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Factors affecting the yield of virus in a cloned cell line of Trichoplusia ni infected with a nuclear polyhedrosis virus
JOURNAL
OF INVERTEBRATE
PATHOLOGY
26,251-257
(1975)
Factors Affecting the Yield of Virus in a Cloned Cell Line of Trichoplusia ni Infected With a Nuclear Polyhedrosis Virus MARTHA Department
BROWN
AND
P. FAULKNER
of Microbiology and Immunology, Queen’s University, Kings ton, Ontario, Canada Received
December
27, 1974
The TN-368 tissue culture line of the cabbage looper, Trichoplusiu ni, has been cloned. The doubling times of three clones at 27°C were 27.6 f 3.4 hr, 21.9 + I .7 hr, and 27.4 + 5.9 hr and that of the uncloned culture was 15.8 i 1.5 hr. Growth of cells in all cultures was arrested after infection with a nuclear polyhedrosis virus of T. ni. There was little difference in the yield of polyhedra from cultures of uncloned or cloned cells infected at a multiplicity of infection (m.o.i) = 4. Yields of polyhedra were about the same when a m.o.i. was in the range of O.OlL 4.0, but the yield tripled in the range m.o.i. = 20-30. At higher multiplicities, up to m.o.i.= 500 the yield of polyhedra progressively fell. It is concluded that the observed variation in numbers of polyhedra borne by individual cells in culture is not due to genetic variability among cells, nor can it be accounted for as a consequence of differing m.o.i. by virus. It is postulated that variation in polyhedra yield among cells in culture may be due to such factors as (I) strain differences in the virus, (2) the stage in the cell cycle at which a particular cell is present when infected.
INTRODUCTION The nuclear polyhedrosis virus (NPV) of several insects has been passed in tissue culture (Goodwin et al., 1970, 1973; Faulkner and Henderson, 1972; Sohi and Cunningham, 1972; Vail et al., 1973; Knudson and Tinsley, 1974). In the case of the NPV of Trichoplusia ni, infected cultures release nonoccluded viruses (NOV) and nuclei of cells become filled with polyhedron-shaped inclusion bodies (Faulkner and Henderson, 1972). Polyhedra are resistant to inactivation under field conditions, and they represent the form of the virus that is transmitted under natural conditions (Stairs, 1968). The yield of polyhedra obtained from tissue cultures is variable. Average yields of 64 polyhedra/cell (Vail et al., 1973) were reported for the NPV of Autographa californica grown in T. ni cells. Spodoptera frugiperda cultures infected with S. frugiperda NPV yielded 12-40 polyhedra per cell (Knudson and Tinsley, 1974). Individual cells of infected cultures exhibit great variation in numbers of polyhedra observed in nuclei. Thus a range of 5-200 polyhedra/cell was seen in T. ni NPV infecCopyright All rights
D 1975 by Academic Press, Inc. of reproduction in any form reserved.
tions (Faulkner and Henderson, 1972) and some cells contained over 100 polyhedra when infected with A. californica NPV (Vail et al., 1973). The yield of polyhedra falls dramatically with serial undiluted passage of T. ni NPV in culture. MacKinnon et al. (1974) found that average yield of polyhedra fell from 28/tell to <5/tell after 12 passages in tissue culture. In the experiments reported here we examined two factors that may contribute to variation in polyhedra yield. We found that variation persists after cells are cloned and that the number of inclusion bodies is influenced by the input multiplicity of infection. MATERIALS
AND METHODS
Cells and Virus
The uncloned cell culture derived from adult ovaries of T. ni was obtained in 1972 from Dr. F. W. Hink, Ohio State University, Ohio, USA. Cells were adapted to a modified growth medium, BML/TC 10 (TClOO), (Gardiner and Stockdale, 1973). The medium was prepared complete, sterilized by pressure filtration sequentially 251
252
BROWN
AND
through a series of membrane filters with pore sizes of 1.2, 0.8, 0.45, and 0.22 pm and stored at 4°C. Before use, gentamicin was added at a concentration of 5 mg/lOO ml medium. The cells were grown routinely at 27°C as monolayers in Falcon plastic flasks or glass bottles. They were subcultured twice weekly using a split ratio of 1:4. The virus used was originally obtained from the hemolymph of NPV infected T. ni (Faulkner and Henderson, 1972), and was of the MEV type (MacKinnon et al, 1974). It had been serially passaged in tissue culture at a m.o.i. approximately 1.0. In these experiments, it was used at tissue culture passage level 4 (NPV4) and the fifty-first passage level (NPVSI). Unless otherwise stated, the virus preparation was not concentrated. Medium was removed from infected cultures 3-4 days postinfection and centrifuged at 2000 g for 30 min. The supernatant was assayed for infectivity and stored at 4” C as a source of virus inoculum. Concentration
of Virus
Trichopfusia ni NPV (at passage level 3) was seeded at m.o.i. = 2 into roller bottles (670 sq. cm. surface area) containing approximately 1 x lo7 cells in 200 ml medium. Four days postinfection, the culture fluid was removed and clarified by centrifugation at 2000 g for 25 min to remove cells and debris. The supernatant was centrifuged at 30,000 g for 1 hr at 4°C. The viral pellet was resuspended in medium using a sterile needle and syringe and was left overnight at 4” C. It was then clarified by centrifugation three times (15 min at 2000 g). Cloning of Cells
The technique of Cooper (1970) was applied to TN-368 cells as follows: each well of a Microtest plate (Falcon Plastics) was seeded with 10 ~1 cell suspension diluted to contain one to ten cells. The plate was incubated in a humid box at 27°C. Two hours after seeding, wells were observed by phase contrast microscopy and scored for presence of single cells. Wells containing single cells were examined over the next few
FAULKNER
days and at the 8-10 cell stage, developing clones were transferred to 30-ml Falcon flasks in 5 ml medium. The cells were detached from wells by aspirating medium using a micropipette. Twice weekly, cells were transferred to a new flask with fresh medium. After reaching confluency, cells were passaged four times before being stored in liquid nitrogen. Approximately 1 x lo6 cells were frozen in 1 ml TClOO containing 10% (v/v) dimethylsulfoxide. Cloned cells used in these experiments were always between the fifth and thirtieth passage after selection. Assay of NO V
The virus assay used was an end point titration in Microtest plates (Falcon Plastics). Each of 60 wells in the plate was seeded with 2 x lo3 cells in 5~1 of medium. Six wells were treated as control wells and received no virus inoculum. The remaining nine wells in each of six rows were inoculated with 5 ~1 of a serial tenfold dilution of the virus. The inoculated plates were incubated at 27°C in a humid container. Seventy-two hr postinfection, wells were scored for the presence or absence of polyhedra. Presence of polyhedra was indicated by a focus of at least three cells, each containing a minimum of three inclusion bodies. The TCID,, titer was calculated by the statistical method derived by Reed and Muench (1938). For convenience, this was expressed as “infectious units per milliliter,” assuming that 1 TCID,, = 0.7 infectious unit (PIU). The validity of the relationship has been confirmed in the end point tissue culture assay of S. frugiperda NPV (Knudson and Tinsley, 1974). RESULTS Nonoccluded Virus and Polyhedra Yield of Cloned Cell Lines
Individual NPV infected cells of an uncloned culture yield varying numbers of inclusion bodies ranging from 5 to 200 polyhedra/cell (Faulkner and Henderson, 1972). One reason for this variation in yield could have been that the tissue culture population of T. ni cells consisted of several
NPV
OF
Trichoplusia
sublines each with differing capacity to support viral growth. Accordingly, three clones were isolated by dilution in Microtest plates. After 1 month the clones were growing to confluency in a 30-ml size plastic Falcon flask. The growth curves of the uncloned line and the three new clones were determined as shown in Figure 1. Doubling times were calculated and are given in Table 1. They ranged from 15.8 + 1.5 hr in the uncloned culture to 27.4 f 5.9 hr in clone 3. Cloned and uncloned cells were infected with virus (NPV4) at a m.o.i. = 4. Free virus (NOV) and polyhedra were harvested at 65 hr postinfection. The data in Table 2 shows that there were no major differences in yield of inclusion bodies or final titer of NOV among the cultures.
100 Cdl Number 90
253
ni IN CELL CULTURE
TABLE 1 Doubling Time of Cloned and Uncloned Cultures Cells Uncloned Clone l/5
Clone 215
Clone 3/5
Time period (hr) O-16.5 5.5-27.0 O-16.5 O-27.0 16.543.5 27.043.5 O-16.5 O-27 .O 16.5-43.5 27.0-43.5 O-16.5 16.5-51.5
Doubling timea (hr) 14.3 17.3 22.6 34.2 32.4 21.2 19.9 26.6 22.1 18.8 21.5 33.3
Mean doubling time (hr)
f 0.9 f 1.0 f 1.0 ?: 1.5 f 1.4 * 0.9 + 1.0 f 1.5 f 1.4 t 1.3 ?: 1.2 f 1.8
15.8 ?: 1.5 27.6 f 3.4
21.9 + 1.7
2714 + 5.9 Cloned and uncloned cells, which had been passaged four times since isolation, were seeded (-5 X lo4 cells in 5 ml medium) in 30 ml Falcon plastic flasks sand incubated at 27°C. After 1 hr, during which time the cells attached to the flask, a cell count was taken and this was designated time 0. Cells were counted in situ, using a graticule in the eyepiece of the microscope, at intervals during a 51.5-hr period. For each flask, ten random fields were counted at each time period (t). ‘The doubling times given in the table for each time interval are the means of 100 values calculated by applying the equation,
DE
At
3.3 1ogblB
to each possible pair of values (B,b) resulting from ten values of each, for a given time period. In the equation, D = doubling time of culture At = t -
to
B = cell number at to h = cell number at f. Pairs in which B was greater than b were rejected. Such cases accounted for 29% (SD 13%) of possible combinations. The variation in doubling times is expressed as standard error. 10
20
30
40
50 Time
60
(hr)
FIG. 1. Cloned and uncloned cells, which had been passaged four times since isolation, were seeded (- 5 x lo1 cells in 5 ml medium) in 30 ml Falcon plastic flasks and incubated at 27°C. After 1 hr, during which time the cells attached to the flask, a cell count was taken and this was designated time 0. Cells were counted in situ. using a graticule in the eyepiece of the microscope, at intervals during a 5 1.5 hr period. For each flask, ten random fields were counted at each time period (t). The data is presented as the mean value l standard error.
Individual cells in infected cultures were observed in the microscope and found to contain variable numbers of polyhedra as has been reported for uncloned cultures (Faulkner and Henderson, 1972). Statistical considerations predict that at a m.o.i. of 4.1, >98% of cells are infected. Observation of cultures at 24 hr postinfection indicated < 10% of uninfected cells. The yield of NOV released from cells ranged from 159 to 380 PIU/cell over the 65-hr period (Table 2).
254
BROWN
AND
TABLE 2 Comparison of NOV and Polyhedra Yield of Cloned Cell@
FAULKNER
TABLE 3 Inhibition of Cellular Multiplication Virus Infection4
NOV yield Cells Uncloned Clone l/l9 Clone 2/l 9 Clone 3/19
Polyhedra/cell 20* 19* 17r 19+ 25~ 21 f 152
3 3 2 3 3 5 2
PIU/ml
PIU/cell
3.2X 107 3.2 x 107 6.1 X 107
186* 19 140 f 14 380 t 38
2.7 X 107 2.5 x 107 2.5 x 107
159 + 16 194 c 19 173 f 17
‘6 X 10s cells were seeded in a 30 ml Falcon plastic flask in a volume
Inhibition of Cell Multiplication Following Virus Infection
General observations of virus infected cell cultures had indicated that there was a marked reduction in cell growth in experiments in which all cells were infected at seeding. The data in Table 3 show the effect on cell numbers of a culture growing in flasks which had been seeded at a m.o.i. = 4. Growth continued in the control flasks containing uncloned or clone 2 cells, but was
by
Time after seeding Cells Uncloned Control NPVs -m.o.i. NPVs -m.o.i. NPVst -m.o.i. Clone 2 Control NPVs --m.o.i. NPVS -m.o.i. NPVsl -m.o.i.
Ohr
21hr
45hr
69 hr
=1 =4 =1
3+1 5+2 4+1 4+2
7+5 41t3 5+3 4*2
16*8 7+4 3k2 5+2
26+11 lo+ 4 5+ 2 5+ 3
=1 =4 =1
6*3 6*2 6+3 5+2
9+4 8*4 8+3 7*1
15*8 9+4 8+3 754
21* 8+ 7+ 6+
9 3 6 3
“Cells (approx 5 X 10s) were seeded in a volume
arrested in cells infected both with early passage (NPVS) or extensively passaged (NPVS 1) virus. E#ect of Viral Input Multiplicity of Infection on Yield of Polyhedra
Flasks of cells were infected with concentrated virus at a m.0.i. of 0.01-500 in order to determine the effect on inclusion body yield. Three days postinfection cells were counted and lysed. The yield of polyhedra/cell is shown graphically in Figure 2 as a function of input m.o.i. Input multiplicities below 4.0 yielded about 20 inclusion bodies/cell. Above m.o.i. = 4 there was a marked increase in polyhedra yield which reached a maximum of approximately 60 polyhedra/cell at m.o.i. = 20-30 PIU/ cell. At a still higher m.o.i. the yield of polyhedra fell off, but did not return to the lowest value. The yield of NOV/clone 2 cell was variable and ranged from 2 to 105 PIU/cell (Table 4). When the data on input multiplicity is grouped as shown in Table 5 it ap-
NPV
OF
~richophsia
ni IN CELL
255
CULTURE
Polyhedra per cell 60
10
0’ -2.0
-1.0
0.0
1.0
2.0
3.0 Log MOI
FIG. 2. 5 x IO5 clone 2 cells, between the 5th and 30th passage after cloning were seeded in 30 ml Falcon flasks. After the cells had attached, the medium was decanted, and virus inoculum (< I ml) was added. The concentrated virus preparation used had a titer of 2.5 x IO8 PIU/ml. After I hr adsorption period, fresh medium was added to yield a total volume per flask of 5 ml. The experiment was terminated 72 hr postinfection. Polyhedra yield per cell was determined by the method described in the Legend to Table 2.
pears that the maximum yield of NOV/cell at a m.o.i. of 0.01-4.00 coincides with minimum yield of polyhedra/cell. The relationship does not appear to hold however, at m.0.i. = 400 f. Relationship Number of experiments 1 1 4 3 6 2 2 3 3 2 1 1 1 1
TABLE 4 Between NOV Yield of Infection
m.0.i. 0.01 0.1 1.0 4 IO 20 30 40 75 100 200 300 400 500
and Multiplicity Average number NOV/cell 10.5 c 432 76 + 45 * 24* lo* 16 * 8i 39* 2+ 5+ 17+ 45* 58*
11 4 32 14 11 9 15 6 20 1 1 2 5 6
‘The average NOV yield was determined as described in the legend to Table 2. When more than one experiment was done, the variation is expressed as standard error of the mean calculated from yields in individual experiments. When only one experiment was done, the variation results from error in cell number used in calculation.
DISCUSSION Inclusion bodies produced as a consequence of infecting tissue cultures with NPV of T. ni have been shown to possess similar virulence to those obtained from diseased insects. This similarity holds for polyhedra tested in laboratory feeding experiments (Faulkner and Henderson, 1972) and for polyhedra tested for efficacy in field tests (Ignoffo et al, 1974). However, extensive serial passage of the same virus in tissue culture leads to a marked reduction in numbers of polyhedra produced. Those that are formed contain aberrant particles and are not infectious per OS (MacKinnon et al., 1974). With these considerations in mind the
Relationship Production Input
m.0.i.
0.01-4 10-40 75-300 400-500
TABLE 5 Between NOV Yield, Polyhedra and Multiplicity of Infectiona PIU/cell 67+ IS 15 f 10 16+ 6 S2+ 6
Polyhedra/cell 23 +4 55 r 9 41 f 7 42t 6
‘Data were taken from Table 4 and Figure 2. The values of PIU/cell and polyhedra/cell were calculated as the mean of the respective values for each multiplicity of infection within each group.
256
BROWN
AND
experiments reported here were undertaken to examine some of the factors affecting polyhedra yield at early passage level of virus. We started our investigation by recognizing that individual cells of an uncloned culture can contain as few as 5 and as many as 200 polyhedra/nucleus (Faulkner and Henderson, 1972). Data given in Table 2 show that those clones selected from an extensively passaged line of TN368 cells did not appear to yield significantly different quantities of inclusion bodies. The experiments were performed at m.o.i. = 4 which, from statistical considerations should have ensured that each cell was exposed to at least 1 PIU of virus at the beginning of the experiment. Infected cells in the cloned cultures contained the same extreme variation of polyhedra in individual cells as had been observed in uncloned cultures. It is shown in Table 2 that cell division is arrested soon after virus infection both in cloned and uncloned cultures. Arrest of cellular growth has also been observed following infection of T. ni cells with NPV of Autographa californica (Vail et al., 1973) and infection of S. frugiperda cells with homologous NPV (Knudson and Tinsley, 1974). Variability in polyhedra production may result from infection of a nonsynchronous culture with the NPV virus. Efficiency of virus production in individual cells in culture may be greatly influenced by the stage of cell cycle at which a cell is infected. We found that the average yield of polyhedra was affected by the multiplicity of infection of the input virus (Fig. 2). At multiplicities from 0.01 to 4 the average yield of polyhedra was about the same, but average yields of polyhedra tripled at m.o.i. 20-30. Still higher multiplicities up to m.o.i. = 500 lead to progressive reduction of average polyhedra yield. The existence of an optimum multiplicity of infection probably indicates that a large number of extracellular and intracellular sites need to be saturated to maximize polyhedra production. The particle/II-l ratio has not been determined for the NPV of T. ni but is in the range
FAULKNER
266 h 177 particles/PFU for another baculovirus, NPV of S. frugiperda (Knudson and Tinsley, 1974). At very high multiplicities of infection the average yield of polyhedra fell (Fig. 2). Under these conditions cells would have been exposed to an enormous number of particles which could have resulted in competition between polyhedron yielding infectious particles and others not yielding polyhedra. Attachment of a large number of particles may damage cell membranes and result in suboptimal conditions for polyhedra production. The yield of NOV/cell varies from ‘2 to 105 PIU/cell (Table 4) and seems to be maximal when polyhedra yield is minimal (Table 5). Working with the NPV of S. frugiperda, Knudson and Tinsley (1974) obtained values of 2-7 III/cell at m.o.i. = 0.2 -25. In the T. ni system, the increase observed in polyhedra yield in response to an increase in multiplicity of infection may be greater than the increase in virion production. This would result in a greater number of virions being occluded and a lower yield of NOV observed. However, at input m.o.i. above 400, the yield of NOV increases while the polyhedra yield does not change. The stimulation of polyhedra synthesis at these multiplicities may be lessened by suboptimal conditions for protein synthesis in the cytoplasm, due to damage to cell membranes caused by attachment of many particles. Virion synthesis, which occurs in the nucleus, may be less affected by cell membrane changes. Thus, infectious particles which reach the nucleus replicate, but since polyhedra production is not optimal, a large number of virions may not be occluded. Hence, an increase in NOV yield is observed at m.0.i. = 400 +. In conclusion, it does not appear that the observed variation in numbers of polyhedra in individual cells can be due solely to the presence in cultures of clones of high and low yielding strains of cells nor can it be ascribed to the consequence of cells in culture being infected with vastly different numbers of infecting particles. We have still to investigate
NPV
OF
~richo/hsia
whether our presently uncloned virus contains high polyhedra yielding strains and whether cells infected at various stages in their growth yield differing quantities of virus. ACKNOWLEDGMENTS Some of this work was done at the Natural Environment Research Council, Unit of Invertebrate Virology, Oxford, England. We thank Dr. T. W. Tinsley for providing space, and acknowledge much valuable discussion with the staff of the Unit. We also thank Roger R. Moore and John Palmer for help in the statistical analyses.
REFERENCES COOPER, J. E. K. 1970. “Rapid” isolation of single-cell clones of mammalian cell cultures. Texas Rep. Biol. Med., 28,29. FAULKNER, P. AND HENDERSON, J. F. 1972. Serial passage of a nuclear polyhedrosis virus of the cabbage looper (Trichoplusiu nil in a continuous tissue culture cell line. Virology. 50,920-924. GARDINER, G. R. AN5 STOCKDALE, H. 1973. Adaptation of an established line of Spodoptera frugiperda to a simple tissue culture medium. Abstract in Proceedings of Vlth Annual Meeting of the Society for Invertebrate Pathology, 20. Oxford, England. GOODWIN, R. H., VAUGHN, J. L., ADAMS, J. R., AND Lou~ou5~s. S. J. 1970. Replication of a nuclear polyhedrosis virus in an established insect cell line. J. Invertebr. Pathol.. 16,284-288.
ni
IN
CELL
CULTURE
257
GOODWIN, R. H., VAUGHN, J: L., ADAMS, J. R., AND LOULOUDES, S. J. 1973. The effect of insect cell lines and tissue culture media on Baculovirus polyhedra production. Misc. Publ. Entomol. Sot. Amer., 66-72. HINK, W. F. 1970. Established insect cell line from the cabbage looper, Trichoplusia ni. Nature (London), 226,466-467. IGNOFFO, C. M., HOSTETTER, D. L., AND SHAPIRO, M. 1974. Efficacy of insect viruses propagated in vivo and in vitro. .I. Invertebr. Pathol., 24, 184-187. KNUDSON, D. L. AND TINSLEY, T. W. 1974. Replication of a nuclear polyhedrosis.viruH in a continuous cell culture of Spodoptero Jrugiperda: Purification, assay of infectivity and growth characteristics of the virus. J. Viral., 14,934944. MACKINNON, E. A., HENDERSON, J. F., STOLTZ, D. B., AND FAULKNER, P. 1974. Morphogenesis of Nuclear Polyhedrosis Virus under conditions of prolonged passage in vitro. J. Ultrastruc~. Res.. 49.419-435. REED, L. J. AND MUENCH, H. 1938. A simple method of estimating fifty percent end points. Amer. J. Hyg.. 27,493-497. SOHI, S. S. AND CUNNINGHAM, J. C. 1972. Replication of a nuclear polyhedrosis virus in serially transferred insect hemocyte cultures. J. Inverrebr. Parhol.. 19, 51-61. STAIRS, G. R. 1968. Inclusion-type insect viruses. turr. Top. Microbial. Immunol.. 42, l-24. VAIL, P. V., JAY, D. L., AND HINK, W. F. 1973. Replication and infectivity of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica. produced in cells grown in vitro. J. Invertebr. Pathol., 22,231-237.
OF INVERTEBRATE
PATHOLOGY
26,251-257
(1975)
Factors Affecting the Yield of Virus in a Cloned Cell Line of Trichoplusia ni Infected With a Nuclear Polyhedrosis Virus MARTHA Department
BROWN
AND
P. FAULKNER
of Microbiology and Immunology, Queen’s University, Kings ton, Ontario, Canada Received
December
27, 1974
The TN-368 tissue culture line of the cabbage looper, Trichoplusiu ni, has been cloned. The doubling times of three clones at 27°C were 27.6 f 3.4 hr, 21.9 + I .7 hr, and 27.4 + 5.9 hr and that of the uncloned culture was 15.8 i 1.5 hr. Growth of cells in all cultures was arrested after infection with a nuclear polyhedrosis virus of T. ni. There was little difference in the yield of polyhedra from cultures of uncloned or cloned cells infected at a multiplicity of infection (m.o.i) = 4. Yields of polyhedra were about the same when a m.o.i. was in the range of O.OlL 4.0, but the yield tripled in the range m.o.i. = 20-30. At higher multiplicities, up to m.o.i.= 500 the yield of polyhedra progressively fell. It is concluded that the observed variation in numbers of polyhedra borne by individual cells in culture is not due to genetic variability among cells, nor can it be accounted for as a consequence of differing m.o.i. by virus. It is postulated that variation in polyhedra yield among cells in culture may be due to such factors as (I) strain differences in the virus, (2) the stage in the cell cycle at which a particular cell is present when infected.
INTRODUCTION The nuclear polyhedrosis virus (NPV) of several insects has been passed in tissue culture (Goodwin et al., 1970, 1973; Faulkner and Henderson, 1972; Sohi and Cunningham, 1972; Vail et al., 1973; Knudson and Tinsley, 1974). In the case of the NPV of Trichoplusia ni, infected cultures release nonoccluded viruses (NOV) and nuclei of cells become filled with polyhedron-shaped inclusion bodies (Faulkner and Henderson, 1972). Polyhedra are resistant to inactivation under field conditions, and they represent the form of the virus that is transmitted under natural conditions (Stairs, 1968). The yield of polyhedra obtained from tissue cultures is variable. Average yields of 64 polyhedra/cell (Vail et al., 1973) were reported for the NPV of Autographa californica grown in T. ni cells. Spodoptera frugiperda cultures infected with S. frugiperda NPV yielded 12-40 polyhedra per cell (Knudson and Tinsley, 1974). Individual cells of infected cultures exhibit great variation in numbers of polyhedra observed in nuclei. Thus a range of 5-200 polyhedra/cell was seen in T. ni NPV infecCopyright All rights
D 1975 by Academic Press, Inc. of reproduction in any form reserved.
tions (Faulkner and Henderson, 1972) and some cells contained over 100 polyhedra when infected with A. californica NPV (Vail et al., 1973). The yield of polyhedra falls dramatically with serial undiluted passage of T. ni NPV in culture. MacKinnon et al. (1974) found that average yield of polyhedra fell from 28/tell to <5/tell after 12 passages in tissue culture. In the experiments reported here we examined two factors that may contribute to variation in polyhedra yield. We found that variation persists after cells are cloned and that the number of inclusion bodies is influenced by the input multiplicity of infection. MATERIALS
AND METHODS
Cells and Virus
The uncloned cell culture derived from adult ovaries of T. ni was obtained in 1972 from Dr. F. W. Hink, Ohio State University, Ohio, USA. Cells were adapted to a modified growth medium, BML/TC 10 (TClOO), (Gardiner and Stockdale, 1973). The medium was prepared complete, sterilized by pressure filtration sequentially 251
252
BROWN
AND
through a series of membrane filters with pore sizes of 1.2, 0.8, 0.45, and 0.22 pm and stored at 4°C. Before use, gentamicin was added at a concentration of 5 mg/lOO ml medium. The cells were grown routinely at 27°C as monolayers in Falcon plastic flasks or glass bottles. They were subcultured twice weekly using a split ratio of 1:4. The virus used was originally obtained from the hemolymph of NPV infected T. ni (Faulkner and Henderson, 1972), and was of the MEV type (MacKinnon et al, 1974). It had been serially passaged in tissue culture at a m.o.i. approximately 1.0. In these experiments, it was used at tissue culture passage level 4 (NPV4) and the fifty-first passage level (NPVSI). Unless otherwise stated, the virus preparation was not concentrated. Medium was removed from infected cultures 3-4 days postinfection and centrifuged at 2000 g for 30 min. The supernatant was assayed for infectivity and stored at 4” C as a source of virus inoculum. Concentration
of Virus
Trichopfusia ni NPV (at passage level 3) was seeded at m.o.i. = 2 into roller bottles (670 sq. cm. surface area) containing approximately 1 x lo7 cells in 200 ml medium. Four days postinfection, the culture fluid was removed and clarified by centrifugation at 2000 g for 25 min to remove cells and debris. The supernatant was centrifuged at 30,000 g for 1 hr at 4°C. The viral pellet was resuspended in medium using a sterile needle and syringe and was left overnight at 4” C. It was then clarified by centrifugation three times (15 min at 2000 g). Cloning of Cells
The technique of Cooper (1970) was applied to TN-368 cells as follows: each well of a Microtest plate (Falcon Plastics) was seeded with 10 ~1 cell suspension diluted to contain one to ten cells. The plate was incubated in a humid box at 27°C. Two hours after seeding, wells were observed by phase contrast microscopy and scored for presence of single cells. Wells containing single cells were examined over the next few
FAULKNER
days and at the 8-10 cell stage, developing clones were transferred to 30-ml Falcon flasks in 5 ml medium. The cells were detached from wells by aspirating medium using a micropipette. Twice weekly, cells were transferred to a new flask with fresh medium. After reaching confluency, cells were passaged four times before being stored in liquid nitrogen. Approximately 1 x lo6 cells were frozen in 1 ml TClOO containing 10% (v/v) dimethylsulfoxide. Cloned cells used in these experiments were always between the fifth and thirtieth passage after selection. Assay of NO V
The virus assay used was an end point titration in Microtest plates (Falcon Plastics). Each of 60 wells in the plate was seeded with 2 x lo3 cells in 5~1 of medium. Six wells were treated as control wells and received no virus inoculum. The remaining nine wells in each of six rows were inoculated with 5 ~1 of a serial tenfold dilution of the virus. The inoculated plates were incubated at 27°C in a humid container. Seventy-two hr postinfection, wells were scored for the presence or absence of polyhedra. Presence of polyhedra was indicated by a focus of at least three cells, each containing a minimum of three inclusion bodies. The TCID,, titer was calculated by the statistical method derived by Reed and Muench (1938). For convenience, this was expressed as “infectious units per milliliter,” assuming that 1 TCID,, = 0.7 infectious unit (PIU). The validity of the relationship has been confirmed in the end point tissue culture assay of S. frugiperda NPV (Knudson and Tinsley, 1974). RESULTS Nonoccluded Virus and Polyhedra Yield of Cloned Cell Lines
Individual NPV infected cells of an uncloned culture yield varying numbers of inclusion bodies ranging from 5 to 200 polyhedra/cell (Faulkner and Henderson, 1972). One reason for this variation in yield could have been that the tissue culture population of T. ni cells consisted of several
NPV
OF
Trichoplusia
sublines each with differing capacity to support viral growth. Accordingly, three clones were isolated by dilution in Microtest plates. After 1 month the clones were growing to confluency in a 30-ml size plastic Falcon flask. The growth curves of the uncloned line and the three new clones were determined as shown in Figure 1. Doubling times were calculated and are given in Table 1. They ranged from 15.8 + 1.5 hr in the uncloned culture to 27.4 f 5.9 hr in clone 3. Cloned and uncloned cells were infected with virus (NPV4) at a m.o.i. = 4. Free virus (NOV) and polyhedra were harvested at 65 hr postinfection. The data in Table 2 shows that there were no major differences in yield of inclusion bodies or final titer of NOV among the cultures.
100 Cdl Number 90
253
ni IN CELL CULTURE
TABLE 1 Doubling Time of Cloned and Uncloned Cultures Cells Uncloned Clone l/5
Clone 215
Clone 3/5
Time period (hr) O-16.5 5.5-27.0 O-16.5 O-27.0 16.543.5 27.043.5 O-16.5 O-27 .O 16.5-43.5 27.0-43.5 O-16.5 16.5-51.5
Doubling timea (hr) 14.3 17.3 22.6 34.2 32.4 21.2 19.9 26.6 22.1 18.8 21.5 33.3
Mean doubling time (hr)
f 0.9 f 1.0 f 1.0 ?: 1.5 f 1.4 * 0.9 + 1.0 f 1.5 f 1.4 t 1.3 ?: 1.2 f 1.8
15.8 ?: 1.5 27.6 f 3.4
21.9 + 1.7
2714 + 5.9 Cloned and uncloned cells, which had been passaged four times since isolation, were seeded (-5 X lo4 cells in 5 ml medium) in 30 ml Falcon plastic flasks sand incubated at 27°C. After 1 hr, during which time the cells attached to the flask, a cell count was taken and this was designated time 0. Cells were counted in situ, using a graticule in the eyepiece of the microscope, at intervals during a 51.5-hr period. For each flask, ten random fields were counted at each time period (t). ‘The doubling times given in the table for each time interval are the means of 100 values calculated by applying the equation,
DE
At
3.3 1ogblB
to each possible pair of values (B,b) resulting from ten values of each, for a given time period. In the equation, D = doubling time of culture At = t -
to
B = cell number at to h = cell number at f. Pairs in which B was greater than b were rejected. Such cases accounted for 29% (SD 13%) of possible combinations. The variation in doubling times is expressed as standard error. 10
20
30
40
50 Time
60
(hr)
FIG. 1. Cloned and uncloned cells, which had been passaged four times since isolation, were seeded (- 5 x lo1 cells in 5 ml medium) in 30 ml Falcon plastic flasks and incubated at 27°C. After 1 hr, during which time the cells attached to the flask, a cell count was taken and this was designated time 0. Cells were counted in situ. using a graticule in the eyepiece of the microscope, at intervals during a 5 1.5 hr period. For each flask, ten random fields were counted at each time period (t). The data is presented as the mean value l standard error.
Individual cells in infected cultures were observed in the microscope and found to contain variable numbers of polyhedra as has been reported for uncloned cultures (Faulkner and Henderson, 1972). Statistical considerations predict that at a m.o.i. of 4.1, >98% of cells are infected. Observation of cultures at 24 hr postinfection indicated < 10% of uninfected cells. The yield of NOV released from cells ranged from 159 to 380 PIU/cell over the 65-hr period (Table 2).
254
BROWN
AND
TABLE 2 Comparison of NOV and Polyhedra Yield of Cloned Cell@
FAULKNER
TABLE 3 Inhibition of Cellular Multiplication Virus Infection4
NOV yield Cells Uncloned Clone l/l9 Clone 2/l 9 Clone 3/19
Polyhedra/cell 20* 19* 17r 19+ 25~ 21 f 152
3 3 2 3 3 5 2
PIU/ml
PIU/cell
3.2X 107 3.2 x 107 6.1 X 107
186* 19 140 f 14 380 t 38
2.7 X 107 2.5 x 107 2.5 x 107
159 + 16 194 c 19 173 f 17
‘6 X 10s cells were seeded in a 30 ml Falcon plastic flask in a volume
Inhibition of Cell Multiplication Following Virus Infection
General observations of virus infected cell cultures had indicated that there was a marked reduction in cell growth in experiments in which all cells were infected at seeding. The data in Table 3 show the effect on cell numbers of a culture growing in flasks which had been seeded at a m.o.i. = 4. Growth continued in the control flasks containing uncloned or clone 2 cells, but was
by
Time after seeding Cells Uncloned Control NPVs -m.o.i. NPVs -m.o.i. NPVst -m.o.i. Clone 2 Control NPVs --m.o.i. NPVS -m.o.i. NPVsl -m.o.i.
Ohr
21hr
45hr
69 hr
=1 =4 =1
3+1 5+2 4+1 4+2
7+5 41t3 5+3 4*2
16*8 7+4 3k2 5+2
26+11 lo+ 4 5+ 2 5+ 3
=1 =4 =1
6*3 6*2 6+3 5+2
9+4 8*4 8+3 7*1
15*8 9+4 8+3 754
21* 8+ 7+ 6+
9 3 6 3
“Cells (approx 5 X 10s) were seeded in a volume
arrested in cells infected both with early passage (NPVS) or extensively passaged (NPVS 1) virus. E#ect of Viral Input Multiplicity of Infection on Yield of Polyhedra
Flasks of cells were infected with concentrated virus at a m.0.i. of 0.01-500 in order to determine the effect on inclusion body yield. Three days postinfection cells were counted and lysed. The yield of polyhedra/cell is shown graphically in Figure 2 as a function of input m.o.i. Input multiplicities below 4.0 yielded about 20 inclusion bodies/cell. Above m.o.i. = 4 there was a marked increase in polyhedra yield which reached a maximum of approximately 60 polyhedra/cell at m.o.i. = 20-30 PIU/ cell. At a still higher m.o.i. the yield of polyhedra fell off, but did not return to the lowest value. The yield of NOV/clone 2 cell was variable and ranged from 2 to 105 PIU/cell (Table 4). When the data on input multiplicity is grouped as shown in Table 5 it ap-
NPV
OF
~richophsia
ni IN CELL
255
CULTURE
Polyhedra per cell 60
10
0’ -2.0
-1.0
0.0
1.0
2.0
3.0 Log MOI
FIG. 2. 5 x IO5 clone 2 cells, between the 5th and 30th passage after cloning were seeded in 30 ml Falcon flasks. After the cells had attached, the medium was decanted, and virus inoculum (< I ml) was added. The concentrated virus preparation used had a titer of 2.5 x IO8 PIU/ml. After I hr adsorption period, fresh medium was added to yield a total volume per flask of 5 ml. The experiment was terminated 72 hr postinfection. Polyhedra yield per cell was determined by the method described in the Legend to Table 2.
pears that the maximum yield of NOV/cell at a m.o.i. of 0.01-4.00 coincides with minimum yield of polyhedra/cell. The relationship does not appear to hold however, at m.0.i. = 400 f. Relationship Number of experiments 1 1 4 3 6 2 2 3 3 2 1 1 1 1
TABLE 4 Between NOV Yield of Infection
m.0.i. 0.01 0.1 1.0 4 IO 20 30 40 75 100 200 300 400 500
and Multiplicity Average number NOV/cell 10.5 c 432 76 + 45 * 24* lo* 16 * 8i 39* 2+ 5+ 17+ 45* 58*
11 4 32 14 11 9 15 6 20 1 1 2 5 6
‘The average NOV yield was determined as described in the legend to Table 2. When more than one experiment was done, the variation is expressed as standard error of the mean calculated from yields in individual experiments. When only one experiment was done, the variation results from error in cell number used in calculation.
DISCUSSION Inclusion bodies produced as a consequence of infecting tissue cultures with NPV of T. ni have been shown to possess similar virulence to those obtained from diseased insects. This similarity holds for polyhedra tested in laboratory feeding experiments (Faulkner and Henderson, 1972) and for polyhedra tested for efficacy in field tests (Ignoffo et al, 1974). However, extensive serial passage of the same virus in tissue culture leads to a marked reduction in numbers of polyhedra produced. Those that are formed contain aberrant particles and are not infectious per OS (MacKinnon et al., 1974). With these considerations in mind the
Relationship Production Input
m.0.i.
0.01-4 10-40 75-300 400-500
TABLE 5 Between NOV Yield, Polyhedra and Multiplicity of Infectiona PIU/cell 67+ IS 15 f 10 16+ 6 S2+ 6
Polyhedra/cell 23 +4 55 r 9 41 f 7 42t 6
‘Data were taken from Table 4 and Figure 2. The values of PIU/cell and polyhedra/cell were calculated as the mean of the respective values for each multiplicity of infection within each group.
256
BROWN
AND
experiments reported here were undertaken to examine some of the factors affecting polyhedra yield at early passage level of virus. We started our investigation by recognizing that individual cells of an uncloned culture can contain as few as 5 and as many as 200 polyhedra/nucleus (Faulkner and Henderson, 1972). Data given in Table 2 show that those clones selected from an extensively passaged line of TN368 cells did not appear to yield significantly different quantities of inclusion bodies. The experiments were performed at m.o.i. = 4 which, from statistical considerations should have ensured that each cell was exposed to at least 1 PIU of virus at the beginning of the experiment. Infected cells in the cloned cultures contained the same extreme variation of polyhedra in individual cells as had been observed in uncloned cultures. It is shown in Table 2 that cell division is arrested soon after virus infection both in cloned and uncloned cultures. Arrest of cellular growth has also been observed following infection of T. ni cells with NPV of Autographa californica (Vail et al., 1973) and infection of S. frugiperda cells with homologous NPV (Knudson and Tinsley, 1974). Variability in polyhedra production may result from infection of a nonsynchronous culture with the NPV virus. Efficiency of virus production in individual cells in culture may be greatly influenced by the stage of cell cycle at which a cell is infected. We found that the average yield of polyhedra was affected by the multiplicity of infection of the input virus (Fig. 2). At multiplicities from 0.01 to 4 the average yield of polyhedra was about the same, but average yields of polyhedra tripled at m.o.i. 20-30. Still higher multiplicities up to m.o.i. = 500 lead to progressive reduction of average polyhedra yield. The existence of an optimum multiplicity of infection probably indicates that a large number of extracellular and intracellular sites need to be saturated to maximize polyhedra production. The particle/II-l ratio has not been determined for the NPV of T. ni but is in the range
FAULKNER
266 h 177 particles/PFU for another baculovirus, NPV of S. frugiperda (Knudson and Tinsley, 1974). At very high multiplicities of infection the average yield of polyhedra fell (Fig. 2). Under these conditions cells would have been exposed to an enormous number of particles which could have resulted in competition between polyhedron yielding infectious particles and others not yielding polyhedra. Attachment of a large number of particles may damage cell membranes and result in suboptimal conditions for polyhedra production. The yield of NOV/cell varies from ‘2 to 105 PIU/cell (Table 4) and seems to be maximal when polyhedra yield is minimal (Table 5). Working with the NPV of S. frugiperda, Knudson and Tinsley (1974) obtained values of 2-7 III/cell at m.o.i. = 0.2 -25. In the T. ni system, the increase observed in polyhedra yield in response to an increase in multiplicity of infection may be greater than the increase in virion production. This would result in a greater number of virions being occluded and a lower yield of NOV observed. However, at input m.o.i. above 400, the yield of NOV increases while the polyhedra yield does not change. The stimulation of polyhedra synthesis at these multiplicities may be lessened by suboptimal conditions for protein synthesis in the cytoplasm, due to damage to cell membranes caused by attachment of many particles. Virion synthesis, which occurs in the nucleus, may be less affected by cell membrane changes. Thus, infectious particles which reach the nucleus replicate, but since polyhedra production is not optimal, a large number of virions may not be occluded. Hence, an increase in NOV yield is observed at m.0.i. = 400 +. In conclusion, it does not appear that the observed variation in numbers of polyhedra in individual cells can be due solely to the presence in cultures of clones of high and low yielding strains of cells nor can it be ascribed to the consequence of cells in culture being infected with vastly different numbers of infecting particles. We have still to investigate
NPV
OF
~richo/hsia
whether our presently uncloned virus contains high polyhedra yielding strains and whether cells infected at various stages in their growth yield differing quantities of virus. ACKNOWLEDGMENTS Some of this work was done at the Natural Environment Research Council, Unit of Invertebrate Virology, Oxford, England. We thank Dr. T. W. Tinsley for providing space, and acknowledge much valuable discussion with the staff of the Unit. We also thank Roger R. Moore and John Palmer for help in the statistical analyses.
REFERENCES COOPER, J. E. K. 1970. “Rapid” isolation of single-cell clones of mammalian cell cultures. Texas Rep. Biol. Med., 28,29. FAULKNER, P. AND HENDERSON, J. F. 1972. Serial passage of a nuclear polyhedrosis virus of the cabbage looper (Trichoplusiu nil in a continuous tissue culture cell line. Virology. 50,920-924. GARDINER, G. R. AN5 STOCKDALE, H. 1973. Adaptation of an established line of Spodoptera frugiperda to a simple tissue culture medium. Abstract in Proceedings of Vlth Annual Meeting of the Society for Invertebrate Pathology, 20. Oxford, England. GOODWIN, R. H., VAUGHN, J. L., ADAMS, J. R., AND Lou~ou5~s. S. J. 1970. Replication of a nuclear polyhedrosis virus in an established insect cell line. J. Invertebr. Pathol.. 16,284-288.
ni
IN
CELL
CULTURE
257
GOODWIN, R. H., VAUGHN, J: L., ADAMS, J. R., AND LOULOUDES, S. J. 1973. The effect of insect cell lines and tissue culture media on Baculovirus polyhedra production. Misc. Publ. Entomol. Sot. Amer., 66-72. HINK, W. F. 1970. Established insect cell line from the cabbage looper, Trichoplusia ni. Nature (London), 226,466-467. IGNOFFO, C. M., HOSTETTER, D. L., AND SHAPIRO, M. 1974. Efficacy of insect viruses propagated in vivo and in vitro. .I. Invertebr. Pathol., 24, 184-187. KNUDSON, D. L. AND TINSLEY, T. W. 1974. Replication of a nuclear polyhedrosis.viruH in a continuous cell culture of Spodoptero Jrugiperda: Purification, assay of infectivity and growth characteristics of the virus. J. Viral., 14,934944. MACKINNON, E. A., HENDERSON, J. F., STOLTZ, D. B., AND FAULKNER, P. 1974. Morphogenesis of Nuclear Polyhedrosis Virus under conditions of prolonged passage in vitro. J. Ultrastruc~. Res.. 49.419-435. REED, L. J. AND MUENCH, H. 1938. A simple method of estimating fifty percent end points. Amer. J. Hyg.. 27,493-497. SOHI, S. S. AND CUNNINGHAM, J. C. 1972. Replication of a nuclear polyhedrosis virus in serially transferred insect hemocyte cultures. J. Inverrebr. Parhol.. 19, 51-61. STAIRS, G. R. 1968. Inclusion-type insect viruses. turr. Top. Microbial. Immunol.. 42, l-24. VAIL, P. V., JAY, D. L., AND HINK, W. F. 1973. Replication and infectivity of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica. produced in cells grown in vitro. J. Invertebr. Pathol., 22,231-237.