENCES FOGH, J. (1961). Filamentous organization of poliovirus particles. Virology 14, 495-497. FOGH, J., and STUART, D. C., JR. (1960). Intracellular crystals of polioviruses in HeLa cells. Virology 11, 308-311. HOWES, D. W., and MELNICK, J. L. (1957). The growth cycle of poliovirus in monkey kidney cells. I. Maturation and release of virus in monolayer cultures. Virology 4, 97-108. LUFT, L. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol.
9, 409414. MAYOR, H. D. (1961). Cytochemical and fluorescent antibody studies on the growth of poliovirus in tissue culture. Texas Repts. Biol. and Med. 19, 106-122. MELSICK, J. L. (1956). Tissue culture methods for
FORMATION
3x1
the cultivation of poliomyelitis and other viruses. In “Diagnostic Procedures for Virus and Rickettsial Diseases,” 2nd ed., pp. 97-152. American Public Health Association. MELNICK, J. L., HSIUNG, G. D., RAPPAPORT, C., HOWES, D., and REISSIG, M. (1957). Factors influencing the proliferation of viruses. Texas Repts. Biol. and Med. 15, 496-533. NELSOX, E., HAGER, H., and Kovacs, E. (1969). Intracellular virus crystals in the central nervous system of mice following rodent polioencephalitis (MM strain) infection. J. Biophys. Biochem. Cytol. 8, 825-828. NGREz-MONTIEL, and WEIBEL, J. (1960). Electron microscope study of ECHO 19 virus infection in monkey kidney cells. J. Biophys. Biochem. Cytol. 8, 29-295. RIFKIND, R. A., GODMAN, G. C., HOWE, C., MORGAX, C., and ROSE, H. M. (1961). Structure and development of viruses as observed in the electron microscope. VI. ECHO viruses, type 9. J. Exptl. Med. 114, 1-11. ROIZMAN, B. (1959). Preparation of high potenq poliovirus in FL cell cultures at room temperat,ure. Proc. Sot. Exptl. Biol. Med. 101, 41&411. STUART, D. C., JR., and FOGH, J. (1959). Electron microscopic demonstration of intracellular poliovirus crystals. Exptl. Cell Research 18, 378-381. STUART, D. C., JR., and FOGH, J. (1961). Micromorphology of FL cells infected with polio and 13, 177-190. Coxsackie viruses. Virology TAYLOR, A. R. and MCCORDIICK, M. J. (1956). Electron microscopy of poliomyelitis virus. I’nle J Biol. and Med. 28, 589-597.
FIG. 8. Ultrathin section showing portion of a cytoplasmic array of poliovirus particles (VC) grouped around a vacuole (Vu). Magnification: X 55,999. FIG. 9. Ultrathin section showing virus particles and cell organelles in extracellular space. Numerous virus particles (VP) are observed either contained within a “bleb” (VB) or associated with a membrane component (M). Mitochondria (Mi) and vacuoles (Vu) are present outside the plasma membrane (PM). Magnification: X 35,009. FIG. 10. Ultrathin section again showing virus particles (VP), membrane components (M), mitochondria (Mi), and vacuoles (Vu), in extracellular space. A large bleb (VB) containing many virus particles is clearly evident. Magnification: X 55.000.