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Cell-virus interactions with the shope fibroma virus on cultures of rabbit and rat cells
VIROLOGY
23,
-Qirus
473-485
(1964)
interactions
with
the
Shape
of Rabbit ELLA Section
of Genetics,
and
Rat
Virus
on
Cells’
A&-D LEO SACIIS
ISRAELI Weiznzann
~i~r~~~
Institute
Accepted
April
of Science,
Rehovoth,
Israel
3, 196Q
The cell-virus interaations in vitro of the Shope fib:ona virus (SFV) w?re st~~diad on rabbit and rat tissue cultures. With both rabbit and rat primary or s::undary monolayers, SFlrproduced a cytopathic effest (CPE) in cultures infected at virus:eeli ratios of 1 or above, and pocks in cultures infected with about 330 pock-forming units (= plaque forming units) (PFU) per plate. The pocks on rabbit cultures lysed t.o form plaques, while those on rat cultures remained intact wit,h only a, partial cell lysis at the periphery of the pock. An inhibitor in the agar overlay, which could be precipitated by protamine sulfate, caused a delay in the time of appearance of pocks in rat cultures, and affected the transition of pocks to plaques in rabbit cult.ures. The one-step growth curves in rabbit. and rat cells were similar, but rat cultures generally had a lower percentage of virus-yielding cells and a lower virus yield in such cells. The virus yields per virus-yielding cell were always low, the figures ranging from 2 to 36 PFU for rabbit and 1 to 13 PFU for rat cells. The average virus yield per pock was 5.5 X lo3 PFU in rabbit, and 1.8 X 10’ PFU in rat cultures. The nature of t,he pocks produced in vitro was studied in rabbit cultures. In contrast to the results obtained after infection of primary or secondary monolayers, pocks were not produced after infection of sparse cultures, or after cloning of infected cells. Monolayers derived from 28 single cell rabbit embryo clones, also did not form typical pocks on virus infection, but exhibited CPE and plaque formation. In secondary cultures that formed pocks, a higher mitotic frequency than in noninfected cultures, coinciding with the time of pock appearance, was observed after infection of rabbit testes cells but not after infect,ion of rabbit or rat embryo cells. The data are discussed in relation to the possibilities that pock formation by SFV in vitro might result (I) from cell aggregation in either mixed cell populations or special types of cells, or (2) from a stimulation of cell division.
injected with smaller amounts of virus or in adult rabbits treated with cortisone or X-rays, and proliferative-regressive in adult rabbits. The ability to react differen@y in animals of differing ages is reminiscent of t-he situation with the Rous sarcoma virus (Duran-Reynals, 1940b, 1959). There is also some analogy to polyoma virus in Lhat both SFV and pol3Toma can produce regressive fibromas in rabbits, although in the ease of polyoma, this reaction occurs after virus inoculation into newborns (Eddy e2 al., 1959; Fogel and Sachs, 1960). It is: therefore, of interest to study cell--virus interactions with
IKTRODUCTION
The Shope fibroma virus (SFV) of rabbits (Shope, 1932a, b), one of the pox group of viruses, ean produce tumors in the rabbit. The state of the rabbit at the time of virus injection seems to determine the type of cellular reaction of the host (Duran-Reynals, 1940a, 1945; Hare1 and Constantin, 1954). The reaction is hemorrhagic-destructive in newborn rabbits injected with high doses of virus, proliferative-invasive in baby rabbits 1 Aided by Research Grant CA-05266 from the I\iationai Cancer Institute, United States Public Health Service. 473
474
ISRAELI
AND
SFV in order to determine in what way they may be different or similar to those occurring with the other pox or tumor viruses. This study might lead to a better understanding of the mechanism of tumor induction by viruses. SFV was first cultured in vitro as early as 1933 (Rous et al.), and some aspects of its growth in vivo and in vitro were studied by Hare1 (1956) and Constantin et al. (1956). The virus causesa cytopathic effect in tissue culture (Chaproniere and Andrewes, 1957; Kilham, 1959), and the cell-virus interaction in vitro can lead to pock formation (Padgett et al., 1962; and personal observation). The cytopathogenic properties of SFV were recently used to develop an accurate method for virus titration in vitro (Padgett et al., 1962; Verna and Eylar, 1962, and personal observation). The aim of the present work was to analyze the cell-virus interactions in two different cell systems. One system was from the rabbit, an animal that is susceptible to SFV tumor formation in vivo, and the other from the rat, an animal that so far seemsto be resistant to tumor formation in vivo. Experiments were also carried out on the pocks formed in vitro, with a view to determining whether they are produced by cell proliferation or cell aggregation. MATERIALS
AND
METHODS
Cell Cultures Including Medium and Incubation Cells were obtained from noninbred domestic rabbits and noninbred albino rats. Rabbit embryos were excised on the 17th to 18th day of gestation and rat embryos on the 15th to 17th day. The procedure for preparing primary and secondary cultures was as described by Winocour and Sachs (1960). For primary embryo cultures, 1 X lo7 cells were seededin loo-mm petri dishes. After 5 or 6 days the cultures were trypsinized and 60-n-m petri dishes were seededwith 2.4 X lo6 cellsper plate to make secondary cultures that were used for virus inoculation 48 hours later, when complete monolayers had formed. Rabbit kidney cultures were prepared from kidneys taken from lo-day-old rabbits.
SACHS
The kidneys were trypsinized after decapsulation and removal of the hilus; confluent monolayers, either primary or secondary, which were formed after inoculating 1.6 X 10” cells per 60 mm petri dish, were used for virus inoculation. Testes for cultures were taken from l-month-old rabbits. After removal of the capsule, and trypsinization, 1 X lo6 cells were seeded in 35-mm petri dishes; cultures were used after about 4 days, when monolayers had formed. All cultures were made in Falcon plastic petri dishes. Eagle’s medium with a fourfold concentration of amino acids and vitamins (EM), and 5 % of calf serum was used throughout. The cultures before virus infection were incubated at 37°C. After infection, they were incubated at 35°C (Kilham, 1959). All the incubators contained a constant flow of 10 % COZ in air and a humidified atmosphere. Virus and Virus Stocks SFV (Patuxent strain) was obtained through the courtesy of Dr. L. Kilham. Three successive single-plaque isolations were made on rabbit embryo cultures, and the final isolate was used to initiate the virus stocks for the present experiments. Stocks were obtained by infecting secondary rabbit monolayers at virus: cell ratios of 1:2 or 1: 1 pock-forming units (= plaque forming units) (PFU) per cell. After 4 days of incubation, the cultures were frozen and thawed once, the cells were scraped off the dish with a rubber wedge and sonically vibrated in a Raytheon 200-watt lo-kc magnetostrictive oscillator at 9000 cycles per second for 3 minutes. The cell debris was spun down at 1400 g for 30 minutes, and the supernatant was distributed into tubes and kept at -50°C until use. To test the fibroma-producing properties in vivo, different stock sampleswere injected intracutaneously to either adult or baby rabbits. No lossof fibroma-producing properties was detected during 12 passagesof the virus in rabbit tissue cultures. Pock and Plaque Assays The medium was removed from the secondary cultures; 0.1 ml of virus, appropri-
CELL-VIRUS
INTERACTIONS
ately diluted, was added to the monolayer. After 3 hours of adsorption at 35”C, during which the plates were tilted twice to ensure virus redistribution, the cultures were covered with 5 ml of EM containing 0.9 % agar and 5 % calf serum. For the routine assays the agar overlay on rat cultures contained, in addition, 400 pg/ml of protamine sulfate (Nutritional Biochemical Corporation). Pocks appeared 5 days after infection of rabbit cultures and 7 days after infection of rat cultures. Although they could be counted at that time by illuminating the cultures with an oblique light, it was preferable to add a second overlay of 2.5 ml agar medium which contained 0.01% (w/v) neutral red to facilitate counting. Pock counts were made twice, the first time at 6-7 days after infecting rabbit cultures, and 10 or II days after infecting rat cultures. The second time was 16-21 and 21-2<5 days after infection of rabbit and rat cultures, respectively. This second count proved to be valuable, since some of the pocks appeared or became more pronounced only later, owing to their development into plaques, in the case of rabbit, and enlargement, in the case of rat cultures. Assays for Vim.9Producing Cells IcfectiVe center assay. Cultures containing I to 2 X 105 cells per plate, were infected at virus: cell ratios of 3-22 PFU per cell and after 1 hour’s adsorption they were washed twice with phosphate-buffered saline (PBS). The cells were then suspended with the aid of trypsin, and after centrifugation, were diluted in medium. The cell dilutions were inoculated in amounts of 2 ml on monolayers of rabbit cells. After overnight incubation, the medium was removed, and the cultures were covered with agar medium and further incubated as for pock and plaque assays. Gounts oj cells containing inclusion bodies. Cultures containing 1 to 2 X lo5 cells per plate, were infected at virus:cell ratios of 3-25 PFU per cell. After 1 hour’s adsorption, the cultures were washed twice with PBS and covered with growth medium. After further incubation for 24 hours, the medium was removed; the cultures were washed once with PBS and then fixed with methanol and stained with May-Grunwald-Giemsa. Cells
WITH
SHOFE
FIBROMA
VIRUS
475
were counted for the presence of inclusion bodies, under X 1500 magnification, as a percentage of the total number of cells. Growth Curves Cultures containing 1 to 2 X IO5 cells per plate were infected at virus:cell ratios of l-25 PFU per cell. After 1 hour’s adsorption, the plates were washed twice with PBS and growth medium was added. Two plates were frozen immediately at -20°C for zero time titration. The remaining cultures were incubated further, and at appropriate time intervals, 4 plates were removed, 2 of which were frozen at -20°C for total virus titrations, and 2 others were fixed and stained for counts of cells with inclusion bodies. For free virus titrations, 0.5 ml of medium from each of two plates was pooled, and the supernatent, after spinning down cell debris at 1400 g for 30 minutes, was kept at -20°C. At the end of the experiment the samples were thawed, both plates of each point mere pooled, and all the samples, both for free and total virus, were disrupted by sonic vibration. The cell debris from the tot.al virus samples was then spun down at 1400 g for 30 minutes. Titrations of the virus were made on rat or rabbit embryo monolayers. Counts of Mitotic Figures in Infected and I:12i7zfectedCultures 1.0 X lo6 testes cells and 1.2 X 10” rat and rabbit embryo cells were plated in 35-mm plastic petri dishes to form monolayers. These monolayers were infected with 150, 300, or 500 PFU. Medium (0.1 ml) instead of virus was added to control cultures for the adsorption period. At desired times, samples of 2 or 3 plates from each group were fixed and stained with May-GrunwaldGiemsa. Mitotic figures were counted at X 1500 magnification in almost the entire plate, i.e., an area of about 3.4 cmZfor rabbit monolayers, and in half the plate, i.e., an area of about I.7 cm2for rat monolayers. Feeder Layers Rat embryo secondary culture cells mere used for feeder layers. The cultures were irradiated with 5000 r, and the cells then seededat 8 X IO* cells per 60-mm petri dish.
ISRAELI
476
AXD
Isolation 0.f Clones Clones of rabbit embryo cells were isolated from plates seeded with about 250 cells of either primary or secondary cultures in 60-mm petri dishes containing irradiated rat feeder cells. The cloneswere isolated by trypsinizing the cells in small cylinders placed over the clones; the cells of each clone were then transferred to a plate with feeder cells. When a clone had formed a monolayer, the cells were transferred to 2 petri dishes (without a feeder layer), one of which was later infected, the other serving as a control. RESULTS
CPE and Pock Formation Monolayers of both rabbit and rat embryo cells showed 2 different types of cell-virus interactions, depending on whether the cultures were infected at high or low virus: cell ratios. At virus: cell ratios of 1 PFU per cell or above, there was a general cytopathic effect (CPE). At low virus: cell ratios, such as to assure countable areas of infection, i.e., up to 300 PFU per plate, the first change observed was the formation of pocks, and CPE and cell destruction was found only in later stages. Rat and rabbit cultures differed in the type of pocks and in the nature of the CPE.
SACHS
such infected cultures through succeeding passageseither at 35°C or 37”C, were unsuccessful, and after 2 to 4 passages,almost no cells remained. Infection with about 300 PFU per plate. At low virus:cell ratios, the cultures at 4 days after infection showed areas which consisted of elongated refractile cells. During the following days, bundles of more or lessparallel cells, protruding from the monolayer, were found in these areas, and these will be called ‘Lpocks” (Fig. IA). At later stages, a lytic process took place. It usually began about 6-7 days after infection on one side of the pock and the bundles of cells became less crowded becauseof detachment of dead cells. Finally, holes developed with heaped-up cells at the periphery in place of the pocks (Fig. 1B). This continuing process led to the formation of plaques having dimensions of 24 mm at about the third week after infection in cultures that were covered with agar. Under a fluid medium the entire culture eventually underwent CPE, due to spread of the virus. Medium change or passaging of the cultures strikingly enhanced the CPE. This could be due to the washing out of interferon, as found in vaccinia-infected cultures (Glasgow and Habel, 1962).
Rabbit TestesCultures Since Kilham (1956) described poor propagation of the virus with no CPE in domestic Rabbit Embryo Cultures rabbit testes explants, monolayers of rabbit Virus :cell ratios above 1. When virus: cell testes cells were also infected. It was found ratios above 1 were used, a general change that when these cultures were infected with took place in the culture 2-3 days after in300 PFU, pocks appeared after 4-5 days, fection. At the beginning, cells became more which later gave way to lysis, as in monorefractile, and later on, at about the 4th day, layers of embryo cells. The pocks were differmany of the cells died and detached from ent from those on embryo cultures, being the plate. The cells elongated and the cullarger and showing pronounced random oritures showed areasof thickness reminiscent of entation of the cells. The onset of CPE ocpocks in their structure, but with no definite curred several days later than in embryo boundaries. Side by side with these areas cultures, i.e., about the 7th to 9th day after there were areas of lysis. Cells containing infection. It began on one side of the pock inclusion bodies showed an increase in volperiphery, forming a hole in the monolayer ume, which preceded their lysis. In later near the original intact pock. The CPE stages, clumping of cells occurred here and spread to the center of the pock only a few there, accompanied by cell detachment. This days later. CPE seemed to involve most of the cells from the beginning, but complete destruc- Rat Cultures tion of the culture resulted only about 3 The general CPE which appeared after weeks after infection. Attempts to maintain infection at virus:cell ratios above 1 was
CELL-VIRUS
INTERACTIONS
somewhat similar to that found in rabbit embryo cultures. Many of the cells contained inclusion bodies, although no enlargement of cell volume occurred, and there was less clumping. These cultures could be kept through succeeding passages, when incubated at 37°C. At this temperature they seemed to establish carrier cultures, and counts made during the course of 9 passages indicated that 15 % of the cells had inclusion bodies. The pocks which were formed at low virus:cell ratios, i.e., about 300 PFU per plate, differed from the rabbit pocks. They began to appear 5 days after infection as areas with more refractile cells, many of which contained dark cytoplasmic granules, which were seen only in living cultures. On succeeding days, they consisted of piled-up, rounded cells. Although these cells died later on, as was shown by their inability to take up the neutral red stain, they did not disintegrate, and the pock never developed into a plaque. It enlarged during the first days of its existence and acquired an irregular shape as a result of uneven spreading of the piled-up cells (Fig. lC, D). The periphery of the pock became sparse, owing to lysis of part of the cells. These pocks were usually smaller than the rabbit pocks. In a fluid medium new pocks continued to appear; after about a month most of the cells died awing to confluency of pocks. In this case, as in rabbit cultures, this process was enhanced by changing the medium or by passaging the culture at 35°C. Figure 2 shows photographs of plates with pocks on rat and plaques on rabbit monolayers. The Effect on Pock and Plaque Formation of Protanzine Sulj”ate Inclusion in the Agar It was noticed that when rat embryo cultures inoculated for a pock assay were covered with an agar overlay, pocks appeared later than in similar cultures with a fluid medium. Since this might be due to the presence of inhibitors in the agar, it was of interest to see whether the polysaccharide inhibitor protamine sulfate (Takemoto and Liebhaber, 1961) effected any change. Table 1 summarizes an experiment, carried out to study the effect on pock development of
WITH
SHOPE
FIBROMA
477
VIRUS
varying concentrations of protamine in the overlay. In overlays containing no protamine, the appearance of pocks was delayed for about a week, and their course of development was retarded. The number of pocks under agar with protamine was somewhat higher than under agar without prot’amine. The optimal concentration of protamine was found to be 400 pg/ml, the same as described by Takemoto and Liebhaber (1961). When rabbit embryo monolayers were used, different’ results were obtained. There was no retardation of pock appearance in the presence of the agar inhibitor, but it had an effect on further development of the plaques, delaying the lysis of dead cells in them. In contrast, when rabbit, kidney cu.. tures were infected, the addition of agar arrested the development of pocks at’ their early stages, giving them the shape of undeveloped pocks seen on rat embryo cutures. In this case, when protamine was incorporated into the agar, the pocks developed v&h a shape similar to &at found on rabbit embryo cells, and 5 days after their appearance complete lysis took place, the plaque area becoming a hole, fret from cell debris. Assays
for
Virus-Yielding
Cells
Since SFV produces cytoplasmic inclusion bodies in cells in which it replicates, the presence of inclusion bodies can be used to estimate the proportion of virus-producing cells in a culture. Table 2 shows the results of a comparison between counts of cells with inclusion bodies and infective center assays. The data indicate that the method of inclusion body count gives about a 2% times higher estimate of the proportion of virus-yielding cells. Since the counting of cells with inclusion bodies was more convenient than an infective center assay, it Ivas used in all the subsequent experimenrs. Virus-Yielding Cells as a Functior: sorption Time
~1.iAd-
Cultures containing 1.0 X IO5 rabbit embryo cells, or 1.4 X 105 rat, cells, per dish were infected with 2.2 X 106 PFU of virus and incubated. At l-hour int’ervalsJ 2 plates were washed twice with PI38 and
FIG. 1 478
CELL-VIRUS
FIG. 2. Plaques on rabbit (A) infection. The plates are covered was taken on a white background,
IXTERACTIONS
and pocks with agar and that
WITH
SHOPE
on rat (B) monolayers, containing neutral red. of rat pocks on a black
covered with EM. All the plates were incubated for an additional 24 hours, then washed twice with PBS, fixed, and stained for counts of cells containing inclusion bodies. Figure 3 summarizes the results of such experiments. The data show that the proportion of virus-yielding ceils was higher in rabbit than in rat. but in both cultures it increased as a function of adsorption time. At, the time the experiment was terminated only 50% of the rat cells contained inclusion bodies as compared to 88 % of rabbit cells. A higher percentage of virus yielding cells for rabbit cultures was also found in other experiments.
FIBROMA
VIRUS
479
9 and 13 days, respect,ively, The photograph of rabbit background. Magnification TABLE
after plaques : X 0.9.
1
THE EFFECT ON POCK FORMATIOX ox RAT Ezinn~o MONOLAYERS OF PROTAMINE SCLFATE INCLUSION IN THE AGAR OVERLAY Protamine concentration (dml) 0
200 400
Number of pocks” Days after infection __-.._. 8 IQ 12
-6
i
zt
+
+
75
+
++
85
a Average of 3 to 4 assay plates. b In the first days after their appearance, pocks were very small and difficult to count. number of pocks at these times has therefore designated as - to ++.
21
63 94 106
the The been
Vims Adsorption on Monolayers h’xpressed by Pock and Plaque Counts rat cultures. Pock and plaque assays made One hundred PFU in 0.1 ml were added on rabbit and rat monolayers gave eomparof cultures to a monolayer. At l-hour intervals, groups able results, so that both types can be equally used for virus assay. of 4 plates were covered with an agar overlay and incubated for the appearance of pocks. The results of such experiments are repre- Growth Curves sented in Fig. 4. Xo significant differences Growth curves of WV were made on in adsorption rate on the monolayers were rabbit and rat embryo cells. Figure 5 gives found by this method, between rabbit and representative growth curves after infection
FIG. 1. SFV-infected rabbit and rat monolayers, covered with agar: (B) with neutral red; (A, C, and D) without neutral red. (A) Pock on rabbit embryo monolayer, 6 days after infection. Elongated piledup cells can be seen. Magnification: X 85. (B) Later stage than (A). The pock has developed into a plaque, 10 days after infection. An edge of heaped-up cells can be seen at one side of the plaque. Magnification: X 175. (C) Pock on rat embryo monolayer, 7 days after infection. The pock is composedof piledup rounded cells. Magnification: X 85. (D) The edge of a pock of about the same age as C, shearing elongated refractile cells at the periphery. Magnification: X 175.
480
ISRAELI
AND
SACHS
TABLE ESTIMATION COUNTS
OF THE NUMBER OF CELLS WITH
2
OF VIRUS-YIELDING INCLUSION BODIES
RABBIT AND RAT EMBRYO AND BY INFECTIVE CENTER Virus-yielding
Expt.
no.
Cells
Virus: cell ratioa (PFU:ceil)
Inclusion Total
R47
Rabbit Rat
22 4 16 3
bodies
x x x x
Infective
‘%
10” 104 104 104
BY
cells per plate according count6
number
7.0 7.0 7.1 5.7
CELLS ASSAY
Total
70.0 70.0 44.6 35.4
3.8 1.9 1.4 1.7
to
center
assay”
number
%
104 104 104 104
38.0 19.0 10.0 12.1
x x x x
a Cultures containing 1.0 or 1.4 X lo5 rabbit or rat cells, respectively, were infected with virus centrations of 2.2 X lo6 PFU and 4.4 X lo5 PFU per plate. b At 24 hours after virus infection, the proportion of cells with inclusion bodies was determined counting about 1000 cells in 2 plates, and their total number in the entire plate was then calculated. c The titrations for infective centers were all made on rabbit embryo secondary monolayers.
at virus: cell ratios of 25 PFU per cell for rabbits, and 17 for rats. Figure 6 shows the correlation between total and free virus, after infection at virus:cell ratios of 2 PFU per cell for rabbits and 1 for rats. It can be seen that a considerable amount of virus is cell associated. The rise in virus titer sometimes began after 12 hours (Fig. 5) and at other times after 8 hours (Fig. 6) in rabbit cultures, and after 12, or 15 hours in rat cultures (Figs. 5 and 6). In the growth curves shown in Fig. 5, counts of cells with inclusion bodies were made at intervals during the 24 hours of the experiment. Inclusion bodies were found as soon as 4 hours after infection. The final percentage of cells with inclusion bodies was 90 % in rabbits and 77 % in rats, so that the curves can be regarded as onestep growth curves. The proportion of virusyielding rat cells in this experiment was somewhat higher than that found in other experiments (Table 2). From the results in Fig. 5 the total virus yield per virus-yielding cell can be calculated as 18 PFU per cell for rabbits and 1 PFU per cell for rats. A generally higher virus yield per cell in rabbits was also found in other experiments. In all cases the total virus yield per virusyielding cell was low. The yield per cell in 10 different experiments ranged from 2 to 36 PFU for rabbits, and 1 to 13 PFU for rats. The finding of a well-developed inclusion
conby
100 I
c 2
60-
Y d
50-
0 IL 0 2
./
4o
/ /
z E
30-
a
20-
---o
/---0 /
P
0’
/ O’
IO I;, 2
, 3 ,HOURS
FIG. 3. Percentage of virus-yielding function of adsorption time, estimated ing cells with inclusion bodies. Each average of counts made on 2 plates. cultures; 0, rat cultures.
, 4
I. 5
cells, as a by countpoint is an 0, Rabbit
CELL-VIRUS
ISTERACTIOXS
WITH
100; sot
SHOPE
FIBROMA
I-IRES
481
to a lower yield per cell or are due also to a lower number of virus-yielding cells in t#hc pock is so far unknown.
It was of interest to determine whether pocks are formed after infection of sparse cultures. Sparse layers of rabbit testes and embryo cells containing 2 to 3 X 105 cells per plate were infected with 3 X 1Q5PFIJ or
6
5 I
i
I
L
123456
HOURS FIG. 3. The adsorption of SFV on confluent monolayers. In calculating the percentage PFU adsorbed the average number of PFU at 6 hours was taken as 100%. Each point is an average of counts made on 4 plates. @, Rabbit cultures; 0, rat cultures.
body in cells producing only 1 or 2 PFU suggest,sthe possibility that the sensitivity of the virus assay is very low and/or that a considerable amount of noninfectious virus material is being produced. As a further comparison between the 2 species, the virus titer was determined in single pocks. Pocks were isolated from cultures with agar overlays, with a thin-bore pipette, at 5-7 days after their appearance, i.e., at lo-14 days after virus infection. They were suspendedin growth medium and subjected t,o 3 cycles of freezing and thawing before titration. The results (Table 3) indicate t’hat the titers obtained from rabbit pocks were generally higher than those obtained from rat pocks. Whether the low figures obtained for rat pocks are due only
%18-R % IB-R’
ri
50
67 75 71
90
28
63
77
I 4
76
I 8
12
16
20
24
HOURS FIG. 5. One-step growth curves of SFV on rabbit and rat cultures, infected at virus:ceii ratios of 25 and 17 PFIJ per cell, respectively. Each point is based on a pool of 2 plates. Time zero is after virus adsorption. @, rabbit cultures; X, rat cultures, yi IB - R = percentage of rabbit cells with inclusion bodies. 7; IB - Rt = percentage of rat cells with inclusion bodies.
482
ISRAELI
\ J ,d 12
AND SACHS
I I I 60 $2 96 120 HOURS FIG. 6. Relation between free and total virus in growth curves of SFV on rabbit and rat cultures infected at virus: cell ratios of 2 and 1 PFU per cell, respectively. Each point is based on a pool of 2 plates. 0, rabbit total; 0, rabbit free; A rat total; a, rat free. By July, 858, 4 tabs., 6 figs., Israeli 2
0
1 24
A6
I 46
200 PFU. Although there was a CPE, there was no pock formation in cultures infected at either of these virus inputs, even though confluent cell layers were formed in later stages. There was also no development of pocks when 6 X lo4 testes and 4 X lo4 embryo cells from infected cultures were plated at 1 to 3 X lo3 per plate for cloning. The existence of a monolayer at the time of infection thus seemsto be an essential requirement for pock formation. The Frequency of Mitoses in Injected and Uninfected Cultures Since the results of the above experiments suggested that separate cells do not proliferate to form a pock in vitro, it was of interest to determine whether pocks are formed on monolayers as a result of cell proliferation. Monolayers of rabbit and rat embryo cells were infected with 150 or 300 PFU, and monolayers of rabbit testis cells with 150, 300, or 500 PFU. The number of mitoses was counted at various times after infection in an area of 3.4 cm2for rabbit monolayers and 1.7 cm2 for rat monolayers. In the first experiment with rat cultures there were cells
TABLE VIRUS
YIELD RAT
PER POCK ON RABBIT EMBRYO CULTURES
Cell type
Pock no.
Rabbit
1 2 3 4 5 6 7 1 2 3
Rat
3 AND
PFU per pock 3.2 1.6 1.0 1.3 1.0 8.5 2.7 5.0 2.7 2.2
X X x x x x X x X x
lo3 lo2 103 104 104 103 lo3 101 lo2 102
on the fifth and sixth day after infection with unclear figures, which could have been either in early prophase or in a stage of pycnosis. In the second experiment, therefore, 0.1 pg colchicine per milliliter was added to the cultures for 8 hours prior to fixation. The results (Table 4) show that there was no significant increase in mitotic frequency in rabbit and rat embryo cultures, but that in infected testes cultures there was a significant increase in mitoses which coincided
CELL-YIRT;S
INTERACTIOSS
WITH TABLE
SHOPE
FIBROMA
VIRUS
483
4
NUMBER OF MITOSES IN INFECTED AXD NONINFECTED RABBIT AND RAT MONOLAYEIZS Virus Cell
type
input
(PFU P" plate)
R52BI
(days)
300
3 4 r
R52BII
300
R52BIII
300
s 5 4 5 1 3 4 5 4 5 4 5 4 5 6 5 6
R52C
Rabbit embryo
Time
inf~~~~n
Rabbit testes
150
R52E
300
R70A
500
Rt5A
Rt5Be
Rat embryo
300
300
Kumber Control
of mitoses” Infected
549 429 561 375, 302 177, 182, 200 440, 438 560, 621 785, 937 85, 104 47, 23 52, 33, 27 138, 188, 230 116, 126, 141 141, 189 86, 88 669 591, 762 565 2047 2155, 1976
471 277, 324, 211 262, 278 465, 575, 450 211, 228, 187 303, 341 339, 293 709, 706 80, 134 101, 77 84, 145, 146 432, 411, 527 318, 284, 322 459, 415 251, 368, 320 586 639 + 290" 599 + 219" 2117, 2019 2582, 1890
a Counted in an area of 3.4 cm2 in rabbit and 1.7 cm2 in rat cultures. Each sample. b Calculated according to student’s t test. c Figttres after + are unclear figures, which may be either early prophase d Numbers in parentheses are ratios including the unclear figures. o Colchicine (0.1 pg/ml) was added for 8 hours before fixation.
with the time of pock appearance. In another experiment with rabbit testis cultures, which is not given in Table 4, the CPE appeared, for unknown reasons, earlier than usual (on the 4th to 5th day after infection), and in this experiment there was no difference in the mitotic frequency of the infected and uninfected cultures. This could suggest that the cytopathic process may mask the proliferative reaction. It could then be argued that there is a proliferative reaction in both rabbit testes and embryo cells, but that the increase in mitoses can be observed only in rabbit testes cultures which show a slow CPE, but not in rabbit embryo cultures where the CPE is more rapid. This suggestion, however, could not account for the results with rat. embryo cultures, since with these cells the CPE is even slower than with rabbit testes cells. In this latter case, there-
Ratio of mitoses in infected to mitoses in control
0.86 0.63 0.48 1.47 1.12 0.73 0.53 0.82 i.10 2.53 3.37 2.47 2.41 2.04 3.59 0.88 0.94 1.06 I.00 1.08 value
or first
P valuest’
CO.05 <0.005
represents
a separate
stages
of pyeuosis.
fore, it can be suggestedthat pock formation in v&o may be due to cell aggregation rather than to a virus-induced cell proliferation. In cultures infected with small amounts of virus, no dividing cells were found to contain inclusion bodies. In cultures infected with high dosesof virus about 4 % of the dividing cells contained some atypical inclusions, although further tests are required to be certain that these were true inclusion bodies. Reaction oj Cell Clones to T’irus Pafectiorz In order to determine whether the progeny of individual cells from rabbit embryo were equally susceptible to pock formation, cultures derived from 28 single cell clones were infected as monolayers with 200 PFU per plate. Pocklike structures were formed in only 6 out’ of the 28 clones tested, but these were not like the typical large pocks ap-
484
ISRAELI
AND
pearing on secondary mixed cultures. There were also differences in the time after infection at which a CPE appeared. In 23 of the 28 clones a CPE was observed after 5-7 days, in one after 9 days, and in 4 after 12 days. Twenty of the clones showed a strong CPE on the 12th day, and 8 showed only limited areas of slight CPE at this time. To rule out the possibility of the presence of an interfering virus in the clones, extracts of cells from 2 other clones, one of which gave only CPE and the other neither CPE nor pocks, were prepared by sonic vibration and centrifugation to remove cell debris. These were added to normal secondary cultures, which were then incubated for 6 days before infection with SFV. No interference with pock production was found in these extracts, and the secondary cultures developed the typical SFV pocks. DISCUSSION
The present in vitro experiments with two cell systems, one from rabbits and one from rats, can be divided into the kinetics of virus reproduction, and the analysis of pock formation. The experiments in the first category showed that there was a similar course of virus multiplication in both rabbit and rat cells, the only difference being a lower percentage of virus-yielding cells and yield per cell for rat cells. It should be mentioned, however, that, in other experiments, rat cells were for a period of several months almost completely resistant to SFV infection, perhaps owing to an interfering rat virus. A comparison of SFV to the related vaccinia virus indicates that the CPE appears earlier after vaccinia (Glasgow and Habel, 1962) than after SFV infection. But both viruses seem to have about similar growth curves (Furness and Youngner, 1959; Cairns, 1960), adsorption rates (Youngner, 1956), and proportion of free to total virus (Sheek and Magee, 1961). Vaccinia has also been reported to produce pocklike areas on mouse monolayers and, like SFV, gives a general CPE in cultures infected with a high dose of virus and pocklike areas in cultures infected with low doses of virus (Glasgow and Habel, 1962).
SACHS
In relation to the pocks produced by SFV in v&o, a difference between rabbit and rat cultures has been found in the morphological appearance of the pocks and their development. However, in both species, no pocks were produced after infection of secondary monolayers with a virus: cell ratio of 1 PFU per cell or higher. In further studies with rabbit cultures, infection of sparse layers, cloning of infected cells, or infection of monolayers derived from 28 embryo cell clones also did not result in formation of pocks. These results indicate the following requirements for pock production in vitro: Virus input should be low, the culture should be in the form of a monolayer at the time of infection’, and this monolayer may have to be either composedof a mixed cell population or presumably contains special types of cells that can form pocks. Further cloning experiments are being carried out to clarify whether it is special cell types or mixed cell populations that are required for pock formation. The data on mitotic frequency in infected monolayers that formed pocks indicated that in infected testes cultures there was an increase in mitotic frequency that coincided with the time of pock development. No such increase was observed in infected rabbit or rat embryo cultures. The present results suggest two explanations for pock formation by SFV in vitro: (1) Pocks are formed as a result of cell aggregation in both embryo and testes cultures, but rabbit testes cultures can show in addition a higher mitotic frequency. (2) There is a difference in the mechanism leading to two types of pocks, one type being the result of cell aggregation and the other type the result of a proliferative process. Further experiments are required to distinguish between these possibilities and to determine the relationship between the mechanism of pock formation in vitro and tumor formation in viva. 2 It has further been observed, that pocks were not formed after infection of confluent rabbit embryo secondary monolayers obtained after seeding 2 to 3 X 105 cells, in contrast to the pocks formed after infection of monolayers obtained from the routine seeding of 2.4 X 106 cells.
CELL-VIRUS
INTERACTIONS
WITH
ACKNOWLEDGMENT We to Mr.
are indebted to Mrs. Wilma Montian Eliyahu Nava for technical assistance.
and
REFEREECES CAIRNS, J. (1960). The initiation of vaccinia infection. Virology II, 603623. CHBPRONIERE, I). M., and ANDREWES, C. H. (1957). Cultivation of rabbit myxoma and fibroma viruses in tissues of nonsusceptible hosts. Vi/.ozogy 4, 351-365. Co~sra~~rrs, T., FEBVRE, H., and HAREL, J. (1956). Cycle de multiplication du virus du fibrome de Shope in vitro (Souche OA). Conzpt. Rend. sot. Biol. 150, 347-348. DURAX-REYNALS, F. (1940a). Production of degenerative or neoplastic effect in the newborn rabbit by the Shape tibroma virus. Yule J. Biol. Med. 13? 99-110. Dr-RAX-REIN-&LB, F. (1940b). A hemorrhagic disease occurring in chick inoculated with the Rous and Fujinami viruses. Yale J. Biol. Med. 13, 77-98. DURAX-REYNALS, F. (1945). Immunological fact,ors that influence the neoplastic effects of the rabbit fibroma virus. Cancer Res. 5, 25-39. DUR~N~RE~NALS, F. (1959). Virus induced tumors and the virus theory of cancer. In “The Physiopathology of Cancer” (F. Homburger and W. H. Fishman, eds.), 2nd ed., pp. 298-337. HoeberHarper. Sew York. RUDY, I). TE., STEWART, S. E., KIRSCHSTEIN, R. L., and YOUNG, R. D. (1959). Induction of subcutaneous nodules in rabbits with SE polyoma virus. Nature 184, 7666767. FOGEL, M., and SACHS, 1;. (1960). The in vitro and in lyiz-0 analysis of mammalian tumor viruses. I\,‘. Antibody response and tumor induction with polyoma virus in different species. J. Natl. Cuncer Inst. .%, 839-857. FURNESS, G., and YOUNGNER, J. S. (1959). Onestep growth curves for vaccinia virus in cultures of monkey kidney cells. Viro7ogy 9, 3866395. GLASG~X~, L. A. and HABEL, K. (1962). The role of interferon in vaccinia virus infection in mouse embryo tissue culture. J. Exptl. Med. 115, 503512.
SHOPE
FIBROMA
VIRUS
486
HAREL, J. (1956). Titration et etude du cycle de multiplication in vivo du virus fibromsteux de Shope dans les tumeurs malignes du laperau. Compt. hhd. Sot. Biol, 150, 139-142. HAREL, J., and COXSTANTIN, T. (1954j. Sur ia malignite des tumeurs provoqubes par !e virus fibromateux de Shope chew le lapin nouveau-n6 et le lapin adult,e trait6 par des doses massives de cort,isone. Bull. Cancer 41, 482-497. KII,HAM, L. (1956). Propagation of fibroma virus in tissue culture of cotton tail t,estes. Prcc. Sot. Expll. Biol. Xed. 92, 739%7q2. KILHA~V, L. (1959). Relation of thermoresi&.ree to virulence among fibroma and mysoma viruses. Virology 9, 186~48’7. PADGETT, B. L., MOORE, M. S., and WALICER, I). L. (1962). Plaque assays for myxoma and fibroma viruses and differentiation of the viruses by plaque form. Virology 17, 462-469. Revs, P., MCMASTER, P. I)., and Hrrniics, S, S. (1933). The fixation of certain viruses on the cells of susceptible animals and protection afforded by such cells. Proc. Sot. Exptl. Biol. Med.31,90-91. SHEEK, M. R., ~~~MAGEE, W. E. (1961). An aubo radiographic study on. the intracellular develop ment of vaccinia virus. Virology 13, l-C&163. SHOPE, R. E. (1932a). A transmissible tumor-like condition in rabbits. J. Exptl. &fed. 56, 79HO2. SHOPE, R. E. (1932b). A filtrable virus causing a tumor-like condition in rabbits and its relation ship to virus myxomatosum. J. Ezptl. Xetl. 56, 803-822. Tax~~oro, K. K., and LIEBHABER, N. (EH). Alteration of plaque morphology of EMC virus with polycations. Virology I-i, 502-504. VERXB, J. E., and EYI,~R, 0. R. (1962). Rabbit fibroma virus plaque assay and in vifro studies. Virology 18, 266-273. WINVOCOUR, E., and SACHS, L. (1960). Cell-virus interactions with the poiyoma virus. I. Stud.ie~ on the lytic interactions in the mouse embryo system. Virology 11, 699-721. YOUSGNER, J. S. (1956). \%us adsorption and plaque formation in monolayer cultures of trypsin-dispersed monkey kidney. J. Immund. '76, 28%-292.
23,
-Qirus
473-485
(1964)
interactions
with
the
Shape
of Rabbit ELLA Section
of Genetics,
and
Rat
Virus
on
Cells’
A&-D LEO SACIIS
ISRAELI Weiznzann
~i~r~~~
Institute
Accepted
April
of Science,
Rehovoth,
Israel
3, 196Q
The cell-virus interaations in vitro of the Shope fib:ona virus (SFV) w?re st~~diad on rabbit and rat tissue cultures. With both rabbit and rat primary or s::undary monolayers, SFlrproduced a cytopathic effest (CPE) in cultures infected at virus:eeli ratios of 1 or above, and pocks in cultures infected with about 330 pock-forming units (= plaque forming units) (PFU) per plate. The pocks on rabbit cultures lysed t.o form plaques, while those on rat cultures remained intact wit,h only a, partial cell lysis at the periphery of the pock. An inhibitor in the agar overlay, which could be precipitated by protamine sulfate, caused a delay in the time of appearance of pocks in rat cultures, and affected the transition of pocks to plaques in rabbit cult.ures. The one-step growth curves in rabbit. and rat cells were similar, but rat cultures generally had a lower percentage of virus-yielding cells and a lower virus yield in such cells. The virus yields per virus-yielding cell were always low, the figures ranging from 2 to 36 PFU for rabbit and 1 to 13 PFU for rat cells. The average virus yield per pock was 5.5 X lo3 PFU in rabbit, and 1.8 X 10’ PFU in rat cultures. The nature of t,he pocks produced in vitro was studied in rabbit cultures. In contrast to the results obtained after infection of primary or secondary monolayers, pocks were not produced after infection of sparse cultures, or after cloning of infected cells. Monolayers derived from 28 single cell rabbit embryo clones, also did not form typical pocks on virus infection, but exhibited CPE and plaque formation. In secondary cultures that formed pocks, a higher mitotic frequency than in noninfected cultures, coinciding with the time of pock appearance, was observed after infection of rabbit testes cells but not after infect,ion of rabbit or rat embryo cells. The data are discussed in relation to the possibilities that pock formation by SFV in vitro might result (I) from cell aggregation in either mixed cell populations or special types of cells, or (2) from a stimulation of cell division.
injected with smaller amounts of virus or in adult rabbits treated with cortisone or X-rays, and proliferative-regressive in adult rabbits. The ability to react differen@y in animals of differing ages is reminiscent of t-he situation with the Rous sarcoma virus (Duran-Reynals, 1940b, 1959). There is also some analogy to polyoma virus in Lhat both SFV and pol3Toma can produce regressive fibromas in rabbits, although in the ease of polyoma, this reaction occurs after virus inoculation into newborns (Eddy e2 al., 1959; Fogel and Sachs, 1960). It is: therefore, of interest to study cell--virus interactions with
IKTRODUCTION
The Shope fibroma virus (SFV) of rabbits (Shope, 1932a, b), one of the pox group of viruses, ean produce tumors in the rabbit. The state of the rabbit at the time of virus injection seems to determine the type of cellular reaction of the host (Duran-Reynals, 1940a, 1945; Hare1 and Constantin, 1954). The reaction is hemorrhagic-destructive in newborn rabbits injected with high doses of virus, proliferative-invasive in baby rabbits 1 Aided by Research Grant CA-05266 from the I\iationai Cancer Institute, United States Public Health Service. 473
474
ISRAELI
AND
SFV in order to determine in what way they may be different or similar to those occurring with the other pox or tumor viruses. This study might lead to a better understanding of the mechanism of tumor induction by viruses. SFV was first cultured in vitro as early as 1933 (Rous et al.), and some aspects of its growth in vivo and in vitro were studied by Hare1 (1956) and Constantin et al. (1956). The virus causesa cytopathic effect in tissue culture (Chaproniere and Andrewes, 1957; Kilham, 1959), and the cell-virus interaction in vitro can lead to pock formation (Padgett et al., 1962; and personal observation). The cytopathogenic properties of SFV were recently used to develop an accurate method for virus titration in vitro (Padgett et al., 1962; Verna and Eylar, 1962, and personal observation). The aim of the present work was to analyze the cell-virus interactions in two different cell systems. One system was from the rabbit, an animal that is susceptible to SFV tumor formation in vivo, and the other from the rat, an animal that so far seemsto be resistant to tumor formation in vivo. Experiments were also carried out on the pocks formed in vitro, with a view to determining whether they are produced by cell proliferation or cell aggregation. MATERIALS
AND
METHODS
Cell Cultures Including Medium and Incubation Cells were obtained from noninbred domestic rabbits and noninbred albino rats. Rabbit embryos were excised on the 17th to 18th day of gestation and rat embryos on the 15th to 17th day. The procedure for preparing primary and secondary cultures was as described by Winocour and Sachs (1960). For primary embryo cultures, 1 X lo7 cells were seededin loo-mm petri dishes. After 5 or 6 days the cultures were trypsinized and 60-n-m petri dishes were seededwith 2.4 X lo6 cellsper plate to make secondary cultures that were used for virus inoculation 48 hours later, when complete monolayers had formed. Rabbit kidney cultures were prepared from kidneys taken from lo-day-old rabbits.
SACHS
The kidneys were trypsinized after decapsulation and removal of the hilus; confluent monolayers, either primary or secondary, which were formed after inoculating 1.6 X 10” cells per 60 mm petri dish, were used for virus inoculation. Testes for cultures were taken from l-month-old rabbits. After removal of the capsule, and trypsinization, 1 X lo6 cells were seeded in 35-mm petri dishes; cultures were used after about 4 days, when monolayers had formed. All cultures were made in Falcon plastic petri dishes. Eagle’s medium with a fourfold concentration of amino acids and vitamins (EM), and 5 % of calf serum was used throughout. The cultures before virus infection were incubated at 37°C. After infection, they were incubated at 35°C (Kilham, 1959). All the incubators contained a constant flow of 10 % COZ in air and a humidified atmosphere. Virus and Virus Stocks SFV (Patuxent strain) was obtained through the courtesy of Dr. L. Kilham. Three successive single-plaque isolations were made on rabbit embryo cultures, and the final isolate was used to initiate the virus stocks for the present experiments. Stocks were obtained by infecting secondary rabbit monolayers at virus: cell ratios of 1:2 or 1: 1 pock-forming units (= plaque forming units) (PFU) per cell. After 4 days of incubation, the cultures were frozen and thawed once, the cells were scraped off the dish with a rubber wedge and sonically vibrated in a Raytheon 200-watt lo-kc magnetostrictive oscillator at 9000 cycles per second for 3 minutes. The cell debris was spun down at 1400 g for 30 minutes, and the supernatant was distributed into tubes and kept at -50°C until use. To test the fibroma-producing properties in vivo, different stock sampleswere injected intracutaneously to either adult or baby rabbits. No lossof fibroma-producing properties was detected during 12 passagesof the virus in rabbit tissue cultures. Pock and Plaque Assays The medium was removed from the secondary cultures; 0.1 ml of virus, appropri-
CELL-VIRUS
INTERACTIONS
ately diluted, was added to the monolayer. After 3 hours of adsorption at 35”C, during which the plates were tilted twice to ensure virus redistribution, the cultures were covered with 5 ml of EM containing 0.9 % agar and 5 % calf serum. For the routine assays the agar overlay on rat cultures contained, in addition, 400 pg/ml of protamine sulfate (Nutritional Biochemical Corporation). Pocks appeared 5 days after infection of rabbit cultures and 7 days after infection of rat cultures. Although they could be counted at that time by illuminating the cultures with an oblique light, it was preferable to add a second overlay of 2.5 ml agar medium which contained 0.01% (w/v) neutral red to facilitate counting. Pock counts were made twice, the first time at 6-7 days after infecting rabbit cultures, and 10 or II days after infecting rat cultures. The second time was 16-21 and 21-2<5 days after infection of rabbit and rat cultures, respectively. This second count proved to be valuable, since some of the pocks appeared or became more pronounced only later, owing to their development into plaques, in the case of rabbit, and enlargement, in the case of rat cultures. Assays for Vim.9Producing Cells IcfectiVe center assay. Cultures containing I to 2 X 105 cells per plate, were infected at virus: cell ratios of 3-22 PFU per cell and after 1 hour’s adsorption they were washed twice with phosphate-buffered saline (PBS). The cells were then suspended with the aid of trypsin, and after centrifugation, were diluted in medium. The cell dilutions were inoculated in amounts of 2 ml on monolayers of rabbit cells. After overnight incubation, the medium was removed, and the cultures were covered with agar medium and further incubated as for pock and plaque assays. Gounts oj cells containing inclusion bodies. Cultures containing 1 to 2 X lo5 cells per plate, were infected at virus:cell ratios of 3-25 PFU per cell. After 1 hour’s adsorption, the cultures were washed twice with PBS and covered with growth medium. After further incubation for 24 hours, the medium was removed; the cultures were washed once with PBS and then fixed with methanol and stained with May-Grunwald-Giemsa. Cells
WITH
SHOFE
FIBROMA
VIRUS
475
were counted for the presence of inclusion bodies, under X 1500 magnification, as a percentage of the total number of cells. Growth Curves Cultures containing 1 to 2 X IO5 cells per plate were infected at virus:cell ratios of l-25 PFU per cell. After 1 hour’s adsorption, the plates were washed twice with PBS and growth medium was added. Two plates were frozen immediately at -20°C for zero time titration. The remaining cultures were incubated further, and at appropriate time intervals, 4 plates were removed, 2 of which were frozen at -20°C for total virus titrations, and 2 others were fixed and stained for counts of cells with inclusion bodies. For free virus titrations, 0.5 ml of medium from each of two plates was pooled, and the supernatent, after spinning down cell debris at 1400 g for 30 minutes, was kept at -20°C. At the end of the experiment the samples were thawed, both plates of each point mere pooled, and all the samples, both for free and total virus, were disrupted by sonic vibration. The cell debris from the tot.al virus samples was then spun down at 1400 g for 30 minutes. Titrations of the virus were made on rat or rabbit embryo monolayers. Counts of Mitotic Figures in Infected and I:12i7zfectedCultures 1.0 X lo6 testes cells and 1.2 X 10” rat and rabbit embryo cells were plated in 35-mm plastic petri dishes to form monolayers. These monolayers were infected with 150, 300, or 500 PFU. Medium (0.1 ml) instead of virus was added to control cultures for the adsorption period. At desired times, samples of 2 or 3 plates from each group were fixed and stained with May-GrunwaldGiemsa. Mitotic figures were counted at X 1500 magnification in almost the entire plate, i.e., an area of about 3.4 cmZfor rabbit monolayers, and in half the plate, i.e., an area of about I.7 cm2for rat monolayers. Feeder Layers Rat embryo secondary culture cells mere used for feeder layers. The cultures were irradiated with 5000 r, and the cells then seededat 8 X IO* cells per 60-mm petri dish.
ISRAELI
476
AXD
Isolation 0.f Clones Clones of rabbit embryo cells were isolated from plates seeded with about 250 cells of either primary or secondary cultures in 60-mm petri dishes containing irradiated rat feeder cells. The cloneswere isolated by trypsinizing the cells in small cylinders placed over the clones; the cells of each clone were then transferred to a plate with feeder cells. When a clone had formed a monolayer, the cells were transferred to 2 petri dishes (without a feeder layer), one of which was later infected, the other serving as a control. RESULTS
CPE and Pock Formation Monolayers of both rabbit and rat embryo cells showed 2 different types of cell-virus interactions, depending on whether the cultures were infected at high or low virus: cell ratios. At virus: cell ratios of 1 PFU per cell or above, there was a general cytopathic effect (CPE). At low virus: cell ratios, such as to assure countable areas of infection, i.e., up to 300 PFU per plate, the first change observed was the formation of pocks, and CPE and cell destruction was found only in later stages. Rat and rabbit cultures differed in the type of pocks and in the nature of the CPE.
SACHS
such infected cultures through succeeding passageseither at 35°C or 37”C, were unsuccessful, and after 2 to 4 passages,almost no cells remained. Infection with about 300 PFU per plate. At low virus:cell ratios, the cultures at 4 days after infection showed areas which consisted of elongated refractile cells. During the following days, bundles of more or lessparallel cells, protruding from the monolayer, were found in these areas, and these will be called ‘Lpocks” (Fig. IA). At later stages, a lytic process took place. It usually began about 6-7 days after infection on one side of the pock and the bundles of cells became less crowded becauseof detachment of dead cells. Finally, holes developed with heaped-up cells at the periphery in place of the pocks (Fig. 1B). This continuing process led to the formation of plaques having dimensions of 24 mm at about the third week after infection in cultures that were covered with agar. Under a fluid medium the entire culture eventually underwent CPE, due to spread of the virus. Medium change or passaging of the cultures strikingly enhanced the CPE. This could be due to the washing out of interferon, as found in vaccinia-infected cultures (Glasgow and Habel, 1962).
Rabbit TestesCultures Since Kilham (1956) described poor propagation of the virus with no CPE in domestic Rabbit Embryo Cultures rabbit testes explants, monolayers of rabbit Virus :cell ratios above 1. When virus: cell testes cells were also infected. It was found ratios above 1 were used, a general change that when these cultures were infected with took place in the culture 2-3 days after in300 PFU, pocks appeared after 4-5 days, fection. At the beginning, cells became more which later gave way to lysis, as in monorefractile, and later on, at about the 4th day, layers of embryo cells. The pocks were differmany of the cells died and detached from ent from those on embryo cultures, being the plate. The cells elongated and the cullarger and showing pronounced random oritures showed areasof thickness reminiscent of entation of the cells. The onset of CPE ocpocks in their structure, but with no definite curred several days later than in embryo boundaries. Side by side with these areas cultures, i.e., about the 7th to 9th day after there were areas of lysis. Cells containing infection. It began on one side of the pock inclusion bodies showed an increase in volperiphery, forming a hole in the monolayer ume, which preceded their lysis. In later near the original intact pock. The CPE stages, clumping of cells occurred here and spread to the center of the pock only a few there, accompanied by cell detachment. This days later. CPE seemed to involve most of the cells from the beginning, but complete destruc- Rat Cultures tion of the culture resulted only about 3 The general CPE which appeared after weeks after infection. Attempts to maintain infection at virus:cell ratios above 1 was
CELL-VIRUS
INTERACTIONS
somewhat similar to that found in rabbit embryo cultures. Many of the cells contained inclusion bodies, although no enlargement of cell volume occurred, and there was less clumping. These cultures could be kept through succeeding passages, when incubated at 37°C. At this temperature they seemed to establish carrier cultures, and counts made during the course of 9 passages indicated that 15 % of the cells had inclusion bodies. The pocks which were formed at low virus:cell ratios, i.e., about 300 PFU per plate, differed from the rabbit pocks. They began to appear 5 days after infection as areas with more refractile cells, many of which contained dark cytoplasmic granules, which were seen only in living cultures. On succeeding days, they consisted of piled-up, rounded cells. Although these cells died later on, as was shown by their inability to take up the neutral red stain, they did not disintegrate, and the pock never developed into a plaque. It enlarged during the first days of its existence and acquired an irregular shape as a result of uneven spreading of the piled-up cells (Fig. lC, D). The periphery of the pock became sparse, owing to lysis of part of the cells. These pocks were usually smaller than the rabbit pocks. In a fluid medium new pocks continued to appear; after about a month most of the cells died awing to confluency of pocks. In this case, as in rabbit cultures, this process was enhanced by changing the medium or by passaging the culture at 35°C. Figure 2 shows photographs of plates with pocks on rat and plaques on rabbit monolayers. The Effect on Pock and Plaque Formation of Protanzine Sulj”ate Inclusion in the Agar It was noticed that when rat embryo cultures inoculated for a pock assay were covered with an agar overlay, pocks appeared later than in similar cultures with a fluid medium. Since this might be due to the presence of inhibitors in the agar, it was of interest to see whether the polysaccharide inhibitor protamine sulfate (Takemoto and Liebhaber, 1961) effected any change. Table 1 summarizes an experiment, carried out to study the effect on pock development of
WITH
SHOPE
FIBROMA
477
VIRUS
varying concentrations of protamine in the overlay. In overlays containing no protamine, the appearance of pocks was delayed for about a week, and their course of development was retarded. The number of pocks under agar with protamine was somewhat higher than under agar without prot’amine. The optimal concentration of protamine was found to be 400 pg/ml, the same as described by Takemoto and Liebhaber (1961). When rabbit embryo monolayers were used, different’ results were obtained. There was no retardation of pock appearance in the presence of the agar inhibitor, but it had an effect on further development of the plaques, delaying the lysis of dead cells in them. In contrast, when rabbit, kidney cu.. tures were infected, the addition of agar arrested the development of pocks at’ their early stages, giving them the shape of undeveloped pocks seen on rat embryo cutures. In this case, when protamine was incorporated into the agar, the pocks developed v&h a shape similar to &at found on rabbit embryo cells, and 5 days after their appearance complete lysis took place, the plaque area becoming a hole, fret from cell debris. Assays
for
Virus-Yielding
Cells
Since SFV produces cytoplasmic inclusion bodies in cells in which it replicates, the presence of inclusion bodies can be used to estimate the proportion of virus-producing cells in a culture. Table 2 shows the results of a comparison between counts of cells with inclusion bodies and infective center assays. The data indicate that the method of inclusion body count gives about a 2% times higher estimate of the proportion of virus-yielding cells. Since the counting of cells with inclusion bodies was more convenient than an infective center assay, it Ivas used in all the subsequent experimenrs. Virus-Yielding Cells as a Functior: sorption Time
~1.iAd-
Cultures containing 1.0 X IO5 rabbit embryo cells, or 1.4 X 105 rat, cells, per dish were infected with 2.2 X 106 PFU of virus and incubated. At l-hour int’ervalsJ 2 plates were washed twice with PI38 and
FIG. 1 478
CELL-VIRUS
FIG. 2. Plaques on rabbit (A) infection. The plates are covered was taken on a white background,
IXTERACTIONS
and pocks with agar and that
WITH
SHOPE
on rat (B) monolayers, containing neutral red. of rat pocks on a black
covered with EM. All the plates were incubated for an additional 24 hours, then washed twice with PBS, fixed, and stained for counts of cells containing inclusion bodies. Figure 3 summarizes the results of such experiments. The data show that the proportion of virus-yielding ceils was higher in rabbit than in rat. but in both cultures it increased as a function of adsorption time. At, the time the experiment was terminated only 50% of the rat cells contained inclusion bodies as compared to 88 % of rabbit cells. A higher percentage of virus yielding cells for rabbit cultures was also found in other experiments.
FIBROMA
VIRUS
479
9 and 13 days, respect,ively, The photograph of rabbit background. Magnification TABLE
after plaques : X 0.9.
1
THE EFFECT ON POCK FORMATIOX ox RAT Ezinn~o MONOLAYERS OF PROTAMINE SCLFATE INCLUSION IN THE AGAR OVERLAY Protamine concentration (dml) 0
200 400
Number of pocks” Days after infection __-.._. 8 IQ 12
-6
i
zt
+
+
75
+
++
85
a Average of 3 to 4 assay plates. b In the first days after their appearance, pocks were very small and difficult to count. number of pocks at these times has therefore designated as - to ++.
21
63 94 106
the The been
Vims Adsorption on Monolayers h’xpressed by Pock and Plaque Counts rat cultures. Pock and plaque assays made One hundred PFU in 0.1 ml were added on rabbit and rat monolayers gave eomparof cultures to a monolayer. At l-hour intervals, groups able results, so that both types can be equally used for virus assay. of 4 plates were covered with an agar overlay and incubated for the appearance of pocks. The results of such experiments are repre- Growth Curves sented in Fig. 4. Xo significant differences Growth curves of WV were made on in adsorption rate on the monolayers were rabbit and rat embryo cells. Figure 5 gives found by this method, between rabbit and representative growth curves after infection
FIG. 1. SFV-infected rabbit and rat monolayers, covered with agar: (B) with neutral red; (A, C, and D) without neutral red. (A) Pock on rabbit embryo monolayer, 6 days after infection. Elongated piledup cells can be seen. Magnification: X 85. (B) Later stage than (A). The pock has developed into a plaque, 10 days after infection. An edge of heaped-up cells can be seen at one side of the plaque. Magnification: X 175. (C) Pock on rat embryo monolayer, 7 days after infection. The pock is composedof piledup rounded cells. Magnification: X 85. (D) The edge of a pock of about the same age as C, shearing elongated refractile cells at the periphery. Magnification: X 175.
480
ISRAELI
AND
SACHS
TABLE ESTIMATION COUNTS
OF THE NUMBER OF CELLS WITH
2
OF VIRUS-YIELDING INCLUSION BODIES
RABBIT AND RAT EMBRYO AND BY INFECTIVE CENTER Virus-yielding
Expt.
no.
Cells
Virus: cell ratioa (PFU:ceil)
Inclusion Total
R47
Rabbit Rat
22 4 16 3
bodies
x x x x
Infective
‘%
10” 104 104 104
BY
cells per plate according count6
number
7.0 7.0 7.1 5.7
CELLS ASSAY
Total
70.0 70.0 44.6 35.4
3.8 1.9 1.4 1.7
to
center
assay”
number
%
104 104 104 104
38.0 19.0 10.0 12.1
x x x x
a Cultures containing 1.0 or 1.4 X lo5 rabbit or rat cells, respectively, were infected with virus centrations of 2.2 X lo6 PFU and 4.4 X lo5 PFU per plate. b At 24 hours after virus infection, the proportion of cells with inclusion bodies was determined counting about 1000 cells in 2 plates, and their total number in the entire plate was then calculated. c The titrations for infective centers were all made on rabbit embryo secondary monolayers.
at virus: cell ratios of 25 PFU per cell for rabbits, and 17 for rats. Figure 6 shows the correlation between total and free virus, after infection at virus:cell ratios of 2 PFU per cell for rabbits and 1 for rats. It can be seen that a considerable amount of virus is cell associated. The rise in virus titer sometimes began after 12 hours (Fig. 5) and at other times after 8 hours (Fig. 6) in rabbit cultures, and after 12, or 15 hours in rat cultures (Figs. 5 and 6). In the growth curves shown in Fig. 5, counts of cells with inclusion bodies were made at intervals during the 24 hours of the experiment. Inclusion bodies were found as soon as 4 hours after infection. The final percentage of cells with inclusion bodies was 90 % in rabbits and 77 % in rats, so that the curves can be regarded as onestep growth curves. The proportion of virusyielding rat cells in this experiment was somewhat higher than that found in other experiments (Table 2). From the results in Fig. 5 the total virus yield per virus-yielding cell can be calculated as 18 PFU per cell for rabbits and 1 PFU per cell for rats. A generally higher virus yield per cell in rabbits was also found in other experiments. In all cases the total virus yield per virusyielding cell was low. The yield per cell in 10 different experiments ranged from 2 to 36 PFU for rabbits, and 1 to 13 PFU for rats. The finding of a well-developed inclusion
conby
100 I
c 2
60-
Y d
50-
0 IL 0 2
./
4o
/ /
z E
30-
a
20-
---o
/---0 /
P
0’
/ O’
IO I;, 2
, 3 ,HOURS
FIG. 3. Percentage of virus-yielding function of adsorption time, estimated ing cells with inclusion bodies. Each average of counts made on 2 plates. cultures; 0, rat cultures.
, 4
I. 5
cells, as a by countpoint is an 0, Rabbit
CELL-VIRUS
ISTERACTIOXS
WITH
100; sot
SHOPE
FIBROMA
I-IRES
481
to a lower yield per cell or are due also to a lower number of virus-yielding cells in t#hc pock is so far unknown.
It was of interest to determine whether pocks are formed after infection of sparse cultures. Sparse layers of rabbit testes and embryo cells containing 2 to 3 X 105 cells per plate were infected with 3 X 1Q5PFIJ or
6
5 I
i
I
L
123456
HOURS FIG. 3. The adsorption of SFV on confluent monolayers. In calculating the percentage PFU adsorbed the average number of PFU at 6 hours was taken as 100%. Each point is an average of counts made on 4 plates. @, Rabbit cultures; 0, rat cultures.
body in cells producing only 1 or 2 PFU suggest,sthe possibility that the sensitivity of the virus assay is very low and/or that a considerable amount of noninfectious virus material is being produced. As a further comparison between the 2 species, the virus titer was determined in single pocks. Pocks were isolated from cultures with agar overlays, with a thin-bore pipette, at 5-7 days after their appearance, i.e., at lo-14 days after virus infection. They were suspendedin growth medium and subjected t,o 3 cycles of freezing and thawing before titration. The results (Table 3) indicate t’hat the titers obtained from rabbit pocks were generally higher than those obtained from rat pocks. Whether the low figures obtained for rat pocks are due only
%18-R % IB-R’
ri
50
67 75 71
90
28
63
77
I 4
76
I 8
12
16
20
24
HOURS FIG. 5. One-step growth curves of SFV on rabbit and rat cultures, infected at virus:ceii ratios of 25 and 17 PFIJ per cell, respectively. Each point is based on a pool of 2 plates. Time zero is after virus adsorption. @, rabbit cultures; X, rat cultures, yi IB - R = percentage of rabbit cells with inclusion bodies. 7; IB - Rt = percentage of rat cells with inclusion bodies.
482
ISRAELI
\ J ,d 12
AND SACHS
I I I 60 $2 96 120 HOURS FIG. 6. Relation between free and total virus in growth curves of SFV on rabbit and rat cultures infected at virus: cell ratios of 2 and 1 PFU per cell, respectively. Each point is based on a pool of 2 plates. 0, rabbit total; 0, rabbit free; A rat total; a, rat free. By July, 858, 4 tabs., 6 figs., Israeli 2
0
1 24
A6
I 46
200 PFU. Although there was a CPE, there was no pock formation in cultures infected at either of these virus inputs, even though confluent cell layers were formed in later stages. There was also no development of pocks when 6 X lo4 testes and 4 X lo4 embryo cells from infected cultures were plated at 1 to 3 X lo3 per plate for cloning. The existence of a monolayer at the time of infection thus seemsto be an essential requirement for pock formation. The Frequency of Mitoses in Injected and Uninfected Cultures Since the results of the above experiments suggested that separate cells do not proliferate to form a pock in vitro, it was of interest to determine whether pocks are formed on monolayers as a result of cell proliferation. Monolayers of rabbit and rat embryo cells were infected with 150 or 300 PFU, and monolayers of rabbit testis cells with 150, 300, or 500 PFU. The number of mitoses was counted at various times after infection in an area of 3.4 cm2for rabbit monolayers and 1.7 cm2 for rat monolayers. In the first experiment with rat cultures there were cells
TABLE VIRUS
YIELD RAT
PER POCK ON RABBIT EMBRYO CULTURES
Cell type
Pock no.
Rabbit
1 2 3 4 5 6 7 1 2 3
Rat
3 AND
PFU per pock 3.2 1.6 1.0 1.3 1.0 8.5 2.7 5.0 2.7 2.2
X X x x x x X x X x
lo3 lo2 103 104 104 103 lo3 101 lo2 102
on the fifth and sixth day after infection with unclear figures, which could have been either in early prophase or in a stage of pycnosis. In the second experiment, therefore, 0.1 pg colchicine per milliliter was added to the cultures for 8 hours prior to fixation. The results (Table 4) show that there was no significant increase in mitotic frequency in rabbit and rat embryo cultures, but that in infected testes cultures there was a significant increase in mitoses which coincided
CELL-YIRT;S
INTERACTIOSS
WITH TABLE
SHOPE
FIBROMA
VIRUS
483
4
NUMBER OF MITOSES IN INFECTED AXD NONINFECTED RABBIT AND RAT MONOLAYEIZS Virus Cell
type
input
(PFU P" plate)
R52BI
(days)
300
3 4 r
R52BII
300
R52BIII
300
s 5 4 5 1 3 4 5 4 5 4 5 4 5 6 5 6
R52C
Rabbit embryo
Time
inf~~~~n
Rabbit testes
150
R52E
300
R70A
500
Rt5A
Rt5Be
Rat embryo
300
300
Kumber Control
of mitoses” Infected
549 429 561 375, 302 177, 182, 200 440, 438 560, 621 785, 937 85, 104 47, 23 52, 33, 27 138, 188, 230 116, 126, 141 141, 189 86, 88 669 591, 762 565 2047 2155, 1976
471 277, 324, 211 262, 278 465, 575, 450 211, 228, 187 303, 341 339, 293 709, 706 80, 134 101, 77 84, 145, 146 432, 411, 527 318, 284, 322 459, 415 251, 368, 320 586 639 + 290" 599 + 219" 2117, 2019 2582, 1890
a Counted in an area of 3.4 cm2 in rabbit and 1.7 cm2 in rat cultures. Each sample. b Calculated according to student’s t test. c Figttres after + are unclear figures, which may be either early prophase d Numbers in parentheses are ratios including the unclear figures. o Colchicine (0.1 pg/ml) was added for 8 hours before fixation.
with the time of pock appearance. In another experiment with rabbit testis cultures, which is not given in Table 4, the CPE appeared, for unknown reasons, earlier than usual (on the 4th to 5th day after infection), and in this experiment there was no difference in the mitotic frequency of the infected and uninfected cultures. This could suggest that the cytopathic process may mask the proliferative reaction. It could then be argued that there is a proliferative reaction in both rabbit testes and embryo cells, but that the increase in mitoses can be observed only in rabbit testes cultures which show a slow CPE, but not in rabbit embryo cultures where the CPE is more rapid. This suggestion, however, could not account for the results with rat. embryo cultures, since with these cells the CPE is even slower than with rabbit testes cells. In this latter case, there-
Ratio of mitoses in infected to mitoses in control
0.86 0.63 0.48 1.47 1.12 0.73 0.53 0.82 i.10 2.53 3.37 2.47 2.41 2.04 3.59 0.88 0.94 1.06 I.00 1.08 value
or first
P valuest’
CO.05 <0.005
represents
a separate
stages
of pyeuosis.
fore, it can be suggestedthat pock formation in v&o may be due to cell aggregation rather than to a virus-induced cell proliferation. In cultures infected with small amounts of virus, no dividing cells were found to contain inclusion bodies. In cultures infected with high dosesof virus about 4 % of the dividing cells contained some atypical inclusions, although further tests are required to be certain that these were true inclusion bodies. Reaction oj Cell Clones to T’irus Pafectiorz In order to determine whether the progeny of individual cells from rabbit embryo were equally susceptible to pock formation, cultures derived from 28 single cell clones were infected as monolayers with 200 PFU per plate. Pocklike structures were formed in only 6 out’ of the 28 clones tested, but these were not like the typical large pocks ap-
484
ISRAELI
AND
pearing on secondary mixed cultures. There were also differences in the time after infection at which a CPE appeared. In 23 of the 28 clones a CPE was observed after 5-7 days, in one after 9 days, and in 4 after 12 days. Twenty of the clones showed a strong CPE on the 12th day, and 8 showed only limited areas of slight CPE at this time. To rule out the possibility of the presence of an interfering virus in the clones, extracts of cells from 2 other clones, one of which gave only CPE and the other neither CPE nor pocks, were prepared by sonic vibration and centrifugation to remove cell debris. These were added to normal secondary cultures, which were then incubated for 6 days before infection with SFV. No interference with pock production was found in these extracts, and the secondary cultures developed the typical SFV pocks. DISCUSSION
The present in vitro experiments with two cell systems, one from rabbits and one from rats, can be divided into the kinetics of virus reproduction, and the analysis of pock formation. The experiments in the first category showed that there was a similar course of virus multiplication in both rabbit and rat cells, the only difference being a lower percentage of virus-yielding cells and yield per cell for rat cells. It should be mentioned, however, that, in other experiments, rat cells were for a period of several months almost completely resistant to SFV infection, perhaps owing to an interfering rat virus. A comparison of SFV to the related vaccinia virus indicates that the CPE appears earlier after vaccinia (Glasgow and Habel, 1962) than after SFV infection. But both viruses seem to have about similar growth curves (Furness and Youngner, 1959; Cairns, 1960), adsorption rates (Youngner, 1956), and proportion of free to total virus (Sheek and Magee, 1961). Vaccinia has also been reported to produce pocklike areas on mouse monolayers and, like SFV, gives a general CPE in cultures infected with a high dose of virus and pocklike areas in cultures infected with low doses of virus (Glasgow and Habel, 1962).
SACHS
In relation to the pocks produced by SFV in v&o, a difference between rabbit and rat cultures has been found in the morphological appearance of the pocks and their development. However, in both species, no pocks were produced after infection of secondary monolayers with a virus: cell ratio of 1 PFU per cell or higher. In further studies with rabbit cultures, infection of sparse layers, cloning of infected cells, or infection of monolayers derived from 28 embryo cell clones also did not result in formation of pocks. These results indicate the following requirements for pock production in vitro: Virus input should be low, the culture should be in the form of a monolayer at the time of infection’, and this monolayer may have to be either composedof a mixed cell population or presumably contains special types of cells that can form pocks. Further cloning experiments are being carried out to clarify whether it is special cell types or mixed cell populations that are required for pock formation. The data on mitotic frequency in infected monolayers that formed pocks indicated that in infected testes cultures there was an increase in mitotic frequency that coincided with the time of pock development. No such increase was observed in infected rabbit or rat embryo cultures. The present results suggest two explanations for pock formation by SFV in vitro: (1) Pocks are formed as a result of cell aggregation in both embryo and testes cultures, but rabbit testes cultures can show in addition a higher mitotic frequency. (2) There is a difference in the mechanism leading to two types of pocks, one type being the result of cell aggregation and the other type the result of a proliferative process. Further experiments are required to distinguish between these possibilities and to determine the relationship between the mechanism of pock formation in vitro and tumor formation in viva. 2 It has further been observed, that pocks were not formed after infection of confluent rabbit embryo secondary monolayers obtained after seeding 2 to 3 X 105 cells, in contrast to the pocks formed after infection of monolayers obtained from the routine seeding of 2.4 X 106 cells.
CELL-VIRUS
INTERACTIONS
WITH
ACKNOWLEDGMENT We to Mr.
are indebted to Mrs. Wilma Montian Eliyahu Nava for technical assistance.
and
REFEREECES CAIRNS, J. (1960). The initiation of vaccinia infection. Virology II, 603623. CHBPRONIERE, I). M., and ANDREWES, C. H. (1957). Cultivation of rabbit myxoma and fibroma viruses in tissues of nonsusceptible hosts. Vi/.ozogy 4, 351-365. Co~sra~~rrs, T., FEBVRE, H., and HAREL, J. (1956). Cycle de multiplication du virus du fibrome de Shope in vitro (Souche OA). Conzpt. Rend. sot. Biol. 150, 347-348. DURAX-REYNALS, F. (1940a). Production of degenerative or neoplastic effect in the newborn rabbit by the Shape tibroma virus. Yule J. Biol. Med. 13? 99-110. Dr-RAX-REIN-&LB, F. (1940b). A hemorrhagic disease occurring in chick inoculated with the Rous and Fujinami viruses. Yale J. Biol. Med. 13, 77-98. DURAX-REYNALS, F. (1945). Immunological fact,ors that influence the neoplastic effects of the rabbit fibroma virus. Cancer Res. 5, 25-39. DUR~N~RE~NALS, F. (1959). Virus induced tumors and the virus theory of cancer. In “The Physiopathology of Cancer” (F. Homburger and W. H. Fishman, eds.), 2nd ed., pp. 298-337. HoeberHarper. Sew York. RUDY, I). TE., STEWART, S. E., KIRSCHSTEIN, R. L., and YOUNG, R. D. (1959). Induction of subcutaneous nodules in rabbits with SE polyoma virus. Nature 184, 7666767. FOGEL, M., and SACHS, 1;. (1960). The in vitro and in lyiz-0 analysis of mammalian tumor viruses. I\,‘. Antibody response and tumor induction with polyoma virus in different species. J. Natl. Cuncer Inst. .%, 839-857. FURNESS, G., and YOUNGNER, J. S. (1959). Onestep growth curves for vaccinia virus in cultures of monkey kidney cells. Viro7ogy 9, 3866395. GLASG~X~, L. A. and HABEL, K. (1962). The role of interferon in vaccinia virus infection in mouse embryo tissue culture. J. Exptl. Med. 115, 503512.
SHOPE
FIBROMA
VIRUS
486
HAREL, J. (1956). Titration et etude du cycle de multiplication in vivo du virus fibromsteux de Shope dans les tumeurs malignes du laperau. Compt. hhd. Sot. Biol, 150, 139-142. HAREL, J., and COXSTANTIN, T. (1954j. Sur ia malignite des tumeurs provoqubes par !e virus fibromateux de Shope chew le lapin nouveau-n6 et le lapin adult,e trait6 par des doses massives de cort,isone. Bull. Cancer 41, 482-497. KII,HAM, L. (1956). Propagation of fibroma virus in tissue culture of cotton tail t,estes. Prcc. Sot. Expll. Biol. Xed. 92, 739%7q2. KILHA~V, L. (1959). Relation of thermoresi&.ree to virulence among fibroma and mysoma viruses. Virology 9, 186~48’7. PADGETT, B. L., MOORE, M. S., and WALICER, I). L. (1962). Plaque assays for myxoma and fibroma viruses and differentiation of the viruses by plaque form. Virology 17, 462-469. Revs, P., MCMASTER, P. I)., and Hrrniics, S, S. (1933). The fixation of certain viruses on the cells of susceptible animals and protection afforded by such cells. Proc. Sot. Exptl. Biol. Med.31,90-91. SHEEK, M. R., ~~~MAGEE, W. E. (1961). An aubo radiographic study on. the intracellular develop ment of vaccinia virus. Virology 13, l-C&163. SHOPE, R. E. (1932a). A transmissible tumor-like condition in rabbits. J. Exptl. &fed. 56, 79HO2. SHOPE, R. E. (1932b). A filtrable virus causing a tumor-like condition in rabbits and its relation ship to virus myxomatosum. J. Ezptl. Xetl. 56, 803-822. Tax~~oro, K. K., and LIEBHABER, N. (EH). Alteration of plaque morphology of EMC virus with polycations. Virology I-i, 502-504. VERXB, J. E., and EYI,~R, 0. R. (1962). Rabbit fibroma virus plaque assay and in vifro studies. Virology 18, 266-273. WINVOCOUR, E., and SACHS, L. (1960). Cell-virus interactions with the poiyoma virus. I. Stud.ie~ on the lytic interactions in the mouse embryo system. Virology 11, 699-721. YOUSGNER, J. S. (1956). \%us adsorption and plaque formation in monolayer cultures of trypsin-dispersed monkey kidney. J. Immund. '76, 28%-292.