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GAL virus: Its growth cycle in tissue culture and some of its properties
VIROLOGY
31.5322 (1961)
13,
GAL
Virus:
Its Growth Some
GEORGE
Viral
and
Cycle
in Tissue
Culture
of Its Properties
R. SHARPLESS, SEYMOUR LEVINE, AND MARY E. ENGLERT Rickettsial
and
Biochemistry
American CyanamidCompany, Accepted
and
M. C. DAVIES,
Research Sections, Lederle Laboratories, Pearl River, New York
November
30, 1960
The GAL virus growth cycle hasbeendeterminedby plaqueassayand correlated with electronmicroscopeobservationsof infected cells. The virus was found to have a longlatent periodandgrowth cycle,to be producedonly within the cellnucleus,andto accumulatethere. It alsowasfound to be polyhedralin shapeand ether stableand to resemble adenovirus in its intranuclear site of development, its morphology, and its ether stability. Data concerning its stability under various conditions of storage are included. INTRODUCTION
A virus isolated in tissue cultures from filtrates of chicken livers infected with the RPL-12 strain of lymphomatosis, and referred to in a number of reports as the lymphomatosis virus (Fontes et al., 1958; Sharpless et al., 1958; Levine and Sharpless, 1959; Stoker, 1959; Buthala, 1960; Defendi and Sharpless, 1958; Davies et ah, 1959; Defendi and Kritchevsky, 1960), has been named gallus adeno-!ike (GAL) virus (Burmester et al., 1960). GAL virus had been thought to be related to lymphomatosis because earlier studies showed that (1) the disease was produced by dilutions of the virus far exceeding the known infectivity of the original material-eighth to fourteenth tissue culture passages; (2) the virus was recovered from diseasedbirds; and (3) it was neutralized by antiserum known to neutralize lymphomatosis virus. However, later studies showed that (a) GAL antiserum did not neutralize lymphomatosis virus; (b) subsequent tissue-culture passages and plaque isolates of GAL virus did not induce lymphomatosis in susceptible chicks, and (,c) progeny of GAL-immunized dams were as susceptible to lymphomatosis as were
progeny from nonimmunized dams (Burmester et al., 1960). This report on GAL virus describesits development within the cell and its morphology, as determined by plaque assay or by electron microscopy, and its stability in ether and at various storage temperatures. MATERIALS
SND
METHODS
Stock virus was propagated in chick embryo liver monolayers in Medium 199 (Morgan et al., 1950) and harvested after 5 days of incubation. One of the plaque methods used for assay utilized trypsinized preparations of g-day-old chick embryos suspended in an agar medium containing 5% cow serum (Levine and Sharpless, 1959). The other was a modification of the procedure reported by Stoker (1959) adapted to permit development of macroscopic plaques. With the modified procedure, monolayers were washed once with 3 ml phosphatebuffered saline (PBS), and to each was added an appropriate virus dilution in 1.0 ml Eagle’s medium (1955) enriched with 3% cow serum (CSE) . An attachment period of 2 hours at 37” in air containing 4% COZ was adopted as a result of the experiment summarized in Fig. 1.
315
316
SHARPLESS,
4 1.0.I0
1
Auachment
2
Perxsd
3
LEVINE,
DAVIES,
4
before (Hours)
Addttion
of Aqar.
FIG. 1. Demonstration of attachment period required for maximum number of plaques of GAL virus on chick embryo liver monolayers. Virus was added to each of 20 monolayers as described in the text and the plates were placed at 37”. At each of the indicated times, 5 plates were overlaid with the agar medium without removing the virus.
AND
ENGLERT
after virus infection, plates were stained with 1: 12,000 neutral red. For electron microscopy, cell layers were washed with isotonic saline at, 37”, fixed with 1.0% osmic acid buffered at pH 7.3, and dehydrated with graded concentrations of alcohol. For solvent changes, decantation was aided by low speed centrifugation. Cells were embedded in 75% butyl, 25% methyl methacrylate polymerized by heating at at 65-70” overnight. Embedded cells were cut into sections with a Porter-Blum ultramicrotome, using either glass or diamond knives, and picked up on carbon-filmed 200-mesh copper grids for examination in a microscope (RCA EMU-3C) equipped with a 50-p objective aperture. Photomicrographs were taken at instrument magnifications of 300&12,000 and enlarged photographically. To reveal virus ultrastructure more clearly, some mounted sections were stained with phosphotungstic acid (PTA). Grids were floated face down for 15-30 minutes on a solution of 2% PTA in 40% alcohol and rinsed once with water. EXPERIMENTAL
Growth
FIG.
chick
2. One-step embryo liver
growth curve monolayers.
for
GAL
virus
in
After attachment, each plate was overlaid with 3 ml CSE containing 0.9% agar washed according to the method of Dulbecco and Vogt (1954) and incubated at, 37”. After 34 days a second layer of the same agar medium was added. Seven days
Cycle Determined
by Plaque Assay
The purpose of the first experiment was to determine the duration of a single cycle of virus growth on chick embryo liver monolayers. Three monolayers were each exposed to 3.5 x lo8 plaque-forming units (PFU) of virus in CSE. The plates were incubated at room temperature 3 hours to permit virus to attach, washed twice with 3 ml PBS, overlaid with 4 ml CSE, and then incubated at 37”. Whole chick embryo cells suspended in agar were used to assay the medium for virus content. The various times at which medium was removed for assay and replaced with fresh virus-growth medium are shown in Fig. 2, a curve representing cumulative production of virus. The assumption that a single cycle of infection is represented is justified by the high multiplicity of the virus added (greater than 50) and by the long attachment-period. The outstanding feature of the cycle appears to be its long duration. The latent
GAL
VIRUS
GROWTH
period was more than 20 hours and the entire cycle required as many as 72 hours. The next experiment was designed to determine the relationship between the extracellular and cell-associated virus. Chick embryo liver monolayers were exposed to 7 x lo* PFU of virus, allowed 2 hours at room temperature for attachment, washed twice with 3 ml PBS, and overlaid with 4 ml CSE. A count at the time of infection indicated that representative monolayers contained 6.0 x 10B cells per plate. In Fig. 3, the points represent the times when the medium from a single plate was removed for assay and the monolayer disrupted by
CYCLE
freezing and thawing once in 2 ml distilled water. Each of these samples was assayed on chick embryo liver monolayers and the data obtained were plotted as virus yield per plate versus time. The dotted line represents the average cell-count per plate at time of infection. Again the growth cycle was a long one and the latent period, as shown by the curve for the yield of cell-associated virus, was prolonged. Virus first appeared some time after 16 hours and then increased precipitously. No extracellular virus appeared until 20 hours after infection. The unquestionable accumulation of virus within
1o'O
10’
Av.Number of --w--------------‘--------cell*,piatRs. -ece ll* ,piatRs.
A cell associated aseociatcd virus 0 extracellular tit-us
10'
FIG.
3. Growth
317
cycle of GAL virus in chick embryo liver monolayers.
318
SHARPLESS,
LEVINE,
the cell also is evident from the curve. At 30 hours the concentration of extracellular virus was only 3% of that associated with the cell. Results of other experiments were essentially similar. About 16 hours after infection cell-associated virus was present. Extracellular virus appeared between 20 and 30 hours after infection and amounted to only a small fraction of the cell-associated virus present at the time. Electron Microscopy Growth Cycle
of Stages of the Virus
For these experiments, chick embryo liver cultures were prepared in serum bottles as previously described (Davis and Sharpless, 1959) and were incubated for 48 hours at 37”. Then the growth was replaced with Medium 199. Eight of the bottles were inoculated, each receiving about lo8 tissue culture-infectious doses of virus. (Since this approximates 5-10 infectious doses per cell, observed results can be assumed to represent a single cycle of infection.) At each 6-hour interval after infection, one bottle was processed for electron microscopy as described under Methods. Figure 4 shows a portion of a representative culture of uninfected chick embryo liver cells. In such cultures nucleoli are quite large and usually single, mitochondria vary considerably in size and shape, the endoplasmic reticulum is abundant throughout the cytoplasm, and lipid droplets are sometimes apparent. Signs of infection were first seen within 16 to 20 hours. Figures 5 and 6 are cells 18 hours after infection. In their nuclei are formations typically associated with the beginning of virus synthesis. Most often these formations occurred as a ring or partial ring. During the next few hours, the intranuclear formations enlarged rapidly and the nucleus became distorted and expanded to occupy a progressively larger portion of the cell. Between 20 and 30 hours after infection, rows of virus particles began to appear within the nucleus. An example of this stage of infection is shown in Fig. 7. The nuclear membrane is much distorted, and cyto-
DAVIES,
AND
ENGLERT
plasmic structures are undergoing necrotic changes. These changes were usually accompanied by the appearance at the periphery of the nucleus of a virus-free mass which retained its identity until late stages of infection. For the next 18 to 24 hours active synthesis of virus continued, accompanied by continued enlargement of the nucleus and organization of the virus particles. Figure 8 shows a typical nucleus 42 hours after infection. Although the nuclear membrane is still identifiable, viral arrays occupy most of the nucleus and the entire cell obviously is undergoing necrosis. By 48 hours after infection (Fig. 9) the cytoplasm was reduced to debris, t,he nucleus was entirely replaced by virus, and the three-dimensional packing of virus particles in lattices of several different orientations was clearly evident. Part of a viral matrix is shown at high magnification in Fig. IO. To accentuate virus ultrastructure, sections were stained with PTA. Intranuclear particles averaged 86 my in diameter, with a centroid about 44 rnp in diameter. Both centroid and complet,e particle showed an electron-dense outer layer. The particles in the intranuclear arrays appeared hexagonal. These hexagonal cross sections led us to re-examine photographs of the virus in spray droplets (Davies and Sharpless, 1959). Most of those particles appeared spherical, but a significant number were found so oriented that a polyhedral shape could be seen. Figure 11 shows such a particle. By negative staining, the particles were subsequently found t.o be icosahedral (Davies and Englert, 1961). Stability
of the Virus
Franklin (1958) suggested that the ether stability of a mature virus particle is related to the virus maturation mechanism, ether-sensitive viruses maturing only at the membrane of the host cell and being continuously released, ether-stable viruses maturing within the cell until released en masse. Growth curves and electron micrographs showed that GAL virus develops in the nucleus and accumulates within the cell. To see whether it fitted Franklin’s hypothe-
GAL
VIRUS
GROWTH
CYCLE
4. Portion of uninfected chick embryo liver cell layer; cm, cell membrane; nm, numembrane ; n, nucleolus; m, mitochondria. FIG. 5. Nucleus of chick embryo liver cell 18 hours after infection with GAL virus. A typical intranuclear formation associated with infection is shown. FIG. 6. Nucleus of chick embryo liver cell 18 hours after infection with GAL virus showing ring shape of intranuclear mass-a formation that occurred frequently. FIG. 7. Nucleus and portion of cytoplasm of chick embryo liver cell 30 hours after infection with GAL virus. Virus particles are beginning to array themselves within nucleus. Mitochondria, as shown at upper right, are degenerating. Arrow indicates nuclear peripheral mass typical of this stage of infection and believed to be reoriented nucleolar material. FIG.
clear
SHARPLESS,
LEVINE,
DAVIES,
AND
ENGLERT
FIG. 8. Chick embryo liver cell 42 hours after infection with GAL virus. Viral arrays almor 3t m fill nucleus. Nuclear membrane is still intact and peripheral mass still visible. Cytoplas shows advanced necrosis. FIG. 9. Chick embryo liver cell 48 hours after infection with GAL virus, the terminal stag :e. Nucleus is filled with crystal-like virus arrays in several orientations. Nuclear membrane is breaking down. Only fragments of cytoplasm remain. FIG. 10. Higher magnification of part of intranuclear array of GAL virus 48 hours after il IIfection of chick embryo liver cell, showing hexagonal cross sections of viruses and, where a central section was obtained, the internal structure. FIG. 11. A single GAL virus in a shadowed spray drop, showing polyhedral shape,
GAL
TABLE ETHER
STABILITY
GAL Control
with
b
GROWTH
321
CYCLE
DISCUSSION
1 OF GAL
Plaque-forming
Material
VIRUS
VIRUS~ units
per ml
Virus
alone
Virus
9.G x 3.1 x
108 108
1.8
a Five cubic centimeters 5 cc of ether and placed b Newcastle disease virus.
of virus overnight
+ ether
x 103
108
were mixed at 5”.
sis, its ether stability was determined. For this experiment, an aliquot of virus was shaken with an equal volume of ether and the mixture was capped and refrigerated overnight. An ether-sensitive agent, an allantoic-fluid harvest of Newcastle disease virus, was tested simultaneously as a control. Results, shown in Table 1, indicate that GAL is relatively ether stable. The stability of the virus under various conditions of storage was also ascertained. The titer, as determined by dilution end point assay, dropped from 10Q.5 ID50 of virus per milliliter to 105.5 in 9 days at 37”, 4 months at room temperature, and more than 15 months at 4”.
The most striking characteristics of GAL virus are its prolonged growth cycle and its tendency to accumulate within the cell. Completion of the cycle required a minimum of 48 hours. Extracellular virus as late as 30 hours after infection amounted to only 3-200/o of that in the cells. The correlation between the virus growth curve and observed morphologic changes in infected cells is represented diagrammatically in Fig. 12. The earliest changes in nuclear morphology were seen at about the same time that infectious virus was first detected within the cells (14-18 hours after infection). Electron micrographs show that the virus accumulates in the nucleus and is released only when the nuclear membrane disintegrates. In the growth curve, the first appearance of virus was followed by a period of exceedingly rapid virus synthesis. This was seen in thin sections as the development of an intranuclear crystal-like lattice of virus particles and resembles the observation in cells infected with adenovirus reported by Lagermalm et al. (1957) and Morgan et al. (1960).
A Cell Associated Virus . FreeVirus
I
I
I
I
I 6p Hours
I
FIG.
I 70
12. Correlation
of morphologic
changes
in cell and growth
curves
of GAL
virus.
322
SHARPLESS,
LEVINE,
The electron-microscope observations also correlate well with the light-microscope observations of Defendi and Sharpless (1958). They found intranuclear inclusions that were basophilic and Feulgen positive, and reported that with the appearance of these inclusions the nucleolus became diffuse and shifted toward the nuclear membrane. By electron microscopy these inclusions were seen to be the site of virus synthesis. A mass found by light microscopy at the periphery of the nucleus was assumed to be reoriented nucleolar material because RNAase reversed its pyroninophilic reaction. The nuclear mass consistently seen by electron microscopy near the membrane at 2&30 hours after infection is believed to be the same material. No evidence of virus in the cytoplasm was seen, although Atanasiu and Lepine (1960) reported such findings in thin sections of infected chick embryo liver cells. It is not surprising that for maximum plaque titer, a particle the size of GAL virus must be allowed time for attachment before the addition of agar, since it would not readily diffuse through the agar. The ether stability of the virus supports the hypothesis of Franklin (1958) which relates ether stability to the site of virus maturation and the virus release mechanism. ACKNOWLEDGMENTS The authors William Boss Esther Chasan manuscript.
are grateful to Walter Olson and for technical assistance, and to for help in preparation of the
REFERENCES ATANASIU, P., and LEPINE, P. (1960). Electron microscope morphological studies on a virus (GAL) associated with the RPL-I2 strain of fowl leucosis. Ann. inst. Pasteur 98, 915-919. BURMESTER, B. R., SHARPLESS, G. R., and FONTES, A. K. (1960). Virus isolated from avian lymphomas unrelated to lymphomatosis virus. J. Natl. Cancer Inst. 24, 1443-1447. BUTHALA, D. A. (1960). Plaque formation by visceral lymphomatosis virus in chicken embryo kidney cells. Virology 10,382-384. DAVIES, M. C., and ENGLERT, M. E. (1961). The icosahedral shape of GAL virus. Virology 13, 143-144.
DAVIES,
Ah-D
ENGLERT
DAVIES, M. C., and SHARPLESS, G. R. (1959). Electron microscopy of avian lymphomatosis virus propagated in tissue culture. Cancer Reseurck
19,233-235. DAVIES, M. C., SHARPLESS, G. R., and ENGLERT, M. E. (1959). Avian visceral lymphomatosis virus in tissue culture. Proc. 17th Ann. Meeting Electron Microscope Sot. Am., in J. Applied Physics 30(4), 2025 (Abstract B-7). DEFEXDI, V., and KRITCHEVSKY, D. (1960). A histoautoradiographic study of deoxyribonucleic acid synthesis in tissue cultures of chicken embryo liver cells infected with RPL-12 lymphomatosis virus. J. Biophys. Biochem. Cytol. 7, 201-204. DEFENDI, V., and SHARPLESS, G. R. (1958). The RPL-12 lymphomatosis virus in chicken embryo and in chicken-embryo tissue culture. Morphological and cytochemical observations. J. Natl. Cancer Inst. 21, 925-959. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines of poliomyelitis viruses. J. Exptl. Med. 99, 167-182. EAGLE, H. (1955). Nutritional needs of mammalian cells in tissue culture. Science 122, 501604. FONTES, A. K., BURMESTER, B. R., WALTER, W. G., and ISELER, P. E. (1958). Growth in tissue culture of cytopathogenic agent from strain of virus which produces avian lymphomatosis. Proc. Sot. Exptl. Biol. Med. 97, 854-857. FRANKLIN, R. M. (1958). An hypothesis to explain the relation between the synthesis and release of animal viruses from infected cells and the lipid content of the viruses. Ezperientia 14, 346348. LAGERMALM, G., KJELLEN, L., THORSSON, K. G., and SVEDMYR, A. (1957). Electron microscopy of HeLa cells infected with agents of the adenovirus (APC-RI-ARD) group. Arch. ges. Virusforsch. 7, 221-241. LEVINE, S., and SHARPLESS, G. R. (1959). Plaque formation by lymphomatosis virus. Virology 8,
265-267. MORGAX, C., GODMAN, G. C., BREITENFELD, P. M., and ROSE, H. M. (1960). A correlative study by electron and light microscopy of the development of adeno 5 virus. I. Electron microscopy. J. Exptl. Med. 112, 373-382. MORGAN, J. F., MORTON, H. J., and PARKER, R. C. (1950). Nutrition of animal cells in tissue culture. I. Proc. Sot. Exptl. Biol. Med. 73, l-8. SH.QRPLESS, G. R., DEFENDI, V., and Cox, H. R. (1958). Cultivation in tissue culture of the virus of avian lymphomatosis. Proc. Sot. Exptl. Biol. Med. 97,755-757. STOKER, M. G. P. (1959). Studies on avian l.vmphoma virus in tissue culture. Virology 8, 250-261.
31.5322 (1961)
13,
GAL
Virus:
Its Growth Some
GEORGE
Viral
and
Cycle
in Tissue
Culture
of Its Properties
R. SHARPLESS, SEYMOUR LEVINE, AND MARY E. ENGLERT Rickettsial
and
Biochemistry
American CyanamidCompany, Accepted
and
M. C. DAVIES,
Research Sections, Lederle Laboratories, Pearl River, New York
November
30, 1960
The GAL virus growth cycle hasbeendeterminedby plaqueassayand correlated with electronmicroscopeobservationsof infected cells. The virus was found to have a longlatent periodandgrowth cycle,to be producedonly within the cellnucleus,andto accumulatethere. It alsowasfound to be polyhedralin shapeand ether stableand to resemble adenovirus in its intranuclear site of development, its morphology, and its ether stability. Data concerning its stability under various conditions of storage are included. INTRODUCTION
A virus isolated in tissue cultures from filtrates of chicken livers infected with the RPL-12 strain of lymphomatosis, and referred to in a number of reports as the lymphomatosis virus (Fontes et al., 1958; Sharpless et al., 1958; Levine and Sharpless, 1959; Stoker, 1959; Buthala, 1960; Defendi and Sharpless, 1958; Davies et ah, 1959; Defendi and Kritchevsky, 1960), has been named gallus adeno-!ike (GAL) virus (Burmester et al., 1960). GAL virus had been thought to be related to lymphomatosis because earlier studies showed that (1) the disease was produced by dilutions of the virus far exceeding the known infectivity of the original material-eighth to fourteenth tissue culture passages; (2) the virus was recovered from diseasedbirds; and (3) it was neutralized by antiserum known to neutralize lymphomatosis virus. However, later studies showed that (a) GAL antiserum did not neutralize lymphomatosis virus; (b) subsequent tissue-culture passages and plaque isolates of GAL virus did not induce lymphomatosis in susceptible chicks, and (,c) progeny of GAL-immunized dams were as susceptible to lymphomatosis as were
progeny from nonimmunized dams (Burmester et al., 1960). This report on GAL virus describesits development within the cell and its morphology, as determined by plaque assay or by electron microscopy, and its stability in ether and at various storage temperatures. MATERIALS
SND
METHODS
Stock virus was propagated in chick embryo liver monolayers in Medium 199 (Morgan et al., 1950) and harvested after 5 days of incubation. One of the plaque methods used for assay utilized trypsinized preparations of g-day-old chick embryos suspended in an agar medium containing 5% cow serum (Levine and Sharpless, 1959). The other was a modification of the procedure reported by Stoker (1959) adapted to permit development of macroscopic plaques. With the modified procedure, monolayers were washed once with 3 ml phosphatebuffered saline (PBS), and to each was added an appropriate virus dilution in 1.0 ml Eagle’s medium (1955) enriched with 3% cow serum (CSE) . An attachment period of 2 hours at 37” in air containing 4% COZ was adopted as a result of the experiment summarized in Fig. 1.
315
316
SHARPLESS,
4 1.0.I0
1
Auachment
2
Perxsd
3
LEVINE,
DAVIES,
4
before (Hours)
Addttion
of Aqar.
FIG. 1. Demonstration of attachment period required for maximum number of plaques of GAL virus on chick embryo liver monolayers. Virus was added to each of 20 monolayers as described in the text and the plates were placed at 37”. At each of the indicated times, 5 plates were overlaid with the agar medium without removing the virus.
AND
ENGLERT
after virus infection, plates were stained with 1: 12,000 neutral red. For electron microscopy, cell layers were washed with isotonic saline at, 37”, fixed with 1.0% osmic acid buffered at pH 7.3, and dehydrated with graded concentrations of alcohol. For solvent changes, decantation was aided by low speed centrifugation. Cells were embedded in 75% butyl, 25% methyl methacrylate polymerized by heating at at 65-70” overnight. Embedded cells were cut into sections with a Porter-Blum ultramicrotome, using either glass or diamond knives, and picked up on carbon-filmed 200-mesh copper grids for examination in a microscope (RCA EMU-3C) equipped with a 50-p objective aperture. Photomicrographs were taken at instrument magnifications of 300&12,000 and enlarged photographically. To reveal virus ultrastructure more clearly, some mounted sections were stained with phosphotungstic acid (PTA). Grids were floated face down for 15-30 minutes on a solution of 2% PTA in 40% alcohol and rinsed once with water. EXPERIMENTAL
Growth
FIG.
chick
2. One-step embryo liver
growth curve monolayers.
for
GAL
virus
in
After attachment, each plate was overlaid with 3 ml CSE containing 0.9% agar washed according to the method of Dulbecco and Vogt (1954) and incubated at, 37”. After 34 days a second layer of the same agar medium was added. Seven days
Cycle Determined
by Plaque Assay
The purpose of the first experiment was to determine the duration of a single cycle of virus growth on chick embryo liver monolayers. Three monolayers were each exposed to 3.5 x lo8 plaque-forming units (PFU) of virus in CSE. The plates were incubated at room temperature 3 hours to permit virus to attach, washed twice with 3 ml PBS, overlaid with 4 ml CSE, and then incubated at 37”. Whole chick embryo cells suspended in agar were used to assay the medium for virus content. The various times at which medium was removed for assay and replaced with fresh virus-growth medium are shown in Fig. 2, a curve representing cumulative production of virus. The assumption that a single cycle of infection is represented is justified by the high multiplicity of the virus added (greater than 50) and by the long attachment-period. The outstanding feature of the cycle appears to be its long duration. The latent
GAL
VIRUS
GROWTH
period was more than 20 hours and the entire cycle required as many as 72 hours. The next experiment was designed to determine the relationship between the extracellular and cell-associated virus. Chick embryo liver monolayers were exposed to 7 x lo* PFU of virus, allowed 2 hours at room temperature for attachment, washed twice with 3 ml PBS, and overlaid with 4 ml CSE. A count at the time of infection indicated that representative monolayers contained 6.0 x 10B cells per plate. In Fig. 3, the points represent the times when the medium from a single plate was removed for assay and the monolayer disrupted by
CYCLE
freezing and thawing once in 2 ml distilled water. Each of these samples was assayed on chick embryo liver monolayers and the data obtained were plotted as virus yield per plate versus time. The dotted line represents the average cell-count per plate at time of infection. Again the growth cycle was a long one and the latent period, as shown by the curve for the yield of cell-associated virus, was prolonged. Virus first appeared some time after 16 hours and then increased precipitously. No extracellular virus appeared until 20 hours after infection. The unquestionable accumulation of virus within
1o'O
10’
Av.Number of --w--------------‘--------cell*,piatRs. -ece ll* ,piatRs.
A cell associated aseociatcd virus 0 extracellular tit-us
10'
FIG.
3. Growth
317
cycle of GAL virus in chick embryo liver monolayers.
318
SHARPLESS,
LEVINE,
the cell also is evident from the curve. At 30 hours the concentration of extracellular virus was only 3% of that associated with the cell. Results of other experiments were essentially similar. About 16 hours after infection cell-associated virus was present. Extracellular virus appeared between 20 and 30 hours after infection and amounted to only a small fraction of the cell-associated virus present at the time. Electron Microscopy Growth Cycle
of Stages of the Virus
For these experiments, chick embryo liver cultures were prepared in serum bottles as previously described (Davis and Sharpless, 1959) and were incubated for 48 hours at 37”. Then the growth was replaced with Medium 199. Eight of the bottles were inoculated, each receiving about lo8 tissue culture-infectious doses of virus. (Since this approximates 5-10 infectious doses per cell, observed results can be assumed to represent a single cycle of infection.) At each 6-hour interval after infection, one bottle was processed for electron microscopy as described under Methods. Figure 4 shows a portion of a representative culture of uninfected chick embryo liver cells. In such cultures nucleoli are quite large and usually single, mitochondria vary considerably in size and shape, the endoplasmic reticulum is abundant throughout the cytoplasm, and lipid droplets are sometimes apparent. Signs of infection were first seen within 16 to 20 hours. Figures 5 and 6 are cells 18 hours after infection. In their nuclei are formations typically associated with the beginning of virus synthesis. Most often these formations occurred as a ring or partial ring. During the next few hours, the intranuclear formations enlarged rapidly and the nucleus became distorted and expanded to occupy a progressively larger portion of the cell. Between 20 and 30 hours after infection, rows of virus particles began to appear within the nucleus. An example of this stage of infection is shown in Fig. 7. The nuclear membrane is much distorted, and cyto-
DAVIES,
AND
ENGLERT
plasmic structures are undergoing necrotic changes. These changes were usually accompanied by the appearance at the periphery of the nucleus of a virus-free mass which retained its identity until late stages of infection. For the next 18 to 24 hours active synthesis of virus continued, accompanied by continued enlargement of the nucleus and organization of the virus particles. Figure 8 shows a typical nucleus 42 hours after infection. Although the nuclear membrane is still identifiable, viral arrays occupy most of the nucleus and the entire cell obviously is undergoing necrosis. By 48 hours after infection (Fig. 9) the cytoplasm was reduced to debris, t,he nucleus was entirely replaced by virus, and the three-dimensional packing of virus particles in lattices of several different orientations was clearly evident. Part of a viral matrix is shown at high magnification in Fig. IO. To accentuate virus ultrastructure, sections were stained with PTA. Intranuclear particles averaged 86 my in diameter, with a centroid about 44 rnp in diameter. Both centroid and complet,e particle showed an electron-dense outer layer. The particles in the intranuclear arrays appeared hexagonal. These hexagonal cross sections led us to re-examine photographs of the virus in spray droplets (Davies and Sharpless, 1959). Most of those particles appeared spherical, but a significant number were found so oriented that a polyhedral shape could be seen. Figure 11 shows such a particle. By negative staining, the particles were subsequently found t.o be icosahedral (Davies and Englert, 1961). Stability
of the Virus
Franklin (1958) suggested that the ether stability of a mature virus particle is related to the virus maturation mechanism, ether-sensitive viruses maturing only at the membrane of the host cell and being continuously released, ether-stable viruses maturing within the cell until released en masse. Growth curves and electron micrographs showed that GAL virus develops in the nucleus and accumulates within the cell. To see whether it fitted Franklin’s hypothe-
GAL
VIRUS
GROWTH
CYCLE
4. Portion of uninfected chick embryo liver cell layer; cm, cell membrane; nm, numembrane ; n, nucleolus; m, mitochondria. FIG. 5. Nucleus of chick embryo liver cell 18 hours after infection with GAL virus. A typical intranuclear formation associated with infection is shown. FIG. 6. Nucleus of chick embryo liver cell 18 hours after infection with GAL virus showing ring shape of intranuclear mass-a formation that occurred frequently. FIG. 7. Nucleus and portion of cytoplasm of chick embryo liver cell 30 hours after infection with GAL virus. Virus particles are beginning to array themselves within nucleus. Mitochondria, as shown at upper right, are degenerating. Arrow indicates nuclear peripheral mass typical of this stage of infection and believed to be reoriented nucleolar material. FIG.
clear
SHARPLESS,
LEVINE,
DAVIES,
AND
ENGLERT
FIG. 8. Chick embryo liver cell 42 hours after infection with GAL virus. Viral arrays almor 3t m fill nucleus. Nuclear membrane is still intact and peripheral mass still visible. Cytoplas shows advanced necrosis. FIG. 9. Chick embryo liver cell 48 hours after infection with GAL virus, the terminal stag :e. Nucleus is filled with crystal-like virus arrays in several orientations. Nuclear membrane is breaking down. Only fragments of cytoplasm remain. FIG. 10. Higher magnification of part of intranuclear array of GAL virus 48 hours after il IIfection of chick embryo liver cell, showing hexagonal cross sections of viruses and, where a central section was obtained, the internal structure. FIG. 11. A single GAL virus in a shadowed spray drop, showing polyhedral shape,
GAL
TABLE ETHER
STABILITY
GAL Control
with
b
GROWTH
321
CYCLE
DISCUSSION
1 OF GAL
Plaque-forming
Material
VIRUS
VIRUS~ units
per ml
Virus
alone
Virus
9.G x 3.1 x
108 108
1.8
a Five cubic centimeters 5 cc of ether and placed b Newcastle disease virus.
of virus overnight
+ ether
x 103
108
were mixed at 5”.
sis, its ether stability was determined. For this experiment, an aliquot of virus was shaken with an equal volume of ether and the mixture was capped and refrigerated overnight. An ether-sensitive agent, an allantoic-fluid harvest of Newcastle disease virus, was tested simultaneously as a control. Results, shown in Table 1, indicate that GAL is relatively ether stable. The stability of the virus under various conditions of storage was also ascertained. The titer, as determined by dilution end point assay, dropped from 10Q.5 ID50 of virus per milliliter to 105.5 in 9 days at 37”, 4 months at room temperature, and more than 15 months at 4”.
The most striking characteristics of GAL virus are its prolonged growth cycle and its tendency to accumulate within the cell. Completion of the cycle required a minimum of 48 hours. Extracellular virus as late as 30 hours after infection amounted to only 3-200/o of that in the cells. The correlation between the virus growth curve and observed morphologic changes in infected cells is represented diagrammatically in Fig. 12. The earliest changes in nuclear morphology were seen at about the same time that infectious virus was first detected within the cells (14-18 hours after infection). Electron micrographs show that the virus accumulates in the nucleus and is released only when the nuclear membrane disintegrates. In the growth curve, the first appearance of virus was followed by a period of exceedingly rapid virus synthesis. This was seen in thin sections as the development of an intranuclear crystal-like lattice of virus particles and resembles the observation in cells infected with adenovirus reported by Lagermalm et al. (1957) and Morgan et al. (1960).
A Cell Associated Virus . FreeVirus
I
I
I
I
I 6p Hours
I
FIG.
I 70
12. Correlation
of morphologic
changes
in cell and growth
curves
of GAL
virus.
322
SHARPLESS,
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The electron-microscope observations also correlate well with the light-microscope observations of Defendi and Sharpless (1958). They found intranuclear inclusions that were basophilic and Feulgen positive, and reported that with the appearance of these inclusions the nucleolus became diffuse and shifted toward the nuclear membrane. By electron microscopy these inclusions were seen to be the site of virus synthesis. A mass found by light microscopy at the periphery of the nucleus was assumed to be reoriented nucleolar material because RNAase reversed its pyroninophilic reaction. The nuclear mass consistently seen by electron microscopy near the membrane at 2&30 hours after infection is believed to be the same material. No evidence of virus in the cytoplasm was seen, although Atanasiu and Lepine (1960) reported such findings in thin sections of infected chick embryo liver cells. It is not surprising that for maximum plaque titer, a particle the size of GAL virus must be allowed time for attachment before the addition of agar, since it would not readily diffuse through the agar. The ether stability of the virus supports the hypothesis of Franklin (1958) which relates ether stability to the site of virus maturation and the virus release mechanism. ACKNOWLEDGMENTS The authors William Boss Esther Chasan manuscript.
are grateful to Walter Olson and for technical assistance, and to for help in preparation of the
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