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Infection of synchronized TN-368 cell cultures with alfalfa looper nuclear polyhedrosis virus
JOURNAL
OF INVERTEBRATE
PATHOLOGY
32, l-5 (1978)
Infection of Synchronized TN-368 Cell Cultures with Alfalfa Looper Nuclear Polyhedrosis Virus D. E. LYNN Department of Entomology,
AND W. F. HINK
The Ohio State University, Columbus, Ohio 43210
Received September 19, 1977 Synchronized cultures of the TN-368 insect cell line were infected with a nuclear polyhedrosis virus from the alfalfa looper, Autographa californica, during different phases of the cell cycle. Cultures exposed to virus during the middle and late S phase have higher percentages of infected cells than cultures inoculated with virus in the Gz phase. The amount of virus produced from each infected cell (polyhedra and plaque forming units) is not significantly different between cultures infected at aJl phases of the cell cycle. KEY WORDS: Autographa californica; Trichoplusia ni; nuclear polyhedrosis virus.
INTRODUCTION
FH medium (Hink and Strauss, 1976a). Conditioned medium (C-TNM-FH) used in the studies was prepared from TNM-FH by growing cells in a monolayer from an initial density of 2 x lo5 cells/ml for 12 hr. After 12 hr, the medium was poured off the monolayers and filtered through a 0.22~pm filter (Millipore Corp., Bedford, Massachusetts). Synchronization of the TN-368 cells was accomplished with the double thymidine block procedure described previously (Lynn and Hink, 1978). This technique results in over 90% of the cells entering DNA synthesis immediately following removal of the block with the degree of synchrony being 42% at 14 hr following the block. Virus infection studies. The virus used in these studies was AcNPV as described by Vail et al. (1971) and was obtained from Dr. Vail in 1972. The inoculation used had been passed through T. ni larvae and twice through TN-368 cells and was stored at 4°C in TNMFH. It was characterized by 80% MP variant as described by Hink and Vail (1973) and Ramoska and Hink (1974). Virus titers were determined with a plaque assay technique (Hink and Strauss, 1977). Assays were performed in triplicate. Infection studies with synchronized cultures consisted of two somewhat varied procedures. Cultures in 25cm2 glass tissue cul-
Insect tissue culture is being considered as a method of producing insect viruses for use as microbial insecticides. To reduce costs, the virus yields from cell cultures must be optimized to obtain as many virus inclusion bodies as possible. The alfalfa looper, Autographa californica, nuclear polyhedrosis virus (AcNPV) and the cabbage looper, Trichoplusia ni, insect cell line (TN-368) is one system receiving consideration. Variability in nuclear polyhedrosis virus yields depends upon the phase of growth (lag, exponential, or stationary) that cultures are in at the time of infection (Volkman and Summers, 1975; Vaughn, 1976). It has also been proposed that variation in virus yield could be a result of the cell cycle phase that individual cells are in at the time of infection (Brown and Faulkner, 1975). This study was undertaken to test this latter hypothesis in the AcNPV/TN-368 system. METHODS
AND MATERIALS
Cells and media. The ovarian insect cell line, TN-368, developed from a virgin adult T. ni (Hink, 1970) was used in these studies. The line had been subcultured over 1000 times at the beginning of these experiments, and the cells were grown in modified TNMl
0022-2011/78/0321-0001$01.00/0 Copyright Q 1978 by Academic Press, Inc. AU ri@Us of reproduction in any form reserved.
2
LYNN AND HINK
ture flasks (type T flasks, Bellco Glass, Vineland, New Jersey) were synchronized by the double thymidine block method of Puck (1964) as modified by Lynn and Hink (1978). At 1.5 hr intervals from times 0 through 13.5 hr post-block, four replicate monolayer cultures were infected with a multiplicity of infection (m.0.i.) of five plaque forming units (pfu) per cell over a I-hr adsorption period, with tilting of the cultures at 15-min intervals. After the adsorption period, the virus was poured off the cultures, and 5 ml of C-TNM-FH was added. The infected cells were incubated at 28°C for 25 hr. At 25 hr post-infection, cells were suspended by shaking, and the percentage of cells containing polyhedra in their nuclei (percentage infection) was determined by observing 150250 cells from each flask under phase microscopy on a hemacytometer. Cell density was also determined at this time by hemacytometer counts. The number of pfu/infected cell was determined by plaque assay. Polyhedral inclusion bodies (PIBs)/infected cell were determined by sonifying the 25-hr-old infected cultures and counting the number of PIBs with a hemacytometer. In a second procedure, double thymidine block-synchronized cultures in 12.5 ml of
FIG. 1. Percentage infection in synchronized TN-368 cultures assayed 25 hr post-infection. X axis is the time following the second synchronization block with thymidine when the cultures were inoculated with virus. Each point is the mean of four replicate cultures; bars equal 1 SD. S, G2, and M + G, are the phases of the cell cycle at the time of infection.
medium in 65-cm2 glass tissue culture flasks (type B flasks, Bellco Glass, Vineland, New Jersey) were infected with an m.o.i. of 20 pfu/cell at times 3, 8, and 13 hr post-block. In addition, asynchronous cultures were infected also with an m.o.i. of 20 (three replicate cultures were used for each treatment). Following 1 hr of adsorption with tilting at 15-min intervals, the virus was decanted, and 12.5 ml of C-TNM-FH was placed over each culture. Virus replication curves were obtained by removing 0.3-ml samples of the supernatant from each infected culture at times over the following 72 hr. The samples were diluted 1:lO in fresh TNM-FH, centrifuged at 121g in a Sorvall RC2-B refrigerated centrifuge to remove cells and debris and were then frozen at -80°C until they were plaque-assayed. At 72 hr post-infection, the cells were suspended by shaking and assayed for percentage infection and PIBs/cell as described earlier. The statistical analysis in all experiments was made with Newman-Keul’s mean comparison test at the 0.05 level of significance (Newman, 1939). RESULTS
AND DISCUSSION
In the first series of experiments, the cultures were examined at 25 hr post-infection. Cells exposed to virus in middle and late S phase have a statistically higher percentage infection than cells in Gz (Fig. 1, Table 1). Cells infected at 6 hr post-block, shown in synchronization studies to be about 30% S and 70% G, (Lynn and Hink, 1978), have a mean percentage infection which is not statistically different from S or G, infected cells. This was also the case with cultures infected 13.5 hr post-block. Cells infected immediately following the second thymidine block have a significantly lower percentage infection as compared to all other infection times. This may be a result of conditions occurring in the early S phase or it could be the result of some type of recovery to the thymidine block occurring in the cultures at the time. No significant differences were ob-
NPV IN SYNCHRONIZED TABLE INFECTION
1
OF SYNCHRONIZED
Percentage infection Time of infection (hr of post-block) 0 1.5 3 4.5 6 7.5 9 10.5 12 13.5
Mean* 62.3 85.3 84.2 87.7 81.0 75.5 75.1 76.4 74.6 81.1
a c c c bc b b b b bc
3
CELL CULTURES
CULTURES~
PIB/cell SD
Mean”
3.5 4.0 1.6 2.0 3.9 5.0 5.4 3.1 2.8 4.3
168.9 138.1 131.0 98.9 93.3 99.8 120.5 99.8 112.3 99.5
d e e e e e e e e e
pfu/cell SD
Mean*
25.8 26.9 26.2 16.9 24.5 21.2 21.7 13.1 15.6 10.9
180.4 162.4 168.5 168.6 194.7 229.7 214.7 220.9 246.6 222.7
SD f f f f f f f f f f
67.6 37.8 31.1 41.0 55.0 67.8 63.1 73.4 83.3 71.2
(2Culture evaluated 25 hr post-infection. * Means followed by the same letter are not significantly different at the 0.05 level with Newman-Keul’s mean comparison test.
served in the pfu/infected cell data. In the PIB/infected cell data, only the 0 time period showed a significantly higher value. This may be an actual result of infection during early S phase, but when compared with the percentage infection data for time 0, which was lower than other times, and the pfu/infected cell data which was not significantly different at time 0, it is believed that this is an artifact due to the thymidine block and not due to cells being in the S phase. A possible explanation for the higher percentage infection at 25 hr post-infection in cells infected during the S phase is that the biosynthetic activity occurring in the cells during the S phase may provide necessary conditions for more rapid replication of virus. Such factors could include DNA synthesizing enzymes or cellular pools of necessary precursors for virus replication. These kinds of factors have been proposed in other virus/ cell systems, such as equine abortion virus in KB cells (Lawrence, 1971), SV40 in CV, cells (Pages et al., 1973), SV40 in transformed hamster cells (Kaplan et al., 1975), and Epstein-Barr virus in Raji cells (Hampar et al., 1976). In each of these cases, a DNA virus was replicated in greater quantities when the cells were infected (or the
virus activated) during the S phase of the host cell cycle. To test this possibility further in the TN36WAcNPV system, a second infection study was made. One-step virus replication curves were produced from cells infected in the S,
TIME
SHOWS
Post
lnfaction~
FIG. 2. Virus replication curves for cells infected during different cell cycle phases. Each point is the mean of three replicate cultures. 0, Asynchronous; A, S phase; 0, G, phase; A, M + G, period.
LYNN AND HINK TABLE INFECTION
Cell cycle phase when inoculated with virus (hr post-block) s (3) G (8) M + G, (13) Asynchronous
2
OF SYNCHRONIZED
Percentage infection Meanb 99.03 93.68 97.46 95.26
a
c ab bc
CULTURES~
PIB/cell
SD
Mean
SD
0.86 2.14 0.47 2.08
125.84 150.15 137.07 103.12
35.06 3.80 13.98 11.05
a Culture evaluated 72 hr post-infection. * Means followed by the same letter are not significantly different at the 0.05 level with Newman-Keul’s mean comparison test.
Gz, and M + G, phases and as asynchronous populations. If the virus replicated more rapidly in S phase cells as proposed above, it would be expected that an increase in virus titer would occur at an earlier time postinfection. The results of the one-step virus replication studies are presented in Figure 2. All of the virus growth curves have the same general shape and are similar to one presented by Hink and Strauss (1976b) for this virus/cell system. In all four growth curves, the initial increase in the amount of released virus is between 10 and 12.5 hr post-infection. This indicates that no difference occurs in the replication and release of nucleocapsids for cells infected in the different cell cycle phases. In addition to the virus replication curves, PIB/cell and percentage infection data were collected for these cultures at 72 hr postinfection and are presented in Table 2. No significant difference was detected in the PIB/infected cell ratio. The percentage infection data are similar to the 25hr post-infection data in that the S phase infected cells had a significantly higher percentage infection than did Gz or asynchronous cultures (0.05 level of significance with Newman-Keul’s mean comparison test; Newman, 1939). M + G, infected cultures did not differ from S phase or asynchronous infected cultures, but had higher values than those for G, infected cultures. From these results, it appears that the virus
is not produced more rapidly in S phase cells. Instead, the S phase TN-368 cells must be more susceptible to AcNPV infection than are cells in other phases of the cell cycle. This may be a result of conditions at the cell membrane during the S phase. These conditions may be a change in surface charge as found to occur in human cell lines, RPM1 number 41 (Mayhew and O’Grady, 1965; Mayhew, 1966), and HeLa (Brent and Forrester, 1967), which could affect virus adsorption. Another possibility is that an actual physical change in the cell membrane, as observed in Chinese hamster cells (Enlander et al., 1974)) may result in increased adsorption and/or penetration of the virus infective units. The observations made in this study may provide insight to the previous findings reported concerning the variability in virus yields with NPVs in cultured cells. Volkman and Summers (1975) and Vaughn (1976) repored higher virus yields from cultures infected during exponential growth as compared to the lag and stationary growth phases. It is possible that cultures in these nondividing periods of growth are primarily in a noncycling period of growth (G,). Cells in Go may be less susceptible and produce fewer polyhedra in the same manner as the cells in G, phase in this study. Further studies are necessary to determine the feasibility of using synchronized cultures to increase virus yields. It may be possible to utilize suspension culture techniques developed for TN-368 (Hink and Strauss, 1976a) in association with an automatic synchronizer such as described by Okada and Shonohara (1974) to provide a method for increasing the efficiency of virus production. ACKNOWLEDGMENTS This research was supported, in part, by U. S. Environmental Protection Agency Grant R-802516.
REFERENCES BRENT, T. P., AND FORKESTER, J. A. 1967. Changes in surface charge of Hela cells during the cell cycle. Nature (London), 215, 92-93.
NPV IN SYNCHRONIZED BROWN, M., AND FAULKNER, P. 1975. Factors affecting the yield of virus in a cloned cell line of Trichoplusin ni infected with a nuclear polyhedrosis virus. .Z. Znvertebr. Pathol., 26, 251-257. ENLANDER, D., SCOTT, T., AND TOBEY, R. A. 1974. Observations of the surface of synchronized Chinese hamster ovary cells in suspension culture. Zn “Scanning Electron Microscopy11974.” ITT Research Institute, Chicago, Illinois. HAMPAR, B., LENOIR, G., NONOYAMA, M., DERGE, J. G., AND CHANG, S. 1976. Cell cycle dependence for activation of Epstein-Barr virus by inhibitors of protein synthesis or medium deficient in arginine. Virology,
69, 660-668.
HINK, W. F. 1970. Established insect cell line from the cabbage looper, Trichoplusia ni. Nature (London),
226, 466-467.
HINK, W. F., AND STRAUSS, E. 1976a. Growth of the Trichoplusia ni (TN-368) cell line in suspension cultdre. In “Invertebrate Tissue Culture, Applications in Medicine, Biology, and Agriculture” (E. Kurstak and K. Maramorsch, eds.), pp. 297-300. Academic Press, New York. HINK, W. F., AND STRAUSS, E. 1976b. Replication of alfalfa looper nuclear polyhedrosis virus in the Trichoplusia ni (TN-368) cell line. In “Invertebrate Tissue Culture, Applications in Medicine, Biology, and Agriculture.” (E. Kurstak and K. Maramorsch, eds.), pp. 369-374. Academic Press, New York. HINK, W. F., AND STRAUSS, E. M. 1977. An improved technique for plaque assay ofAutographa californica nuclear polyhedrosis virus on TN-368 cells. J. Znvertebr.
Pathol.,
29, 390-391.
HINK, W. F., AND VAIL, P. V. 1973. A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line. J. Znvertebr. Pathol., 22, 168- 174. KAPLAN, J. C., Kleinman, L. F., AND BLACK, P. H. 1975. Cell cycle dependence of simian virus 40 induction from transformed hamster cells by ultraviolet irradiation. Virology, 68, 215-220. LAWRENCE, W. C. 1971. Evidence for a relationship
CELL CULTURES
5
between equine abortion. (herpes) virus deoxyribonucleic acid synthesis and the S phase of the KB cell cycle.
J. Virol.,
7, 736-748,
LYNN, D. E., AND HINK, W. F. 1978. Cell cycle amdysis and synchronization of the TN-368 insect cell line. In vitro, 14, 236-238. MAYHEW, E. 1966. Cellular electrophoretic mobility and the mitotic cycle. J. Gen. Physiol., 49,717-725. MAYHEW, E., AND O’GRADY, E. A. 1965. Electrophoretic mobihties of tissue culture cells in exponential and parasynchronous growth. Nature (London), 2437, 86-87. NEWMAN, D. 1939. The distribution ofrange in samples from a normal population, expressed in terms of an independent estimate of standard deviation. Biometrika, 31, 20-30. OKADA, S., AND SHONOHARA, K. 1974. An automatic synchronizer for suspension culture and L5178Y cells. Ceil Tissue Kinet., 7, 195-199. PAGES, J., Manteuil, S., Stehehn, D., Fiszman, M., Marx, M., AND Girard, M. 1973. Relationship between replication of simian virus 40 DNA and specific events of the host cell cycle. J. Virol., 12,99- 107. PUCK, T. T. 1964. Phasing, mitotic delay, and chromosomal abberrations in mammalian cells. Science, 144, 565-566. RAMOSKA, W. A., AND HINK, W. F. 1974. Electron microscope examination of two plaque variants from a nuclear polyhedrosis virus of the alfalfa looper, Autographa
californica.
J. Znvertebr.
Pathol.,
23,
197-201. VAIL, P. V., SUTTTER, G., JAY, D. L., AND GOUGI-I, D. 197 1. Reciprocal infectivity of nuclear polyhedrosis viruses of the cabbage looper and alfalfa looper. J. Znvertebr. Pathol., 17, 383-388. VAUGHN, J. L. 1976. The production of nuclear polyhedrosis viruses in large-volume cell cultures. J. Znvertebr.
Pathol..
28, 233-237.
VOLKMAN, L. E., AND SUMMERS, M. D. 1975. Nuclear polyhedrosis virus detection: relative capabilities of clones developed from Trichoplusia ni ovarian cell line TN-368 to serve as indicator cells in a plaque assay. J. Virol., 16, 1630-1637.
OF INVERTEBRATE
PATHOLOGY
32, l-5 (1978)
Infection of Synchronized TN-368 Cell Cultures with Alfalfa Looper Nuclear Polyhedrosis Virus D. E. LYNN Department of Entomology,
AND W. F. HINK
The Ohio State University, Columbus, Ohio 43210
Received September 19, 1977 Synchronized cultures of the TN-368 insect cell line were infected with a nuclear polyhedrosis virus from the alfalfa looper, Autographa californica, during different phases of the cell cycle. Cultures exposed to virus during the middle and late S phase have higher percentages of infected cells than cultures inoculated with virus in the Gz phase. The amount of virus produced from each infected cell (polyhedra and plaque forming units) is not significantly different between cultures infected at aJl phases of the cell cycle. KEY WORDS: Autographa californica; Trichoplusia ni; nuclear polyhedrosis virus.
INTRODUCTION
FH medium (Hink and Strauss, 1976a). Conditioned medium (C-TNM-FH) used in the studies was prepared from TNM-FH by growing cells in a monolayer from an initial density of 2 x lo5 cells/ml for 12 hr. After 12 hr, the medium was poured off the monolayers and filtered through a 0.22~pm filter (Millipore Corp., Bedford, Massachusetts). Synchronization of the TN-368 cells was accomplished with the double thymidine block procedure described previously (Lynn and Hink, 1978). This technique results in over 90% of the cells entering DNA synthesis immediately following removal of the block with the degree of synchrony being 42% at 14 hr following the block. Virus infection studies. The virus used in these studies was AcNPV as described by Vail et al. (1971) and was obtained from Dr. Vail in 1972. The inoculation used had been passed through T. ni larvae and twice through TN-368 cells and was stored at 4°C in TNMFH. It was characterized by 80% MP variant as described by Hink and Vail (1973) and Ramoska and Hink (1974). Virus titers were determined with a plaque assay technique (Hink and Strauss, 1977). Assays were performed in triplicate. Infection studies with synchronized cultures consisted of two somewhat varied procedures. Cultures in 25cm2 glass tissue cul-
Insect tissue culture is being considered as a method of producing insect viruses for use as microbial insecticides. To reduce costs, the virus yields from cell cultures must be optimized to obtain as many virus inclusion bodies as possible. The alfalfa looper, Autographa californica, nuclear polyhedrosis virus (AcNPV) and the cabbage looper, Trichoplusia ni, insect cell line (TN-368) is one system receiving consideration. Variability in nuclear polyhedrosis virus yields depends upon the phase of growth (lag, exponential, or stationary) that cultures are in at the time of infection (Volkman and Summers, 1975; Vaughn, 1976). It has also been proposed that variation in virus yield could be a result of the cell cycle phase that individual cells are in at the time of infection (Brown and Faulkner, 1975). This study was undertaken to test this latter hypothesis in the AcNPV/TN-368 system. METHODS
AND MATERIALS
Cells and media. The ovarian insect cell line, TN-368, developed from a virgin adult T. ni (Hink, 1970) was used in these studies. The line had been subcultured over 1000 times at the beginning of these experiments, and the cells were grown in modified TNMl
0022-2011/78/0321-0001$01.00/0 Copyright Q 1978 by Academic Press, Inc. AU ri@Us of reproduction in any form reserved.
2
LYNN AND HINK
ture flasks (type T flasks, Bellco Glass, Vineland, New Jersey) were synchronized by the double thymidine block method of Puck (1964) as modified by Lynn and Hink (1978). At 1.5 hr intervals from times 0 through 13.5 hr post-block, four replicate monolayer cultures were infected with a multiplicity of infection (m.0.i.) of five plaque forming units (pfu) per cell over a I-hr adsorption period, with tilting of the cultures at 15-min intervals. After the adsorption period, the virus was poured off the cultures, and 5 ml of C-TNM-FH was added. The infected cells were incubated at 28°C for 25 hr. At 25 hr post-infection, cells were suspended by shaking, and the percentage of cells containing polyhedra in their nuclei (percentage infection) was determined by observing 150250 cells from each flask under phase microscopy on a hemacytometer. Cell density was also determined at this time by hemacytometer counts. The number of pfu/infected cell was determined by plaque assay. Polyhedral inclusion bodies (PIBs)/infected cell were determined by sonifying the 25-hr-old infected cultures and counting the number of PIBs with a hemacytometer. In a second procedure, double thymidine block-synchronized cultures in 12.5 ml of
FIG. 1. Percentage infection in synchronized TN-368 cultures assayed 25 hr post-infection. X axis is the time following the second synchronization block with thymidine when the cultures were inoculated with virus. Each point is the mean of four replicate cultures; bars equal 1 SD. S, G2, and M + G, are the phases of the cell cycle at the time of infection.
medium in 65-cm2 glass tissue culture flasks (type B flasks, Bellco Glass, Vineland, New Jersey) were infected with an m.o.i. of 20 pfu/cell at times 3, 8, and 13 hr post-block. In addition, asynchronous cultures were infected also with an m.o.i. of 20 (three replicate cultures were used for each treatment). Following 1 hr of adsorption with tilting at 15-min intervals, the virus was decanted, and 12.5 ml of C-TNM-FH was placed over each culture. Virus replication curves were obtained by removing 0.3-ml samples of the supernatant from each infected culture at times over the following 72 hr. The samples were diluted 1:lO in fresh TNM-FH, centrifuged at 121g in a Sorvall RC2-B refrigerated centrifuge to remove cells and debris and were then frozen at -80°C until they were plaque-assayed. At 72 hr post-infection, the cells were suspended by shaking and assayed for percentage infection and PIBs/cell as described earlier. The statistical analysis in all experiments was made with Newman-Keul’s mean comparison test at the 0.05 level of significance (Newman, 1939). RESULTS
AND DISCUSSION
In the first series of experiments, the cultures were examined at 25 hr post-infection. Cells exposed to virus in middle and late S phase have a statistically higher percentage infection than cells in Gz (Fig. 1, Table 1). Cells infected at 6 hr post-block, shown in synchronization studies to be about 30% S and 70% G, (Lynn and Hink, 1978), have a mean percentage infection which is not statistically different from S or G, infected cells. This was also the case with cultures infected 13.5 hr post-block. Cells infected immediately following the second thymidine block have a significantly lower percentage infection as compared to all other infection times. This may be a result of conditions occurring in the early S phase or it could be the result of some type of recovery to the thymidine block occurring in the cultures at the time. No significant differences were ob-
NPV IN SYNCHRONIZED TABLE INFECTION
1
OF SYNCHRONIZED
Percentage infection Time of infection (hr of post-block) 0 1.5 3 4.5 6 7.5 9 10.5 12 13.5
Mean* 62.3 85.3 84.2 87.7 81.0 75.5 75.1 76.4 74.6 81.1
a c c c bc b b b b bc
3
CELL CULTURES
CULTURES~
PIB/cell SD
Mean”
3.5 4.0 1.6 2.0 3.9 5.0 5.4 3.1 2.8 4.3
168.9 138.1 131.0 98.9 93.3 99.8 120.5 99.8 112.3 99.5
d e e e e e e e e e
pfu/cell SD
Mean*
25.8 26.9 26.2 16.9 24.5 21.2 21.7 13.1 15.6 10.9
180.4 162.4 168.5 168.6 194.7 229.7 214.7 220.9 246.6 222.7
SD f f f f f f f f f f
67.6 37.8 31.1 41.0 55.0 67.8 63.1 73.4 83.3 71.2
(2Culture evaluated 25 hr post-infection. * Means followed by the same letter are not significantly different at the 0.05 level with Newman-Keul’s mean comparison test.
served in the pfu/infected cell data. In the PIB/infected cell data, only the 0 time period showed a significantly higher value. This may be an actual result of infection during early S phase, but when compared with the percentage infection data for time 0, which was lower than other times, and the pfu/infected cell data which was not significantly different at time 0, it is believed that this is an artifact due to the thymidine block and not due to cells being in the S phase. A possible explanation for the higher percentage infection at 25 hr post-infection in cells infected during the S phase is that the biosynthetic activity occurring in the cells during the S phase may provide necessary conditions for more rapid replication of virus. Such factors could include DNA synthesizing enzymes or cellular pools of necessary precursors for virus replication. These kinds of factors have been proposed in other virus/ cell systems, such as equine abortion virus in KB cells (Lawrence, 1971), SV40 in CV, cells (Pages et al., 1973), SV40 in transformed hamster cells (Kaplan et al., 1975), and Epstein-Barr virus in Raji cells (Hampar et al., 1976). In each of these cases, a DNA virus was replicated in greater quantities when the cells were infected (or the
virus activated) during the S phase of the host cell cycle. To test this possibility further in the TN36WAcNPV system, a second infection study was made. One-step virus replication curves were produced from cells infected in the S,
TIME
SHOWS
Post
lnfaction~
FIG. 2. Virus replication curves for cells infected during different cell cycle phases. Each point is the mean of three replicate cultures. 0, Asynchronous; A, S phase; 0, G, phase; A, M + G, period.
LYNN AND HINK TABLE INFECTION
Cell cycle phase when inoculated with virus (hr post-block) s (3) G (8) M + G, (13) Asynchronous
2
OF SYNCHRONIZED
Percentage infection Meanb 99.03 93.68 97.46 95.26
a
c ab bc
CULTURES~
PIB/cell
SD
Mean
SD
0.86 2.14 0.47 2.08
125.84 150.15 137.07 103.12
35.06 3.80 13.98 11.05
a Culture evaluated 72 hr post-infection. * Means followed by the same letter are not significantly different at the 0.05 level with Newman-Keul’s mean comparison test.
Gz, and M + G, phases and as asynchronous populations. If the virus replicated more rapidly in S phase cells as proposed above, it would be expected that an increase in virus titer would occur at an earlier time postinfection. The results of the one-step virus replication studies are presented in Figure 2. All of the virus growth curves have the same general shape and are similar to one presented by Hink and Strauss (1976b) for this virus/cell system. In all four growth curves, the initial increase in the amount of released virus is between 10 and 12.5 hr post-infection. This indicates that no difference occurs in the replication and release of nucleocapsids for cells infected in the different cell cycle phases. In addition to the virus replication curves, PIB/cell and percentage infection data were collected for these cultures at 72 hr postinfection and are presented in Table 2. No significant difference was detected in the PIB/infected cell ratio. The percentage infection data are similar to the 25hr post-infection data in that the S phase infected cells had a significantly higher percentage infection than did Gz or asynchronous cultures (0.05 level of significance with Newman-Keul’s mean comparison test; Newman, 1939). M + G, infected cultures did not differ from S phase or asynchronous infected cultures, but had higher values than those for G, infected cultures. From these results, it appears that the virus
is not produced more rapidly in S phase cells. Instead, the S phase TN-368 cells must be more susceptible to AcNPV infection than are cells in other phases of the cell cycle. This may be a result of conditions at the cell membrane during the S phase. These conditions may be a change in surface charge as found to occur in human cell lines, RPM1 number 41 (Mayhew and O’Grady, 1965; Mayhew, 1966), and HeLa (Brent and Forrester, 1967), which could affect virus adsorption. Another possibility is that an actual physical change in the cell membrane, as observed in Chinese hamster cells (Enlander et al., 1974)) may result in increased adsorption and/or penetration of the virus infective units. The observations made in this study may provide insight to the previous findings reported concerning the variability in virus yields with NPVs in cultured cells. Volkman and Summers (1975) and Vaughn (1976) repored higher virus yields from cultures infected during exponential growth as compared to the lag and stationary growth phases. It is possible that cultures in these nondividing periods of growth are primarily in a noncycling period of growth (G,). Cells in Go may be less susceptible and produce fewer polyhedra in the same manner as the cells in G, phase in this study. Further studies are necessary to determine the feasibility of using synchronized cultures to increase virus yields. It may be possible to utilize suspension culture techniques developed for TN-368 (Hink and Strauss, 1976a) in association with an automatic synchronizer such as described by Okada and Shonohara (1974) to provide a method for increasing the efficiency of virus production. ACKNOWLEDGMENTS This research was supported, in part, by U. S. Environmental Protection Agency Grant R-802516.
REFERENCES BRENT, T. P., AND FORKESTER, J. A. 1967. Changes in surface charge of Hela cells during the cell cycle. Nature (London), 215, 92-93.
NPV IN SYNCHRONIZED BROWN, M., AND FAULKNER, P. 1975. Factors affecting the yield of virus in a cloned cell line of Trichoplusin ni infected with a nuclear polyhedrosis virus. .Z. Znvertebr. Pathol., 26, 251-257. ENLANDER, D., SCOTT, T., AND TOBEY, R. A. 1974. Observations of the surface of synchronized Chinese hamster ovary cells in suspension culture. Zn “Scanning Electron Microscopy11974.” ITT Research Institute, Chicago, Illinois. HAMPAR, B., LENOIR, G., NONOYAMA, M., DERGE, J. G., AND CHANG, S. 1976. Cell cycle dependence for activation of Epstein-Barr virus by inhibitors of protein synthesis or medium deficient in arginine. Virology,
69, 660-668.
HINK, W. F. 1970. Established insect cell line from the cabbage looper, Trichoplusia ni. Nature (London),
226, 466-467.
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