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The incorporation of neutral red and acridine orange into developing poliovirus particles making them photosensitive
VIROLOGY
14, 11-21 (1961)
The Incorporation of Neutral Red and Developing Poliovirus Particles Making DEREK Department
CROWTHER2
of Virology
AND
and Epidemiology, Houston Accepted
JOSEPH
Acridine Orange into Them Photosensitive’ L. MELNICK
Baylor University College of Medicine, 25, Texas
January
3, 1961
This study concerns the effects of neutral red, acridine orange, and light on the growth of virulent (Mahoney) and attenuated (LSc) polioviruses in monkey kidney cell cultures. Mature, infective virus incubated with the dyes for 1 hour in the absence of cells and then exposed to white light showed no reduction in titer. Virus grown in cells containing neutral red or acridine orange may be subsequently inactivated by light, thus indicating an incorporation of the dye into the developing virus. The effect of neutral red and light was the same for the virulent and the attenuated strains. The biological and chemical similarities between the quinonimide dyes (neutral red, toluidine blue) and the acridines (acridine orange, proflavine) are discussed. INTRODUCTION
cell monolayers Melnick, 1955).
In the presence of the vital dye neutral red, virus plaques may be reduced in size and number, but to a variable degree (Darnell et al., 1958; McClain and Hackett, 1958; Waterson, 1959; Green and Opton, 1959). Work described in this paper was designed to elucidate the part played by light in the growth of poliovirus in cells containing neutral red and also to determine whether cells treated with neutral red and light might selectively favor the growth of virulent as against attenuated poliovirus. MATERIALS
in bottles
(Hsiung
and
Application of light. Cell monolayers were exposed under 20-watt “daylight” type fluorescent lighting tubes in a 37” incubator. Three lighting tubes were attached to the shelf above so that the monolayers were approximately 8-9 inches from the light source. Careful arrangement of the cell layers under the light insured that all the cells were approximately the same distance from the light. The tubes were placed under the light on wooden wedges so that the tubes were inclined at 5”. A careful check on the temperature showed that this did not vary more than 0.5” from the reading obtained in the incubator above the light. An attempt was made to keep the developing virus and the harvested virus in the dark as much as possible when the light was not being applied. Thus, the experiments were performed and titrated in subdued lighting, and aluminum foil was used to envelop the tissue culture racks to prevent any extraneous light from reaching the cells. (Yell counting. The cells in each tube culture were washed and incubated for approxi-
AND METHODS
Monkey kidney cultures were grown and maintained in lactalbumin medium as described by Melnick (1956). Viral assays were performed by the plaque technique on ‘This study was aided by a grant from The National Foundation, and by a training grant (23-74) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service. ‘On leave from the University of Cambridge, England, as a Predoctoral Trainee in Virology, 1959-1960. 11
12
CROWTHER AND MELNICK
mately 20 minutes with 1 ml Versene solution (Melnick, 1956). For counting the total number of cells, 0.1% crystal violet in 0.1 M aqueous citric acid was used. For counting live cells, 1.0 ml of a 0.01% solution of Janus green B was added to 1 ml of cell suspension and the mixture was incubated at 37” for 30 minutes. Dead cells were counted by mixing 1 ml of a 1.0% trypan blue solution with 1 ml of cell suspension. The cell count was then done immediately because trypan blue is toxic to cells. Cells unstained by trypan blue can also be counted and used to indicate the percentage of live cells. Neutral red. The neutral red which was added to the cells for conditioning was added to the agar in the case of the bottles. When used in tubes, the dye was added to the maintenance medium. In the tube experiments, 2-3 hours were allowed for the neutral red to be taken in by the cells before any light was applied. Viruses. The virulent strains of poliovirus were those in use for preparing inactivated poliovaccine (Mahoney, MEF-1, and Saukett) ; the attenuated strains were those in use for preparing live poliovaccine (LSc, P712, and Leon). In the 8-hour yield experiments, 105a5 PFU in 1 ml were added to each tube culture, allowed to absorb for 1 hour, washed off with warm medium (three or four times with 4 ml fluid), and allowed to incubate in 1 ml of maintenance medium for 8 hours before harvesting. Harvesting. Each tube was subjected to two cycles of freezing (-20”) and thawing. After the second thawing, 5 tubes at each variable were combined and centrifuged (for 30 minutes at 2000 rpm) to remove cell debris; the supernates were stored at -20” for later titration by the plaque technique. Acridine orange staining for nucleic acids. Cultures were grown on coverslips in flatsided (Leighton) tubes for these experiments. At the intervals indicated below, they were transferred quickly to Carnoy’s fluid (ethanol-chloroform-acetic acid) at room temperature for a fixation period of 5 minutes. After this they were passed into
absolute ethanol, hydrated through an ethanol-water series, placed for 5 minutes in a pH 4 citric acid-phosphate buffer, and stained for 5 minutes with 0.014% acridine orange made up in the same buffer. After rinsing for 3 minutes in the buffer, the coverslips were blotted gently and mounted on microscope slides, using the pH 4 buffer as mounting medium. RNA digestion. Carnoy-fixed coverslips of infected and control cultures were incubated with 0.05% ribonuclease (Worthington, 5~ recrystallized) at pH 4 for 1 hour at 37”. Fluorescent antibody. Antipolio globulin labeled with fluorescein isothiocyanate was used as described in another paper from this laboratory (Mayor, 1961). As controls, normal cells were treated with the labeled globulin, and infected cells were treated first with unlabeled blocking antiserum and then with the labeled globulin3 RESULTS a. Determination of Amounts of Neutral Red and Light Tolerated by Cells Photosensitivity to white light of cells containing small concentrations of neutral red was readily demonstrated, as has been described by Klein and Goodgal (1959). Cells exposed to light and neutral red in sufficient amounts became enlarged and globoid. Some cells became four to five times larger in diameter than normal monkey kidney cells. The dye was seen in the cytoplasm in small discrete areas rather similar to those seen after vital staining with acridine orange (Mayor, 1961). The over-all picture of a gradual transformation to round refractile cells was similar to a viral cytopathic effect. The possibility that such changes were induced by a virus which had been activated by the treatment was considered, but no agent could be isolated. Similar changes in cells in vivo have been described by pathologists and may be induced by a variety of causes, e.g., radiation, and poisons such as carbon tetrachloride. 3 We wish to acknowledge Dr. Heather Donald Mayor’s participation in these experiments.
POLIOVIRUS
INCORPORATION
In the tube experiments to be described, values for neutral red and light were chosen that gave no degeneration of the monolayer after 1 week of incubation (1:240,000 neutral red plus 1 hour of light). For monolayers under agar containing 1: 60,000 neutral red, 15 minutes of exposure to white light applied directly to the cell sheet through the glass could be tolerated without producing any subsequent disruption of the cell sheet. For controls, half the treated bottle was covered with tape and in addition separate control bottles with untreated cells were used. b. Efficiency of Plating Agar plus Light
under Neutral
Red
Experiments were carried out to determine the degree to which monolayers treated under the conditions of the preceding paragraph supported plaque production by virulent and attenuated polioviruses. Bottle cultures were inoculated with approximately 20 plaque-forming units (PFU) of virus, allowed to absorb for 1 hour, overlaid, and then exposed to light for 5, 10, and 15 minutes. Three bottles were used for each variable. There was no significant difference as a result of exposing the bottles at any time within 24 hours after overlaying. In each set of bottles there was a reduction in plaque size and number, which was most pronounced in those bottles exposed for 15 minutes. In the exposed series the plaques were not only smaller and less numerous, but also hazier and more difficult to count than those in the control series. The differences between the control and treated cultures were most pronounced when read on the second day and progressively became less with longer incubation time, as shown in Table 1. The final plaque counts showed a similar reduction for both LSc and Mahoney-approximately 50%. As the plaques produced by Mahoney and LSc strains differ in size on macaque kidney cell monolayers, it is difficult to assess any plaque difference there may be between these strains on treated rhesus cells. Kidney cell cultures from green t monkeys (Cercopithecus aethiops sabaeusl are known to be somewhat more susceptible to poliovirus
13
OF DYES TABLE
1
REDUCTION IN PLAQUE COUNT CAUSED BY NEUTRAL RED AND LIGHTC Days when plaque counts were made Attenuatedb 2 3 4 Virulent 2 3 4
Time of exposure to light during first 24 hours None
5 min
10 min
15 min
10 20 24
1 9 12
0 7c 12c
0 Bc 1oc
10 23 26
4 16 19
0 9” 15c
0 9c 15”
~~_
a PFU readings were averaged from 12 to 15 bottle cultures exposed to light for the stated time. * Plaques produced by the attenuated strain were smaller than those produced by the virulent one. c In general, smaller, hazier, and more difficult to read.
(Hsiung and Melnick, 1957). However, the results described in Table 1 were also reproduced in cultures of green monkey kidney cells in which LSc and Mahoney strains produce plaques of nearly equa1 size. The effect of light on developing plaques was next studied. After 5 days of incubation, plaques of Mahoney and LSc were chosen in control bottles and exposed to light for 15 minutes. The subsequent increase in size was smaller than that observed in the controls, but again there was no difference between the behavior of Mahoney and LSc strains in this respect. c. Single-Cycle (S-hour) Yields of Mahoney and LSc with Different Concentrations of Neutral Red In these experiments, 105.5 PFU were added to each tissue culture tube with different concentrations of neutral red in the medium, ranging from 0 to 1:30,000. An attempt was made to keep the tubes in the dark as much as possible; however, small amounts of light were present at times during the experiment. The resulting inhibitions of the g-hour yields are shown in Fig.
14
CROWTHER
AND MELNICK
d. Effect of Pre- and Posttreatment with Neutral Red and Light on the Titration of Poliovirus in Tube Cultures under Fluid Medium
Comantrotion
of Neutral
Red
FIG. 1. Inhibition of S-hour yields of poliovirus (Mahoney) by neutral red at different concentrations. TABLE
2
TITERS AFTER INOCULATION OF PRETREATED CELLS IN TUBE CULTUREW Experi- Experi- Average ment Ib ment IP
Virus strain LSc (treated) Mahoney (treated) LSc (controls)
Mahoney (controls)
6.75 7.5 6.75 8.0
6.5 7.5 6.75 7.5
6.63 7.5 6.75 7.75
5 Values: log TCDSo per milliliter. b Experiment I: 1:240,000 neutral
red plus 1
hour light. c Experiment II: 1:480,000 neutral red plus 135 hours light.
0
I
2
3
4 HOURS
5
6
7
6
FIG. 2. Growth curves of Mahoney and LSc strains on pretreated and control cells. The pretreatment consisted of 1:240,000 neutral red plus 1 hour of light. 1. No differences were found in the behavior of virulent and attenuated virus.
Two degrees of pretreatment were used on cells upon which virus titrations were subsequently carried out, namely 1: 240,000 dye plus 1 hour of light, and 1: 480,000 dye plus 1M hours of light. The end point titers may be seen in Table 2. There was no significant difference in the response of pretreated and control cells, either in the rate of development of cytopathic changes or in the final titers which were reached by the fifth to sixth day. In the posttreatment experiment, the tube cultures were incubated with a 1: 240,000 dilution of neutral red for 2 hours and then inoculated with dilutions of virus. Three hours after inoculation the tubes were exposed for 1 hour to white light in the usual manner. Again, no difference in the production of cytopathic change between treated cells and control cells for either Mahoney or LSc was observed, and the final end point titers were identical with those of the controls. e. Virus Cells
Growth
Curves
on Pretreated
Pretreatment was carried out on half the tubes using 1:240,000 neutral red and 1 hour of light. Tubes for control growth curves were kept in the dark without neutral red. Virus was inoculated into each tube so that the final concentration of virus in each tube was lo5 PFU/ml. The virus was allowed to adsorb for 1 hour and then the unadsorbed virus was removed by washing four times with 5 ml of warm maintenance medium. Finally 1 ml of medium was left in contact with the cells and the tubes were incubated. At the appropriate time interval, 5 tubes for each point on the curve were removed and harvested in the manner described (each culture in 1 ml), and the virus was estimated by the plaque technique. The growth curves obtained are shown in Fig. 2. This degree of pretreatment gave an average depression throughout an g-hour growth cycle of approximately 96% for both viruses. This ex-
POLIOVIRUS
INCORPORATION
15
OF DYES
periment, as well as others, demonstrated that the viruses adsorbed equally well onto treated and control cells, hence the effect of the treatment is occurring on the intracellular development of infective poliovirus. f. Effect of Different Amounts of Pretreatment on Single-Cycle Yields of Poliovirus In these experiments the amount of pretreatment was varied to show up any effects which may have been missed by using only one degree of treatment. Figure 3 shows how the concentration of neutral red affects the inhibition of LSc and Mahoney in pretreated cells in which the amount of light is held constant. The single-cycle (8hour) yields were obtained in the usual manner, inoculating 105.5 PFU into each tube. Again, each variable was covered by 5 tubes. The similarity between Fig. 3 and Fig. 1 (in which the action of neutral red alone was plotted) is quite apparent. It seems that the exposure to light does not alter the yield very much when the cells are exposed before inoculation of virus. This was supported by results of &hour yield experiments in which cell cultures containing 1:240,000 neutral red were treated with light for periods from 30 to 90 minutes prior to inoculation of virus. This led to the suggestion that the growth curves shown in Fig. 2 were influenced mainly by the neutral red and small amounts of subsequent exposure to stray light, rather than by the effects of the pretreatment light. In comparing virulent and attenuated polioviruses, it should be noted that in these experiments Mahoney and LSc strains behaved similarly, within the limits of experimental error.
1:400,000 1:600.000 t : eoo.wo 1:600,000 t: 300,000 I: 10Q000 Concantrotion of Neutral Red
FIG. 3. Influence of concentration of neutral red on the inhibition of LSc and Mahoney strains in pretreated cells.
5 2 J ;= 03 1
Nsutral
Red Constant
3: 00 Pernod
in Growth
: 1240,000
HOURS
Cycle
During
cc.nttnu.a~,ty
5-6
p,e,.n,
6-7
Which Ltght Was Apptkd
FIG. 4. Inhibition of Mahoney and LSc strains by neutral red and light. Cultures containing neutral red and virus were irradiated with light as indicated during the growth cycle.
had been allowed to absorb 1: 240,000 neutral red for 2 hours. After washing, the infected cells were exposed to light in the usual manner for 1 hour, at different periods in the S-hour growth period of the virus. All tubes were harvested after 8 hours g. Single-Cycle Yields of Poliovirus in and titrated by the plaque technique, 5 Neutral Red-Treated Cells Applying the tubes being harvested for each variable. Light at Different Times during the The results with LSc and Mahoney are Growth Cycle shown in Fig. 4. It may be seen that the The S-hour yields were obtained in the inhibition is approximately the same when usual manner, using an inoculum of 105.5 light is applied during the first hour after PFU per tube culture, which previously adsorption as it is during the sixth hour.
16
CROWTHER
AND
The differences between the attenuated and virulent strains are not significant. The inhibition is greater than that expected for 1:240,000 neutral red alone; hence at 6 hours when inhibition of the 8hour yield would be expected to be only 1 log, a subsequent exposure to light for the next hour results in a reduction in titer of 2-3 logs. This indicates that the effect of neutral red and light may be on the fully formed virus as well as on the developmental stages. The following experiments were conducted to elucidate whether it is the combined action of neutral red plus light on fully developed virus which reduces its titer, or whether virus grown in cells containing neutral red is subsequently inactivated by light. h. The Effect of Neutral on Whole Virus
TABLE
Time allowed for adsorption of N.R. before exposure to light (hr)
AFTER DIFFERENT PH 7.5-No
Cont. of N.R.
harvested
immediately;
i. Experiments Demonstrating Incorporation of Neutral Red into the Developing Poliovirus Single-cycle @-hour) yields of LSc and Mahoney strains were grown in the dark in different concentrations of neutral red ranging from 1:30,000 to 1:300,000. After harvesting, l-ml aliquots were exposed to light in the usual manner for 1 hour. Control l-ml aliquots from the same source were incubated in the dark. Both sets were 3
EXPOSURES TO NEUTRAL CELLS PRESENT
Time of exposure to white light W
0 1:30,000 0 1:240,000 1:240,000 1:30,000 1:30,000 1:30,000 1:30,000 A: not incubated, in the light.
l-ml aliquots. The amount of treatment applied is shown in Table 3. The new virus was freshly harvested on the day of the experiment, while the old virus was taken from stock which had been stored for several months at -20”. There was no difference in titer between treated and untreated virus. The experiment was performed at pH 7.5. Another experiment performed at pH 8.6 gave similar results. It appears then that the reduction in infective virus described in Section f above was due to the effects of light on virus developed in the presence of neutral red, since the dye and light have no apparent effect on the mature virus grown in the absence of neutral red. In order to determine whether such a hypothesis is tenable, the following further experiments were carried out.
Red Plus Light
The factors studied were as follows: (1) amount of light; (2) time of adsorption of neutral red to virus ; (3) pH ; (4) age and storage of virus harvest. Yamamoto (1958) has shown that each of the first three factors plays an important role in the inactivation of phage by active dyes. Virus in concentrations of approximately lo5 PFU/ml was distributed into tubes in
TITERS OF VIRUS (PFU/ML)
MELNICK
RED (N.R.)
AND LIGHT AT
LSC (fresh harvest)
LSC (stored virus)
Mahoney (fresh harvest)
Mahoney (stored virus)
5.3 5.2 5.3 5.6 5.3 5.2 5.2
5.0
5.2 5.3
5.1 5.1 5.1 4.9 5.2
5.1
5.0
5.4 5.4 5.3 5.2 5.3 5.3 5.4 5.4 5.2
B to I: 2 hours’
incubation
at 37” with
5.1 5.0 5.2 5.3 5.2 5.2 different
periods
POLIOVIRUS
INCORPORATION TABLE
4
EFFECT OF LIGHT ON VIRUS GROWN IN PRESENCE OF NEUTRAL
-
N.K. concentration
I
Average
Log10 inhibition, dark
5.9
5.8
5.54 4.75
4.3 4.0 2.8 1.2
4.5 3.6 2.9 1.0
5.75 4.75 4.4 3.7 2.85 1.1
5.2
5.0 -
5.1 4.4 3.7 3.35 1.5
4.0 3.3 1.5 -
-
Expt. III
4.1 3.4
3.5
4.4 3.0
RED IN THE DARK
in dark 1 hou rI ‘FU titers after incubation
1Xxpt. II --
1Expt.
Mahoney 0 1:300,000 1:240,000 1:120,000 1:60,000 1:30,000 LSC 0 1:300,000 1:120,000 1:90,000 1:30,000 a Inhibition in the dark.
‘FU titers after incubation
17
OF DYES
Expt. I
Expt. II
Expt. III
0 1.00 1.35 2.05 2.90 4.65
6.0
5.7
5.56 4.0
0 0.7 1.4 1.75 3.6
5.0
5.2
2.1 2.1
2.1 2.1
due to light over and above that due to neutral
then titrated by the plaque technique; the results are shown in Table 4. There was a very marked reduction in infectivity upon exposure to light of these viruses which had been developed in the dark in cells containing neutral red. The reduction in infectivity attributed to the light increases with increasing concentration of dye only up to a point. At higher concentrations of neutral red, the inhibition due to the dye alone (plus the small amount of light unavoidably applied during the course of the experiment) is so great that it tends to obscure any reduction due to subsequent exposure to light. It appears that neutral red becomes incorporated into poliovirus developing in its presence. For example, with Mahoney virus at 1: 240,000 concentration of dye-well below the cytotoxic level-the infectivity of the recovered virus was relatively close to that of the control harvest. However, subsequent exposure to light reduced the infectivity an additional thousandfold. The neutral red is not just on the outside of the virus particles, since the addition of dye plus light to mature virus is not effective; however, the evidence is indirect. This topic is considered further in relation to other work, in the Discussion of this paper.
1.0
Average
in light 1 hou
T
Log10 inhibition light-darka
Log,, inhibition, light
0 1.73 >4.73 4.73 >4.73 4.73
0 0.73 >3.38 2.68 >1.83 -
0 1.7 3.35 3.0 >4.1
0 1.0 1.95 1.25
1. red alone plus l-hour
-
incubation
at 37”
j. Acridine Orange and Fluorescent Antibody Staining of Cells in Which Virus is Developing in the Presence of Neutral Red Monolayer cultures on coverslips were inoculated with 0.1 ml undiluted MEF-1 (titer lOa PFU/ml). One hour was allowed for adsorption and the excess was washed off. After 5 and 8 hours, 2 tubes at each period were harvested for titration, and several others were stained with acridine orange or with fluorescent antibody by the direct method. Three sets of tube cultures were inoculated and kept in the dark: virus alone, virus plus l:lOO,OOO neutral red, and controls without either virus or dye. As found with the type 1 strains, neutral red in the dark produced a decrease of 1.5 log units in the g-hour yield. Very clear results were obtained with fluorescent antibody staining. Virus grown for 8 hours in neutral red stained with intensity equal to that of virus in control cultures. Controls without virus were uniformly negative. Prior treatment of infected cultures with type 2 polio antiserum blocked the staining. With acridine orange staining, the control cells without virus were uniformly
18
CROWTHER
NEUTRAL RED (TOLUYLE NE RED)
PROFLAVIN (AN ACRIDINE)
GENERAL FORMULA OF AN ACTIVE DYE
FIG. 5. Structural formulas of photodynamically active dyes.
TABLE
5
PHOTOINACTIVATION OF MAHONEY POLIOVIRUS GROWN IN THE PRESENCE OF ACRIDINE ORANGE IN THE DARK Concentration Subsequent of acrid& treatment E$$orange present during growth bar%ing (“g$T”/ of virus in dark virus
0 1: 100,000
1:200,000 ~__ 1:300,000
5.5 5.6
ExperiAverage ment II lop reduc(‘Og$Tu/ ~~f;i’$~
Dark Light __-__ Dark Light
3.3 1.0
5.5 5.6 ~__ 2.3 1.2
Dark Light
3.5 1.6
3.2 1.4
Dark Light
4.0 1.0
3.6 1.6
None ---1.7 __-__ 1.9 2.5
stained : light green nucleus, reddish-tinged nucleolus, and red cytoplasm. In virus cultures harvested at 5 hours, the red staining of the cytoplasm became brilliant, indicating a production of RNA over and above that of controls, which has been described in detail by Mayor (1961). Virus grown in cultures in the presence of neutral red gave a picture similar to that of virus grown in the absence of the dye, indicating that RNA synthesis was not inhibited to a great extent by neutral red. In assessing the results of the acridine
AND MELNICK
orange staining, it must be remembered that both cellular RNA and virus RNA have been reported to increase during growth of poliovirus (Ackermann et al., 1959). It is not possible, therefore, to say for certain that viral RNA is not inhibited, because it may well be overwhelmed in amount by the cellular RNA increase. However, it may be said that the bulk of the RNA increase is not inhibited to any great extent. As mentioned, the titer of the virus was reduced about 1.5 log units when virus was grown for 8 hours in 1: 100,000 neutral red. However, the extent and intensity of fluorescent, antibody staining and of acridine orange staining were identical with controls containing more than ten times as much infectious virus. Hence, the poliovirus antigens and the cell and virus RNA increases, as revealed by acridine orange staining, are not inhibited radically by the amount of neutral red used. I%. The Inhibitory Effects of Compounds Structurally Related to Neutral Red The structural similarities between an acridine and neutral red may be seen in Fig. 5. These suggested the possibility that acridine orange might act in a similar manner to neutral red. Experiments attempting to demonstrate the incorporation of acridine orange into developing poliovirus were performed, analogous to those with neutral red, as outlined in Section i above. Table 5 shows the results of two such experiments. As in the experiments with neutral red, those viruses which had developed in the dark in cells containing acridine orange were markedly reduced in infectivity upon exposure to light, as compared with control samples kept in the incubator in the dark. Controls in which the mature virus was incubated in the presence of acridine orange and subsequently exposed to light for 1 hour showed no reduction in titer. The results of these experiments indicate that acridine orange can also become incorporated into developing poliovirus and that the resulting infective poliovirus may be inactivated by subsequent exposure to light. Not all heterotricyclic compounds are active in this way, however. Ribofla-
POLIOVIRUS
INCORPORATION
vine, at a concentration of 1: 100,000, caused neither inhibition of plating efficiency nor a reduction in the production of infective virus by cells incubated with riboflavine, with or without light. DISCUSSION
Neutral red-at high dilutions which have no visible influence on the cells nor on the cytopathic response to virus-together with only small amounts of light, reduces the normal yields of infective poliovirus considerably. To be effective the light must be applied after virus inoculation. However, the time at which the light is applied after inoculation does not appear to be of overriding importance. The experiments included both Mahoney, as an example of a virulent poliovirus, and LSc, as an attenuated poliovirus. However, both viruses behaved similarly in all the experiments described; the inhibition produced in yields of both viruses was approximately the same. Hence neutral red and light under the conditions of our experiments could not be used to distinguish virulent and attenuated strains. Opton and Green (1960) described experiments in which fully developed LSc and Mahoney viruses were exposed to light in the presence of neutral red. Whereas in the experiments described in our paper the titers of mature virus were the same before and after exposure to light, Opton described a small reduction in titer with Mahoney and a slight rise in titer with LSc. He considered the small differences to be significant because they were repeatable in his hands. The experiments which have been described in this paper, when compared with previous work of other investigators, indicate a similarity between the biological effects of neutral red and of other photodynamically active dyes such as proflavine, toluidine blue, and acridine orange. This is perhaps not surprising when one considers the formulas of these compounds (Fig. 5). Proflavine is an acridine and has the formula shown with a carbon in the X-position. Neutral red, a quinonimide dye, has a nitrogen in the X-position. Toluidine blue is yet another example of a quinon-
OF DYES
19
imide dye. The similarities between the formulas are obvious. Tricyclic photodynamitally active dyes have the general formula shown at the bottom of Fig. 5, “X” indicating carbon or nitrogen and “Y” indicating sulfur, oxygen, or nitrogen. All are basic heterotricyclic compounds with an affinity for nucleic acid. Yamamoto (1958) showed that neutral red, acridine orange, toluidine blue and other active dyes can inactivate certain phages, but LoGrippo and Basinski (1960) found that acridine orange has no effect on poliovirus and Coxsackie virus. Similarly, Hiatt et al. (1960) have found that these two enteroviruses cannot be inactivated by toluidine blue and white light, whereas vaccinia and adenoviruses are readily inactivated under the same conditions. Helprin and Hiatt (1959) have pointed out that susceptibility of phage to the action of an active dye and light appears to be linked with the permeability of the virus since there is a correlation between these experiments and osmotic permeability of the phage as measured by osmotic disruption experiments. Schaffer (1960) has recently found that proflavine cannot attach to mature poliovirus particles, whereas it can be incorporated into developing poliovirus. In the course of this study, we have independently found that a similar sort of phenomenon occurs with neutral red: poliovirus is not inactivated by neutral red and light in the absence of cells, but virus grown in cells containing neutral red may be inactivated by light, indicating that an incorporation of the dye into developing poliovirus may occur. We have also shown in this paper that acridine orange yields similar results, and in an associated study, Mayor and Diwan (1961) have obtained direct microscopic evidence of the incorporation of the dye into the virus. Ledinko (1958) demonstrated the cytopathogenicity of poliovirus to be unaffected by proflavine, and we have observed that the same is true using neutral red, unless near-toxic levels of the dye are used. Both proflavine and neutral red are capable of inhibiting the production of infective poliovirus even in subdued lighting. It is not
20
CROWTHER
known as yet exactly what part the dye alone and what part added light play in the production of noninfective virus. In the experiments of Ledinko (1958) and of Schaffer (1960)) virus produced in the presence of proflavine, and shown by Schaffer (1960) to contain the dye, was largely noninfective, but laboratory lighting was not excluded. Schaffer (unpublished) has now found that proflavine in the dark produces only slight inhibition in the production of infective poliovirus. The action of proflavine is generally thought to be on maturation alone (Ledinko, 1958). The basic nature of the dye would seem to support this contention, the positively charged molecule combining with the nucleic acid and inhibiting the proper aggregation of the nucleic acid with its protein coat. Our experiments with neutral red and the similarities noted between the acridines and quinonimide dyes suggest the production of noninfective virus by these dyes is not mainly due to a prohibition of the aggregation of nucleic acid with its protein coat, but rather to a subsequent inactivation of an infective virus containing the active dye. This is supported by the fact that electron micrographs of poliovirus grown in the presence of proflavine are not significantly different from controls; however, the infectivity of the proflavine virus was considerably lower (Horne and Nagington, 1959). Probably the former mechanism does play some part in the production of noninfective virus. Also, it is likely that some virus particles containing dye together with protein around their nucleic acid will be noninfective even without light. The experiments performed with proflavine and other acridines as viral inhibitors have been carried out, until recently, without reference to the part that light may play in this inhibition. Eaton et al. (1951) demonstrated the inhibition in production of infective virus by certain dyes of the acridine group. Inhibition was found to be effective against meningopneumonitis, feline pneumonitis, mumps, and influenza viruses. These workers came to the conclusion that the inhibition of virus was not caused by the action of the dye on extracellular virus because very little inactivation oc-
AND MELNICK
curred in the absence of cells. They did not consider the possibility of incorporation of the dyes into the virus particle and its subsequent inactivation by light. Our recent experiments as described in this paper have shown that although acridine orange can reduce somewhat the yields of infective poliovirus grown on monkey kidney cells in the dark, a considerable further reduction may be obtained by exposure of the harvested virus to white light. The variable amounts of inhibition due to the active dyes, obtained in different laboratories, and also the variable results of experiments done in the same laboratory (for example, the earlier neutral red experiments in our own laboratory) may now be explained by the previously unconsidered part that light can play in the production of infective virus. A quantitative comparison between the effects of the different active dyes cannot be made at present since the conditions under which the experiments have been conducted in different laboratories vary. REFERENCES ACKERMANN,W. W., LOH, P. C., and PAYNE, F. E. ( 1959). Studies of the biosynthesis of protein and ribonucleic acid in HeLa cells infected with poliovirus. Virology 7,179-183. DARNELL,J. E., JR., LOCKHART,R. Z., JR., and SAWYER, T. K. (1958). The effect of neutral red on plaque formation in two virus-cell systems. Virology 6,567-568. EATON,M. D., CHEEVER,F. S., and LEVENSON,C. G. (1951). Further observations of the effect of acridines on the growth of viruses. J. Immunol. 66,463-476. GREEN,R. H., and OPTON,E. M. (1959). Photosensitization of tissue culture cells and its effect on viral plaque formation. Proc. Sot. Ezpt2. Biol. Med. 102, 519-521. HELPRIN, J. J., and HIATT, C. W. (1959). Photosensitization of T2 coliphage with toluidine blue. J. Bacterial. 77, 502-565. HIATT, C. W., KAUFMAN, E., HELPRIN, J. J., and BARON, S. (1960). Inactivation of viruses by photodynamic action of toluidine blue. J. Immunol. 84,48@484. HORNE,R. W., and NAGINGTON,J. (1959). Electron microscope studies of the development and structure of poliomyelitis virus. J. Mol. Biol. 1, 333338. HSIUXG, G. D., and MELNICK, J. L. (1955). Plaque formation with poliomyelitis, Coxsackie, and
POLIOVIRUS
INCORPORATION
orphan (Echo) viruses in bottle cultures of monkey epithelial cells. Virology 1, 533-535. HSIUNG, G. D., and MELNICK, J. L. (1957). Comparative susceptibility of kidney cells from different monkey species to enteric viruses (poliomyelitis, Coxsackie and ECHO groups). .I. Immunol. 78, 137-146. KLEIN, S. W., and GOODGAL,S. H. (1959). Photodynamic inactivation of monkey kidney cell monolayers. Science 130,629. LEDINKO, N. (1958). Production of noninfectious complement-fixing poliovirus particles in HeLa cells treated with proflavine. Virology 6, 512524. LOGRIPPO,’G., and BASINSKI, D. H. (1960). The value of acridine orange for sterilizing viruses in plasma and whole blood. Pederation Proc. 19, 56. MAYOR, H. (1961). Cytochemical and fluorescent antibody studies on the growth of poliovirus in tissue culture. l’exas Repts. Biol. and Med. 19, 106-122. MAYOR, H. D., and DIWAX, A. R. (1961). Studies on the acridine orange staining of two purified
OF DYES
21
RNA viruses: poliovirus and tobacco mosaic virus. Virology 14,7482. MCCLAIN, M., and HACKETT, A. J. (1958). A comparative study of the growth of vesicular stomatitis virus in five tissue culture systems. J. Immunol. 80,356361. MELNICK, J. L. (1956). Tissue culture methods fat the cultivation of poliomyelitis and other viruses. In “Diagnostic Procedures for Virus and Rickettsial Diseases,” 2nd ed., pp. 97-152. Am. Publ. Health Assn., New York. OPTON, E. M., and GREEN, R. H. (1960). Effects of neutral red and light on polioviruses. Federation Proc. 19, 408. SCHAFFER, F. L. (1960). The nature of noninfectious particles produced by poliovirus infected tissue cultures treated with proflavin. Federation Proc. 19, 405, and personal communication. WATERSON, A. P. (1959). Effect of neutral red in plaque formation of fowl plague virus. Nature 183,628. YAMAMOTO,N. (1958). Photodynamic inactivation of bacteriophage and its inhibition. J. Bacterial. 75,443448.
14, 11-21 (1961)
The Incorporation of Neutral Red and Developing Poliovirus Particles Making DEREK Department
CROWTHER2
of Virology
AND
and Epidemiology, Houston Accepted
JOSEPH
Acridine Orange into Them Photosensitive’ L. MELNICK
Baylor University College of Medicine, 25, Texas
January
3, 1961
This study concerns the effects of neutral red, acridine orange, and light on the growth of virulent (Mahoney) and attenuated (LSc) polioviruses in monkey kidney cell cultures. Mature, infective virus incubated with the dyes for 1 hour in the absence of cells and then exposed to white light showed no reduction in titer. Virus grown in cells containing neutral red or acridine orange may be subsequently inactivated by light, thus indicating an incorporation of the dye into the developing virus. The effect of neutral red and light was the same for the virulent and the attenuated strains. The biological and chemical similarities between the quinonimide dyes (neutral red, toluidine blue) and the acridines (acridine orange, proflavine) are discussed. INTRODUCTION
cell monolayers Melnick, 1955).
In the presence of the vital dye neutral red, virus plaques may be reduced in size and number, but to a variable degree (Darnell et al., 1958; McClain and Hackett, 1958; Waterson, 1959; Green and Opton, 1959). Work described in this paper was designed to elucidate the part played by light in the growth of poliovirus in cells containing neutral red and also to determine whether cells treated with neutral red and light might selectively favor the growth of virulent as against attenuated poliovirus. MATERIALS
in bottles
(Hsiung
and
Application of light. Cell monolayers were exposed under 20-watt “daylight” type fluorescent lighting tubes in a 37” incubator. Three lighting tubes were attached to the shelf above so that the monolayers were approximately 8-9 inches from the light source. Careful arrangement of the cell layers under the light insured that all the cells were approximately the same distance from the light. The tubes were placed under the light on wooden wedges so that the tubes were inclined at 5”. A careful check on the temperature showed that this did not vary more than 0.5” from the reading obtained in the incubator above the light. An attempt was made to keep the developing virus and the harvested virus in the dark as much as possible when the light was not being applied. Thus, the experiments were performed and titrated in subdued lighting, and aluminum foil was used to envelop the tissue culture racks to prevent any extraneous light from reaching the cells. (Yell counting. The cells in each tube culture were washed and incubated for approxi-
AND METHODS
Monkey kidney cultures were grown and maintained in lactalbumin medium as described by Melnick (1956). Viral assays were performed by the plaque technique on ‘This study was aided by a grant from The National Foundation, and by a training grant (23-74) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service. ‘On leave from the University of Cambridge, England, as a Predoctoral Trainee in Virology, 1959-1960. 11
12
CROWTHER AND MELNICK
mately 20 minutes with 1 ml Versene solution (Melnick, 1956). For counting the total number of cells, 0.1% crystal violet in 0.1 M aqueous citric acid was used. For counting live cells, 1.0 ml of a 0.01% solution of Janus green B was added to 1 ml of cell suspension and the mixture was incubated at 37” for 30 minutes. Dead cells were counted by mixing 1 ml of a 1.0% trypan blue solution with 1 ml of cell suspension. The cell count was then done immediately because trypan blue is toxic to cells. Cells unstained by trypan blue can also be counted and used to indicate the percentage of live cells. Neutral red. The neutral red which was added to the cells for conditioning was added to the agar in the case of the bottles. When used in tubes, the dye was added to the maintenance medium. In the tube experiments, 2-3 hours were allowed for the neutral red to be taken in by the cells before any light was applied. Viruses. The virulent strains of poliovirus were those in use for preparing inactivated poliovaccine (Mahoney, MEF-1, and Saukett) ; the attenuated strains were those in use for preparing live poliovaccine (LSc, P712, and Leon). In the 8-hour yield experiments, 105a5 PFU in 1 ml were added to each tube culture, allowed to absorb for 1 hour, washed off with warm medium (three or four times with 4 ml fluid), and allowed to incubate in 1 ml of maintenance medium for 8 hours before harvesting. Harvesting. Each tube was subjected to two cycles of freezing (-20”) and thawing. After the second thawing, 5 tubes at each variable were combined and centrifuged (for 30 minutes at 2000 rpm) to remove cell debris; the supernates were stored at -20” for later titration by the plaque technique. Acridine orange staining for nucleic acids. Cultures were grown on coverslips in flatsided (Leighton) tubes for these experiments. At the intervals indicated below, they were transferred quickly to Carnoy’s fluid (ethanol-chloroform-acetic acid) at room temperature for a fixation period of 5 minutes. After this they were passed into
absolute ethanol, hydrated through an ethanol-water series, placed for 5 minutes in a pH 4 citric acid-phosphate buffer, and stained for 5 minutes with 0.014% acridine orange made up in the same buffer. After rinsing for 3 minutes in the buffer, the coverslips were blotted gently and mounted on microscope slides, using the pH 4 buffer as mounting medium. RNA digestion. Carnoy-fixed coverslips of infected and control cultures were incubated with 0.05% ribonuclease (Worthington, 5~ recrystallized) at pH 4 for 1 hour at 37”. Fluorescent antibody. Antipolio globulin labeled with fluorescein isothiocyanate was used as described in another paper from this laboratory (Mayor, 1961). As controls, normal cells were treated with the labeled globulin, and infected cells were treated first with unlabeled blocking antiserum and then with the labeled globulin3 RESULTS a. Determination of Amounts of Neutral Red and Light Tolerated by Cells Photosensitivity to white light of cells containing small concentrations of neutral red was readily demonstrated, as has been described by Klein and Goodgal (1959). Cells exposed to light and neutral red in sufficient amounts became enlarged and globoid. Some cells became four to five times larger in diameter than normal monkey kidney cells. The dye was seen in the cytoplasm in small discrete areas rather similar to those seen after vital staining with acridine orange (Mayor, 1961). The over-all picture of a gradual transformation to round refractile cells was similar to a viral cytopathic effect. The possibility that such changes were induced by a virus which had been activated by the treatment was considered, but no agent could be isolated. Similar changes in cells in vivo have been described by pathologists and may be induced by a variety of causes, e.g., radiation, and poisons such as carbon tetrachloride. 3 We wish to acknowledge Dr. Heather Donald Mayor’s participation in these experiments.
POLIOVIRUS
INCORPORATION
In the tube experiments to be described, values for neutral red and light were chosen that gave no degeneration of the monolayer after 1 week of incubation (1:240,000 neutral red plus 1 hour of light). For monolayers under agar containing 1: 60,000 neutral red, 15 minutes of exposure to white light applied directly to the cell sheet through the glass could be tolerated without producing any subsequent disruption of the cell sheet. For controls, half the treated bottle was covered with tape and in addition separate control bottles with untreated cells were used. b. Efficiency of Plating Agar plus Light
under Neutral
Red
Experiments were carried out to determine the degree to which monolayers treated under the conditions of the preceding paragraph supported plaque production by virulent and attenuated polioviruses. Bottle cultures were inoculated with approximately 20 plaque-forming units (PFU) of virus, allowed to absorb for 1 hour, overlaid, and then exposed to light for 5, 10, and 15 minutes. Three bottles were used for each variable. There was no significant difference as a result of exposing the bottles at any time within 24 hours after overlaying. In each set of bottles there was a reduction in plaque size and number, which was most pronounced in those bottles exposed for 15 minutes. In the exposed series the plaques were not only smaller and less numerous, but also hazier and more difficult to count than those in the control series. The differences between the control and treated cultures were most pronounced when read on the second day and progressively became less with longer incubation time, as shown in Table 1. The final plaque counts showed a similar reduction for both LSc and Mahoney-approximately 50%. As the plaques produced by Mahoney and LSc strains differ in size on macaque kidney cell monolayers, it is difficult to assess any plaque difference there may be between these strains on treated rhesus cells. Kidney cell cultures from green t monkeys (Cercopithecus aethiops sabaeusl are known to be somewhat more susceptible to poliovirus
13
OF DYES TABLE
1
REDUCTION IN PLAQUE COUNT CAUSED BY NEUTRAL RED AND LIGHTC Days when plaque counts were made Attenuatedb 2 3 4 Virulent 2 3 4
Time of exposure to light during first 24 hours None
5 min
10 min
15 min
10 20 24
1 9 12
0 7c 12c
0 Bc 1oc
10 23 26
4 16 19
0 9” 15c
0 9c 15”
~~_
a PFU readings were averaged from 12 to 15 bottle cultures exposed to light for the stated time. * Plaques produced by the attenuated strain were smaller than those produced by the virulent one. c In general, smaller, hazier, and more difficult to read.
(Hsiung and Melnick, 1957). However, the results described in Table 1 were also reproduced in cultures of green monkey kidney cells in which LSc and Mahoney strains produce plaques of nearly equa1 size. The effect of light on developing plaques was next studied. After 5 days of incubation, plaques of Mahoney and LSc were chosen in control bottles and exposed to light for 15 minutes. The subsequent increase in size was smaller than that observed in the controls, but again there was no difference between the behavior of Mahoney and LSc strains in this respect. c. Single-Cycle (S-hour) Yields of Mahoney and LSc with Different Concentrations of Neutral Red In these experiments, 105.5 PFU were added to each tissue culture tube with different concentrations of neutral red in the medium, ranging from 0 to 1:30,000. An attempt was made to keep the tubes in the dark as much as possible; however, small amounts of light were present at times during the experiment. The resulting inhibitions of the g-hour yields are shown in Fig.
14
CROWTHER
AND MELNICK
d. Effect of Pre- and Posttreatment with Neutral Red and Light on the Titration of Poliovirus in Tube Cultures under Fluid Medium
Comantrotion
of Neutral
Red
FIG. 1. Inhibition of S-hour yields of poliovirus (Mahoney) by neutral red at different concentrations. TABLE
2
TITERS AFTER INOCULATION OF PRETREATED CELLS IN TUBE CULTUREW Experi- Experi- Average ment Ib ment IP
Virus strain LSc (treated) Mahoney (treated) LSc (controls)
Mahoney (controls)
6.75 7.5 6.75 8.0
6.5 7.5 6.75 7.5
6.63 7.5 6.75 7.75
5 Values: log TCDSo per milliliter. b Experiment I: 1:240,000 neutral
red plus 1
hour light. c Experiment II: 1:480,000 neutral red plus 135 hours light.
0
I
2
3
4 HOURS
5
6
7
6
FIG. 2. Growth curves of Mahoney and LSc strains on pretreated and control cells. The pretreatment consisted of 1:240,000 neutral red plus 1 hour of light. 1. No differences were found in the behavior of virulent and attenuated virus.
Two degrees of pretreatment were used on cells upon which virus titrations were subsequently carried out, namely 1: 240,000 dye plus 1 hour of light, and 1: 480,000 dye plus 1M hours of light. The end point titers may be seen in Table 2. There was no significant difference in the response of pretreated and control cells, either in the rate of development of cytopathic changes or in the final titers which were reached by the fifth to sixth day. In the posttreatment experiment, the tube cultures were incubated with a 1: 240,000 dilution of neutral red for 2 hours and then inoculated with dilutions of virus. Three hours after inoculation the tubes were exposed for 1 hour to white light in the usual manner. Again, no difference in the production of cytopathic change between treated cells and control cells for either Mahoney or LSc was observed, and the final end point titers were identical with those of the controls. e. Virus Cells
Growth
Curves
on Pretreated
Pretreatment was carried out on half the tubes using 1:240,000 neutral red and 1 hour of light. Tubes for control growth curves were kept in the dark without neutral red. Virus was inoculated into each tube so that the final concentration of virus in each tube was lo5 PFU/ml. The virus was allowed to adsorb for 1 hour and then the unadsorbed virus was removed by washing four times with 5 ml of warm maintenance medium. Finally 1 ml of medium was left in contact with the cells and the tubes were incubated. At the appropriate time interval, 5 tubes for each point on the curve were removed and harvested in the manner described (each culture in 1 ml), and the virus was estimated by the plaque technique. The growth curves obtained are shown in Fig. 2. This degree of pretreatment gave an average depression throughout an g-hour growth cycle of approximately 96% for both viruses. This ex-
POLIOVIRUS
INCORPORATION
15
OF DYES
periment, as well as others, demonstrated that the viruses adsorbed equally well onto treated and control cells, hence the effect of the treatment is occurring on the intracellular development of infective poliovirus. f. Effect of Different Amounts of Pretreatment on Single-Cycle Yields of Poliovirus In these experiments the amount of pretreatment was varied to show up any effects which may have been missed by using only one degree of treatment. Figure 3 shows how the concentration of neutral red affects the inhibition of LSc and Mahoney in pretreated cells in which the amount of light is held constant. The single-cycle (8hour) yields were obtained in the usual manner, inoculating 105.5 PFU into each tube. Again, each variable was covered by 5 tubes. The similarity between Fig. 3 and Fig. 1 (in which the action of neutral red alone was plotted) is quite apparent. It seems that the exposure to light does not alter the yield very much when the cells are exposed before inoculation of virus. This was supported by results of &hour yield experiments in which cell cultures containing 1:240,000 neutral red were treated with light for periods from 30 to 90 minutes prior to inoculation of virus. This led to the suggestion that the growth curves shown in Fig. 2 were influenced mainly by the neutral red and small amounts of subsequent exposure to stray light, rather than by the effects of the pretreatment light. In comparing virulent and attenuated polioviruses, it should be noted that in these experiments Mahoney and LSc strains behaved similarly, within the limits of experimental error.
1:400,000 1:600.000 t : eoo.wo 1:600,000 t: 300,000 I: 10Q000 Concantrotion of Neutral Red
FIG. 3. Influence of concentration of neutral red on the inhibition of LSc and Mahoney strains in pretreated cells.
5 2 J ;= 03 1
Nsutral
Red Constant
3: 00 Pernod
in Growth
: 1240,000
HOURS
Cycle
During
cc.nttnu.a~,ty
5-6
p,e,.n,
6-7
Which Ltght Was Apptkd
FIG. 4. Inhibition of Mahoney and LSc strains by neutral red and light. Cultures containing neutral red and virus were irradiated with light as indicated during the growth cycle.
had been allowed to absorb 1: 240,000 neutral red for 2 hours. After washing, the infected cells were exposed to light in the usual manner for 1 hour, at different periods in the S-hour growth period of the virus. All tubes were harvested after 8 hours g. Single-Cycle Yields of Poliovirus in and titrated by the plaque technique, 5 Neutral Red-Treated Cells Applying the tubes being harvested for each variable. Light at Different Times during the The results with LSc and Mahoney are Growth Cycle shown in Fig. 4. It may be seen that the The S-hour yields were obtained in the inhibition is approximately the same when usual manner, using an inoculum of 105.5 light is applied during the first hour after PFU per tube culture, which previously adsorption as it is during the sixth hour.
16
CROWTHER
AND
The differences between the attenuated and virulent strains are not significant. The inhibition is greater than that expected for 1:240,000 neutral red alone; hence at 6 hours when inhibition of the 8hour yield would be expected to be only 1 log, a subsequent exposure to light for the next hour results in a reduction in titer of 2-3 logs. This indicates that the effect of neutral red and light may be on the fully formed virus as well as on the developmental stages. The following experiments were conducted to elucidate whether it is the combined action of neutral red plus light on fully developed virus which reduces its titer, or whether virus grown in cells containing neutral red is subsequently inactivated by light. h. The Effect of Neutral on Whole Virus
TABLE
Time allowed for adsorption of N.R. before exposure to light (hr)
AFTER DIFFERENT PH 7.5-No
Cont. of N.R.
harvested
immediately;
i. Experiments Demonstrating Incorporation of Neutral Red into the Developing Poliovirus Single-cycle @-hour) yields of LSc and Mahoney strains were grown in the dark in different concentrations of neutral red ranging from 1:30,000 to 1:300,000. After harvesting, l-ml aliquots were exposed to light in the usual manner for 1 hour. Control l-ml aliquots from the same source were incubated in the dark. Both sets were 3
EXPOSURES TO NEUTRAL CELLS PRESENT
Time of exposure to white light W
0 1:30,000 0 1:240,000 1:240,000 1:30,000 1:30,000 1:30,000 1:30,000 A: not incubated, in the light.
l-ml aliquots. The amount of treatment applied is shown in Table 3. The new virus was freshly harvested on the day of the experiment, while the old virus was taken from stock which had been stored for several months at -20”. There was no difference in titer between treated and untreated virus. The experiment was performed at pH 7.5. Another experiment performed at pH 8.6 gave similar results. It appears then that the reduction in infective virus described in Section f above was due to the effects of light on virus developed in the presence of neutral red, since the dye and light have no apparent effect on the mature virus grown in the absence of neutral red. In order to determine whether such a hypothesis is tenable, the following further experiments were carried out.
Red Plus Light
The factors studied were as follows: (1) amount of light; (2) time of adsorption of neutral red to virus ; (3) pH ; (4) age and storage of virus harvest. Yamamoto (1958) has shown that each of the first three factors plays an important role in the inactivation of phage by active dyes. Virus in concentrations of approximately lo5 PFU/ml was distributed into tubes in
TITERS OF VIRUS (PFU/ML)
MELNICK
RED (N.R.)
AND LIGHT AT
LSC (fresh harvest)
LSC (stored virus)
Mahoney (fresh harvest)
Mahoney (stored virus)
5.3 5.2 5.3 5.6 5.3 5.2 5.2
5.0
5.2 5.3
5.1 5.1 5.1 4.9 5.2
5.1
5.0
5.4 5.4 5.3 5.2 5.3 5.3 5.4 5.4 5.2
B to I: 2 hours’
incubation
at 37” with
5.1 5.0 5.2 5.3 5.2 5.2 different
periods
POLIOVIRUS
INCORPORATION TABLE
4
EFFECT OF LIGHT ON VIRUS GROWN IN PRESENCE OF NEUTRAL
-
N.K. concentration
I
Average
Log10 inhibition, dark
5.9
5.8
5.54 4.75
4.3 4.0 2.8 1.2
4.5 3.6 2.9 1.0
5.75 4.75 4.4 3.7 2.85 1.1
5.2
5.0 -
5.1 4.4 3.7 3.35 1.5
4.0 3.3 1.5 -
-
Expt. III
4.1 3.4
3.5
4.4 3.0
RED IN THE DARK
in dark 1 hou rI ‘FU titers after incubation
1Xxpt. II --
1Expt.
Mahoney 0 1:300,000 1:240,000 1:120,000 1:60,000 1:30,000 LSC 0 1:300,000 1:120,000 1:90,000 1:30,000 a Inhibition in the dark.
‘FU titers after incubation
17
OF DYES
Expt. I
Expt. II
Expt. III
0 1.00 1.35 2.05 2.90 4.65
6.0
5.7
5.56 4.0
0 0.7 1.4 1.75 3.6
5.0
5.2
2.1 2.1
2.1 2.1
due to light over and above that due to neutral
then titrated by the plaque technique; the results are shown in Table 4. There was a very marked reduction in infectivity upon exposure to light of these viruses which had been developed in the dark in cells containing neutral red. The reduction in infectivity attributed to the light increases with increasing concentration of dye only up to a point. At higher concentrations of neutral red, the inhibition due to the dye alone (plus the small amount of light unavoidably applied during the course of the experiment) is so great that it tends to obscure any reduction due to subsequent exposure to light. It appears that neutral red becomes incorporated into poliovirus developing in its presence. For example, with Mahoney virus at 1: 240,000 concentration of dye-well below the cytotoxic level-the infectivity of the recovered virus was relatively close to that of the control harvest. However, subsequent exposure to light reduced the infectivity an additional thousandfold. The neutral red is not just on the outside of the virus particles, since the addition of dye plus light to mature virus is not effective; however, the evidence is indirect. This topic is considered further in relation to other work, in the Discussion of this paper.
1.0
Average
in light 1 hou
T
Log10 inhibition light-darka
Log,, inhibition, light
0 1.73 >4.73 4.73 >4.73 4.73
0 0.73 >3.38 2.68 >1.83 -
0 1.7 3.35 3.0 >4.1
0 1.0 1.95 1.25
1. red alone plus l-hour
-
incubation
at 37”
j. Acridine Orange and Fluorescent Antibody Staining of Cells in Which Virus is Developing in the Presence of Neutral Red Monolayer cultures on coverslips were inoculated with 0.1 ml undiluted MEF-1 (titer lOa PFU/ml). One hour was allowed for adsorption and the excess was washed off. After 5 and 8 hours, 2 tubes at each period were harvested for titration, and several others were stained with acridine orange or with fluorescent antibody by the direct method. Three sets of tube cultures were inoculated and kept in the dark: virus alone, virus plus l:lOO,OOO neutral red, and controls without either virus or dye. As found with the type 1 strains, neutral red in the dark produced a decrease of 1.5 log units in the g-hour yield. Very clear results were obtained with fluorescent antibody staining. Virus grown for 8 hours in neutral red stained with intensity equal to that of virus in control cultures. Controls without virus were uniformly negative. Prior treatment of infected cultures with type 2 polio antiserum blocked the staining. With acridine orange staining, the control cells without virus were uniformly
18
CROWTHER
NEUTRAL RED (TOLUYLE NE RED)
PROFLAVIN (AN ACRIDINE)
GENERAL FORMULA OF AN ACTIVE DYE
FIG. 5. Structural formulas of photodynamically active dyes.
TABLE
5
PHOTOINACTIVATION OF MAHONEY POLIOVIRUS GROWN IN THE PRESENCE OF ACRIDINE ORANGE IN THE DARK Concentration Subsequent of acrid& treatment E$$orange present during growth bar%ing (“g$T”/ of virus in dark virus
0 1: 100,000
1:200,000 ~__ 1:300,000
5.5 5.6
ExperiAverage ment II lop reduc(‘Og$Tu/ ~~f;i’$~
Dark Light __-__ Dark Light
3.3 1.0
5.5 5.6 ~__ 2.3 1.2
Dark Light
3.5 1.6
3.2 1.4
Dark Light
4.0 1.0
3.6 1.6
None ---1.7 __-__ 1.9 2.5
stained : light green nucleus, reddish-tinged nucleolus, and red cytoplasm. In virus cultures harvested at 5 hours, the red staining of the cytoplasm became brilliant, indicating a production of RNA over and above that of controls, which has been described in detail by Mayor (1961). Virus grown in cultures in the presence of neutral red gave a picture similar to that of virus grown in the absence of the dye, indicating that RNA synthesis was not inhibited to a great extent by neutral red. In assessing the results of the acridine
AND MELNICK
orange staining, it must be remembered that both cellular RNA and virus RNA have been reported to increase during growth of poliovirus (Ackermann et al., 1959). It is not possible, therefore, to say for certain that viral RNA is not inhibited, because it may well be overwhelmed in amount by the cellular RNA increase. However, it may be said that the bulk of the RNA increase is not inhibited to any great extent. As mentioned, the titer of the virus was reduced about 1.5 log units when virus was grown for 8 hours in 1: 100,000 neutral red. However, the extent and intensity of fluorescent, antibody staining and of acridine orange staining were identical with controls containing more than ten times as much infectious virus. Hence, the poliovirus antigens and the cell and virus RNA increases, as revealed by acridine orange staining, are not inhibited radically by the amount of neutral red used. I%. The Inhibitory Effects of Compounds Structurally Related to Neutral Red The structural similarities between an acridine and neutral red may be seen in Fig. 5. These suggested the possibility that acridine orange might act in a similar manner to neutral red. Experiments attempting to demonstrate the incorporation of acridine orange into developing poliovirus were performed, analogous to those with neutral red, as outlined in Section i above. Table 5 shows the results of two such experiments. As in the experiments with neutral red, those viruses which had developed in the dark in cells containing acridine orange were markedly reduced in infectivity upon exposure to light, as compared with control samples kept in the incubator in the dark. Controls in which the mature virus was incubated in the presence of acridine orange and subsequently exposed to light for 1 hour showed no reduction in titer. The results of these experiments indicate that acridine orange can also become incorporated into developing poliovirus and that the resulting infective poliovirus may be inactivated by subsequent exposure to light. Not all heterotricyclic compounds are active in this way, however. Ribofla-
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vine, at a concentration of 1: 100,000, caused neither inhibition of plating efficiency nor a reduction in the production of infective virus by cells incubated with riboflavine, with or without light. DISCUSSION
Neutral red-at high dilutions which have no visible influence on the cells nor on the cytopathic response to virus-together with only small amounts of light, reduces the normal yields of infective poliovirus considerably. To be effective the light must be applied after virus inoculation. However, the time at which the light is applied after inoculation does not appear to be of overriding importance. The experiments included both Mahoney, as an example of a virulent poliovirus, and LSc, as an attenuated poliovirus. However, both viruses behaved similarly in all the experiments described; the inhibition produced in yields of both viruses was approximately the same. Hence neutral red and light under the conditions of our experiments could not be used to distinguish virulent and attenuated strains. Opton and Green (1960) described experiments in which fully developed LSc and Mahoney viruses were exposed to light in the presence of neutral red. Whereas in the experiments described in our paper the titers of mature virus were the same before and after exposure to light, Opton described a small reduction in titer with Mahoney and a slight rise in titer with LSc. He considered the small differences to be significant because they were repeatable in his hands. The experiments which have been described in this paper, when compared with previous work of other investigators, indicate a similarity between the biological effects of neutral red and of other photodynamically active dyes such as proflavine, toluidine blue, and acridine orange. This is perhaps not surprising when one considers the formulas of these compounds (Fig. 5). Proflavine is an acridine and has the formula shown with a carbon in the X-position. Neutral red, a quinonimide dye, has a nitrogen in the X-position. Toluidine blue is yet another example of a quinon-
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imide dye. The similarities between the formulas are obvious. Tricyclic photodynamitally active dyes have the general formula shown at the bottom of Fig. 5, “X” indicating carbon or nitrogen and “Y” indicating sulfur, oxygen, or nitrogen. All are basic heterotricyclic compounds with an affinity for nucleic acid. Yamamoto (1958) showed that neutral red, acridine orange, toluidine blue and other active dyes can inactivate certain phages, but LoGrippo and Basinski (1960) found that acridine orange has no effect on poliovirus and Coxsackie virus. Similarly, Hiatt et al. (1960) have found that these two enteroviruses cannot be inactivated by toluidine blue and white light, whereas vaccinia and adenoviruses are readily inactivated under the same conditions. Helprin and Hiatt (1959) have pointed out that susceptibility of phage to the action of an active dye and light appears to be linked with the permeability of the virus since there is a correlation between these experiments and osmotic permeability of the phage as measured by osmotic disruption experiments. Schaffer (1960) has recently found that proflavine cannot attach to mature poliovirus particles, whereas it can be incorporated into developing poliovirus. In the course of this study, we have independently found that a similar sort of phenomenon occurs with neutral red: poliovirus is not inactivated by neutral red and light in the absence of cells, but virus grown in cells containing neutral red may be inactivated by light, indicating that an incorporation of the dye into developing poliovirus may occur. We have also shown in this paper that acridine orange yields similar results, and in an associated study, Mayor and Diwan (1961) have obtained direct microscopic evidence of the incorporation of the dye into the virus. Ledinko (1958) demonstrated the cytopathogenicity of poliovirus to be unaffected by proflavine, and we have observed that the same is true using neutral red, unless near-toxic levels of the dye are used. Both proflavine and neutral red are capable of inhibiting the production of infective poliovirus even in subdued lighting. It is not
20
CROWTHER
known as yet exactly what part the dye alone and what part added light play in the production of noninfective virus. In the experiments of Ledinko (1958) and of Schaffer (1960)) virus produced in the presence of proflavine, and shown by Schaffer (1960) to contain the dye, was largely noninfective, but laboratory lighting was not excluded. Schaffer (unpublished) has now found that proflavine in the dark produces only slight inhibition in the production of infective poliovirus. The action of proflavine is generally thought to be on maturation alone (Ledinko, 1958). The basic nature of the dye would seem to support this contention, the positively charged molecule combining with the nucleic acid and inhibiting the proper aggregation of the nucleic acid with its protein coat. Our experiments with neutral red and the similarities noted between the acridines and quinonimide dyes suggest the production of noninfective virus by these dyes is not mainly due to a prohibition of the aggregation of nucleic acid with its protein coat, but rather to a subsequent inactivation of an infective virus containing the active dye. This is supported by the fact that electron micrographs of poliovirus grown in the presence of proflavine are not significantly different from controls; however, the infectivity of the proflavine virus was considerably lower (Horne and Nagington, 1959). Probably the former mechanism does play some part in the production of noninfective virus. Also, it is likely that some virus particles containing dye together with protein around their nucleic acid will be noninfective even without light. The experiments performed with proflavine and other acridines as viral inhibitors have been carried out, until recently, without reference to the part that light may play in this inhibition. Eaton et al. (1951) demonstrated the inhibition in production of infective virus by certain dyes of the acridine group. Inhibition was found to be effective against meningopneumonitis, feline pneumonitis, mumps, and influenza viruses. These workers came to the conclusion that the inhibition of virus was not caused by the action of the dye on extracellular virus because very little inactivation oc-
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curred in the absence of cells. They did not consider the possibility of incorporation of the dyes into the virus particle and its subsequent inactivation by light. Our recent experiments as described in this paper have shown that although acridine orange can reduce somewhat the yields of infective poliovirus grown on monkey kidney cells in the dark, a considerable further reduction may be obtained by exposure of the harvested virus to white light. The variable amounts of inhibition due to the active dyes, obtained in different laboratories, and also the variable results of experiments done in the same laboratory (for example, the earlier neutral red experiments in our own laboratory) may now be explained by the previously unconsidered part that light can play in the production of infective virus. A quantitative comparison between the effects of the different active dyes cannot be made at present since the conditions under which the experiments have been conducted in different laboratories vary. REFERENCES ACKERMANN,W. W., LOH, P. C., and PAYNE, F. E. ( 1959). Studies of the biosynthesis of protein and ribonucleic acid in HeLa cells infected with poliovirus. Virology 7,179-183. DARNELL,J. E., JR., LOCKHART,R. Z., JR., and SAWYER, T. K. (1958). The effect of neutral red on plaque formation in two virus-cell systems. Virology 6,567-568. EATON,M. D., CHEEVER,F. S., and LEVENSON,C. G. (1951). Further observations of the effect of acridines on the growth of viruses. J. Immunol. 66,463-476. GREEN,R. H., and OPTON,E. M. (1959). Photosensitization of tissue culture cells and its effect on viral plaque formation. Proc. Sot. Ezpt2. Biol. Med. 102, 519-521. HELPRIN, J. J., and HIATT, C. W. (1959). Photosensitization of T2 coliphage with toluidine blue. J. Bacterial. 77, 502-565. HIATT, C. W., KAUFMAN, E., HELPRIN, J. J., and BARON, S. (1960). Inactivation of viruses by photodynamic action of toluidine blue. J. Immunol. 84,48@484. HORNE,R. W., and NAGINGTON,J. (1959). Electron microscope studies of the development and structure of poliomyelitis virus. J. Mol. Biol. 1, 333338. HSIUXG, G. D., and MELNICK, J. L. (1955). Plaque formation with poliomyelitis, Coxsackie, and
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orphan (Echo) viruses in bottle cultures of monkey epithelial cells. Virology 1, 533-535. HSIUNG, G. D., and MELNICK, J. L. (1957). Comparative susceptibility of kidney cells from different monkey species to enteric viruses (poliomyelitis, Coxsackie and ECHO groups). .I. Immunol. 78, 137-146. KLEIN, S. W., and GOODGAL,S. H. (1959). Photodynamic inactivation of monkey kidney cell monolayers. Science 130,629. LEDINKO, N. (1958). Production of noninfectious complement-fixing poliovirus particles in HeLa cells treated with proflavine. Virology 6, 512524. LOGRIPPO,’G., and BASINSKI, D. H. (1960). The value of acridine orange for sterilizing viruses in plasma and whole blood. Pederation Proc. 19, 56. MAYOR, H. (1961). Cytochemical and fluorescent antibody studies on the growth of poliovirus in tissue culture. l’exas Repts. Biol. and Med. 19, 106-122. MAYOR, H. D., and DIWAX, A. R. (1961). Studies on the acridine orange staining of two purified
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RNA viruses: poliovirus and tobacco mosaic virus. Virology 14,7482. MCCLAIN, M., and HACKETT, A. J. (1958). A comparative study of the growth of vesicular stomatitis virus in five tissue culture systems. J. Immunol. 80,356361. MELNICK, J. L. (1956). Tissue culture methods fat the cultivation of poliomyelitis and other viruses. In “Diagnostic Procedures for Virus and Rickettsial Diseases,” 2nd ed., pp. 97-152. Am. Publ. Health Assn., New York. OPTON, E. M., and GREEN, R. H. (1960). Effects of neutral red and light on polioviruses. Federation Proc. 19, 408. SCHAFFER, F. L. (1960). The nature of noninfectious particles produced by poliovirus infected tissue cultures treated with proflavin. Federation Proc. 19, 405, and personal communication. WATERSON, A. P. (1959). Effect of neutral red in plaque formation of fowl plague virus. Nature 183,628. YAMAMOTO,N. (1958). Photodynamic inactivation of bacteriophage and its inhibition. J. Bacterial. 75,443448.