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The prolonged persistence of Western equine encephalomyelitis virus in cultures of strain L cells
VIROLOGY
3, 62-75 (1957)
The Prolonged Encephalomyelitis
Persistence of Western Equine Virus in Cultures of Strain L Cells’ VELMA
Department
of Microbiology,
C. CHAMBERS
University of Washington Seattle, Washington
School of Medicine,
Accepted October 17, 1966 Inoculation of cultures of strain L cells with Western equine encephalomyelitis (WEE) virus was followed by multiplication of virus and varying degrees of cellular degeneration. Large viral inocula frequently caused less degeneration than smaller inocula. Surviving cells proliferated and eventually produced thickly populated cultures. Some of these cultures continued to produce virus over a period of many months. As long as virus persisted in cultures, the cells were immune to mass destruction by superinfection from newly added virus. After virus disappeared from cultures, either spontaneously or following a period of exposure to antiserum, cultures reverted to increased susceptibility to small inocula. The hypothesis proposed to explain these observations postulates that noninfectious virus, either inactivated or incomplete, protected cells in infected cultures by autointerference. Periodic loss of interfering activity presumably rendered individual cells susceptible to infection from time to time, thereby providing a continuing supply of susceptible cells in infected cultures over long periods of time. INTRODUCTION
The prolonged persistence of viruses in tissue culture systems has been reported on many occasions. Examples of these reports include the persistence of vaccinia virus in cultures of chick embryonic tissue for 9 weeks (Feller et al., 1940), the presence of mumps virus in cultures of amniotic membrane of chick embryos for 32 days (Weller and Enders, 1948), the finding of Newcastle disease virus in cultures of chick muscle fibroblasts for several weeks after inoculation (Gey and Bang, 1951), and the persistence of dengue virus in cultures of monkey testicular tissue for 11 weeks (Hotta and Evans, 1956). 1 This work was supported in part by a contract with the Office of Naval Research, and by grants from the Eli Lilly Company and the State of Washington Initiative 171 Fund for Research in Biology and Medicine. 62
PERSISTENCE
OF WEE
VIRUS
IN
CULTURES
OF STRAIN
L CELLS
03
The present paper describes the prolonged persistence of Western equine encephalomyelitis (WEE) virus in cultures of strain L cells. The following mechanisms were given consideration in attempting to explain the prolonged infection of these cultures: (1) destruction of susceptible cells and selection of genetically resistant cells; (2) a state of continuing autointerference and (3) a virus-cell relationship comparable to lysogenicity (Chambers and Evans, 1953). The present paper provides evidence which supports the second mechanism. A hypothesis is, therefore, proposed which utilizes the autointerference phenomenon t,o explain the persistence of WEE virus in cultures of &rain I, cells. MATERIALS
AND
METHODS
Virus. WEE virus was obtained from Dr. Carl Eklund in April, 1948, as a lyophilized suspension of mouse brain. It had undergone 85 mouse passages in the Rocky Mountain Laboratory and 7 to 13 mouse passages in our laboratory. Tissue culture system. Strain L cells are fibroblast-type cells which originated from the subcutaneous and adipose tissue of a mouse and were started in tissue culture by Dr. Earle in 1940 (Earle, 1943). Later ma1 gnancy developed in some of these cultures after exposure to a carcinogenic agent (Earle, 1943; Earle and Nettleship, 1943). In 1948 the present line of strain L cells was started from a single cell from one of the mal’gnant cultures (Sanford et al., 1948). ,1 culture of strain L cells was received from Dr. Earle in February, 19$L. The cell line has been maintained in this laboratory by cult’ivation directly on the inner glass surface of tubes or flasks with a nutrient medium consisting of 20% chick embryo ext,ract, 40% horse serum, and 40% Hanks’ balanced salt solution, and containing 100 units of penicillin and 100 pg of strept’omycin per milliliter. The medium was renewed at 2- to 4-day intervals and subcultures were initiated at frequent intervals by scraping the cells from the glass surface and transferring them by pipet to new tubes or flasks. Cultures were incubated at 35 to 37”. Rapid proliferation of cells produced a thickly populated cultsure within a few days. Such a culture was found t,o contain between three million and five million cells as determined by the method of enumeration of cell nuclei developed by Sanford and co-workers (1951). Cultures were inoculated with WEE virus by allowing 0.1 ml of the inoculum to flow over the cells after the fluid medium had been removed. Nine-tenths milliliter of tissue culture medium were then added. Inoculated cult,ures were maintained in the same manner as t,he st#ock cultures.
64
VELMA
C. CHAMBERS
Age of cultures up to 6 months did not appear to influence the degenerat:on of cells after viral inoculation. Cuhures which had been maintained longer than a year before inoculation showed less degeneration due to virus than did younger cultures. Assay of virus. At appropriate intervals following the inoculation of cultures of strain L cells with WEE virus, the tissue culture fluids were harvested and injectBed into mice by the int’racranial route to determine the presence of virus. When it was desirable to determine the amount of virus present, tenfold serial dilutions of tissue culture fluids in Ringer’s solution were injected. The LD6o titer was calculated according t,o t,he method of Reed and Muench (1938). The fluids were frequent’ly preserved in the frozen state at -20” to -70” for periods ranging from 1 day to several weeks before they were injected into mice. When fluids from cultures which had produced virus for long periods of time were inoculated into mice, the incubation period was sometimes prolonged several days. After 11 to 14 mont,hs in tissue culture, identification of the infectious agent as WEE virus was confirmed by t,he Communicable Disease Center at, Mont’gomery, Alabama. RESULTS
Multiplication qf Western equine encephalomyelitis virus in cultures of strain L cells. WEE virus was propagated through four serial passages in strain L cells. Fluid harvested from the fourth passage represented a lO-3o dilution of the original inoculum and was infectious for all 3 mice inoculated. This dilution was brought about by frequent renewal of tissue culture fluid and by dilution of the inocula for serial subinoculat on. Figures 1 and 2 show the changes which took place in the concentrat,ion of virus in the fluids of two tissue cult,ures, one of which was inocuated with a lo-? dilut,ion of virus-infected mouse brain and the other with a 10-S dilution. A comparison of the two figures shows that the smaller amount) of virus (lo”.* mouse LDSo) inoculated int,o culture L-144 ncreased more rapidly, reached a higher concent,ration in extracellulal fluid, and produced more extensive cytonecrosis than did the larger inoculum (106.8 mouse lIDso) in culture L-136. The concentration of virus in the fluid phase of culture L-136 remained moderately high (lo3 to IO5 I,Dbo) for more than 3 weeks. Assay of virus in the flu’d of a second cultlure (L-137), which had received the same amount of viral inoculum as L-136, showed similar changes in
PERSISTESCE
OF WEE
CYTONECROSISo LOGI
VIRUS
IN
o
o
CULTURES
OF STRAIN
L CELLS
65
60
234567 DAYS B Estimated f
Endpoint Lowest
Degeneration
endpoint
based on titrotion
of inoculum
not reached dilution of fluid tested was not infective of cells slight 0 ; marked
@ ; severe
for mice
l
FIG. 1. Growth
curve for Western equine encephalomyelitis virus in cult.ure L-136 which was inoculated with 106.8mouse LDjo of infected mouse brain suspension. Medium was changed on the fourth, seventh, and eleventh days and every 3 or 4 days thereafter. Degeneration of cells in these cultures was recorded as “slight, ” “marked,” and “severe” when degeneration involved 250/, or less, 25 to 75$‘&,and 75% or more of the cell population respectively.
virus concentration. These changes occurred about the same time in the two cultures. Virus was still present in both cultures 101 days after inoculation. Virus persisted in culture L-144 for 71 days. No virus was det,ected in this culture on the eighty-eight.h day after inoculation. Another culture (L-145), which was inoculated with the same amount of virus as culture L-144, was completely destroyed by the virus and no virus was present on the twenty-fifth day after inoculation. The rise and decline of virus in the fluid phase of cu:t’ure L-145 during the first week aft,er inoculation was simi’ar t,o t,hat, described for culture L-144.
66
VELMA
CYTONECROSIS LOG 7.0
0
a
C. CHAMBERS
@
l
0’30’ I 2 3 4 5 6 7 m
Estimated
f
Endpoint
not
Lowest
dilution
Degeneration
endpoint
@.
a
0
9 II DAYS
I8
71
based
on titration
Of inOCUlU~
reached of fluid
of cells
slight
tested 0;
was not InfeCtlVe tIWrked
@;
SeWe
for mice
l
FIG. 2. Growth curve for Western equine encephalomyelitis virus in culture L-144 which was inoculated with 103.8mouse LD,a of infected mouse brain suspension. Medium was changed on the fourth, seventh, and eleventh days and every 3 or 4 days thereafter. Degeneration of cells in these cultures was recorded as “slight, ” “marked,” and “severe” when degeneration involved 25y0 or less, 25 to 75%, and 75yc or more of the cell population respectively.
The cellular degeneration observed in cultures of strain L cells infected with WEE virus varied from complete destruction of cells to little or no detectable degeneration. In several severely degenerated cultures a few surviving cells proliferated and completely replaced the lost tissue. Some cultures continued to produce virus and to exhibit cellular growth over long periods of time. Sl:ght cytonecrosis, which was believed to be the result of viral act-vity, was difficult to distinguish from the degeneration which occurred in older uninfect,ed cultures due to overcrowding of cells. The inverse relationsh,ip between size of viral inoculum and extent of
PERSISTENCE
OF WEE
VIRUS
IN
CULTURES
OF STRAIK
L CELLS
67
cellular degeneration. Frequently less degeneration was observed in cultures inoculated with a very high concentration of WEE-infected mouse brain than was observed in cultures inoculated with a much lower concentration of the virus. Figure 3 contains cumulative data from four experiments showing the per cent of cultures in which “marked” and “severe” degeneration of cells occurred following inoculation with various dilutions of WEE-infected mouse brain. The difference in cytonecrosis observed in these cultures was associated with the greater pro90
80 18 /
0-
18
/
iob5 MOUSE BRAIN DILUTION OFINFECTED
FIG. 3. Comparative
n
Severe degeneration
q
Marked
degeneration
degeneration of strain L cells following inoculat,ion with various amounts of WEE virus. The number above each bar is the number of cultures involved and represent’s cumulative data from four experiments. The mouse LL& titer of the mouse brain suspension employed as inoculum in the four experiments ranged from 1O-7.6 to lo- 8 5. “Severe” degeneration refers to cultures in which 75% or more of the cell population was estimated to have undergone degeneration. “Marked” degeneration refers to cultures with 25 to 75% degeneration.
68
VELMA
C. CHAMBERS
TABLE
1
PERSISTENCE OF WESTERN EQUIKE ENCEPHALOMYELITIS STRAIN 1, CELLS Months after
inoculation
VIRUS IN CIJLTURES OF
of culture
Culture
L-2 L-6 L-11 L-lls3e L-136 L-137
+“+++
4
5
6
7
8
+
+
+
+
+
11
+ + +++ +++h
+ +
12
+
14
16
+
+
+
+ f
f
13
+ f
*++ + + ,+
9
+ +
* + Virus present, in tissue culture fluid. f One or two of the three or four inoculated b Culture L-137 was reinoculated with WEE inoculation.
f, mice survived. virus 3 months
If
after
+
the first
liferation of virus in cultures given a small inoculum than in those given a large inoculum as illustrated by Figs: 1 and 2. The prolonged injection of cultures of strain L cells with Western equine encephalomyelitis virus. After inoculation of cultures of strain L cells with WEE virus, the virus disappeared from some cultures within a month but persisted in other cultures for periods ranging from several weeks to many months. A number of infected cultures were selected for observation over an extended period of time. The results of tests for virus in tissue culture fluids of some of these cultures are presented in Table 1. Virus persisted in culture L-11 for 16 months and continued to persist in several subcultures of cells from this culture. A third serial subculture, L-lls3e, produced virus for 9 months, which was 25 months after inoculation of the original culture. The disappearance of WEE virus in the absence of living cells was investigated. Concentrations of infected mouse brain containing lo3 to 106 mouse LD,, were incubated in the tissue culture medium (1) in the absence of cells, (2) in the presence of strain L cells which had been frozen, and (3) with sediment from chick embryo extract. Of fourteen preparations in these three categories, twelve were free of virus at 4 days. A small amount of virus was present in the other two preparations at 4 days, but no virus was detected at 7 days. The persistence of virus in cult,ures of strain 1, cells for several months suggested the continued multiplication of virus over long periods of
PERSISTENCE
OF WEE
VIRUS
IN
CULTURES
OF STRAIS
L CELLS
69
time. The disappearance of virus within 4 to 7 days in the absence of cells or in the presence of frozen cells eliminated the possibility of an initial multiplication of virus and its persistence in extracellular fluids a,s an acceptable explanation for the prolonged persistence of virus in cult’ures of viable cells. Proliferation of cells in chronically infected cultures and in cultures during repeated challenge inoculation. After the initial degeneration of cells in infected cultures, surviving cells began t,o proliferate and event,ually replaced the dead cells. Proliferation of cells cont,inued in chronically infected cultures. In one experiment nine cultures which still contained virus 15 days afber inoculation were exposed to a series of four challenge inoculations with a 10P5 dilution of WEE-infected mouse brain at 3- or J-day intervals. So detectable degeneration occurred and the cells appeared to proliferat’e normally. The proliferation of cells in chronically infected cult,ures and in cult,ures during repeated inoculations suggest,ed the presence of some prot,ective mechanism which prevented infect,ion of most of the cells and which was available to new cells after cell division. 12euersion of cultures to increased susceptibility following spontaneom loss of virus. Six cultures which had carried virus for several weeks or months prior to spontaneous loss of virus were challenged with a second inoculation of WEE virus in order to determine whet,her the loss of virus was due to the development of a population of genet,ically resistant CYA~~. Controls included five cultures not previously exposed t)o virus and t#hree cultures in which virus was still present at the time of challenge, 101 days after the first inoculation. The viral inoculum used for challenge and for cont,rol cultures consisted of 105.j mouse IJI>S0 WICl~;infect’ed mouse brain. The results of tbis experiment appear in Table 2. Challenge of six cultures, mhic*h had spontaneously lost virus following a previous inoculation with WEE virus, caused degeneration graded as “marked” or “severe” in four of t,hem. This is the number of cultures one would expect t,o show degeneration following an init,ial inoculat,ion with WEE virus according to the cwmulatjivc data given in l:ig. 3. Virus assay on the sevent,h day after challenge showed a higher con~entration of virus in the fluid phase of three of t,hese cultures than was found in the fluid phase of t,cvo cultures which still cwntained virus at the time of challenge. The results indicate that the spontaneous loss of virus from cultures was not due to the development of a population of genetically resistant, wlls. Instead a temporary protecti of cells ap-
70
VELMA
C. CHAMBERS
TABLE RESULTS
OF HOMOLOGOUS
CHALLENGE WITH
-
Days etween st and !nd inxlation
Culture
Cultures from which virus disappeared 13 days or more prior to challenge: L-lS2 L-2 L-14Sl L-126 L-129 L-144 Cultures in which virus was still present at t,he time of challenge : L-143 L-136 L-137 Cultures not previously exposed to virus: L-177 L-155 L-107 I,-228 L-232
2
OF STRAIN L CELLS PREVIOUSLY INFECTED WEE VIRUS Tests for virus in tissue culture fluid 7 days after challenge
Degenerationa of cells after challengeb
10-g
748 758 758 101 101 101
Marked Severe Mild Pione Marked Marked
3/3 c 3/3 3/3
3/3 2/3 213
O/3 O/3 O/3
101 101 101
Not significant None Not significant
m 3/3
O/3 o/3
O/3 O/3
Severe Severe Severe Severe Severe
~
i
CLExtent of degenerat,ion of cells was estimated by microscopic examination of cultures: Severe-approximately 75% or more of the cell population was degenerated. Marked-approximately 25-75y0 of the cell population was degenerated. Mild-less than 25y0 of the cell population showed degeneration. Kot significant-patchy degeneration attributed to aging of the culture and overcrowding of cells. None-no degeneration observed. 6 The challenge inoculum for all cultures was 0.1 ml of the same I@-” dilution of WEE-infected mouse brain (LD60 approximately 10-9.0). c The denominator represents the number of mice that were inoculated. The numerat,or represents the number of mice that died.
peared to develop which finally extended to all cells in chronically infected cultures thereby eliminating infectious virus. The cells then reverted to sensitivity. Reversion
of cultures
to increased
susceptibility
following
e.rposure
qf
PERSISTENCE
OF WEE
VIRUS
IiX CULTURES
OF STRAIN
L CELLS
71
cultures to antiserum. The nine cultures described earlier, which were challenged with a lop5 dilution of WEE virus at 3- or 4-day intervals beginning 15 days after the first inoculation and continuing for 2 weeks, were used in this experiment. The cultures were washed three times wit,h warm Hanks’ solution after which they received 0.9 ml of medium containing anti-WEE horse serum of low tit’er. Medium containing similar antiserum was used for each change of fluid for the durat,ion of the test period, which lasted from 21 to 42 days in different cultures. During the interval when cultures were exposed to immune serum, t,issue culture fluids were harvested at 3- or 4-day intervals and were injected intracranially into mice, either individually or as pools of fluids harvested at one time from a number of cultures, or as pools of several frozen fluids harvested at different times from one culture. These tests indicated incomplete neutralization of virus in five of the nine cultures during exposure to ant,iserum (Table 3). Three of these cultures, L-119, L-124, and L-138, and one other culture, L-123, contained virus followmg removal of antiserum. Mild to severe degeneration of cells occurred in cultures L-123, L-124, and L-138 within a week after the removal of antiserum. Assays for virus in cultures L-123 and L-124 one week after removal of antiserum thowed LDCO titers of 10-4.5, and 10-j.5, respect,ively. It is presumed that following removal of antiserum many susceptible cells were present and that these were promptly infected. It appears that exposure of cultures to antiserum not only removed most of the infectious virus but also eliminated mucah if not. all of the prot)ect,ive agent. The loss of virus from cells subcultured in the presence of antiserum. If chronically infected cultures were in a stat,e comparable to that’ of lysogeny in bacteria, it should be possible to subculture cells from infected cult,ures in the presence of ant’iserum and still retain the capacit,y of cells to produce virus after removal of antiserum. However, if chronic infection of a culture depended upon infection and destruction of cells and spread of virus to other cells, successive subcult,ure of the cells to medium containing antiserum should eliminate the virus. Subcultures were prepared from the nine cultures, L-138, L-164, and L-119 to 125 inclusive, which were described in the preceding section and which are shown in the upper part of Table 3. The subcultures were started after the parent cultures had been exposed to antiserum for periods ranging from 11 days to 4 weeks. Further serial subcultures were made in some inst,anres. For example, in Table 3 dat,a are shown for four serial sub-
72
VELMA
C. CHAMBERS
TABLE EFFECT
OF SPECIFIC
ANTISERUM
Days of antiserum treatment
Culture After sub: culture
1 (
cultures” L-119 L-120 L-121 L-122 L-123 12-124 L-125 L-138 L-164 Subcultures Ll19Sl Ll23Sl L123S2 Ll23S3 Ll23S4 L-124Sl Id-12482 I,-12433
3
ON THE PERSISTENCE CULTURES
OF WEE
VIRCJS IN TISSUE
Tests for virus in tissue culture fluids Total lays 0 serlun treatment
Weeks after removal of antiserum
During :xposure to Ser”n
Original
38 21 42 42 21 21 28 21 32 28 11 24 36 42 11 36 42
17 21 18 9 10 31 9 10
45 32 42 45 52 42 45 52
3/3 O/4 -
2/4 z/4
O/3
l/3 O/3 l/4 c o/3 O/4 o/10 o/3 o/4 O/IO
~ o/3 ~ o/3 ’ o/3 o/3 o/3 o/3 ~ o/3
O/3 o/3 o/3 --
I
CLThe original cultures in t,his experiment had received an init,ial infecting dose of virus one month before exposure to antiserum and had received repeated challenge inoculations during the two weeks immediately preceding exposure to nntiserum. Virus was present in all cultures at t,he time antiserum was introduced. b The numerator indicates the number of mice t,hat, died. The denominator indicates the number of mice inoculated. c 1Mouse death occurred on t,he second d:~y after inoculation and was not attribut,ed to viral infection.
cultures from culture L-123 and three serial subcultures from L-124. The nutrient medium of all subcultures contained antiserum for at least 9 days. Subsequently, medium without antiserum was employed. Tests for virus were made both during the time the medium contained antiserum and subsequently when no antiserum was present. Xo virus was detected in the twenty subcult,ures which were tested. These included
PERSISTEKCE
OF WEE
VIRUS
IN
CULTURES
OF STRAIN
L CELLS
78
10 first generation subcult’ures, 6 second generation, 3 third generation, and 1 fourth generation culture. The one mouse death in the group inoculated with specimen L-119Sl was not attribubed to virus. Test’ results of eight of the cultures are shown in Table 3. k’ailure of suhcultures to produce virus after removal of antiserum provides substantial evidence indicating that lysogenicity is not, t,he explanation for prolonged infection of cultures of strain L cells with WEE virus. I>ISCUSSION
In attempting to explain the prolonged persistence of WEE virus in cultures of strain L cells, the following observat’ions are pert’inent : (1) An inverse relationship frequently existed between the size of viral iuoculum and the extent of cellular degeneration. (2) Cells in infected cultures continued to proliferate during repeated challenge inoculations with WEE virus. (3) Cultures revertled t)o increased susceptibility to small inocula following spontaneous loss of virus and following a period of exposure of infect’ed cultures to antiserum. (4) Subcultures of cells from infect’ed cultures during exposure to antiserum resulted in loss of virus and failure of virus to reappear after removal of antiserum. The inverse relationship between the size of viral inoculum and the extent of cellular degeneration has an obvious similarity to the autointerference that has been shown with influenza virus. It was found that undiluted allantoic fluid from infected eggs produced less virus on passage than did diluted virus (Henle and Henle, 1944). Virus rendered noninfectious by treatment with heat or ultraviolet light retained its ability to interfere with the production of infectious virus. In cultures of strain L cells chronically infected mit’h WEE virus, thermal inactivation of virus undoubtedly was a continuous process. The infectivity of WEE virus was shown to disappear in less than 7 days when incubated in fluid medium in the absence of cells or in the presence of nonliving cells. Thermally inactivat,ed virus may have served as the interfering agent that permitted persistence of healthy multiplying cells despite the presence of infectious virus. However, the existence of noninfectious interfering particles of a different origin is a possibility that is suggested by t,he studies with influenza by Van Magnus (1951a,b,c) and others. In any case, it appears that kcatment of tissue cultures with antiserum effectively removed the interfering agent as well as the infectious virus. The persistence of virus in cultures for long periods of time apparently
74
VELMA
C. CHAMBERS
depended upon occasional infection of some of the cells that regained susceptibility as the effectiveness of previously acquired interfering particles waned. Another possibility is that cells subjected to the simultaneous influence of fully active virus and of inactivated virus may have experienced a modified nonfatal infection which resulted in release of less than normal amounts of virus and in the eventual complete loss of virus from the cell. Further studies are necessary to determine more conclusively t#he validity of the proposed hypothesis. The possibility that autointerference may operate in vivo as a mechanism in limiting infection deserves consideration. ACKNOWLEDGMENT The author wishes to thank out the course of this work.
Dr. C. A. Evans for his valuable
advice
through-
REFERENCES CHAMBERS, V. C., and EVANS, C. A. (1953). Some observations on the growth of Western equine encephalomyelitis virus in cultures of L cells. Bacterial. Proc. 1963, 42. EARLE, W. R. (1943). Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in living cells. J. Natl. Cancer Inst. 4, 165-212. EARLE, W. R., and NETTLESHIP, A. (1943). Production of malignancy in vitro. V. Results of injections of cultures into mice. J. Natl. Cancer Inst. 4, 213-227. FELLER, A. E., ENDERS, J. F., and WELLER, T. H. (1940). The prolonged co-existence of vaccinia virus in high titer and living cells in roller tube cultures of chick embryonic tissues. J. Exptl. Med. 73, 367-388. GEY, G. O., and BANG, F. B. (1951). Viruses and cells-a study in tissue culture applications. I. Cells involved-availability and susceptibility. Trans. N. I’. Acad. Sci. 14, 15-24. HENLE, W., and HENLE, G. (1944). Interference between inactive and active viruses of influenza. I. The incidental occurrence and artificial induction of the phenomenon. Am. J. Med. Sri. 207, 705717. HOTTA, S., and EVANS, C. A. (1956). Cultivation of mouse-adapted dengue virus (type 1) in rhesus monkey tissue culture. J. Infectious Diseases 98, 88-97. REED, L. J., and MUENCH, H. (1938). A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493497. SANFORD, K. K., EARLE, W. R., and LIKELY, G. D. (1948). The growth in vitro of single isolated tissue cells. J. N&Z. Cancer Inst. 9, 229-246. SANFORD, K. K., EARLE, W. R., EVANS, V. J., WALTZ, H. K., and SHANNON, J. E. (1951). The measurement of proliferation in tissue cultures by enumeration of cell nuclei. J. N&Z. Cancer Inst. 11, 773-795. VON MAGNUS, P. (1951a). Propagation of the PR8 strain of influenza A virus in chick embryos. I. The influence of various experimental conditions on virus multiplication. Acta Pathol. Microbial. Stand. 28, 25G277.
PERSISTENCE
OF WEE
VIRUS
VON MAGSUS, P. (1951b). Propagation
IN CULTURES
OF STRAIS
L CELLS
75
of the PR8 strain of influenza A virus in chick embryos. II. The formaCon of “incomplete” virus following inoculation of large doses of seed virus. AC&a Pathol. Microbial. &and. 26, 278-293. VON MAGNUS, P. (1951c). Propagation of the PR8 strain of influenza A virus in chick embryos. III. Properties of the incomplete virus produced in serial passages of undiluted virus. Acta Pathol. Microbial. Stand. 29, 157-181. WELLER, T. H., and ENDERS, J. F. (1948). Production of hemagglutinin by mumps and influenza A viruses in suspended cell tissue cultures. Proc. Sot. Exptl. Biol. Med. 69, 124-128.
3, 62-75 (1957)
The Prolonged Encephalomyelitis
Persistence of Western Equine Virus in Cultures of Strain L Cells’ VELMA
Department
of Microbiology,
C. CHAMBERS
University of Washington Seattle, Washington
School of Medicine,
Accepted October 17, 1966 Inoculation of cultures of strain L cells with Western equine encephalomyelitis (WEE) virus was followed by multiplication of virus and varying degrees of cellular degeneration. Large viral inocula frequently caused less degeneration than smaller inocula. Surviving cells proliferated and eventually produced thickly populated cultures. Some of these cultures continued to produce virus over a period of many months. As long as virus persisted in cultures, the cells were immune to mass destruction by superinfection from newly added virus. After virus disappeared from cultures, either spontaneously or following a period of exposure to antiserum, cultures reverted to increased susceptibility to small inocula. The hypothesis proposed to explain these observations postulates that noninfectious virus, either inactivated or incomplete, protected cells in infected cultures by autointerference. Periodic loss of interfering activity presumably rendered individual cells susceptible to infection from time to time, thereby providing a continuing supply of susceptible cells in infected cultures over long periods of time. INTRODUCTION
The prolonged persistence of viruses in tissue culture systems has been reported on many occasions. Examples of these reports include the persistence of vaccinia virus in cultures of chick embryonic tissue for 9 weeks (Feller et al., 1940), the presence of mumps virus in cultures of amniotic membrane of chick embryos for 32 days (Weller and Enders, 1948), the finding of Newcastle disease virus in cultures of chick muscle fibroblasts for several weeks after inoculation (Gey and Bang, 1951), and the persistence of dengue virus in cultures of monkey testicular tissue for 11 weeks (Hotta and Evans, 1956). 1 This work was supported in part by a contract with the Office of Naval Research, and by grants from the Eli Lilly Company and the State of Washington Initiative 171 Fund for Research in Biology and Medicine. 62
PERSISTENCE
OF WEE
VIRUS
IN
CULTURES
OF STRAIN
L CELLS
03
The present paper describes the prolonged persistence of Western equine encephalomyelitis (WEE) virus in cultures of strain L cells. The following mechanisms were given consideration in attempting to explain the prolonged infection of these cultures: (1) destruction of susceptible cells and selection of genetically resistant cells; (2) a state of continuing autointerference and (3) a virus-cell relationship comparable to lysogenicity (Chambers and Evans, 1953). The present paper provides evidence which supports the second mechanism. A hypothesis is, therefore, proposed which utilizes the autointerference phenomenon t,o explain the persistence of WEE virus in cultures of &rain I, cells. MATERIALS
AND
METHODS
Virus. WEE virus was obtained from Dr. Carl Eklund in April, 1948, as a lyophilized suspension of mouse brain. It had undergone 85 mouse passages in the Rocky Mountain Laboratory and 7 to 13 mouse passages in our laboratory. Tissue culture system. Strain L cells are fibroblast-type cells which originated from the subcutaneous and adipose tissue of a mouse and were started in tissue culture by Dr. Earle in 1940 (Earle, 1943). Later ma1 gnancy developed in some of these cultures after exposure to a carcinogenic agent (Earle, 1943; Earle and Nettleship, 1943). In 1948 the present line of strain L cells was started from a single cell from one of the mal’gnant cultures (Sanford et al., 1948). ,1 culture of strain L cells was received from Dr. Earle in February, 19$L. The cell line has been maintained in this laboratory by cult’ivation directly on the inner glass surface of tubes or flasks with a nutrient medium consisting of 20% chick embryo ext,ract, 40% horse serum, and 40% Hanks’ balanced salt solution, and containing 100 units of penicillin and 100 pg of strept’omycin per milliliter. The medium was renewed at 2- to 4-day intervals and subcultures were initiated at frequent intervals by scraping the cells from the glass surface and transferring them by pipet to new tubes or flasks. Cultures were incubated at 35 to 37”. Rapid proliferation of cells produced a thickly populated cultsure within a few days. Such a culture was found t,o contain between three million and five million cells as determined by the method of enumeration of cell nuclei developed by Sanford and co-workers (1951). Cultures were inoculated with WEE virus by allowing 0.1 ml of the inoculum to flow over the cells after the fluid medium had been removed. Nine-tenths milliliter of tissue culture medium were then added. Inoculated cult,ures were maintained in the same manner as t,he st#ock cultures.
64
VELMA
C. CHAMBERS
Age of cultures up to 6 months did not appear to influence the degenerat:on of cells after viral inoculation. Cuhures which had been maintained longer than a year before inoculation showed less degeneration due to virus than did younger cultures. Assay of virus. At appropriate intervals following the inoculation of cultures of strain L cells with WEE virus, the tissue culture fluids were harvested and injectBed into mice by the int’racranial route to determine the presence of virus. When it was desirable to determine the amount of virus present, tenfold serial dilutions of tissue culture fluids in Ringer’s solution were injected. The LD6o titer was calculated according t,o t,he method of Reed and Muench (1938). The fluids were frequent’ly preserved in the frozen state at -20” to -70” for periods ranging from 1 day to several weeks before they were injected into mice. When fluids from cultures which had produced virus for long periods of time were inoculated into mice, the incubation period was sometimes prolonged several days. After 11 to 14 mont,hs in tissue culture, identification of the infectious agent as WEE virus was confirmed by t,he Communicable Disease Center at, Mont’gomery, Alabama. RESULTS
Multiplication qf Western equine encephalomyelitis virus in cultures of strain L cells. WEE virus was propagated through four serial passages in strain L cells. Fluid harvested from the fourth passage represented a lO-3o dilution of the original inoculum and was infectious for all 3 mice inoculated. This dilution was brought about by frequent renewal of tissue culture fluid and by dilution of the inocula for serial subinoculat on. Figures 1 and 2 show the changes which took place in the concentrat,ion of virus in the fluids of two tissue cult,ures, one of which was inocuated with a lo-? dilut,ion of virus-infected mouse brain and the other with a 10-S dilution. A comparison of the two figures shows that the smaller amount) of virus (lo”.* mouse LDSo) inoculated int,o culture L-144 ncreased more rapidly, reached a higher concent,ration in extracellulal fluid, and produced more extensive cytonecrosis than did the larger inoculum (106.8 mouse lIDso) in culture L-136. The concentration of virus in the fluid phase of culture L-136 remained moderately high (lo3 to IO5 I,Dbo) for more than 3 weeks. Assay of virus in the flu’d of a second cultlure (L-137), which had received the same amount of viral inoculum as L-136, showed similar changes in
PERSISTESCE
OF WEE
CYTONECROSISo LOGI
VIRUS
IN
o
o
CULTURES
OF STRAIN
L CELLS
65
60
234567 DAYS B Estimated f
Endpoint Lowest
Degeneration
endpoint
based on titrotion
of inoculum
not reached dilution of fluid tested was not infective of cells slight 0 ; marked
@ ; severe
for mice
l
FIG. 1. Growth
curve for Western equine encephalomyelitis virus in cult.ure L-136 which was inoculated with 106.8mouse LDjo of infected mouse brain suspension. Medium was changed on the fourth, seventh, and eleventh days and every 3 or 4 days thereafter. Degeneration of cells in these cultures was recorded as “slight, ” “marked,” and “severe” when degeneration involved 250/, or less, 25 to 75$‘&,and 75% or more of the cell population respectively.
virus concentration. These changes occurred about the same time in the two cultures. Virus was still present in both cultures 101 days after inoculation. Virus persisted in culture L-144 for 71 days. No virus was det,ected in this culture on the eighty-eight.h day after inoculation. Another culture (L-145), which was inoculated with the same amount of virus as culture L-144, was completely destroyed by the virus and no virus was present on the twenty-fifth day after inoculation. The rise and decline of virus in the fluid phase of cu:t’ure L-145 during the first week aft,er inoculation was simi’ar t,o t,hat, described for culture L-144.
66
VELMA
CYTONECROSIS LOG 7.0
0
a
C. CHAMBERS
@
l
0’30’ I 2 3 4 5 6 7 m
Estimated
f
Endpoint
not
Lowest
dilution
Degeneration
endpoint
@.
a
0
9 II DAYS
I8
71
based
on titration
Of inOCUlU~
reached of fluid
of cells
slight
tested 0;
was not InfeCtlVe tIWrked
@;
SeWe
for mice
l
FIG. 2. Growth curve for Western equine encephalomyelitis virus in culture L-144 which was inoculated with 103.8mouse LD,a of infected mouse brain suspension. Medium was changed on the fourth, seventh, and eleventh days and every 3 or 4 days thereafter. Degeneration of cells in these cultures was recorded as “slight, ” “marked,” and “severe” when degeneration involved 25y0 or less, 25 to 75%, and 75yc or more of the cell population respectively.
The cellular degeneration observed in cultures of strain L cells infected with WEE virus varied from complete destruction of cells to little or no detectable degeneration. In several severely degenerated cultures a few surviving cells proliferated and completely replaced the lost tissue. Some cultures continued to produce virus and to exhibit cellular growth over long periods of time. Sl:ght cytonecrosis, which was believed to be the result of viral act-vity, was difficult to distinguish from the degeneration which occurred in older uninfect,ed cultures due to overcrowding of cells. The inverse relationsh,ip between size of viral inoculum and extent of
PERSISTENCE
OF WEE
VIRUS
IN
CULTURES
OF STRAIK
L CELLS
67
cellular degeneration. Frequently less degeneration was observed in cultures inoculated with a very high concentration of WEE-infected mouse brain than was observed in cultures inoculated with a much lower concentration of the virus. Figure 3 contains cumulative data from four experiments showing the per cent of cultures in which “marked” and “severe” degeneration of cells occurred following inoculation with various dilutions of WEE-infected mouse brain. The difference in cytonecrosis observed in these cultures was associated with the greater pro90
80 18 /
0-
18
/
iob5 MOUSE BRAIN DILUTION OFINFECTED
FIG. 3. Comparative
n
Severe degeneration
q
Marked
degeneration
degeneration of strain L cells following inoculat,ion with various amounts of WEE virus. The number above each bar is the number of cultures involved and represent’s cumulative data from four experiments. The mouse LL& titer of the mouse brain suspension employed as inoculum in the four experiments ranged from 1O-7.6 to lo- 8 5. “Severe” degeneration refers to cultures in which 75% or more of the cell population was estimated to have undergone degeneration. “Marked” degeneration refers to cultures with 25 to 75% degeneration.
68
VELMA
C. CHAMBERS
TABLE
1
PERSISTENCE OF WESTERN EQUIKE ENCEPHALOMYELITIS STRAIN 1, CELLS Months after
inoculation
VIRUS IN CIJLTURES OF
of culture
Culture
L-2 L-6 L-11 L-lls3e L-136 L-137
+“+++
4
5
6
7
8
+
+
+
+
+
11
+ + +++ +++h
+ +
12
+
14
16
+
+
+
+ f
f
13
+ f
*++ + + ,+
9
+ +
* + Virus present, in tissue culture fluid. f One or two of the three or four inoculated b Culture L-137 was reinoculated with WEE inoculation.
f, mice survived. virus 3 months
If
after
+
the first
liferation of virus in cultures given a small inoculum than in those given a large inoculum as illustrated by Figs: 1 and 2. The prolonged injection of cultures of strain L cells with Western equine encephalomyelitis virus. After inoculation of cultures of strain L cells with WEE virus, the virus disappeared from some cultures within a month but persisted in other cultures for periods ranging from several weeks to many months. A number of infected cultures were selected for observation over an extended period of time. The results of tests for virus in tissue culture fluids of some of these cultures are presented in Table 1. Virus persisted in culture L-11 for 16 months and continued to persist in several subcultures of cells from this culture. A third serial subculture, L-lls3e, produced virus for 9 months, which was 25 months after inoculation of the original culture. The disappearance of WEE virus in the absence of living cells was investigated. Concentrations of infected mouse brain containing lo3 to 106 mouse LD,, were incubated in the tissue culture medium (1) in the absence of cells, (2) in the presence of strain L cells which had been frozen, and (3) with sediment from chick embryo extract. Of fourteen preparations in these three categories, twelve were free of virus at 4 days. A small amount of virus was present in the other two preparations at 4 days, but no virus was detected at 7 days. The persistence of virus in cult,ures of strain 1, cells for several months suggested the continued multiplication of virus over long periods of
PERSISTENCE
OF WEE
VIRUS
IN
CULTURES
OF STRAIS
L CELLS
69
time. The disappearance of virus within 4 to 7 days in the absence of cells or in the presence of frozen cells eliminated the possibility of an initial multiplication of virus and its persistence in extracellular fluids a,s an acceptable explanation for the prolonged persistence of virus in cult’ures of viable cells. Proliferation of cells in chronically infected cultures and in cultures during repeated challenge inoculation. After the initial degeneration of cells in infected cultures, surviving cells began t,o proliferate and event,ually replaced the dead cells. Proliferation of cells cont,inued in chronically infected cultures. In one experiment nine cultures which still contained virus 15 days afber inoculation were exposed to a series of four challenge inoculations with a 10P5 dilution of WEE-infected mouse brain at 3- or J-day intervals. So detectable degeneration occurred and the cells appeared to proliferat’e normally. The proliferation of cells in chronically infected cult,ures and in cult,ures during repeated inoculations suggest,ed the presence of some prot,ective mechanism which prevented infect,ion of most of the cells and which was available to new cells after cell division. 12euersion of cultures to increased susceptibility following spontaneom loss of virus. Six cultures which had carried virus for several weeks or months prior to spontaneous loss of virus were challenged with a second inoculation of WEE virus in order to determine whet,her the loss of virus was due to the development of a population of genet,ically resistant CYA~~. Controls included five cultures not previously exposed t)o virus and t#hree cultures in which virus was still present at the time of challenge, 101 days after the first inoculation. The viral inoculum used for challenge and for cont,rol cultures consisted of 105.j mouse IJI>S0 WICl~;infect’ed mouse brain. The results of tbis experiment appear in Table 2. Challenge of six cultures, mhic*h had spontaneously lost virus following a previous inoculation with WEE virus, caused degeneration graded as “marked” or “severe” in four of t,hem. This is the number of cultures one would expect t,o show degeneration following an init,ial inoculat,ion with WEE virus according to the cwmulatjivc data given in l:ig. 3. Virus assay on the sevent,h day after challenge showed a higher con~entration of virus in the fluid phase of three of t,hese cultures than was found in the fluid phase of t,cvo cultures which still cwntained virus at the time of challenge. The results indicate that the spontaneous loss of virus from cultures was not due to the development of a population of genetically resistant, wlls. Instead a temporary protecti of cells ap-
70
VELMA
C. CHAMBERS
TABLE RESULTS
OF HOMOLOGOUS
CHALLENGE WITH
-
Days etween st and !nd inxlation
Culture
Cultures from which virus disappeared 13 days or more prior to challenge: L-lS2 L-2 L-14Sl L-126 L-129 L-144 Cultures in which virus was still present at t,he time of challenge : L-143 L-136 L-137 Cultures not previously exposed to virus: L-177 L-155 L-107 I,-228 L-232
2
OF STRAIN L CELLS PREVIOUSLY INFECTED WEE VIRUS Tests for virus in tissue culture fluid 7 days after challenge
Degenerationa of cells after challengeb
10-g
748 758 758 101 101 101
Marked Severe Mild Pione Marked Marked
3/3 c 3/3 3/3
3/3 2/3 213
O/3 O/3 O/3
101 101 101
Not significant None Not significant
m 3/3
O/3 o/3
O/3 O/3
Severe Severe Severe Severe Severe
~
i
CLExtent of degenerat,ion of cells was estimated by microscopic examination of cultures: Severe-approximately 75% or more of the cell population was degenerated. Marked-approximately 25-75y0 of the cell population was degenerated. Mild-less than 25y0 of the cell population showed degeneration. Kot significant-patchy degeneration attributed to aging of the culture and overcrowding of cells. None-no degeneration observed. 6 The challenge inoculum for all cultures was 0.1 ml of the same I@-” dilution of WEE-infected mouse brain (LD60 approximately 10-9.0). c The denominator represents the number of mice that were inoculated. The numerat,or represents the number of mice that died.
peared to develop which finally extended to all cells in chronically infected cultures thereby eliminating infectious virus. The cells then reverted to sensitivity. Reversion
of cultures
to increased
susceptibility
following
e.rposure
qf
PERSISTENCE
OF WEE
VIRUS
IiX CULTURES
OF STRAIN
L CELLS
71
cultures to antiserum. The nine cultures described earlier, which were challenged with a lop5 dilution of WEE virus at 3- or 4-day intervals beginning 15 days after the first inoculation and continuing for 2 weeks, were used in this experiment. The cultures were washed three times wit,h warm Hanks’ solution after which they received 0.9 ml of medium containing anti-WEE horse serum of low tit’er. Medium containing similar antiserum was used for each change of fluid for the durat,ion of the test period, which lasted from 21 to 42 days in different cultures. During the interval when cultures were exposed to immune serum, t,issue culture fluids were harvested at 3- or 4-day intervals and were injected intracranially into mice, either individually or as pools of fluids harvested at one time from a number of cultures, or as pools of several frozen fluids harvested at different times from one culture. These tests indicated incomplete neutralization of virus in five of the nine cultures during exposure to ant,iserum (Table 3). Three of these cultures, L-119, L-124, and L-138, and one other culture, L-123, contained virus followmg removal of antiserum. Mild to severe degeneration of cells occurred in cultures L-123, L-124, and L-138 within a week after the removal of antiserum. Assays for virus in cultures L-123 and L-124 one week after removal of antiserum thowed LDCO titers of 10-4.5, and 10-j.5, respect,ively. It is presumed that following removal of antiserum many susceptible cells were present and that these were promptly infected. It appears that exposure of cultures to antiserum not only removed most of the infectious virus but also eliminated mucah if not. all of the prot)ect,ive agent. The loss of virus from cells subcultured in the presence of antiserum. If chronically infected cultures were in a stat,e comparable to that’ of lysogeny in bacteria, it should be possible to subculture cells from infected cult,ures in the presence of ant’iserum and still retain the capacit,y of cells to produce virus after removal of antiserum. However, if chronic infection of a culture depended upon infection and destruction of cells and spread of virus to other cells, successive subcult,ure of the cells to medium containing antiserum should eliminate the virus. Subcultures were prepared from the nine cultures, L-138, L-164, and L-119 to 125 inclusive, which were described in the preceding section and which are shown in the upper part of Table 3. The subcultures were started after the parent cultures had been exposed to antiserum for periods ranging from 11 days to 4 weeks. Further serial subcultures were made in some inst,anres. For example, in Table 3 dat,a are shown for four serial sub-
72
VELMA
C. CHAMBERS
TABLE EFFECT
OF SPECIFIC
ANTISERUM
Days of antiserum treatment
Culture After sub: culture
1 (
cultures” L-119 L-120 L-121 L-122 L-123 12-124 L-125 L-138 L-164 Subcultures Ll19Sl Ll23Sl L123S2 Ll23S3 Ll23S4 L-124Sl Id-12482 I,-12433
3
ON THE PERSISTENCE CULTURES
OF WEE
VIRCJS IN TISSUE
Tests for virus in tissue culture fluids Total lays 0 serlun treatment
Weeks after removal of antiserum
During :xposure to Ser”n
Original
38 21 42 42 21 21 28 21 32 28 11 24 36 42 11 36 42
17 21 18 9 10 31 9 10
45 32 42 45 52 42 45 52
3/3 O/4 -
2/4 z/4
O/3
l/3 O/3 l/4 c o/3 O/4 o/10 o/3 o/4 O/IO
~ o/3 ~ o/3 ’ o/3 o/3 o/3 o/3 ~ o/3
O/3 o/3 o/3 --
I
CLThe original cultures in t,his experiment had received an init,ial infecting dose of virus one month before exposure to antiserum and had received repeated challenge inoculations during the two weeks immediately preceding exposure to nntiserum. Virus was present in all cultures at t,he time antiserum was introduced. b The numerator indicates the number of mice t,hat, died. The denominator indicates the number of mice inoculated. c 1Mouse death occurred on t,he second d:~y after inoculation and was not attribut,ed to viral infection.
cultures from culture L-123 and three serial subcultures from L-124. The nutrient medium of all subcultures contained antiserum for at least 9 days. Subsequently, medium without antiserum was employed. Tests for virus were made both during the time the medium contained antiserum and subsequently when no antiserum was present. Xo virus was detected in the twenty subcult,ures which were tested. These included
PERSISTEKCE
OF WEE
VIRUS
IN
CULTURES
OF STRAIN
L CELLS
78
10 first generation subcult’ures, 6 second generation, 3 third generation, and 1 fourth generation culture. The one mouse death in the group inoculated with specimen L-119Sl was not attribubed to virus. Test’ results of eight of the cultures are shown in Table 3. k’ailure of suhcultures to produce virus after removal of antiserum provides substantial evidence indicating that lysogenicity is not, t,he explanation for prolonged infection of cultures of strain L cells with WEE virus. I>ISCUSSION
In attempting to explain the prolonged persistence of WEE virus in cultures of strain L cells, the following observat’ions are pert’inent : (1) An inverse relationship frequently existed between the size of viral iuoculum and the extent of cellular degeneration. (2) Cells in infected cultures continued to proliferate during repeated challenge inoculations with WEE virus. (3) Cultures revertled t)o increased susceptibility to small inocula following spontaneous loss of virus and following a period of exposure of infect’ed cultures to antiserum. (4) Subcultures of cells from infect’ed cultures during exposure to antiserum resulted in loss of virus and failure of virus to reappear after removal of antiserum. The inverse relationship between the size of viral inoculum and the extent of cellular degeneration has an obvious similarity to the autointerference that has been shown with influenza virus. It was found that undiluted allantoic fluid from infected eggs produced less virus on passage than did diluted virus (Henle and Henle, 1944). Virus rendered noninfectious by treatment with heat or ultraviolet light retained its ability to interfere with the production of infectious virus. In cultures of strain L cells chronically infected mit’h WEE virus, thermal inactivation of virus undoubtedly was a continuous process. The infectivity of WEE virus was shown to disappear in less than 7 days when incubated in fluid medium in the absence of cells or in the presence of nonliving cells. Thermally inactivat,ed virus may have served as the interfering agent that permitted persistence of healthy multiplying cells despite the presence of infectious virus. However, the existence of noninfectious interfering particles of a different origin is a possibility that is suggested by t,he studies with influenza by Van Magnus (1951a,b,c) and others. In any case, it appears that kcatment of tissue cultures with antiserum effectively removed the interfering agent as well as the infectious virus. The persistence of virus in cultures for long periods of time apparently
74
VELMA
C. CHAMBERS
depended upon occasional infection of some of the cells that regained susceptibility as the effectiveness of previously acquired interfering particles waned. Another possibility is that cells subjected to the simultaneous influence of fully active virus and of inactivated virus may have experienced a modified nonfatal infection which resulted in release of less than normal amounts of virus and in the eventual complete loss of virus from the cell. Further studies are necessary to determine more conclusively t#he validity of the proposed hypothesis. The possibility that autointerference may operate in vivo as a mechanism in limiting infection deserves consideration. ACKNOWLEDGMENT The author wishes to thank out the course of this work.
Dr. C. A. Evans for his valuable
advice
through-
REFERENCES CHAMBERS, V. C., and EVANS, C. A. (1953). Some observations on the growth of Western equine encephalomyelitis virus in cultures of L cells. Bacterial. Proc. 1963, 42. EARLE, W. R. (1943). Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in living cells. J. Natl. Cancer Inst. 4, 165-212. EARLE, W. R., and NETTLESHIP, A. (1943). Production of malignancy in vitro. V. Results of injections of cultures into mice. J. Natl. Cancer Inst. 4, 213-227. FELLER, A. E., ENDERS, J. F., and WELLER, T. H. (1940). The prolonged co-existence of vaccinia virus in high titer and living cells in roller tube cultures of chick embryonic tissues. J. Exptl. Med. 73, 367-388. GEY, G. O., and BANG, F. B. (1951). Viruses and cells-a study in tissue culture applications. I. Cells involved-availability and susceptibility. Trans. N. I’. Acad. Sci. 14, 15-24. HENLE, W., and HENLE, G. (1944). Interference between inactive and active viruses of influenza. I. The incidental occurrence and artificial induction of the phenomenon. Am. J. Med. Sri. 207, 705717. HOTTA, S., and EVANS, C. A. (1956). Cultivation of mouse-adapted dengue virus (type 1) in rhesus monkey tissue culture. J. Infectious Diseases 98, 88-97. REED, L. J., and MUENCH, H. (1938). A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493497. SANFORD, K. K., EARLE, W. R., and LIKELY, G. D. (1948). The growth in vitro of single isolated tissue cells. J. N&Z. Cancer Inst. 9, 229-246. SANFORD, K. K., EARLE, W. R., EVANS, V. J., WALTZ, H. K., and SHANNON, J. E. (1951). The measurement of proliferation in tissue cultures by enumeration of cell nuclei. J. N&Z. Cancer Inst. 11, 773-795. VON MAGNUS, P. (1951a). Propagation of the PR8 strain of influenza A virus in chick embryos. I. The influence of various experimental conditions on virus multiplication. Acta Pathol. Microbial. Stand. 28, 25G277.
PERSISTENCE
OF WEE
VIRUS
VON MAGSUS, P. (1951b). Propagation
IN CULTURES
OF STRAIS
L CELLS
75
of the PR8 strain of influenza A virus in chick embryos. II. The formaCon of “incomplete” virus following inoculation of large doses of seed virus. AC&a Pathol. Microbial. &and. 26, 278-293. VON MAGNUS, P. (1951c). Propagation of the PR8 strain of influenza A virus in chick embryos. III. Properties of the incomplete virus produced in serial passages of undiluted virus. Acta Pathol. Microbial. Stand. 29, 157-181. WELLER, T. H., and ENDERS, J. F. (1948). Production of hemagglutinin by mumps and influenza A viruses in suspended cell tissue cultures. Proc. Sot. Exptl. Biol. Med. 69, 124-128.