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Effect of X-irradiation on patas monkey kidney cell susceptibility to enteroviruses
7, 140-149(1959)
VIROLOGY
Effect
of X-Irradiation Cell Susceptibility
on Patas Monkey to Enteroviruses’
Kidney
G. D. HSIUNG Section of Epidemiology and Preventive Medicine, Yale University School of Medicine, S33 Cedar St., New Haven II, Connecticut Accepted
October 17, 1958
Enhancement of patas monkey kidney cell susceptibility to certain enteroviruses was induced by X-irradiation. With small inocula Coxsackie A9 virus growth occurred only in irradiated cells. On serial passages, increasing virus titers and cytopathic changes could be obtained only in irradiated cultures. After growth in irradiated cells, Coxsackie A9 virus was able to grow in nonirradiated cells. ECHO virus type 1 was also adapted to patas cells in a similar manner. The adapted strain yielded somewhat higher titers than the parent virus in patas cult,ures and had acquired the ability to give marked cytopathogenic effect (CPE). Attempts to adapt ECHO virus types 6 and 9 to irradiated patas cells were unsuccessful. Increased susceptibility to Coxsackie A9 virus infection was observed in both primary and first-passage patas monkey kidney cultures when such cultures were irradiated during the first 48 hours of in vitro life, a period of rapid growth. However, cultures irradiated at 7-14 days did not show similar responses. INTRODUCTION
In a previous publication, Hsiung and Melnick (1958a) described the behavior of various enteroviruses in tissue culture derived from different monkey species, indicating some of the virus-host relationships in susceptible and resistant cell systems. Rhesus (Mucuca mulatta) cultures were highly sensitive to the three groups of enteroviruses studied, whereas patas (Erythrocebus patus) monkey kidney cells were relatively resistant to Coxsackie A9 and ECHO type 1 viruses. Increased susceptibility to some viral infections has been shown to occur when certain animal species were exposed to X-irradiation (Clemmesen, 1939; de Gara and Furth, 1945; Clemmesen and Andersen, 1948; 1 Aided
by a grant
from the National
Foundation. 140
EFFECT
OF X-IRRADIATION
ON
PATAS
MOSKEY
KIDNEY
CELL
141
Syverton et al., 1952, 1956). Recently Puck and Marcus (1956) and Cieciura et al. (1957) found that giant cells formed in X-irradiated HeLa cultures were highly sensitive to Newcastle disease virus. The purpose of this study was to determine whether the susceptibility of patas monkey kidney cells to certain enteroviruses could be enhanced by such irradiation techniques. MATERIALS
AND
METHODS
Virus. Poliovirus type 1 (Mahoney strain) ; Coxsackie virus type A9 (Grigg strain); ECHO virus type 1 (Farouk strain), type 6 (D’Amori strain), and type 9 (Bourn strain) were used. All viruses were plaquepurified and grown in rhesus tissue cultures except ECHO virus type 6, which was purified by the terminal-dilution technique. Cultures. Primary patas and rhesus monkey kidney cultures in tubes and bottles were prepared as described previously (Hsiung and Melnick, 1957b). First-passage cultures were obtained by trypsinizing the primary cultures when 7-10 days old and reseeding into bottles. Virus assay. All virus inoculations and titrations were made in rhesus bottle cultures by the plaque technique (Hsiung and Melnick, 1957a) except ECHO virus type 6, in which tube cultures were used. X-ray irradiation.2 The X-irradiation was performed with a General Electric Maxitron deep-therapy machine, using 250 kv and 30-ma beam, filtered through l-mm aluminum filter, at a distance of 40 cm from the target. This gave a rate of 500 r per minute as measured with a Victoreen roentgen meter. Unless otherwise specified, patas cell cultures were irradiated at room temperature with 300 r, 48 hours after seeding in 3-ounce prescription bottles. The medium was changed immediately after irradiation to remove any toxic materials that might have been produced in the fluid. All cultures were used 7-10 days after seeding, i.e., 5-8 days after irradiation unless otherwise specified. EXPERIMENTAL
RESULTS
Persistence of Coxsaclcie A9 Virus Infection in Patas Cultures When irradiated or nonirradiated patas cultures were infected at high multiplicit’ies (10) of Coxsackie A9 virus, significant amounts of virus 2 All the irradiations were performed by Dr. E. H. Botany, Yale University. Measurements made by him data) showed that 30y0 of X-ray intensity was absorbed of culture bottles and the actual dosage was only 70% of 300 r was actually 210 r.
Y. Chu, Department of (Chu, 1958, unpublished or scattered by the glass that measured; therefore,
142
HSIUNG
TABLE A9
PERSISTENCE
OF COXSACKIE
1 VIRUS
CULTURES
IN PATAS
Virus recovered--average titer (log PFU per ml) Days of incubation at 37”
Patas cultures Nonirrad.
oa 3 5 15 25 40
7.7 6.0 5.0 3.8
Irrad.
7.7 6.2 5.7 6.5
LIOriginal inocula (multiplicity 10). b Original inoculum 3 X 107 PFU. c Samples were obtained on the thirteenth
Killed patas cultures Nonirrad.
Irrad.
7.5 5.2 4.6 2.2”
7.5 5.3 4.5 2.9c
Without cells
7.56 4.0 0 OC
-
day.
were recovered in the fluid for as long as 40 days (Table l), although no cytopathogenic effect (CPE) was recognizable. In contrast, Coxsackie A9 virus was rapidly inactivat.ed at 37” when there were no cells present in the media. Virus titers decreased in both irradiated and nonirradiated cultures which had been killed by freezing and thawing three times, although not quite as rapidly as in the cell-free samples (Table 1). This suggests that the virus which was recovered in high titers from the irradiated and nonirradiated patas cultures after prolonged incubation was newly produced. In addition, somewhat higher virus titers were found in irradiated cultures than in nonirradiated ones. A set of the irradiated and nonirradiated patas cultures was infected with Coxsackie A9, carried for 25 days without a cytopathogenic effect (CPE), and then challenged with 2.7 X IO6 rhesus PFU (plaque-forming units) of poliovirus type 1 (Mahoney strain). Cytopathic changes were observed 24 hours after the challenge, and destruction was complete in 48 hours. Yields of poliovirus from the control and the Coxsackie A9infected cultures of irradiated and nonirradiated patas cells were virtually the same, indicating no interference phenomena between these two viruses under these conditions. Initiation Cultures
of Growth and Multiplication with Small Inocula
of Coxsackie A9 Virus in Patas
It has been reported (Hsiung and Melnick, 1958a) that newly produced A9 virus could not be detected in either the fluid phase or in the cells of
EFFECT
OF X-IRRADIATION
ON
P;ZTAS
TABLE
MONKEY
KIDNEY
143
2
OF COXSACKIE A9 VIRUS IN CULTURES SMALL INOCULA
MULTIPLWATIOK
CELL
INFECTED
WITH
Virus titer (log, PFU per ml) Day after inoculation
0 1 2 3 7 21
~~ Nonirrad. patas
Irrad. patas
Rhesus
3.9” 2.4 1.9 0 0 0
3.9a 2.2 2.7 2.6 4.2 3.0
1.9” 4.5 7.4 8.4 8.3 5.9
a Original inoculum (multiplicity b Original inoculum (multiplicity
0.002). 0.00002).
patas cultures when inoculs were small (multiplicities less than 0.01). Experiments using such small inocula are shown in Table 2. The virus t,iters in the fluid of nonirradiated patas bottles fell off sharply, and no virus was recovered after 3 days, whereas some virus multiplication was demonstrable in the irradiated patss cultures. Rhesus cultures used for comparison showed a high titer of virus, although the inoculum was only l/100 of that used in the patas cultures. In four separate experiments small doses (multiplicities 0.0013-0.0023) were used to inoculate 31 irradiated cultures prepared from four different patas monkeys (Table 3). Fifty-two per cent (16/31) of the irradiated cultures yielded virus, whereas no virus was obtained from any of the 26 nonirradiated cultures. All cultures were handled and inoculated in the same way except for the irradiation. In experiments 1 and 2 the cultures were irradiated 48 hours after seeding; in experiment 3, 24 hours after seeding; and in experiment 4, 14 hours after reseeding of trypsinized primary patas cells. Increased cell susceptibility to virus infection occurred only when the cultures were irradiated during the period of most rapid growth (14-48 hours after seeding) or the period of maximal mitotic activity. Serial Passages of Cozsackie A9 Virus in Patas Cultures Subcultures of Coxsackie A9 virus in nonirradiated patas cells invariably failed to yield virus after a few serial passages. Titrations are listed in Table 4 for experiment I in which transfers of 0.1 ml of undiluted fluid to a new tube culture containing 0.9 ml of media was made every 3-4
144
HSIUNG
TABLE INITIATION
OF GROWTH
Low
T3tE
Expt. no.
1 2 3 4
3
A9 IN PATAS MULTIPLICITIES
OF COXSACKIE
Age of culture at irrad. (hours)
Multiplicity of infection”
48 48 24 14
0.0023 0.0013 0.0015 0.0015
Primary Primary Primary First passage
Total Per cent of growth a Multiplicit,y
INFECTED
Il’o. Virus
AT
Positive cultures/total tested Nonirradiated
Irradiated
o/2
l/2
O/6 O/16
o/2
11/17 m 3/4h
O/26
16/31
0 =
h Two samples
CIJLTURES
52
inoculated
Average no. cells (3 X 106). in this experiment showed CPE
days. In a similar manner serial passages of the same virus in irradiated bottle cultures (300 r), using the same dilution factors, gave virus in every passage, with titers only slightly lower than those of the original virus. Results of the two parallel series of transfers in irradiated and nonirradiated patas cultures are also shown in Table 4 (experiments II and III). Every 48 hours 1 ml of each culture fluid was transferred to a new culture containing 9 ml of fresh medium. The titer decreased with each passage in the nonirradiated patas cultures and completely disappeared after the fift,h passage. On the other hand, in the irradiated cell system a minimal titer was reached at the third (experiment II) and sixth (experiment III) passages, followed by increasing values. Marked cytopathic changes related to infection became evident in the irradiated cultures by the fifth passage. Titers in subsequent passages were similar to, or slightly higher than, those of the sixth passage. Adaptation
of Coxsackie
A9 Virus
to Nonirradiated
Patas Cells
After fourteen passages in irradiated patas cells, Coxsackie A9 virus was inoculated into nonirradiated patas cultures. Cytopathic changes were now observed in the nonirradiated cultures as well as irradiated ones. Small plaques were obtained in nonirradiated patas bottle cultures with this adapted virus. In contrast, the original (parent) virus, rarely gave plaques on patas cultures even when undiluted material was used. On rhesus monolayers the adapted virus produced smaller plaques (<5
EFFECT
OF
X-IRRADIATION
ON
PATAS
TABLE SERIAL
TRANSFERS
MONKEY
145
CELL
4 A9 VIRUS
OF COXSACKIE
KIDNEY
IN I’ATAS
CULTURES
Virus titer (log, PFU per ml) Kumber of passages
Experiment
I”
Experiment
II”
Experiment
III<
Nonirrad.
IKWl.
Nonirrad.
Irrad.
Nonirrad.
had.
8.4 7.4 5.6 4.7 1.6 0 0
9.1 8.1 7.1 7.5 7.4 7.8 7.8
8.5 7.4 5.9 4.4 2.2 1.7 0
8.5 7.0 6.4 5.5 6.5 7.2 7.2
8.5 7.4 5.9 4.4 2.2 2.0 0
8.5 7.4 6.6 5.7 5.2 4.8 4.6
u Experiment I: serial transfers were made every 3-4 days in irradiat,ed bottle cultures and nonirradiated tube cultures, using the same dilution factors. * Experiment II: serial transfers were made every 48 hours in parallel in irradiated and nonirradiated bottle cultures. Minimal titer in irradiated culture was reached at the third passage and increased thereafter. c Experiment III: serial transfers were made in parallel as in experiment II, except that the cells were obtained from a different animal. Minimal titer in irradiated culture was reached at the sixth passage and increased slightly (5.2) thereafter. d Original inocula.
mm in diameter at 7 days) than the original strain (> 10 mm in diameter at 7 days). The identity of the adapted virus was confirmed by neut,ralization with antiserum from rabbits which had been immunized with the P.B. strain of Coxsackie A9 virus. The ability of the adapted virus to produce paralysis in suckling mice was somewhat reduced; however, variants of two types of plaques were found after propagation of the adapt’ed virus in a single mouse passage. Preliminary results on this variation were reported recently (Hsiung, 1958b). Attempts to Adapt ECHO Viruses into Patas Cultures ECHO viruses types 1, 6, and 9 were transferred serially in patas cultures in a similar manner to that used for Coxsackie A9 virus. A definite CPE was observed in the irradiated cultures of ECHO-l virus after the third passage (Table 5), but there was no sign of cellular dest,ruction in any of the nonirradiated cultures at the time of transfer (48 hours). However, serial passage of ECHO-l in the nonirradiated patas culture series did not completely eliminate the virus, as in the case
146
HSIUNG
SERIAL Number of passages
TRANSFERS
TABLE 5 OF ECHO-l VIRUS
Cytopathic
effect
IN PATAS
CULTURES”
Virus titer (log, PFU per ml)
Nonirrad.
had.
Nonirrad.
had.
f
f + ++ +++ +++
6.8 4.8 2.8 2.6 2.8
7.4 5.8 5.6 5.0 4.1
1 3 5 7 10
0 0 0 0
u Original inocula 2.7 X lo* PFU. Transfers were made every 48 hours.
of Coxsackie A9, even after ten passages. The ECHO-l virus which was obtained by passage in the irradiated patas cultures was also able to produce a CPE in the nonirradiated patas cultures. When serial transfers were made either with ECHO virus type 6 or type 9, virus was not recoverable in either the irradiated or nonirradiated patas cultures after the third passage. No CPE was observed in any of the cultures, although the transfers were kept for as long as 14 days. Effect of Irradiation
on 7-l&Day-Old
Cultures
Serial transfers. When serial passages of Coxsackie A9 virus were made in patas cultures either irradiated on the ninth or fourteenth day after seeding, virus titers decreased both in the irradiated and the nonirradiated cultures almost at the same rate. No virus was recovered by the fifth passage. Plaque formation. Since 9- or 14-day-old patas cultures did not show any effect when exposed to 300 r, higher dosages were applied to fully grown monolayers of rhesus as well as patas cultures in order to obtain any possible changes in plaque formation. Seven-day-old patas and rhesus cultures were inoculated with poliovirus type 1, Coxsackie A9, or ECHO type 1. All cultures were exposed to 1600 r either immediately after infection or 2 hours after adsorption. Bottles were overlaid after the treatment. There was no significant difference in plaque counts in rhesus cultures whether the bottles were irradiated before or after virus adsorption nor was there any difference between irradiated and nonirradiated ones. Plaques appeared only in irradiated and nonirradiated patas bottles inoculated with poliovirus but not with Coxsackie A9 or ECHO-l virus.
EFFECT
OF X-IRRADIATION
ON
PATAS
MONKEY
KIDNEY
CELL
147
In two other experiments various dosages of X-rays were applied. Both rhesus and patas cultures 7 days old were inoculated with Coxsackie A9 virus and exposed to X-ray at different doses (1500-50,000 r). Plaques were not obtained in any of the patas cultures. The number of plaques in the rhesus cultures was not. measurably influenced by X-ray in any dosage of the range t’ested, and the cell sheet remained microscopically unaffected by the irradiation.
Although there were no cyt,opathic changes in the patas cultures infected with Coxsackie A9, moderate titers of virus were recovered for 40 days; whereas only small amounts of virus were detected on the thirteenth day in the control samples containing killed cells. Thus limited inapparent infection of Coxsackie A9 virus in both irradiated and non irradiated patas cultures apparently occurred on first passage when inocula were large. However, enhancement of virus growth was demonstrated in the irradiated cultures by the persistence of virus in higher titer from 25 days onward. With small inocula, initiation of Coxsackie A9 virus growt~h was found only in irradiated patas cells. In addition, Coxsackie A9 virus has been adapt,ed t’o the resistant system only aft,er serial passages in irradiated cultures. It has been noted previously (Hsiung and Melnick, 1958a) that tiny plaques appeared after a delay in cultures from a few patas monkeys when the inocula contained lo5 or more rhesus PFU of Coxsackie A9 or ECHO-l, but that in most lots of patas cultures, plaques failed to appear even with inocula of 10’ rhesus PFU. Subculture of such small plaques in patas cultures was not successful. The quest’ion was raised whether these small plaques were formed by different, virus particles in the inocula or were due to different degrees of cell susceptibility in individual animals. Serial transfers carried out in the present study indicate that the latter factor may be responsible for the virus multiplication; otherwise, selection of patas-positive particles would have occurred on serial passages through nonirradiated patas cultures. Enhancement of patas cell susceptibility to Coxsackie A9 and ECHO-l viruses was repeatedly obtained only when 24-4%hour-old primary cultures were exposed to X-irradiation. A similar response was obtained from first-passage cultures when exposed 14 hours after reseeding. Such irradiation did not affect the viral susceptibility in 7-14-day-old cultures even when high dosages (20,000-50,000 r) were used. It is, therefore,
148
HSIUNG
indicated that the changes in viral susceptibility depended upon the age of the cultures at which the irradiation was undertaken. The time at which the effect was obtained in irradiated cultures corresponded to the peak of mitotic activity (Chu and Giles, 1957; Chu, 1958, unpublished data). Altered susceptibility to viral infection was demonstrated only in the irradiated cells that have capacity and opportunity for further division after treatment. Marcus (1956) and Cieciura et al. (1957) reported that Newcastle disease virus caused selective destruction of giant cells formed by Xirradiation of HeLn cultures. In this study all the irradiated cultures were used 5-8 days after irradiation. Giant cells were not recognizable for at least 10 days, and sometimes up to 19-20 days after treatment. It was, therefore, not. possible to determine whether only the radiation-induced giant cells were responsible for the multiplication of the virus. Ledinko and Melnick (1954) found that interference was not obtained with poliomyelitis and Coxsackie A9 virus in rhesus kidney cultures. In confirmation of their findings in susceptible cell systems, patas cultures resistant to Coxsackie A9 were still susceptible to poliovirus 25 ‘ays after inoculation of A9 virus and yielded an amount of poliovirus equal to that of the control cultures. In a previous publication (Hsiung and Melnick, 1958a), it has been shown that A9 adsorbed on patas cells as well as on rhesus, although no plaques appeared in the patas cultures. These studies indicate that Coxsackie A9 virus and poliovirus can be demonstrated simultaneously in tissue cultures of susceptible cells as well as of semiresistant cells. Although attempts to adapt ECHO virus types 6 and 9 to X-irradiated patas cultures were unsuccessful, adaptation of Coxsackie A9 and ECHO-l viruses to a partially resistant cell culture by growing these viruses first in a host altered by X-irradiation, suggests that this method may be useful in other systems. Furthermore, such irradiated primary kidney cultures hold promise of usefulness in the isolation of new viruses or agents difficult to grow. Early detection of latent virus in monkey kidney cultures may also be possible by this technique. A latent virus has been found in an irradiated patas culture 1 week before it appeared in the nonirradiated cultures of the same lot. ACKNOWLEDGMENTS The author is greatly indebted to Dr. E. H. Y. Chu for carrying out all the irradiations. She also wishes to express her gratitude to Drs. F. L. Black and J. L.
EFFECT
OF X-IRRilDIATION
Melnick for reading and criticizing her excellent technical assistance.
ON
PATAS
MONKEY
the manuscript,
KIDNEY
CELL
and to Mrs. Grace Tucker
149 for
REFERENCISS E. H. Y., and GILES, N. H. (1957). A st,udy of primate chromosome complements. Am. Naturalist 91.273-282. CIE~IIJRA, S. J., MAR~~JS, P. I., and PUCK, T. T. (1957). The use of X-irradiated HeLa cell giants to detect latent virus in mammalian cells. Virology 3, 426-427. CLEMMESEN, J. (1939). The influence of roent,gen radiation on immunity to Shope fibroma virus. Am. J. Cancer 35, 378-385. CLEN~~ESEN, J., and ANIIERSEN, E. I(. (1948). Influence of roentgen radiation on the spread of vaccinia virus in young rabbits. nctn Palhol. Microbial. &and. 26, 615-618. DE GARA, P. F., and FURTH, J. (1945). The relative susceptibility of normal and x-rayed mice of different stocks to pneumotropic viruses. .I. Zmmz~nol. 60, 255 264. HSIUNG, G. D., and MELNICK, J. L. (1957a). Morphologic characteristics of plaques produced on monkey monolayer cultures by enteric viruses (polio78, 128-136. myelitis, Coxsackie and ECHO groups). J. lmnzunol. HSIUNG, G. D., and MELXICK, J. L. (1957b). Comparative susceptibility of kidney cells from different monkey species to enteric viruses (poliomyelit,is, Coxsackie 78, 137-146. and ECHO groups). J. Immunol. HSI~JN~, G. D., and MELSICK, J. L. (1958a). Adsorption, multiplication and cytopathogenicity of enteric viruses (poliomyelitis, Coxsackie and ECHO groups) in suscept,ible and resistant monkey kidney cells. J. Immunol. 80, 45-52. HSIUNG, G. D. (1958b). Variation in viruses after propagation in X-irradiated primary cell cultures. Abstract BZTfh Intern. Congr. for Microbial., Stockholm, Sweden, August 4-9, p. 269. LEDINKO, N., and MELKICK, J. L. (1954). Interference between poliomyelitis viruses in t,issue culture. J. Ezpll. Afed. 100, 247-267. P~.(:K, T. T., and MARCIJS, I?. I. (1956). Act,ion of x-rays on mammalian cells. J. Expll. Ued. 103, 653-666. SYVERTOS, J. T., WERDER, ,4. A., FRIEUI\IAN, J., ROTH, F. J., GRAHAM, .4. B., and MIRA, 0. J. (1952). Cortisone and roentgen radiat,ion in combination as synergistic agents for production of lethal infections. Proc. Sot. Exptl. Biol. Med. 80, 123-128. SYVERTOK, J. T., BRUNNER, K. T., TOBIS, J. O., and COHEN, M. M. (1956). Recovery of viable virus from poliomyelitis vaccine by use of monkeys pretreated with cortisone and x-radiation. Am. J. Hyg. 64, 74-84. CHI:,
VIROLOGY
Effect
of X-Irradiation Cell Susceptibility
on Patas Monkey to Enteroviruses’
Kidney
G. D. HSIUNG Section of Epidemiology and Preventive Medicine, Yale University School of Medicine, S33 Cedar St., New Haven II, Connecticut Accepted
October 17, 1958
Enhancement of patas monkey kidney cell susceptibility to certain enteroviruses was induced by X-irradiation. With small inocula Coxsackie A9 virus growth occurred only in irradiated cells. On serial passages, increasing virus titers and cytopathic changes could be obtained only in irradiated cultures. After growth in irradiated cells, Coxsackie A9 virus was able to grow in nonirradiated cells. ECHO virus type 1 was also adapted to patas cells in a similar manner. The adapted strain yielded somewhat higher titers than the parent virus in patas cult,ures and had acquired the ability to give marked cytopathogenic effect (CPE). Attempts to adapt ECHO virus types 6 and 9 to irradiated patas cells were unsuccessful. Increased susceptibility to Coxsackie A9 virus infection was observed in both primary and first-passage patas monkey kidney cultures when such cultures were irradiated during the first 48 hours of in vitro life, a period of rapid growth. However, cultures irradiated at 7-14 days did not show similar responses. INTRODUCTION
In a previous publication, Hsiung and Melnick (1958a) described the behavior of various enteroviruses in tissue culture derived from different monkey species, indicating some of the virus-host relationships in susceptible and resistant cell systems. Rhesus (Mucuca mulatta) cultures were highly sensitive to the three groups of enteroviruses studied, whereas patas (Erythrocebus patus) monkey kidney cells were relatively resistant to Coxsackie A9 and ECHO type 1 viruses. Increased susceptibility to some viral infections has been shown to occur when certain animal species were exposed to X-irradiation (Clemmesen, 1939; de Gara and Furth, 1945; Clemmesen and Andersen, 1948; 1 Aided
by a grant
from the National
Foundation. 140
EFFECT
OF X-IRRADIATION
ON
PATAS
MOSKEY
KIDNEY
CELL
141
Syverton et al., 1952, 1956). Recently Puck and Marcus (1956) and Cieciura et al. (1957) found that giant cells formed in X-irradiated HeLa cultures were highly sensitive to Newcastle disease virus. The purpose of this study was to determine whether the susceptibility of patas monkey kidney cells to certain enteroviruses could be enhanced by such irradiation techniques. MATERIALS
AND
METHODS
Virus. Poliovirus type 1 (Mahoney strain) ; Coxsackie virus type A9 (Grigg strain); ECHO virus type 1 (Farouk strain), type 6 (D’Amori strain), and type 9 (Bourn strain) were used. All viruses were plaquepurified and grown in rhesus tissue cultures except ECHO virus type 6, which was purified by the terminal-dilution technique. Cultures. Primary patas and rhesus monkey kidney cultures in tubes and bottles were prepared as described previously (Hsiung and Melnick, 1957b). First-passage cultures were obtained by trypsinizing the primary cultures when 7-10 days old and reseeding into bottles. Virus assay. All virus inoculations and titrations were made in rhesus bottle cultures by the plaque technique (Hsiung and Melnick, 1957a) except ECHO virus type 6, in which tube cultures were used. X-ray irradiation.2 The X-irradiation was performed with a General Electric Maxitron deep-therapy machine, using 250 kv and 30-ma beam, filtered through l-mm aluminum filter, at a distance of 40 cm from the target. This gave a rate of 500 r per minute as measured with a Victoreen roentgen meter. Unless otherwise specified, patas cell cultures were irradiated at room temperature with 300 r, 48 hours after seeding in 3-ounce prescription bottles. The medium was changed immediately after irradiation to remove any toxic materials that might have been produced in the fluid. All cultures were used 7-10 days after seeding, i.e., 5-8 days after irradiation unless otherwise specified. EXPERIMENTAL
RESULTS
Persistence of Coxsaclcie A9 Virus Infection in Patas Cultures When irradiated or nonirradiated patas cultures were infected at high multiplicit’ies (10) of Coxsackie A9 virus, significant amounts of virus 2 All the irradiations were performed by Dr. E. H. Botany, Yale University. Measurements made by him data) showed that 30y0 of X-ray intensity was absorbed of culture bottles and the actual dosage was only 70% of 300 r was actually 210 r.
Y. Chu, Department of (Chu, 1958, unpublished or scattered by the glass that measured; therefore,
142
HSIUNG
TABLE A9
PERSISTENCE
OF COXSACKIE
1 VIRUS
CULTURES
IN PATAS
Virus recovered--average titer (log PFU per ml) Days of incubation at 37”
Patas cultures Nonirrad.
oa 3 5 15 25 40
7.7 6.0 5.0 3.8
Irrad.
7.7 6.2 5.7 6.5
LIOriginal inocula (multiplicity 10). b Original inoculum 3 X 107 PFU. c Samples were obtained on the thirteenth
Killed patas cultures Nonirrad.
Irrad.
7.5 5.2 4.6 2.2”
7.5 5.3 4.5 2.9c
Without cells
7.56 4.0 0 OC
-
day.
were recovered in the fluid for as long as 40 days (Table l), although no cytopathogenic effect (CPE) was recognizable. In contrast, Coxsackie A9 virus was rapidly inactivat.ed at 37” when there were no cells present in the media. Virus titers decreased in both irradiated and nonirradiated cultures which had been killed by freezing and thawing three times, although not quite as rapidly as in the cell-free samples (Table 1). This suggests that the virus which was recovered in high titers from the irradiated and nonirradiated patas cultures after prolonged incubation was newly produced. In addition, somewhat higher virus titers were found in irradiated cultures than in nonirradiated ones. A set of the irradiated and nonirradiated patas cultures was infected with Coxsackie A9, carried for 25 days without a cytopathogenic effect (CPE), and then challenged with 2.7 X IO6 rhesus PFU (plaque-forming units) of poliovirus type 1 (Mahoney strain). Cytopathic changes were observed 24 hours after the challenge, and destruction was complete in 48 hours. Yields of poliovirus from the control and the Coxsackie A9infected cultures of irradiated and nonirradiated patas cells were virtually the same, indicating no interference phenomena between these two viruses under these conditions. Initiation Cultures
of Growth and Multiplication with Small Inocula
of Coxsackie A9 Virus in Patas
It has been reported (Hsiung and Melnick, 1958a) that newly produced A9 virus could not be detected in either the fluid phase or in the cells of
EFFECT
OF X-IRRADIATION
ON
P;ZTAS
TABLE
MONKEY
KIDNEY
143
2
OF COXSACKIE A9 VIRUS IN CULTURES SMALL INOCULA
MULTIPLWATIOK
CELL
INFECTED
WITH
Virus titer (log, PFU per ml) Day after inoculation
0 1 2 3 7 21
~~ Nonirrad. patas
Irrad. patas
Rhesus
3.9” 2.4 1.9 0 0 0
3.9a 2.2 2.7 2.6 4.2 3.0
1.9” 4.5 7.4 8.4 8.3 5.9
a Original inoculum (multiplicity b Original inoculum (multiplicity
0.002). 0.00002).
patas cultures when inoculs were small (multiplicities less than 0.01). Experiments using such small inocula are shown in Table 2. The virus t,iters in the fluid of nonirradiated patas bottles fell off sharply, and no virus was recovered after 3 days, whereas some virus multiplication was demonstrable in the irradiated patss cultures. Rhesus cultures used for comparison showed a high titer of virus, although the inoculum was only l/100 of that used in the patas cultures. In four separate experiments small doses (multiplicities 0.0013-0.0023) were used to inoculate 31 irradiated cultures prepared from four different patas monkeys (Table 3). Fifty-two per cent (16/31) of the irradiated cultures yielded virus, whereas no virus was obtained from any of the 26 nonirradiated cultures. All cultures were handled and inoculated in the same way except for the irradiation. In experiments 1 and 2 the cultures were irradiated 48 hours after seeding; in experiment 3, 24 hours after seeding; and in experiment 4, 14 hours after reseeding of trypsinized primary patas cells. Increased cell susceptibility to virus infection occurred only when the cultures were irradiated during the period of most rapid growth (14-48 hours after seeding) or the period of maximal mitotic activity. Serial Passages of Cozsackie A9 Virus in Patas Cultures Subcultures of Coxsackie A9 virus in nonirradiated patas cells invariably failed to yield virus after a few serial passages. Titrations are listed in Table 4 for experiment I in which transfers of 0.1 ml of undiluted fluid to a new tube culture containing 0.9 ml of media was made every 3-4
144
HSIUNG
TABLE INITIATION
OF GROWTH
Low
T3tE
Expt. no.
1 2 3 4
3
A9 IN PATAS MULTIPLICITIES
OF COXSACKIE
Age of culture at irrad. (hours)
Multiplicity of infection”
48 48 24 14
0.0023 0.0013 0.0015 0.0015
Primary Primary Primary First passage
Total Per cent of growth a Multiplicit,y
INFECTED
Il’o. Virus
AT
Positive cultures/total tested Nonirradiated
Irradiated
o/2
l/2
O/6 O/16
o/2
11/17 m 3/4h
O/26
16/31
0 =
h Two samples
CIJLTURES
52
inoculated
Average no. cells (3 X 106). in this experiment showed CPE
days. In a similar manner serial passages of the same virus in irradiated bottle cultures (300 r), using the same dilution factors, gave virus in every passage, with titers only slightly lower than those of the original virus. Results of the two parallel series of transfers in irradiated and nonirradiated patas cultures are also shown in Table 4 (experiments II and III). Every 48 hours 1 ml of each culture fluid was transferred to a new culture containing 9 ml of fresh medium. The titer decreased with each passage in the nonirradiated patas cultures and completely disappeared after the fift,h passage. On the other hand, in the irradiated cell system a minimal titer was reached at the third (experiment II) and sixth (experiment III) passages, followed by increasing values. Marked cytopathic changes related to infection became evident in the irradiated cultures by the fifth passage. Titers in subsequent passages were similar to, or slightly higher than, those of the sixth passage. Adaptation
of Coxsackie
A9 Virus
to Nonirradiated
Patas Cells
After fourteen passages in irradiated patas cells, Coxsackie A9 virus was inoculated into nonirradiated patas cultures. Cytopathic changes were now observed in the nonirradiated cultures as well as irradiated ones. Small plaques were obtained in nonirradiated patas bottle cultures with this adapted virus. In contrast, the original (parent) virus, rarely gave plaques on patas cultures even when undiluted material was used. On rhesus monolayers the adapted virus produced smaller plaques (<5
EFFECT
OF
X-IRRADIATION
ON
PATAS
TABLE SERIAL
TRANSFERS
MONKEY
145
CELL
4 A9 VIRUS
OF COXSACKIE
KIDNEY
IN I’ATAS
CULTURES
Virus titer (log, PFU per ml) Kumber of passages
Experiment
I”
Experiment
II”
Experiment
III<
Nonirrad.
IKWl.
Nonirrad.
Irrad.
Nonirrad.
had.
8.4 7.4 5.6 4.7 1.6 0 0
9.1 8.1 7.1 7.5 7.4 7.8 7.8
8.5 7.4 5.9 4.4 2.2 1.7 0
8.5 7.0 6.4 5.5 6.5 7.2 7.2
8.5 7.4 5.9 4.4 2.2 2.0 0
8.5 7.4 6.6 5.7 5.2 4.8 4.6
u Experiment I: serial transfers were made every 3-4 days in irradiat,ed bottle cultures and nonirradiated tube cultures, using the same dilution factors. * Experiment II: serial transfers were made every 48 hours in parallel in irradiated and nonirradiated bottle cultures. Minimal titer in irradiated culture was reached at the third passage and increased thereafter. c Experiment III: serial transfers were made in parallel as in experiment II, except that the cells were obtained from a different animal. Minimal titer in irradiated culture was reached at the sixth passage and increased slightly (5.2) thereafter. d Original inocula.
mm in diameter at 7 days) than the original strain (> 10 mm in diameter at 7 days). The identity of the adapted virus was confirmed by neut,ralization with antiserum from rabbits which had been immunized with the P.B. strain of Coxsackie A9 virus. The ability of the adapted virus to produce paralysis in suckling mice was somewhat reduced; however, variants of two types of plaques were found after propagation of the adapt’ed virus in a single mouse passage. Preliminary results on this variation were reported recently (Hsiung, 1958b). Attempts to Adapt ECHO Viruses into Patas Cultures ECHO viruses types 1, 6, and 9 were transferred serially in patas cultures in a similar manner to that used for Coxsackie A9 virus. A definite CPE was observed in the irradiated cultures of ECHO-l virus after the third passage (Table 5), but there was no sign of cellular dest,ruction in any of the nonirradiated cultures at the time of transfer (48 hours). However, serial passage of ECHO-l in the nonirradiated patas culture series did not completely eliminate the virus, as in the case
146
HSIUNG
SERIAL Number of passages
TRANSFERS
TABLE 5 OF ECHO-l VIRUS
Cytopathic
effect
IN PATAS
CULTURES”
Virus titer (log, PFU per ml)
Nonirrad.
had.
Nonirrad.
had.
f
f + ++ +++ +++
6.8 4.8 2.8 2.6 2.8
7.4 5.8 5.6 5.0 4.1
1 3 5 7 10
0 0 0 0
u Original inocula 2.7 X lo* PFU. Transfers were made every 48 hours.
of Coxsackie A9, even after ten passages. The ECHO-l virus which was obtained by passage in the irradiated patas cultures was also able to produce a CPE in the nonirradiated patas cultures. When serial transfers were made either with ECHO virus type 6 or type 9, virus was not recoverable in either the irradiated or nonirradiated patas cultures after the third passage. No CPE was observed in any of the cultures, although the transfers were kept for as long as 14 days. Effect of Irradiation
on 7-l&Day-Old
Cultures
Serial transfers. When serial passages of Coxsackie A9 virus were made in patas cultures either irradiated on the ninth or fourteenth day after seeding, virus titers decreased both in the irradiated and the nonirradiated cultures almost at the same rate. No virus was recovered by the fifth passage. Plaque formation. Since 9- or 14-day-old patas cultures did not show any effect when exposed to 300 r, higher dosages were applied to fully grown monolayers of rhesus as well as patas cultures in order to obtain any possible changes in plaque formation. Seven-day-old patas and rhesus cultures were inoculated with poliovirus type 1, Coxsackie A9, or ECHO type 1. All cultures were exposed to 1600 r either immediately after infection or 2 hours after adsorption. Bottles were overlaid after the treatment. There was no significant difference in plaque counts in rhesus cultures whether the bottles were irradiated before or after virus adsorption nor was there any difference between irradiated and nonirradiated ones. Plaques appeared only in irradiated and nonirradiated patas bottles inoculated with poliovirus but not with Coxsackie A9 or ECHO-l virus.
EFFECT
OF X-IRRADIATION
ON
PATAS
MONKEY
KIDNEY
CELL
147
In two other experiments various dosages of X-rays were applied. Both rhesus and patas cultures 7 days old were inoculated with Coxsackie A9 virus and exposed to X-ray at different doses (1500-50,000 r). Plaques were not obtained in any of the patas cultures. The number of plaques in the rhesus cultures was not. measurably influenced by X-ray in any dosage of the range t’ested, and the cell sheet remained microscopically unaffected by the irradiation.
Although there were no cyt,opathic changes in the patas cultures infected with Coxsackie A9, moderate titers of virus were recovered for 40 days; whereas only small amounts of virus were detected on the thirteenth day in the control samples containing killed cells. Thus limited inapparent infection of Coxsackie A9 virus in both irradiated and non irradiated patas cultures apparently occurred on first passage when inocula were large. However, enhancement of virus growth was demonstrated in the irradiated cultures by the persistence of virus in higher titer from 25 days onward. With small inocula, initiation of Coxsackie A9 virus growt~h was found only in irradiated patas cells. In addition, Coxsackie A9 virus has been adapt,ed t’o the resistant system only aft,er serial passages in irradiated cultures. It has been noted previously (Hsiung and Melnick, 1958a) that tiny plaques appeared after a delay in cultures from a few patas monkeys when the inocula contained lo5 or more rhesus PFU of Coxsackie A9 or ECHO-l, but that in most lots of patas cultures, plaques failed to appear even with inocula of 10’ rhesus PFU. Subculture of such small plaques in patas cultures was not successful. The quest’ion was raised whether these small plaques were formed by different, virus particles in the inocula or were due to different degrees of cell susceptibility in individual animals. Serial transfers carried out in the present study indicate that the latter factor may be responsible for the virus multiplication; otherwise, selection of patas-positive particles would have occurred on serial passages through nonirradiated patas cultures. Enhancement of patas cell susceptibility to Coxsackie A9 and ECHO-l viruses was repeatedly obtained only when 24-4%hour-old primary cultures were exposed to X-irradiation. A similar response was obtained from first-passage cultures when exposed 14 hours after reseeding. Such irradiation did not affect the viral susceptibility in 7-14-day-old cultures even when high dosages (20,000-50,000 r) were used. It is, therefore,
148
HSIUNG
indicated that the changes in viral susceptibility depended upon the age of the cultures at which the irradiation was undertaken. The time at which the effect was obtained in irradiated cultures corresponded to the peak of mitotic activity (Chu and Giles, 1957; Chu, 1958, unpublished data). Altered susceptibility to viral infection was demonstrated only in the irradiated cells that have capacity and opportunity for further division after treatment. Marcus (1956) and Cieciura et al. (1957) reported that Newcastle disease virus caused selective destruction of giant cells formed by Xirradiation of HeLn cultures. In this study all the irradiated cultures were used 5-8 days after irradiation. Giant cells were not recognizable for at least 10 days, and sometimes up to 19-20 days after treatment. It was, therefore, not. possible to determine whether only the radiation-induced giant cells were responsible for the multiplication of the virus. Ledinko and Melnick (1954) found that interference was not obtained with poliomyelitis and Coxsackie A9 virus in rhesus kidney cultures. In confirmation of their findings in susceptible cell systems, patas cultures resistant to Coxsackie A9 were still susceptible to poliovirus 25 ‘ays after inoculation of A9 virus and yielded an amount of poliovirus equal to that of the control cultures. In a previous publication (Hsiung and Melnick, 1958a), it has been shown that A9 adsorbed on patas cells as well as on rhesus, although no plaques appeared in the patas cultures. These studies indicate that Coxsackie A9 virus and poliovirus can be demonstrated simultaneously in tissue cultures of susceptible cells as well as of semiresistant cells. Although attempts to adapt ECHO virus types 6 and 9 to X-irradiated patas cultures were unsuccessful, adaptation of Coxsackie A9 and ECHO-l viruses to a partially resistant cell culture by growing these viruses first in a host altered by X-irradiation, suggests that this method may be useful in other systems. Furthermore, such irradiated primary kidney cultures hold promise of usefulness in the isolation of new viruses or agents difficult to grow. Early detection of latent virus in monkey kidney cultures may also be possible by this technique. A latent virus has been found in an irradiated patas culture 1 week before it appeared in the nonirradiated cultures of the same lot. ACKNOWLEDGMENTS The author is greatly indebted to Dr. E. H. Y. Chu for carrying out all the irradiations. She also wishes to express her gratitude to Drs. F. L. Black and J. L.
EFFECT
OF X-IRRilDIATION
Melnick for reading and criticizing her excellent technical assistance.
ON
PATAS
MONKEY
the manuscript,
KIDNEY
CELL
and to Mrs. Grace Tucker
149 for
REFERENCISS E. H. Y., and GILES, N. H. (1957). A st,udy of primate chromosome complements. Am. Naturalist 91.273-282. CIE~IIJRA, S. J., MAR~~JS, P. I., and PUCK, T. T. (1957). The use of X-irradiated HeLa cell giants to detect latent virus in mammalian cells. Virology 3, 426-427. CLEMMESEN, J. (1939). The influence of roent,gen radiation on immunity to Shope fibroma virus. Am. J. Cancer 35, 378-385. CLEN~~ESEN, J., and ANIIERSEN, E. I(. (1948). Influence of roentgen radiation on the spread of vaccinia virus in young rabbits. nctn Palhol. Microbial. &and. 26, 615-618. DE GARA, P. F., and FURTH, J. (1945). The relative susceptibility of normal and x-rayed mice of different stocks to pneumotropic viruses. .I. Zmmz~nol. 60, 255 264. HSIUNG, G. D., and MELNICK, J. L. (1957a). Morphologic characteristics of plaques produced on monkey monolayer cultures by enteric viruses (polio78, 128-136. myelitis, Coxsackie and ECHO groups). J. lmnzunol. HSIUNG, G. D., and MELXICK, J. L. (1957b). Comparative susceptibility of kidney cells from different monkey species to enteric viruses (poliomyelit,is, Coxsackie 78, 137-146. and ECHO groups). J. Immunol. HSI~JN~, G. D., and MELSICK, J. L. (1958a). Adsorption, multiplication and cytopathogenicity of enteric viruses (poliomyelitis, Coxsackie and ECHO groups) in suscept,ible and resistant monkey kidney cells. J. Immunol. 80, 45-52. HSIUNG, G. D. (1958b). Variation in viruses after propagation in X-irradiated primary cell cultures. Abstract BZTfh Intern. Congr. for Microbial., Stockholm, Sweden, August 4-9, p. 269. LEDINKO, N., and MELKICK, J. L. (1954). Interference between poliomyelitis viruses in t,issue culture. J. Ezpll. Afed. 100, 247-267. P~.(:K, T. T., and MARCIJS, I?. I. (1956). Act,ion of x-rays on mammalian cells. J. Expll. Ued. 103, 653-666. SYVERTOS, J. T., WERDER, ,4. A., FRIEUI\IAN, J., ROTH, F. J., GRAHAM, .4. B., and MIRA, 0. J. (1952). Cortisone and roentgen radiat,ion in combination as synergistic agents for production of lethal infections. Proc. Sot. Exptl. Biol. Med. 80, 123-128. SYVERTOK, J. T., BRUNNER, K. T., TOBIS, J. O., and COHEN, M. M. (1956). Recovery of viable virus from poliomyelitis vaccine by use of monkeys pretreated with cortisone and x-radiation. Am. J. Hyg. 64, 74-84. CHI:,