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The multiplication of vaccinia virus in suspended KB cells
VIROLOGY
16, 404-416
(1961)
The Multiplication
of Vaccinia
Virus
in Suspended
KB Cells
K. B. EASTERBROOK Department
of Microbiology, The John Curtin School of Medical Australian National University, Canberra, Australia Accepted
Research,
The
August 15,1961
The multiplication of vaccinia virus was investigated in suspensions of KB cells, assays being made by infectivity titrations and by staining cells with fluorescent antibody. Under the conditions described, 5960% of the input virus was absorbed in 30 minutes, free virus was reduced to less than 1% by a single wash, and virus multiplication was restricted to a single cycle. Adsorbed virus became serum resistant, and soon afterward there was a fall of 8090% in the infectivity titer of cell-associated virus. Assay of single cells in the early stages of the growth cycle indicated the existence of a true eclipse phase. Infectivity began to rise between the fifth and seventh hours, and when all infected cells were producing virus the infectivity titer rose logarithmically between the tenth and thirtieth hour. The maximum titer reached was approximately 150 PFU per infected cell and was independent of the multiplicity of infection between m = 1.5 and m = 15 at least. Most of the newly formed infectious virus was not accessible to antibody, a result showing that maturation was an intracellular process and mature virus remained in an intracellular location. The proportion of infected cells could be determined by a fluorescent cell count at the end of the growth cycle, and from this an estimate of the multiplicity of infection could be made. INTRODUCTION
The infection and maintenance of cells in suspension provides a system in which it is possible to analyze virus-cell interaction in cells exposed to uniform and controlled conditions. The growth cycle of vaccinia virus in tissue culture has been followed under a variety of conditions including cells in suspension (Furness and Younger, 1959; Smith and Sharp, 1960). However, no attempt has been made to determine the number of cells contributing, at a given instant, to the total virus yield. This paper describes the infection of suspended KB cells and their maintenance under conditions which permit a determination of the proportion of infected cells present and an estimate of the multiplicity of infection, and allow repeated sam1 Present address : Department of Microbiology, The University of Western $ustralia.
pling of the cell population and its constituent single cells. The succeeding paper (Easterbrook, 1961) describes the use of this system to analyze the effect of sodium azide on the multiplication of vaccinia virus. MATERIALS
AND
METHODS
Virus. The chorioallantoic membrane (CAM) of 11-day-old chick embryos was infected with a suspension prepared from a single pock of the Mill Hill (V-MH, Fenner, 1958) strain of dermal vaccinia virus. Membranes with confluent lesions were extracted with fluorocarbon (Gessler et al., 1956)) and a stock preparation was distributed into ampules and stored at -60”. Before use thawed preparations were dispersed with an ultrasonic drill. Such preparations contained 108.7 PFU per milliliter and 1O9.5well-dispersed single particles per milliliter when examined with the electron microscope.
VACCINIA
IN SUSPENDED
Media. Many nonimmune sera contain substances inhibitory to vaccinia virus. Some of this activity is destroyed by heating at 56” for 30 minutes, but heat-stable inhibitors that reduce the infectivity by approximately 50% are present in many sera. A few specimens of serum found to be almost free from this inhibitory activity were reserved for the preparation of adsorption medium and noninhibitory growth medium. All sera were heated at 56” for 30 minutes before use. Four types of medium were used for different phases of the experiments. 1. Growth medium for growth of cell monolayers and for growth cycle experiments in which extracellular virus was not assayed. This consisted of 15% human serum and 0.5% lactalbumin hydrolyzate in Hanks’ balanced salt solution (BSS). 2. Noninhibitory growth medium. The human serum used in medium 1 (growth medium) contained heat-stable inhibitors which interfered with the assay of extracellular virus. For experiments involving such assays a medium containing 20% selected noninhibitory calf serum in Eagle’s solution (Eagle, 1955a) was used. 3. Adsorption medium. Adsorption of virus and washing of cells was carried out with more dilute noninhibitory calf serum. Adsorption medium consisted of 0.5% calf serum (noninhibitory) in BSS. 4. Maintenance medium. The yeast-extract maintenance medium of Robertson et al. (1955) was used for certain experiments on adsorption and growth in serum-free medium. This consisted of 0.1% Yeastolate (Difco) and 0.25% glucose in Hanks’ BSS. Cells. KB cells (Eagle, 1955b) were grown as monolayers in flat screw-capped bottles. For the production of cell suspensions a 24-hour culture was usually chosen, half confluent and with few cells in the medium. The monolayer was washed briefly with trypsin-Versene solution (0.025% trypsin, 0.01% EDTA) and incubated for 3-5 minutes at 37” with 5 ml per bottle of the same solution. Cells still adhering to the glass were removed by shaking. An equal volume of adsorption medium at room temperature was added, and the cells were dispersed by expulsion through a wide-bore pipette. Aft’er
KB CELLS
405
two washes, with low speed centrifugation (135 g for 2 minutes), the cells were counted in a Neubauer chamber. Cell viability was assayed by ability to exclude trypan blue (0.5% in phosphate-buffered saline), and on this basis most cell suspensions contained about 95% viable cells. Centrifuge tubes used in cell manipulations were treated with a silicone. Infectivity titrations. Virus was titrated on the CAM of 11-day-old embryonated eggs under the conditions described by Westwood et al. (1957). Results were read on the second day after incubation at 36”. Virus dilutions were made in isotonic saline containing 0.5% gelatin. Cell-associated virus (CAV) was assayed after cell disruption and dispersal of the virus particles by one cycle of freezing and thawing followed by 30 seconds’ treatment with an ultrasonic drill. Staining of antigen. Cell samples containing about lo5 cells were washed in isotonic saline to remove traces of serum protein. Smears prepared from these washed cells were dried at room temperature for 1 hour. After fixation in acetone for 15 minutes, the smears were stained by the Coons’ method, using fluorescein isothiocyanate-conjugated rabbit hyperimmune serum, and examined under a Zeiss microscope equipped for fluorescent observations with an OSRAM HBO 200 mercury vapor lamp as light source. A t,otal of 500 cells were counted in each smear. Complement fixation. Suspensions of infected cells (5.1Oj cells per milliliter) were disrupted by treatment for 30 seconds with an ultrasonic drill. After centrifugation to remove cell debris, the amount of antigen was assayed by complement fixation tests with a hyperimmune rabbit antivaccinia serum. The immune serum, at a dilution of 1: 80, and 3 MHD of complement were used in a dropwise test. Fixation was allowed to occur overnight at 4”. The dilution of antigen that fixed 50% of the complement was taken as the titer. Experimental procedure for single-cycle growth curve. One to two million cells in 1 ml of adsorption medium were transferred to a 3 x ‘A-inch tube, which was closed with a silicone-rubber stopper. The appropriate
406
EASTERBROOK TABLE
1
EVIDENCE FOR THE RETENTION OF CELLULAR INTEGRITY BY INFECTED CELLS AND FOR THE PROLONGED ASSOCIATION OF VIRUS WITH SUCK CELLSO
Medium used for incubationa
40% Human serum 15% Human serum 7% Human serum 20% Calf serum Yeast extract maintenance
Cell count (log no. per ml)
Virus infectivity (log PFU per ml) 24 hours after infection
0 hours
24 hours
Free virus
Cell-associated virus
5.0 5.0 5.0 5.3 5.3
4.9 5.0 5.0 5.3 5.3
3.32 4.48 4.40 4.40 4.48
6.85 6.96 6.82 6.40 6.42
CAV Free virus
3.53 2.48 2.42 2.00 1.94
(1See Methods.
virus dilution was added in a volume of 0.05 ml. Adsorption was allowed to proceed for 30 minutes at room temperature, the cells being stirred by a small magnetic stirrer rotating in contact with the bottom of the tube. Following adsorption, the cells were washed three times with 5-ml volumes of adsorption medium (centrifuged at 135 g for 2 minutes), reducing the free virus to a thousandth of that originally added. The washed cells were then diluted to a concentration of lo6 cells per milliliter in 10-20 ml of warmed growth medium contained in glass-stoppered 6 x l-inch tubes (growth tube). These were flushed with 5% COa-air mixture and incubated over magnetic stirrers in a water bath maintained at 37” ” 0 25”
The concentration of lo6 cells per milliliter in the growth tube was chosen for several reasons. (1) At cell concentrations less than 105/ml the suspended cells did not survive satisfactorily and virus yields fell. (2) Single cells could be isolated from such suspensions without concentrating the sample. (3) Smears for fluorescent staining could be made from a convenient volume of the cell suspension. RESULTS
The Prolonged Association of Vaccinia Virus with Infected Cells
Previous workers (e.g., Overman and Tamm, 1957; Joklik and Rodrick, 1959; Furness and Youngner, 1959) have noted that vaccinia virus produced in infected
cells remains associated with them for prolonged periods. A large number of growthcycle experiments showed that this was true in suspended KB cells also. Table 1 shows typical results. This property allowed (1) estimation of the proportion of infected cells by specific staining of smears made 24 hours after infection and (2) calculation of total virus yield of individual cells and populations of cells by determination of the infectivity released into noninhibitory medium by the disruption of washed cells. Adsorption
of Virus
It was found in preliminary experiments that the most suitable medium for the washing of cells and subsequent adsorption of virus was Hanks’ BSS containing a small amount of serum. To determine the efficiency of adsorption of virus to KB cells suspended in this medium, aliquots containing lo6 cells in 1 ml adsorption medium (which contained 0.5% noninhibitory calf serum) were equilibrated for 30 minutes at 37”, 22”, and 4” and then infected with a virus inoculum of 2 PFU per cell. Samples were removed at intervals, washed, resuspended in growth medium at a concentration of lo6 cells per milliliter and incubated at 37” for a total period of 24 hours. An estimate of the efficiency of virus uptake by cells was obtained from counts of fluorescent cells at the end of this time. Maximum uptake was reached by 30 to 40 minutes at 4” and 22” (Table 2). At 37” no significant in-
VACCINIA
IN SUSPENDED
crease was detected after 10 minutes, but counts decreased after 60 minutes, probably owing to adverse pH conditions produced by cells metabolizing at this concentration. Immediately after an adsorption period of 30 minutes at 22” or 37”, only 75% of the virus input could be recovered as infectious virus, whereas in control tubes lacking only cells the whole of the input was recovered. About one-third of the residual infectivity was in the supernatant fluid and the rest firmly bound to the cells. Subsequent experiments suggested that part at least of the missing 25% of the total inoculum had become noninfectious (gone into “eclipse”) in the cells. It can be inferred that the process responsible for this loss in infectivity starts when the virus is added to the cells, and in all growth curve experiments zero time has been taken as the time of addition of virus to the cells. A period of about 1 hour elapsed between zero time and the commencement of incubation at 37”. In all experiments, 98% of the unadsorbed virus was recovered in the first wash. Penetration
407
KB CELLS TABLE
2
THE EFFECT OF TEMPERATURE AND TIME ON THE INFECTION OF SUSPENDED KB CELLS WITH VACCINIA VIRUS’
Adsorption time (minutes) 10 15 30 40 60 80 150
210
Temperature 4”
22”
37”
40 42 70 62 65 65
35 52 63 66 60 64 70 62
60 52 56 60 45 25 12
(1The results, presented as the percentage of infected cells per sample, are derived from several experiments. Aliquots of cells (lo8 cells per milliliter in adsorption medium) were mixed with virus (2 PFU per cell) at 37”, 22”, and 4”. Samples were removed at intervals, washed, and incubated for 24 hours in growth medium. The percentage of infected cells was determined by fluorescent antibody staining.
05 Virus
The penetration of virus was investigated by determining the rate of acquisition of serum resistance. A population of cells was exposed to virus for 30 minutes at 22”, washed, and diluted in growth medium. After increasing times of incubation at 37”, l-ml aliquots were removed and incubated separately in small tubes in the presence of a 1: 30 dilution of specific hyperimmune serum. At the end of 24 hours’ incubation, samples of cells were removed from all the tubes and stained with fluorescent antibody. Virus rapidly became resistant to neutralization (Fig. l), the 50% end point being reached after only 40 minutes’ incubation of the virus-cell complex (Table 3). In the same experiment, the titer of cellassociated virus at various times after infection was also measured. It is apparent from Table 3, in which the rate of acquisition of serum resistance is compared with the loss of infectivity of cell-associated virus, that the former process occurred at a faster rate than the latter. Thus, on the average, a virus particle associated with a
p: I 0
IQ0 100 T!MEOF ADD”ON Of bMIIB”M ,#AwwE~ or IMWATIOH AT 179
4 yx)
FIG. 1. The acquisition of serum resistance by infected cells. Cells were infected and incubated at 37”. At the times indicated, l-ml samples were removed, transferred to separate growth tubes, and incubated in the presence of 1:30 hyperimmune serum. After 24 hours’ incubation, samples were removed from all tubes and stained with fluorescent antibody.
cell first became resistant to antibody and then became noninfectious. Virus Growth
The development of new viral material was followed by three methods: the specific
408
EASTERBROOK
rescence visible on the surface of the cells at the end of adsorption. This persists until, after about 4-5 hours, new virus antigen appears as small foci in a few cells. These foci Acquisition of Period of Eclipsec of CAV increase in size until the cytoplasm of the serum resistance* incubation log VLlIV (minutes) log Vo’/V’ cell is full of fluorescent material. Concurrently with this, the proportion of cells con0 0.056 0.0 taining antigen increases (Fig. 2). As Cairns 30 0.268 0.076 (1960) found with cells in monolayers, the 60 0.377 0.201 curve of antigen production rises much more 0.745 0.377 90 steeply with higher multiplicities. With a 120 0.745 0.569 viral inoculum of 20 PFU per cell, antigen 1.523 180 0.638 was first seen after 3 hours, and by 11 hours (1Cells were infected and incubated in growth all cells were brilliantly fluorescent. The medium. At the times stated, the titer of CAV was maximum proportion of fluorescing cells measured and l-ml aliquots were removed and was reached by 24 hours and counts were incubated separately in the presence of hyperimroutinely made at this time. mune serum. After a total of 24 hours’ incubation, With multiplicities insufficient to infect samples of cells were removed from all tubes and all cells, the proportion of cells containing stained with fluorescent antibody. antigen at 24 hours is related to the virus * V’ = titer of CAV still accessible to antibody input as shown in Fig. 3. The relation becalculated from the proportion of cells shown by tween the function plotted is linear when fluorescent staining to be uninfected following are multiply antibody treatment (proportion negative = emm). few cells in the population c Eclipse used here to describe the process by infect,ed (i.e., with viral inocula 52 PFU which particles become noninfectious. V = titer per cell) but at higher multiplicities deviates of CAV. somewhat from linearity. This result may be partly due to variation in cell size, but the major factor is probably variation in susceptibility. At high multiplicities all viable cells, as judged by the ability to exclude trypan blue, were infected. The presence of sufficient antigen to fix complement was observed in the first sample TABLE
3
ACQUISITION OF SERUM RESISTANCE AND Loss OF INFECTIVITY OF CELL-ASSOCIATED VIRUS~
FIG. 2. Development of antigen in cells infected with input multiplicities of 20 (O), 8 (A), and 2 (X). Cells removed at the indicated times were stained with fluorescent antibody.
staining of antigen in cells by fluorescent antibody, complement-fixation, and the titration of infectious virus. Development of antigen. In a population of cells receiving a virus inoculum of 2 PFU per cell and stained with fluorescent antibody there is an initial faint diffuse fluo-
FIG 3. Relation between the input multiplicity and the proportion of cells containing antigen at 24 hours.
Cells were
mixed
with
varying
concentra-
tions of virus, adsorption was allowed to proceed in adsorption medium for 30 minutes at room temperature, the cells were washed and then incubated in growth medium for 24 hours at 37”. Cell smears were stained with fluorescent antibody.
409
VACCINIA IN SUSPENDED KB CELLS TABLE 4 THE PRODUCTION OF COMPLEMENT-FIXINQ ANTIQEN IN CELLS INFECTED WITH VACCINIA VIRUSQ Time after infection (hours) 1 6 9 12 18 21 30
antigen titer 0 4 8 16 <32 32 32
a Cells were infected and incubated in growth medium. At the times given 5.105 cells were removed, washed, and resuspended in 1 ml gelatin saline. After disruption of the cells and removal of any particulate material by centrifugation, the resulting antigen preparation was diluted serially and titrated for complement-fixing activity with a final dilution of 1:80 antiserum.
tested, at 6 hours after infection. Thereafter the titer rose rapidly to reach a maximum value of 32 at about 20 hours (Table 4). Development of infectious virus. Alterations in the titer of infectious virus were followed by frequent titrations of the virus content of suspended cell cultures up to 48 hours after the end of the absorption period.
Two virus inocula were used: 2 PFU per cell and 20 PFU per cell. The results will be discussed in terms of changes in the infectious virus content of suspended cell populations and of individual cells and in terms of the relation between antigen stained by fluorescent antibody and infectious virus. 1. Infectious vimcs in cell populations. Figure 4 illustrates typical growth curves obtained from populations of cells suspended in growth medium after infection with viral inocula of 20 and 2 PFU per cell. Titration of unadsorbed virus indicated that the adsorbed multiplicities were 15 and 1.5, respectively, but immediately after the start of the incubation only two-thirds of this could be detected by assay on the CAM. At 2-hourly intervals volumes of 1 ml were removed from the growth tubes. Half of this was used for the preparation of smears for fluorescent staining. The other half was spun down and the supernatant discarded; the cells were resuspended in gelatin saline and stored at -60”. At the completion of the experiment all samples were thawed, treated with the ultrasonic drill, and titrated on the CAM. Cell counts were made on alternate samples and remained constant. The curves for viral infectivity are similar and can be divided into three stages: (1)
iL
FIG. 4. Growth cycle of vaccinia in suspended of 48 hours. Cells infected with input multiplicities
KB cells in growth medium of 20 (0) and 2 (A 1.
over a period
410
EASTERBROOK TABLE
5
ANALYSIS OF THE YIELDS OF 20 SINGLE CELLS ISOLATED AT INTERVALS DURING THE GROWTH CYCLE OF VACCIRIA VIRUS IN SUSPENDED KB CELLS
T-
-
-
Virus yield G’FU)
Number of cells with virus yields (PFU/cell)
-
1 7
11 15 19 27 31 35 49 2
7 11 15 19 27 31 49
- ;
0 -
2- --
0 15 2 4 0 0 1 2 0 -
3 5 0 0 1 2 1 0 1 - _-
20 6 6 7 4 2 2
3 2 2 3 3 3
- - ,3 jfi M :6 9 -- - -- -
$8 2 6 5 3 3 2 -
-7
3 1 8 5 1 1 3 2 3 1 5 7 4 3 2 6 4 3 - _-- -- 1 0 1 1 3 1 - -
-
?
g -
1
5
1 -
28 56 70 77 80 80 80
1 1 4 0
-
3 0 2 -
fall in titer, from 0 to 5 hours, (2) rapid increase, from 5 to 9 or 10 hours, and (3) slower logarithmic increase from 10 to 30 hours. On incubation, the titer of cell-associated virus fell until at 4 hours only lO15% of the virus cell-associated at 0 hours could be detected. From the fifth to the ninth hour, with an inoculum of 20 PFU per cell, and the fifth to the tenth hour with 2 PFU per cell, the infectivity titer rose rapidly to reach a level of about 16 PFU per infected cell. Thereafter there was a slower increase to a maximum of about 150 PFU per infected cell by the thirtieth hour. The titer fell slight,ly during the subsequent 18 hours. 2. Infectious virus in single cells. At intervals single cells were isolated from each of the growth tubes used for the experiment described above. Approximately 20 cells were blown into a small drop of gelatin saline under oil and then transferred individually with a micropipette to tubes containing 0.5 ml gelatin saline. The contents of the tubes were frozen, thawed, and sonicated
1 1 -
_-
0 85 90 95 95 95 95 95 95
8 9 0 0.2! i 35 33 40 37 49 57 50 55 76 72 85 89 71 72 -- --0 16 16 22 38 45 48
0 19 21 24 48 53 I .12
100 25 90 80 100 100 95 90 100 0 70 70 65 80 90 90
-
and the entire sample was titrated on five eggs. The results are shown in Table 5. With an inoculum of 20 PFU per cell all nine cells assayed at the end of the adsorption period yielded virus, the average number of PFU per cell being 9. Six hours later only five out of twenty cells were positive, and each of these yielded only one PFU per cell. By the eleventh hour 90% of the cells sampled yielded virus, and this situation persisted throughout the rest of the experiment. There was a gradual increase in the mean virus yield per cell. With an inoculum of 2 PFU per cell no yielders were found at 7 hours, but at 11 hours 70% of cells yielded a mean of 19 PFU per cell, and the proportion remained about the same for the remainder of the experiment, although the yield per cell rose steadily. Virus yields of yielders in samples of single cells were fairly evenly distributed around the mean value. 3. Relation between development of antigen and infectious virus. In the early stages
of the growth cycle there was a divergence
VACCINIA
IN SUSPENDED
between infectivity and the presence of antigen in cells. With m=15, antigen could first be detected after about 3 hours and was present in a large proportion of cells after 5 hours. In contrast, the first increase in titer of cell-associated virus could not be detected until after 5 hours. Similarly, with the lower multiplicity, 15% of cells contained antigen at 5 hours whereas the infectivity was at its lowest level at this time. Under normal conditions, however, the time of first appearance of new virus is obscured by the persistence of residual uneclipsed virus. The production of new virus is probably better indicated by a change in slope of the eclipse curve than by an absolute increase in titer. This has been shown unequivocably by the use of sodium azide (Easterbrook, 1961). Kinetic experiments with azide allowed an accurate assessment to be made of the production of new antigen and new infectious virus and showed that these events could not be separated in time. Fate of the Infecting
Virus Particle
The first stage of the growth cycle is characterized by a fall in titer of cell-associated virus. To obtain more evidence on the fate of the infecting virus particles, a suspension of cells was infected with an inoculum of 9 PFU per cell (adsorbed m = 4.5) and transferred to a growth tube containing noninhibitory growth medium. Infectivity and fluorescence were measured in the usual way, attention being concentrated on the first 7 hours of the growth cycle. The results are shown in Fig. 5. There was a profound fall in cell-associated virus from 4.5 PFU per cell to 0.04 PFU per cell in the first 3 hours of incubation. The initial fall was very rapid, from 4.5 to 1.2 PFU per cell during the hour occupied by manipulations between addition of virus to cells in the adsorption tube and resuspension of cells in the growth tube. There was a rapid rise in infectivity between the fifth and seventh hour. The infectivity of single cells during this early stage of the growth cycle was measured by two different methods, titration of single cells for cell-associated virus and for their ability to initiate a plaque (Table 6).
KB CELLS
411
The latter property was measured by mixing washed cells with chick embryo fibroblasts and allowing the latter to form a monolayer (P. Abel, personal communication) . The percentage of infected cells at 1 and 5 hours was 90, the same as that determined by single-cell titration and fluorescent antibody staining at the end of the growth period. In contrast, the data from Fig. 5 show that at 5 hours cell-associated virus averaged only 0.04 PFU per cell and none of twenty single cells assayed at this time yielded virus. The virus which has initiated infection in the cells has passed through a stage when its infectivity could not be demonstrated. Virus Release
It has been shown previously (Table 1) that virus produced in cells remains associated with them for long periods. The following experiments were carried out to determine the location of infectious cellassociated virus. After 26 hours’ incubation an infected cell suspension was washed and concentrated to 5 X 1Oj cells per milliliter. An aliquot of 1 ml was exposed to hyperimmune serum (final dilution 1:lOO) for 10 minutes at 37”, then diluted 1:lOO in gelatin saline. After disruption of the cells, residual virus was assayed and compared with a second aliquot treated in the same way but with the omission of antiserum. A third aliquot was sonicated before exposure to antiserum. The same procedure was performed on cells incubated for 48 hours after infection. The results (Table 7) show that at both times the greater part of the total infectious virus was not neutralized by antiserum. An attempt was also made to remove infectious virus from the cell surface by the action of trypsin or Versene. After 28 hours’ incubation, 3 X lo5 cells were washed and divided into three aliquots. These (1) were treated with 0.5% trypsin, (2) were treated with 0.02% Versene, or (3) remained untreated, for 30 minutes at 37”. The cells were then washed twice with gelatin saline and frozen. On titration no difference could be
EASTERBROOK
412
ii FIG. 5. Growth cycle of vaccinia in KB cells suspended in noninhibitory period of 10 hours:
TABLE 6
medium over a
several multiplicities of virus, and after adsorption each preparation was divided into two parts. One of each of these was diluted with an equal volume of uninfected cells so that the final concentration of all preparaPercentage of cells tions was lo5 cells per milliliter. All the Time after addition Initiatof virus to cells samples were then incubated for 24 hours. Yielding Containing ing (hours) virus Examination of stained smears made at the antigen plaques end of this period showed that only half as 88 1 many cells in the preparations mixed with 0 5 90 fresh cells were infected as in the controls. 25 7 With multiplicities of about 1, 60-70s of 92 90 30 cells showed fluorescence by the 24th hour and no more fluoresced on prolonged incudetected between cells exposed to trypsin bation. It was possible that cells not infected initially became insusceptible due to or Versene and those not treated. Both experiments indicate that most of their maintenance in suspension. However, the virus produced remains intracellular. if cells were maintained in suspension in a growth tube, at a concentration of lo5 cells Absence of Secondary Infection in the per milliliter for 24 hours, then concentrated Growth Tube to lo6 cell per milliliter in an adsorption It was important to decide whether sec- tube and infected in parallel with a freshly ondary infection occurred under the condi- prepared cell suspension, they were equally tions described. Cells were infected with susceptible to infection (Fig. 61. TITRATION OF CELLS TAKEN AT INTERVALS DURING A GROWTH CYCLE IN NONINHIBITORY MEDIUM
VACCINIA
IN SUSPENDED
413
KB CELLS
TABLE 7 THE INACCESSIBILITY OF CELL-ASSOCIATED VIRUS TO ANTISERUM" Treatment prior to assay
Virus titer (PFU/ml X 104) 26 Hours
48 Hours
virus
in
1. Treated with antiserum 2. Diluted 1: 100 3. Disrupted
64
96
Antibody-resistant virus disrupted suspension
in
1. Disrupted 2. Treated with antiserum 3. Diluted 1:lOO
3
4
77
128
Antibody-resistant suspension
Total virus content of suspension
1. Disrupted 2. Diluted 1: 100
5 Cells were infected and incubated in growth medium. After 26 and 48 hours, aliquots of cells were removed, washed, and treated prior to assay as shown above.
FIG. 6. Effect of prior incubation of KB cells in suspension for 24 hours on the time of production of virus. 0, Cells incubated for 24 hours before infection. & Control.
414
EASTERBROOK
Both types of experiment indicate that (1960) have shown that in monolayers secondary infection in the growth tube does stained with fluorescent antibody the number of cells stained was proportional to the not occur to a significant extent. amount of virus added. Results reported DISCUSSION here demonstrate a similar relation in the The aim in the experiments described in suspended KB cell system (Fig. 3). With this paper was to obtain an over-all picture small viral inputs, the deviation from a 45% of the growth cycle of vaccinia in suspended slope was slight and the virus multiplicity cells by a method which could be used for (m) can be derived from the expression: quantitative studies on the development of proportion positive = 1 - e-*. With larger poxviruses under conditions of metabolic inputs the estimate obtained in this way beand specific inhibition. For such studies it is comesincreasingly inaccurate and such muldesirable (1) that the multiplicity of the tiplicities have to be calculated approxiinfecting virus should be known, (2) that mately from the stained cell counts obtained the growth of virus should be restricted to with fivefold or tenfold dilutions of the a single cycle, (3) that the virus yields of virus, put up in parallel with the more conindividual cells and populations of cells centrated preparation. This deviation from should be easily determined at intervals linearity has been observed in other virusthroughout the growth cycle, (4) that the cell systems (Marcus, 1959), and it has production of antigen by individual cells been possible to isolate clones of cells havcould be compared with their content of in- ing greater and more uniform susceptibility fectious virus, and (5) that the number of to the viruses studied (NDV: Marcus, 1959; cells responsible for the observed yield of poliovirus: Darnell and Sawyer, 1959). infectious virus should be known. The Doubtless similar clones with high uniform method described satisfied these require- susceptibility to vaccinia virus could be isoments reasonably well. lated, and in these, the high multiplicities The removal of unadsorbed virus from used in recombination experiments might cells in suspension is easily and rapidly be determined with reasonable accuracy. achieved, and at the start of incubation Repeated sampling from a single growth more than 99% of the total virus present is tube obviates the need for time replicates. cell associated. Vaccinia virus is very slowly Individual cells can be isolated without released from infected cells, and this situa- difficulty and their virus assayed at any tion is maintained throughout the growth time in the growth cycle. Since vaccinia vicycle. rus remains cell associated for so long, the Counts of the proportion of cells staining virus released on disruption comprises the with fluorescent antibody at the end of the total infective virus produced in the cell growth cycle provides a ready estimate of prior to sampling. the proportion of cells infected by a given During these experiments, several facts inoculum. It gives information on large emerged which are of general interest in numbers of cells otherwise obtainable only, the consideration of the growth cycle of vacafter dilution, by infected cel1 counts or cinia virus. Adsorbed virus rapidly becomes single-cell assay. The estimate is, of course, resistant to the blocking action of antibody accurate only if virus production is limited and the rate of this reaction exceeds the to those cells initially infected. Direct ex- rate of loss of virus infectivity. Thus an inperiments showed that secondary infection fecting virus particle becomes resistant to did not occur to a significant extent, and antibody neutralization before it becomes the failure of colchicine (50 mg/ml) to affect noninfectious. Such a finding provides evithe proportion of cells stained (unpublished dence that vaccinia virus loses its infectivresult) eliminated the possibility that divi- ity as the result of an intracellular reaction sion of infected and uninfected cells oc- rather than as the result of irreversible curred at different rates during the growth binding to the cell surface. Until recently a cycle. group of workers (Maitland and PostlethNoyes and Watson (1955) and Cairns Waite, 1959) insisted that it had not been
415
VACCINIA IN SUSPENDED KB CELLS satisfactorily demonstrated that vaccinia virus underwent eclipse of the type now regarded as characteristic of virus growth. Since then these workers have obtained evidence of a profound fall in infectivity on incubation of a cell-virus complex (Postlethwaite and Maitland, 1960) as have Furness and Youngner (1959) using monkey kidney cells in monolayers and suspension. In the present experiments, evidence was obtained of the type widely accepted as indicative of an eclipse phase, viz. a fall of titer of cell-associated virus from 4.5 to 0.04 PFU per cell and a failure to recover virus from single cells at a time when the infected cell count was 90%. The fall in
titer of infectious virus observed during this phase was variable under conditions conducive to subsequent virus growth. If, however, new virus growth was inhibited by sodium azide, infectivity fell by 99% and sometimes as much as 99.8% (Easterbrook, 1961). This demonstrates that the apparent extent of eclipse is determined by two processes, the loss of infectivity
evidence of such an eclipse is provided by electron microscopic (Higashi, 1959) and
autoradiographic (Cairns, 1960) observations of the development of DNA pools lacking mature virus particles 6 hours after infection. Cairns (1960) has shown that with the same strain of vaccinia (V-MH) added to KB cell monolayers to give a multiplicity of 6, new antigen and new DNA were first detectable 4 hours after infection. With sus-
pended cells infected with an inoculum of 20 PFU per cell and subsequently stained with newly developed anti-
gen was visible 3 hours after infection and very obvious at 5 hours; sufficient antigen to fix complement was present before 6 hours.
New infectious virus could be detected, at very low concentrations, by the sixth hour and the titer of infectious virus then rose rapidly
until
about the tenth hour. By this
time, with high multiplicities, all cells capable of producing
at a similar rate. With the tested limits, (inputs from 2 to 20 PFU per cell) the final average yield per infected cell was the same. The failure of antibody to neutralize more than a small part of the infectious virus present even at 48 hours after infection,
virus were doing so (as
judged by fluorescent antibody staining). From the tenth until about the thirtieth hour the infectivity titer rose logarithmi-
cally, doubling every 6i/ hours. With lower
and
the failure of trypsin or Versene to remove such virus from cells, show that mature vaccinia virus remains in an intracellular position for a considerable time after its production. The maturation of vaccinia is thus distinguished from that of the myxoviruses. In the latter, assembly and maturation occur at, or near, the cell surface and more than 95% of the infectious virus present at any time during the growth cycle can be neutralized by specific antibody (Franklin and Henry, 1960; Rubin et aE.,1957). REFERENCES
of input virus
and the production of new virus. Conclusive
fluorescent antibody,
multiplicities (inoculum of 2 PFU per cell) asynchrony caused a prolonged and more gradual initial rise, but once again when all infected cells had started to produce virus the infectivity titer rose logarithmically
J. (1960). The initiation of vaccinia infec11,603-623. DARNELL, J. E., JR., and SAWTER, T. K. (1959). Variation in plaque-forming ability among parental and clonal strains of HeLa cells. Virology 8,
CAIRNS,
tion. Virology
223-229.
H. (1955a). The specific ammo acid requirements of a human carcinoma cell (strain HeLa) in tissue culture. J. Exptl. Med. 102, 37-
EAGLE,
48.
H. (195513).Propagation in fluid medium of a human epidermoid carcinoma Strain KB. Proc.
EAGLE,
Sot. Exptl.
Biol. Med. 89,36!&-364.
K. B. (1961). Analysis of the early stages of vaccinia infection in KB cells using sodium azide. Virology 15, 417. FENNER, F. (1958). The biological characters of several strains of vaccinia, cowpox and rabbitpox viruses. Virology $502-529. FRANKLIN, R. M., and HENRY, C. (1960). The multiplication of fowl plague virus in tissue cultures of chick embryo cells. Virology 10,406418. FURNESS, G., and YOUNGNER, J. S. (1959). One-step growth curves for vaccinia virus in cultures of monkey kidney cells. Virology 9,386-395.
EASTERBROOK,
GESSLER,
A. E.,
BENDER,
C. E., and
PARKINSON,
M. C. (1956). A new and rapid method for isolating viruses by selective fluorocarbon deproteinization. Trans. N. Y. Acad. Sci. 18, 701-703. HIGASHI, N. (1959). Electron microscopy of viruses
416
EASTERBROOK
in thin sections of cells grown in culture. Progr. Med. Viral. f&43-72. JOKLIK, W. K., and RODRICK, J. McN. (1959). Biochemical studies on vaccinia virus in cultured cells. I. Incorporation of axlenine-&Cl’ into normal and infected cells. Virology 9, 396416. MAITLAND, H. B., and POSTLETHWAITE, R. (1959). Vaccinia virus in HeLa cells. Symposium Sot. Gen. Microbial. gth, pp. 135-199. MARCUS, P. I. (1959). Host-cell interaction of animal viruses. II. Cell-killing particle enumeration : survival curves at low multiplicities. Virology 9, 546563. NOYES, W. F., and WATSON,B. K. (1955). Studies
on the increase of vaccine virus in cultured human cells by means of the fluorescent antibody technique. .I. Exptl. Med. 102,237-242. OVERMAN, J. R., and TAMM, I. (1957). Multiplication of vaccinia virus in the chorioallantoic membrane in vitro. Virology 3,173-184.
POSTLETHWAITE, R., and MAITLAND, H. B. (1960).
The eclipse phase of vaccinia virus growing in chick embryo monolayers and some technical procedures which affect its demonstration. J. Hyg. 58, 133-145. ROBERTSON, H. E., BRIJNNER, K. T., and SWERTON, J. T. (1955). Propagation in vitro of poliomyelitis
viruses. VII. pH change of HeLa cell cultures for assay. Proc. Sot. Exptl. Biol. Med. 88, 119-122. RUBIN, H., FRANKLIN, R. M., and BALUDA, M. (1957). Infection and growth of Newcastle disease virus (NDV) in cultures of chick embryo lung epithelium. Virology 3,587-600. SMITH, K. O., and SHARP, D. G. (1960). Interaction of virus with cells in tissue culture. I. Adsorption on and growth of vaccinia virus in L cells. Virology 11,51%532. WESTWOOD, J. C. N., PHIPPS, P. H., and BOULTER,
E. A. (1957). The titration of vaccinia virus on the chorioallantoic membrane of the developing chick embryo. J. Hyg. 55,123139.
16, 404-416
(1961)
The Multiplication
of Vaccinia
Virus
in Suspended
KB Cells
K. B. EASTERBROOK Department
of Microbiology, The John Curtin School of Medical Australian National University, Canberra, Australia Accepted
Research,
The
August 15,1961
The multiplication of vaccinia virus was investigated in suspensions of KB cells, assays being made by infectivity titrations and by staining cells with fluorescent antibody. Under the conditions described, 5960% of the input virus was absorbed in 30 minutes, free virus was reduced to less than 1% by a single wash, and virus multiplication was restricted to a single cycle. Adsorbed virus became serum resistant, and soon afterward there was a fall of 8090% in the infectivity titer of cell-associated virus. Assay of single cells in the early stages of the growth cycle indicated the existence of a true eclipse phase. Infectivity began to rise between the fifth and seventh hours, and when all infected cells were producing virus the infectivity titer rose logarithmically between the tenth and thirtieth hour. The maximum titer reached was approximately 150 PFU per infected cell and was independent of the multiplicity of infection between m = 1.5 and m = 15 at least. Most of the newly formed infectious virus was not accessible to antibody, a result showing that maturation was an intracellular process and mature virus remained in an intracellular location. The proportion of infected cells could be determined by a fluorescent cell count at the end of the growth cycle, and from this an estimate of the multiplicity of infection could be made. INTRODUCTION
The infection and maintenance of cells in suspension provides a system in which it is possible to analyze virus-cell interaction in cells exposed to uniform and controlled conditions. The growth cycle of vaccinia virus in tissue culture has been followed under a variety of conditions including cells in suspension (Furness and Younger, 1959; Smith and Sharp, 1960). However, no attempt has been made to determine the number of cells contributing, at a given instant, to the total virus yield. This paper describes the infection of suspended KB cells and their maintenance under conditions which permit a determination of the proportion of infected cells present and an estimate of the multiplicity of infection, and allow repeated sam1 Present address : Department of Microbiology, The University of Western $ustralia.
pling of the cell population and its constituent single cells. The succeeding paper (Easterbrook, 1961) describes the use of this system to analyze the effect of sodium azide on the multiplication of vaccinia virus. MATERIALS
AND
METHODS
Virus. The chorioallantoic membrane (CAM) of 11-day-old chick embryos was infected with a suspension prepared from a single pock of the Mill Hill (V-MH, Fenner, 1958) strain of dermal vaccinia virus. Membranes with confluent lesions were extracted with fluorocarbon (Gessler et al., 1956)) and a stock preparation was distributed into ampules and stored at -60”. Before use thawed preparations were dispersed with an ultrasonic drill. Such preparations contained 108.7 PFU per milliliter and 1O9.5well-dispersed single particles per milliliter when examined with the electron microscope.
VACCINIA
IN SUSPENDED
Media. Many nonimmune sera contain substances inhibitory to vaccinia virus. Some of this activity is destroyed by heating at 56” for 30 minutes, but heat-stable inhibitors that reduce the infectivity by approximately 50% are present in many sera. A few specimens of serum found to be almost free from this inhibitory activity were reserved for the preparation of adsorption medium and noninhibitory growth medium. All sera were heated at 56” for 30 minutes before use. Four types of medium were used for different phases of the experiments. 1. Growth medium for growth of cell monolayers and for growth cycle experiments in which extracellular virus was not assayed. This consisted of 15% human serum and 0.5% lactalbumin hydrolyzate in Hanks’ balanced salt solution (BSS). 2. Noninhibitory growth medium. The human serum used in medium 1 (growth medium) contained heat-stable inhibitors which interfered with the assay of extracellular virus. For experiments involving such assays a medium containing 20% selected noninhibitory calf serum in Eagle’s solution (Eagle, 1955a) was used. 3. Adsorption medium. Adsorption of virus and washing of cells was carried out with more dilute noninhibitory calf serum. Adsorption medium consisted of 0.5% calf serum (noninhibitory) in BSS. 4. Maintenance medium. The yeast-extract maintenance medium of Robertson et al. (1955) was used for certain experiments on adsorption and growth in serum-free medium. This consisted of 0.1% Yeastolate (Difco) and 0.25% glucose in Hanks’ BSS. Cells. KB cells (Eagle, 1955b) were grown as monolayers in flat screw-capped bottles. For the production of cell suspensions a 24-hour culture was usually chosen, half confluent and with few cells in the medium. The monolayer was washed briefly with trypsin-Versene solution (0.025% trypsin, 0.01% EDTA) and incubated for 3-5 minutes at 37” with 5 ml per bottle of the same solution. Cells still adhering to the glass were removed by shaking. An equal volume of adsorption medium at room temperature was added, and the cells were dispersed by expulsion through a wide-bore pipette. Aft’er
KB CELLS
405
two washes, with low speed centrifugation (135 g for 2 minutes), the cells were counted in a Neubauer chamber. Cell viability was assayed by ability to exclude trypan blue (0.5% in phosphate-buffered saline), and on this basis most cell suspensions contained about 95% viable cells. Centrifuge tubes used in cell manipulations were treated with a silicone. Infectivity titrations. Virus was titrated on the CAM of 11-day-old embryonated eggs under the conditions described by Westwood et al. (1957). Results were read on the second day after incubation at 36”. Virus dilutions were made in isotonic saline containing 0.5% gelatin. Cell-associated virus (CAV) was assayed after cell disruption and dispersal of the virus particles by one cycle of freezing and thawing followed by 30 seconds’ treatment with an ultrasonic drill. Staining of antigen. Cell samples containing about lo5 cells were washed in isotonic saline to remove traces of serum protein. Smears prepared from these washed cells were dried at room temperature for 1 hour. After fixation in acetone for 15 minutes, the smears were stained by the Coons’ method, using fluorescein isothiocyanate-conjugated rabbit hyperimmune serum, and examined under a Zeiss microscope equipped for fluorescent observations with an OSRAM HBO 200 mercury vapor lamp as light source. A t,otal of 500 cells were counted in each smear. Complement fixation. Suspensions of infected cells (5.1Oj cells per milliliter) were disrupted by treatment for 30 seconds with an ultrasonic drill. After centrifugation to remove cell debris, the amount of antigen was assayed by complement fixation tests with a hyperimmune rabbit antivaccinia serum. The immune serum, at a dilution of 1: 80, and 3 MHD of complement were used in a dropwise test. Fixation was allowed to occur overnight at 4”. The dilution of antigen that fixed 50% of the complement was taken as the titer. Experimental procedure for single-cycle growth curve. One to two million cells in 1 ml of adsorption medium were transferred to a 3 x ‘A-inch tube, which was closed with a silicone-rubber stopper. The appropriate
406
EASTERBROOK TABLE
1
EVIDENCE FOR THE RETENTION OF CELLULAR INTEGRITY BY INFECTED CELLS AND FOR THE PROLONGED ASSOCIATION OF VIRUS WITH SUCK CELLSO
Medium used for incubationa
40% Human serum 15% Human serum 7% Human serum 20% Calf serum Yeast extract maintenance
Cell count (log no. per ml)
Virus infectivity (log PFU per ml) 24 hours after infection
0 hours
24 hours
Free virus
Cell-associated virus
5.0 5.0 5.0 5.3 5.3
4.9 5.0 5.0 5.3 5.3
3.32 4.48 4.40 4.40 4.48
6.85 6.96 6.82 6.40 6.42
CAV Free virus
3.53 2.48 2.42 2.00 1.94
(1See Methods.
virus dilution was added in a volume of 0.05 ml. Adsorption was allowed to proceed for 30 minutes at room temperature, the cells being stirred by a small magnetic stirrer rotating in contact with the bottom of the tube. Following adsorption, the cells were washed three times with 5-ml volumes of adsorption medium (centrifuged at 135 g for 2 minutes), reducing the free virus to a thousandth of that originally added. The washed cells were then diluted to a concentration of lo6 cells per milliliter in 10-20 ml of warmed growth medium contained in glass-stoppered 6 x l-inch tubes (growth tube). These were flushed with 5% COa-air mixture and incubated over magnetic stirrers in a water bath maintained at 37” ” 0 25”
The concentration of lo6 cells per milliliter in the growth tube was chosen for several reasons. (1) At cell concentrations less than 105/ml the suspended cells did not survive satisfactorily and virus yields fell. (2) Single cells could be isolated from such suspensions without concentrating the sample. (3) Smears for fluorescent staining could be made from a convenient volume of the cell suspension. RESULTS
The Prolonged Association of Vaccinia Virus with Infected Cells
Previous workers (e.g., Overman and Tamm, 1957; Joklik and Rodrick, 1959; Furness and Youngner, 1959) have noted that vaccinia virus produced in infected
cells remains associated with them for prolonged periods. A large number of growthcycle experiments showed that this was true in suspended KB cells also. Table 1 shows typical results. This property allowed (1) estimation of the proportion of infected cells by specific staining of smears made 24 hours after infection and (2) calculation of total virus yield of individual cells and populations of cells by determination of the infectivity released into noninhibitory medium by the disruption of washed cells. Adsorption
of Virus
It was found in preliminary experiments that the most suitable medium for the washing of cells and subsequent adsorption of virus was Hanks’ BSS containing a small amount of serum. To determine the efficiency of adsorption of virus to KB cells suspended in this medium, aliquots containing lo6 cells in 1 ml adsorption medium (which contained 0.5% noninhibitory calf serum) were equilibrated for 30 minutes at 37”, 22”, and 4” and then infected with a virus inoculum of 2 PFU per cell. Samples were removed at intervals, washed, resuspended in growth medium at a concentration of lo6 cells per milliliter and incubated at 37” for a total period of 24 hours. An estimate of the efficiency of virus uptake by cells was obtained from counts of fluorescent cells at the end of this time. Maximum uptake was reached by 30 to 40 minutes at 4” and 22” (Table 2). At 37” no significant in-
VACCINIA
IN SUSPENDED
crease was detected after 10 minutes, but counts decreased after 60 minutes, probably owing to adverse pH conditions produced by cells metabolizing at this concentration. Immediately after an adsorption period of 30 minutes at 22” or 37”, only 75% of the virus input could be recovered as infectious virus, whereas in control tubes lacking only cells the whole of the input was recovered. About one-third of the residual infectivity was in the supernatant fluid and the rest firmly bound to the cells. Subsequent experiments suggested that part at least of the missing 25% of the total inoculum had become noninfectious (gone into “eclipse”) in the cells. It can be inferred that the process responsible for this loss in infectivity starts when the virus is added to the cells, and in all growth curve experiments zero time has been taken as the time of addition of virus to the cells. A period of about 1 hour elapsed between zero time and the commencement of incubation at 37”. In all experiments, 98% of the unadsorbed virus was recovered in the first wash. Penetration
407
KB CELLS TABLE
2
THE EFFECT OF TEMPERATURE AND TIME ON THE INFECTION OF SUSPENDED KB CELLS WITH VACCINIA VIRUS’
Adsorption time (minutes) 10 15 30 40 60 80 150
210
Temperature 4”
22”
37”
40 42 70 62 65 65
35 52 63 66 60 64 70 62
60 52 56 60 45 25 12
(1The results, presented as the percentage of infected cells per sample, are derived from several experiments. Aliquots of cells (lo8 cells per milliliter in adsorption medium) were mixed with virus (2 PFU per cell) at 37”, 22”, and 4”. Samples were removed at intervals, washed, and incubated for 24 hours in growth medium. The percentage of infected cells was determined by fluorescent antibody staining.
05 Virus
The penetration of virus was investigated by determining the rate of acquisition of serum resistance. A population of cells was exposed to virus for 30 minutes at 22”, washed, and diluted in growth medium. After increasing times of incubation at 37”, l-ml aliquots were removed and incubated separately in small tubes in the presence of a 1: 30 dilution of specific hyperimmune serum. At the end of 24 hours’ incubation, samples of cells were removed from all the tubes and stained with fluorescent antibody. Virus rapidly became resistant to neutralization (Fig. l), the 50% end point being reached after only 40 minutes’ incubation of the virus-cell complex (Table 3). In the same experiment, the titer of cellassociated virus at various times after infection was also measured. It is apparent from Table 3, in which the rate of acquisition of serum resistance is compared with the loss of infectivity of cell-associated virus, that the former process occurred at a faster rate than the latter. Thus, on the average, a virus particle associated with a
p: I 0
IQ0 100 T!MEOF ADD”ON Of bMIIB”M ,#AwwE~ or IMWATIOH AT 179
4 yx)
FIG. 1. The acquisition of serum resistance by infected cells. Cells were infected and incubated at 37”. At the times indicated, l-ml samples were removed, transferred to separate growth tubes, and incubated in the presence of 1:30 hyperimmune serum. After 24 hours’ incubation, samples were removed from all tubes and stained with fluorescent antibody.
cell first became resistant to antibody and then became noninfectious. Virus Growth
The development of new viral material was followed by three methods: the specific
408
EASTERBROOK
rescence visible on the surface of the cells at the end of adsorption. This persists until, after about 4-5 hours, new virus antigen appears as small foci in a few cells. These foci Acquisition of Period of Eclipsec of CAV increase in size until the cytoplasm of the serum resistance* incubation log VLlIV (minutes) log Vo’/V’ cell is full of fluorescent material. Concurrently with this, the proportion of cells con0 0.056 0.0 taining antigen increases (Fig. 2). As Cairns 30 0.268 0.076 (1960) found with cells in monolayers, the 60 0.377 0.201 curve of antigen production rises much more 0.745 0.377 90 steeply with higher multiplicities. With a 120 0.745 0.569 viral inoculum of 20 PFU per cell, antigen 1.523 180 0.638 was first seen after 3 hours, and by 11 hours (1Cells were infected and incubated in growth all cells were brilliantly fluorescent. The medium. At the times stated, the titer of CAV was maximum proportion of fluorescing cells measured and l-ml aliquots were removed and was reached by 24 hours and counts were incubated separately in the presence of hyperimroutinely made at this time. mune serum. After a total of 24 hours’ incubation, With multiplicities insufficient to infect samples of cells were removed from all tubes and all cells, the proportion of cells containing stained with fluorescent antibody. antigen at 24 hours is related to the virus * V’ = titer of CAV still accessible to antibody input as shown in Fig. 3. The relation becalculated from the proportion of cells shown by tween the function plotted is linear when fluorescent staining to be uninfected following are multiply antibody treatment (proportion negative = emm). few cells in the population c Eclipse used here to describe the process by infect,ed (i.e., with viral inocula 52 PFU which particles become noninfectious. V = titer per cell) but at higher multiplicities deviates of CAV. somewhat from linearity. This result may be partly due to variation in cell size, but the major factor is probably variation in susceptibility. At high multiplicities all viable cells, as judged by the ability to exclude trypan blue, were infected. The presence of sufficient antigen to fix complement was observed in the first sample TABLE
3
ACQUISITION OF SERUM RESISTANCE AND Loss OF INFECTIVITY OF CELL-ASSOCIATED VIRUS~
FIG. 2. Development of antigen in cells infected with input multiplicities of 20 (O), 8 (A), and 2 (X). Cells removed at the indicated times were stained with fluorescent antibody.
staining of antigen in cells by fluorescent antibody, complement-fixation, and the titration of infectious virus. Development of antigen. In a population of cells receiving a virus inoculum of 2 PFU per cell and stained with fluorescent antibody there is an initial faint diffuse fluo-
FIG 3. Relation between the input multiplicity and the proportion of cells containing antigen at 24 hours.
Cells were
mixed
with
varying
concentra-
tions of virus, adsorption was allowed to proceed in adsorption medium for 30 minutes at room temperature, the cells were washed and then incubated in growth medium for 24 hours at 37”. Cell smears were stained with fluorescent antibody.
409
VACCINIA IN SUSPENDED KB CELLS TABLE 4 THE PRODUCTION OF COMPLEMENT-FIXINQ ANTIQEN IN CELLS INFECTED WITH VACCINIA VIRUSQ Time after infection (hours) 1 6 9 12 18 21 30
antigen titer 0 4 8 16 <32 32 32
a Cells were infected and incubated in growth medium. At the times given 5.105 cells were removed, washed, and resuspended in 1 ml gelatin saline. After disruption of the cells and removal of any particulate material by centrifugation, the resulting antigen preparation was diluted serially and titrated for complement-fixing activity with a final dilution of 1:80 antiserum.
tested, at 6 hours after infection. Thereafter the titer rose rapidly to reach a maximum value of 32 at about 20 hours (Table 4). Development of infectious virus. Alterations in the titer of infectious virus were followed by frequent titrations of the virus content of suspended cell cultures up to 48 hours after the end of the absorption period.
Two virus inocula were used: 2 PFU per cell and 20 PFU per cell. The results will be discussed in terms of changes in the infectious virus content of suspended cell populations and of individual cells and in terms of the relation between antigen stained by fluorescent antibody and infectious virus. 1. Infectious vimcs in cell populations. Figure 4 illustrates typical growth curves obtained from populations of cells suspended in growth medium after infection with viral inocula of 20 and 2 PFU per cell. Titration of unadsorbed virus indicated that the adsorbed multiplicities were 15 and 1.5, respectively, but immediately after the start of the incubation only two-thirds of this could be detected by assay on the CAM. At 2-hourly intervals volumes of 1 ml were removed from the growth tubes. Half of this was used for the preparation of smears for fluorescent staining. The other half was spun down and the supernatant discarded; the cells were resuspended in gelatin saline and stored at -60”. At the completion of the experiment all samples were thawed, treated with the ultrasonic drill, and titrated on the CAM. Cell counts were made on alternate samples and remained constant. The curves for viral infectivity are similar and can be divided into three stages: (1)
iL
FIG. 4. Growth cycle of vaccinia in suspended of 48 hours. Cells infected with input multiplicities
KB cells in growth medium of 20 (0) and 2 (A 1.
over a period
410
EASTERBROOK TABLE
5
ANALYSIS OF THE YIELDS OF 20 SINGLE CELLS ISOLATED AT INTERVALS DURING THE GROWTH CYCLE OF VACCIRIA VIRUS IN SUSPENDED KB CELLS
T-
-
-
Virus yield G’FU)
Number of cells with virus yields (PFU/cell)
-
1 7
11 15 19 27 31 35 49 2
7 11 15 19 27 31 49
- ;
0 -
2- --
0 15 2 4 0 0 1 2 0 -
3 5 0 0 1 2 1 0 1 - _-
20 6 6 7 4 2 2
3 2 2 3 3 3
- - ,3 jfi M :6 9 -- - -- -
$8 2 6 5 3 3 2 -
-7
3 1 8 5 1 1 3 2 3 1 5 7 4 3 2 6 4 3 - _-- -- 1 0 1 1 3 1 - -
-
?
g -
1
5
1 -
28 56 70 77 80 80 80
1 1 4 0
-
3 0 2 -
fall in titer, from 0 to 5 hours, (2) rapid increase, from 5 to 9 or 10 hours, and (3) slower logarithmic increase from 10 to 30 hours. On incubation, the titer of cell-associated virus fell until at 4 hours only lO15% of the virus cell-associated at 0 hours could be detected. From the fifth to the ninth hour, with an inoculum of 20 PFU per cell, and the fifth to the tenth hour with 2 PFU per cell, the infectivity titer rose rapidly to reach a level of about 16 PFU per infected cell. Thereafter there was a slower increase to a maximum of about 150 PFU per infected cell by the thirtieth hour. The titer fell slight,ly during the subsequent 18 hours. 2. Infectious virus in single cells. At intervals single cells were isolated from each of the growth tubes used for the experiment described above. Approximately 20 cells were blown into a small drop of gelatin saline under oil and then transferred individually with a micropipette to tubes containing 0.5 ml gelatin saline. The contents of the tubes were frozen, thawed, and sonicated
1 1 -
_-
0 85 90 95 95 95 95 95 95
8 9 0 0.2! i 35 33 40 37 49 57 50 55 76 72 85 89 71 72 -- --0 16 16 22 38 45 48
0 19 21 24 48 53 I .12
100 25 90 80 100 100 95 90 100 0 70 70 65 80 90 90
-
and the entire sample was titrated on five eggs. The results are shown in Table 5. With an inoculum of 20 PFU per cell all nine cells assayed at the end of the adsorption period yielded virus, the average number of PFU per cell being 9. Six hours later only five out of twenty cells were positive, and each of these yielded only one PFU per cell. By the eleventh hour 90% of the cells sampled yielded virus, and this situation persisted throughout the rest of the experiment. There was a gradual increase in the mean virus yield per cell. With an inoculum of 2 PFU per cell no yielders were found at 7 hours, but at 11 hours 70% of cells yielded a mean of 19 PFU per cell, and the proportion remained about the same for the remainder of the experiment, although the yield per cell rose steadily. Virus yields of yielders in samples of single cells were fairly evenly distributed around the mean value. 3. Relation between development of antigen and infectious virus. In the early stages
of the growth cycle there was a divergence
VACCINIA
IN SUSPENDED
between infectivity and the presence of antigen in cells. With m=15, antigen could first be detected after about 3 hours and was present in a large proportion of cells after 5 hours. In contrast, the first increase in titer of cell-associated virus could not be detected until after 5 hours. Similarly, with the lower multiplicity, 15% of cells contained antigen at 5 hours whereas the infectivity was at its lowest level at this time. Under normal conditions, however, the time of first appearance of new virus is obscured by the persistence of residual uneclipsed virus. The production of new virus is probably better indicated by a change in slope of the eclipse curve than by an absolute increase in titer. This has been shown unequivocably by the use of sodium azide (Easterbrook, 1961). Kinetic experiments with azide allowed an accurate assessment to be made of the production of new antigen and new infectious virus and showed that these events could not be separated in time. Fate of the Infecting
Virus Particle
The first stage of the growth cycle is characterized by a fall in titer of cell-associated virus. To obtain more evidence on the fate of the infecting virus particles, a suspension of cells was infected with an inoculum of 9 PFU per cell (adsorbed m = 4.5) and transferred to a growth tube containing noninhibitory growth medium. Infectivity and fluorescence were measured in the usual way, attention being concentrated on the first 7 hours of the growth cycle. The results are shown in Fig. 5. There was a profound fall in cell-associated virus from 4.5 PFU per cell to 0.04 PFU per cell in the first 3 hours of incubation. The initial fall was very rapid, from 4.5 to 1.2 PFU per cell during the hour occupied by manipulations between addition of virus to cells in the adsorption tube and resuspension of cells in the growth tube. There was a rapid rise in infectivity between the fifth and seventh hour. The infectivity of single cells during this early stage of the growth cycle was measured by two different methods, titration of single cells for cell-associated virus and for their ability to initiate a plaque (Table 6).
KB CELLS
411
The latter property was measured by mixing washed cells with chick embryo fibroblasts and allowing the latter to form a monolayer (P. Abel, personal communication) . The percentage of infected cells at 1 and 5 hours was 90, the same as that determined by single-cell titration and fluorescent antibody staining at the end of the growth period. In contrast, the data from Fig. 5 show that at 5 hours cell-associated virus averaged only 0.04 PFU per cell and none of twenty single cells assayed at this time yielded virus. The virus which has initiated infection in the cells has passed through a stage when its infectivity could not be demonstrated. Virus Release
It has been shown previously (Table 1) that virus produced in cells remains associated with them for long periods. The following experiments were carried out to determine the location of infectious cellassociated virus. After 26 hours’ incubation an infected cell suspension was washed and concentrated to 5 X 1Oj cells per milliliter. An aliquot of 1 ml was exposed to hyperimmune serum (final dilution 1:lOO) for 10 minutes at 37”, then diluted 1:lOO in gelatin saline. After disruption of the cells, residual virus was assayed and compared with a second aliquot treated in the same way but with the omission of antiserum. A third aliquot was sonicated before exposure to antiserum. The same procedure was performed on cells incubated for 48 hours after infection. The results (Table 7) show that at both times the greater part of the total infectious virus was not neutralized by antiserum. An attempt was also made to remove infectious virus from the cell surface by the action of trypsin or Versene. After 28 hours’ incubation, 3 X lo5 cells were washed and divided into three aliquots. These (1) were treated with 0.5% trypsin, (2) were treated with 0.02% Versene, or (3) remained untreated, for 30 minutes at 37”. The cells were then washed twice with gelatin saline and frozen. On titration no difference could be
EASTERBROOK
412
ii FIG. 5. Growth cycle of vaccinia in KB cells suspended in noninhibitory period of 10 hours:
TABLE 6
medium over a
several multiplicities of virus, and after adsorption each preparation was divided into two parts. One of each of these was diluted with an equal volume of uninfected cells so that the final concentration of all preparaPercentage of cells tions was lo5 cells per milliliter. All the Time after addition Initiatof virus to cells samples were then incubated for 24 hours. Yielding Containing ing (hours) virus Examination of stained smears made at the antigen plaques end of this period showed that only half as 88 1 many cells in the preparations mixed with 0 5 90 fresh cells were infected as in the controls. 25 7 With multiplicities of about 1, 60-70s of 92 90 30 cells showed fluorescence by the 24th hour and no more fluoresced on prolonged incudetected between cells exposed to trypsin bation. It was possible that cells not infected initially became insusceptible due to or Versene and those not treated. Both experiments indicate that most of their maintenance in suspension. However, the virus produced remains intracellular. if cells were maintained in suspension in a growth tube, at a concentration of lo5 cells Absence of Secondary Infection in the per milliliter for 24 hours, then concentrated Growth Tube to lo6 cell per milliliter in an adsorption It was important to decide whether sec- tube and infected in parallel with a freshly ondary infection occurred under the condi- prepared cell suspension, they were equally tions described. Cells were infected with susceptible to infection (Fig. 61. TITRATION OF CELLS TAKEN AT INTERVALS DURING A GROWTH CYCLE IN NONINHIBITORY MEDIUM
VACCINIA
IN SUSPENDED
413
KB CELLS
TABLE 7 THE INACCESSIBILITY OF CELL-ASSOCIATED VIRUS TO ANTISERUM" Treatment prior to assay
Virus titer (PFU/ml X 104) 26 Hours
48 Hours
virus
in
1. Treated with antiserum 2. Diluted 1: 100 3. Disrupted
64
96
Antibody-resistant virus disrupted suspension
in
1. Disrupted 2. Treated with antiserum 3. Diluted 1:lOO
3
4
77
128
Antibody-resistant suspension
Total virus content of suspension
1. Disrupted 2. Diluted 1: 100
5 Cells were infected and incubated in growth medium. After 26 and 48 hours, aliquots of cells were removed, washed, and treated prior to assay as shown above.
FIG. 6. Effect of prior incubation of KB cells in suspension for 24 hours on the time of production of virus. 0, Cells incubated for 24 hours before infection. & Control.
414
EASTERBROOK
Both types of experiment indicate that (1960) have shown that in monolayers secondary infection in the growth tube does stained with fluorescent antibody the number of cells stained was proportional to the not occur to a significant extent. amount of virus added. Results reported DISCUSSION here demonstrate a similar relation in the The aim in the experiments described in suspended KB cell system (Fig. 3). With this paper was to obtain an over-all picture small viral inputs, the deviation from a 45% of the growth cycle of vaccinia in suspended slope was slight and the virus multiplicity cells by a method which could be used for (m) can be derived from the expression: quantitative studies on the development of proportion positive = 1 - e-*. With larger poxviruses under conditions of metabolic inputs the estimate obtained in this way beand specific inhibition. For such studies it is comesincreasingly inaccurate and such muldesirable (1) that the multiplicity of the tiplicities have to be calculated approxiinfecting virus should be known, (2) that mately from the stained cell counts obtained the growth of virus should be restricted to with fivefold or tenfold dilutions of the a single cycle, (3) that the virus yields of virus, put up in parallel with the more conindividual cells and populations of cells centrated preparation. This deviation from should be easily determined at intervals linearity has been observed in other virusthroughout the growth cycle, (4) that the cell systems (Marcus, 1959), and it has production of antigen by individual cells been possible to isolate clones of cells havcould be compared with their content of in- ing greater and more uniform susceptibility fectious virus, and (5) that the number of to the viruses studied (NDV: Marcus, 1959; cells responsible for the observed yield of poliovirus: Darnell and Sawyer, 1959). infectious virus should be known. The Doubtless similar clones with high uniform method described satisfied these require- susceptibility to vaccinia virus could be isoments reasonably well. lated, and in these, the high multiplicities The removal of unadsorbed virus from used in recombination experiments might cells in suspension is easily and rapidly be determined with reasonable accuracy. achieved, and at the start of incubation Repeated sampling from a single growth more than 99% of the total virus present is tube obviates the need for time replicates. cell associated. Vaccinia virus is very slowly Individual cells can be isolated without released from infected cells, and this situa- difficulty and their virus assayed at any tion is maintained throughout the growth time in the growth cycle. Since vaccinia vicycle. rus remains cell associated for so long, the Counts of the proportion of cells staining virus released on disruption comprises the with fluorescent antibody at the end of the total infective virus produced in the cell growth cycle provides a ready estimate of prior to sampling. the proportion of cells infected by a given During these experiments, several facts inoculum. It gives information on large emerged which are of general interest in numbers of cells otherwise obtainable only, the consideration of the growth cycle of vacafter dilution, by infected cel1 counts or cinia virus. Adsorbed virus rapidly becomes single-cell assay. The estimate is, of course, resistant to the blocking action of antibody accurate only if virus production is limited and the rate of this reaction exceeds the to those cells initially infected. Direct ex- rate of loss of virus infectivity. Thus an inperiments showed that secondary infection fecting virus particle becomes resistant to did not occur to a significant extent, and antibody neutralization before it becomes the failure of colchicine (50 mg/ml) to affect noninfectious. Such a finding provides evithe proportion of cells stained (unpublished dence that vaccinia virus loses its infectivresult) eliminated the possibility that divi- ity as the result of an intracellular reaction sion of infected and uninfected cells oc- rather than as the result of irreversible curred at different rates during the growth binding to the cell surface. Until recently a cycle. group of workers (Maitland and PostlethNoyes and Watson (1955) and Cairns Waite, 1959) insisted that it had not been
415
VACCINIA IN SUSPENDED KB CELLS satisfactorily demonstrated that vaccinia virus underwent eclipse of the type now regarded as characteristic of virus growth. Since then these workers have obtained evidence of a profound fall in infectivity on incubation of a cell-virus complex (Postlethwaite and Maitland, 1960) as have Furness and Youngner (1959) using monkey kidney cells in monolayers and suspension. In the present experiments, evidence was obtained of the type widely accepted as indicative of an eclipse phase, viz. a fall of titer of cell-associated virus from 4.5 to 0.04 PFU per cell and a failure to recover virus from single cells at a time when the infected cell count was 90%. The fall in
titer of infectious virus observed during this phase was variable under conditions conducive to subsequent virus growth. If, however, new virus growth was inhibited by sodium azide, infectivity fell by 99% and sometimes as much as 99.8% (Easterbrook, 1961). This demonstrates that the apparent extent of eclipse is determined by two processes, the loss of infectivity
evidence of such an eclipse is provided by electron microscopic (Higashi, 1959) and
autoradiographic (Cairns, 1960) observations of the development of DNA pools lacking mature virus particles 6 hours after infection. Cairns (1960) has shown that with the same strain of vaccinia (V-MH) added to KB cell monolayers to give a multiplicity of 6, new antigen and new DNA were first detectable 4 hours after infection. With sus-
pended cells infected with an inoculum of 20 PFU per cell and subsequently stained with newly developed anti-
gen was visible 3 hours after infection and very obvious at 5 hours; sufficient antigen to fix complement was present before 6 hours.
New infectious virus could be detected, at very low concentrations, by the sixth hour and the titer of infectious virus then rose rapidly
until
about the tenth hour. By this
time, with high multiplicities, all cells capable of producing
at a similar rate. With the tested limits, (inputs from 2 to 20 PFU per cell) the final average yield per infected cell was the same. The failure of antibody to neutralize more than a small part of the infectious virus present even at 48 hours after infection,
virus were doing so (as
judged by fluorescent antibody staining). From the tenth until about the thirtieth hour the infectivity titer rose logarithmi-
cally, doubling every 6i/ hours. With lower
and
the failure of trypsin or Versene to remove such virus from cells, show that mature vaccinia virus remains in an intracellular position for a considerable time after its production. The maturation of vaccinia is thus distinguished from that of the myxoviruses. In the latter, assembly and maturation occur at, or near, the cell surface and more than 95% of the infectious virus present at any time during the growth cycle can be neutralized by specific antibody (Franklin and Henry, 1960; Rubin et aE.,1957). REFERENCES
of input virus
and the production of new virus. Conclusive
fluorescent antibody,
multiplicities (inoculum of 2 PFU per cell) asynchrony caused a prolonged and more gradual initial rise, but once again when all infected cells had started to produce virus the infectivity titer rose logarithmically
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