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Photodynamic inactivation of poliovirus
VIROLOGY
21,
332-341 (1963)
Photodynamic CRAIG Department of Virology
Inactivation
WALLIS
JOSEPH
.4ND
and Epide,miology, Houston, Accepted
Baylor Texas
of Poliovirus’ L. MELNICK University
College of Medicine,
July 8,196s
Poliovirus, as usually found in fluids of infected cultures, cannot be inactivated photodynamically in the presence of heterotricyclic dyes. However, if the mature virus is freed of extraneous organic material, dyes can attach to the virus and render it light-sensitive. Optimal conditions for photosensitivity of poliovirus were found to be pH 8.0 in phosphate buffer in lo-’ M dye (neutral red, toluidine blue, or proflavine). Organic buffers (Tris and glycine) prevent photosensitization of poliovirus. When the pH is lowered, the photosensitive virus-dye complex appears to dissociate and the free virus again becomes light-resistant. A cation exchange column adsorbs most of the virusdye complex. That part of the virus which dissociates from the dye on the column is no longer photosensitive. The attachment of dye to vaccinia virus is also reversible, and virus resistant to light can be obtained by chromatographic adsorption of dye from the virus-dye complex. INTRODUCTION
the present study we have found that under certain conditions dye may be bound reversibly to mature virus, making the virus photosensitive.
Several investigators have reported that polioviruses and other enteroviruses are resistant to inactivation by heterotricyclic dyes in the presence of white light (Ledinko, 1958; Hiatt et al., 1960; Opton and Green, and Basinski, 1960; 1960 ; LoGrippo Crowther and Melnick, 1961; Schaffer, 1962). However, if poliovirus is grown in the presence of such dyes, the dye is incorporated into the virus during replication; upon exposure to light, loss of infectivity results (Crowther and Melnick, 1961; Mayor and Diwan, 1961; Schaffer, 1962). On the other hand, several viruses, even
MATERIALS
Monkey kidney (MK) cells. Kidneys from immature rhesus monkeys were trypsinized and grown in M-H la&albumin medium containing 0.2 mM AIC& to suppress adventitious agents and then maintained in M-E medium (Wallis and Melnick, 1962a). Viruses. Virulent strains of poliovirus were plaque-purified lines of type 1, Mahoney; type 2, MEFl; and type 3, P24. Attenuated strains were Sabin’s plaque-purified lines as used in the oral poliovaccine (type 1, LSc; type 2, P712; and type 3, Leon). The WR strain of vaccinia virus was used. All viruses were grown in MK cells maintained in M-E medium, unless otherwise specified in the detailed description of experiments. Virus assays. All virus titrations were
when grown in the absence of dyes, are readily sensitized to photoinactivation. These include members of the adeno-, arbo-, papova-, pox-, and reovirus groups (Hiatt, 1960; Tomita and Prince, 1963). In l Aided tion, and stitute of Institutes Service.
AND METHODS
by a grant from The National Foundaby grant AI-05382 from the National InAllergy and Infectious Diseases, National of Health, United States Public Health 332
INACTIVATION
OF POLIOVIRUS
carried out by determination of plaqueforming units (PFU) using bottle cultures. For poliovirus, MgClz at a concentration of 25 mM was incorporated into the overlay medium to expedite plaque counting (Wallis and Melnick, 196213). All titers are given in logarithms to the base 10. Exposure to light. Virus samples were dispensed into new 13 x loo-mm lime glass tubes having an 0.8-mm wall. Samples were exposed 2 inches from 2 “daylight” type fluorescent lighting tubes (General Electric, Cool White, 15 watts each). At this distance, the temperature of samples increased from an initial temperature of 25” to 30” within 5-10 minutes, but never exceeded 35” even after exposures of over an hour. Up to 20 virus samples could be exposed simultaneously to uniform lighting in a single experiment. Tubes were placed in a rack and backed with aluminum foil for maximum reflection. Virus controls, in the presence or absence of dye, were wrapped in foil to keep them dark and were placed in the rack in locations similar to those of the samples being exposed to light. All virus samples were held in the dark as much as possible when light was not purposely being applied. Thus, experiments were performed and virus was titrated in darkened rooms to minimize the effect of extraneous light. Buffers. Sorensen’s pH 8.0 buffer (0.005 M NaH2P04 and 0.095 M Na2HP04) was used throughout these studies. Other buffers used were glycine (0.05 M) adjusted to pH 4-6 with HCl and to pH 8-9 with NaOH ; phosphate buffer (0.1 M) for pH 6-8; and Tris buffer for pH 8-10. Dyes. These were certified grade (Wational Aniline). They were dissolved in distilled water to make a lop2 M stock (calculated on the basis of an 84% dye content) and sterilized by boiling for 10 minutes. In the alkaline range, neutral red formed a precipitate at concentrations of 1O-3 M. Therefore all dye stocks were diluted to lop3 M in distilled water, and then diluted further in buffer at the pH required for the test. No precipitates formed under these conditions. Exchange resins. Dowex l-X8 (Cl-),
BY LIGHT
333
200400 mesh. This anion exchange resin was prepared by washing repeatedly with phosphate buffer at pH 8 and then was autoclaved. Upon cooling, the resin was washed further with sterile pH 8 buffer until the filtrate obtained was at pH 8. The columns of cation exchange resin, Dowex 50 W-X4 (H+), 50-100 mesh, were prepared in the same way. Excess fluid was removed from columns by positive nitrogen pressure, and virus samples were then passed through the column. RESULTS
The Effect of pH on Photosensitivity Mature Type 1 Poliovirus
of
The earlier experiments on polioviruses were repeated and confirmed-tissue culture fluids containing poliovirus could not be photosensitized. However, when the virus fluids were first dialyzed at 4” for 24 hours against distilled water, and the solution was made alkaline, the virus could be made photosensitive. The details of such experiments are given below. Type 1 (Mahoney) virus having a titer of 108.0 PFU/ml, was dialyzed and diluted lOOO-fold to contain 105.0 PFU/ml in 1O-4 M neutral red from pH 4 to 9. A duplicate series of samples was made in buffers without dye. Samples were exposed to light for 1 hour. The results of two experiments are plotted in Fig. 1. The dark control samples containing virus-dye mixtures and the dyefree samples exposed to light failed to manifest any detectable loss of infectivity and are not plotted in this figure. The virus-dye mixtures exposed to light at pH 4.0-6.0 showed no significant loss of infectivity. However, in 0.1 M phosphate buffer at pH 7.0 the infectivity titer decreased by 1.2 log; at pH 7.5 by 3.0 log; and at pH 8.0, complete inactivation occurred. In the organic buffers (Tris and glycine) at pH 8.0 or higher, only a slight drop in titer occurred (0.1-0.4 log). Thus the organic buffers seemed to prevent the attachment of neutral red to virus, or its penetration into the virus particle. As shown in Fig. 1, pH 8 was optimum for photosensitivity. To determine whether
334
WALLIS
AND
MELNICK
0 EXP. I o EXP. 2
5 1 4
I
I 5
I
I
6
7
, 6
PH FIG. 1. Effect of pH on photodynamic inactivation of type 1 poliovirus. Virus was dialyzed and then diluted to contain 10’ PFU/ml in 10m4M neutral red at different pH levels. Samples were exposed to white light for 1 hour at 2532°C. Control samples in neutral red kept in the dark and samples in dye-free buffers, exposed to light, showed no detectable loss of infectivity and are not plotted.
the virus could be liberated from the dye by changing pH, the following experiment was performed: dialyzed type 1 virus (lo8 PFU/ml) was diluted loo-fold in lop4 neutral red at (A) pH 6.0 and (B) 8.0. Samples were held at 4” for 15 minutes and then samples A and B were each further diluted tenfold in 10e4 M neutral red at (C) pH 6.0 and (D) pH 8.0. All four samples (A + C) (B + C) (A -+ D) and (B + D) were then immediat,ely exposed to light for 1 hour. The results are shown in Table 1. From these results it appears that the virusdye complex formed at pH 8 is readily dissociable on lowering the pH to 6. The virusdye mixture formed and exposed to light at pH 8 was completely inactivated, while an aliquot of this virus-dye mixture shifted to pH 6.0 and then exposed to light did not lose titer. The virus-dye mixture initially
TABLE
1
EFFECT OF PH ON PHOTOSENSITIVITY OF TYPE 1 POLIOVIRUSQ Initial pH adjustment after dialysis of virus
pH during exposure to light
Loglo virus titer (PFU/ml) Exposed to light
Held in dark
PH 6 (-4
G (Cl 8 CD)
4.5 0.0
4.9 4.8
PH 8 03)
6 (Cl 8 CD)
4.6 0.0
4.8 5.0
a Dialyzed Mahoney virus (lo8 PFU/ml) was diluted loo-fold in 10e4 M neutral red at pH 6.0 and at pH 8.0. These samples were held at 4°C for 15 minutes, then were diluted tenfold in 1OW M neutral red at pH (C) 6.0 and (D) 8.0, and immediately exposed to light for 1 hour.
INACTIVATION
OF POLIOVIRUS
at pH 6 remained photoresistant when held at this pH but became extremely photosensitive when the pH was raised to 8. Effect of Tissue Culture Constituents Photoinactivation of Poliovirus
on
Significant photosensitization as described above occurred only if the virus harvests were first dialyzed and then diluted at least loo-fold in the dye using phosphate buffer at pH 8. This indicated that tissue culture harvests contained both dialyzable and nondialyzable constituents that interfered with the attachment of dye to the virus particle. The effect of extraneous material present in the harvest was investigated as follows: Cultures were inoculated with type 1 Mahoney virus at an input of 5-10 PFU per cell. After an adsorption period of 2 hours at 37”, the cells were washed with buffered saline, and then half were changed with M-E lactalbumin hydrolyzate medium and half were changed with Earle’s salt solution (ESS) to reduce the organic constituents in the harvest. After 7 hours’ additional incubation at 37”, all cultures manifested extensive cytopathic changes. They were harvested by freezing and thawing, and the suspension was clarified by centrifugation at 2000 rpm for 15 minutes. A sample of the M-E harvest was dialyzed overnight at 4” against distilled water. A sample of the ESS harvest was passed through an anion exchange column. The virus titer of the M-E harvest was 1O8.5PFU/ml; the M-E dialyzed sample, 1O8.3; the ESS harvest, 10s,4; and the filtrate of the ESS harvest that had been passed through the resin column was 108.2. Each of the samples described was diluted to contain 10s, 107, 106, 105, and lo4 PFU/ml in 1O-4 M neutral red at pH 8.0 and in dye-free buffer at pH 8. Samples were then exposed to light for 1 hour and duplicates were held in the dark. The results are plotted in Fig. 2. Control samples held in the dark and dye-free samples exposed to light showed no loss of infectivity and are not plotted. The sample derived from the M-E harvest showed significant photosensitivity
BY LIGHT
335
only after the virus had been first diluted lOOO-fold. At this dilution, lo5 PFU/ml were present and there was a 2.5 log loss in titer after exposure to light. In the sample diluted lO,OOO-fold, complete photosensitization occurred. On the other hand, the dialyzed M-E harvest could be inactivated after being diluted only lOO-fold in neutral red (2.2 log loss in titer) and complete photosensitization (5 log loss) of the virus was accomplished with the virus diluted lOOOfold. As shown in Fig. 2, the harvest made in salt solution (ESS) had an inactivation curve similar to that of the dialyzed M-E harvest, which suggests that the amino acids and other dialyzable ingredients of the M-E medium interfere with photoinactivation. The anion resin exchange was able to adsorb interfering substances present in the ESS harvests, and yielded the most sensitive virus. When lo* PFU/ml of resinpurified virus was mixed with dye and exposed to light, 1.7 log reduction in titer was achieved. With a tenfold dilution of the virus (to contain lo7 PFU/ml in dye), 4 log reduction in titer resulted, and upon a loo-fold dilution, complete inactivation occurred (6.0 log loss). A series of experiments was carried out to determine which ingredients of the M-E medium prevented photosensitization. The details of a typical experiment follow. Mahoney virus purified by passage through the anion resin was diluted to contain lo5 PFU/ml in pH 8.0 buffer, and 2.4-ml samples were dispensed. One part (0.3 ml) of the various components that constitute M-E medium (as listed in Table 2) was added to 8 parts (2.4 ml) of virus sample, and stored at 4” for 15 minutes. Then 1 part (0.3 ml) of low3 M neutral red was added to each sample, so that the final concentration of dye was 10v4 M. All tubes were further incubated at 4” for 15 minutes and then exposed to light for 1 hour. A duplicat,e series of samples of virus was treated wit’h neutral red before the addition of each of t,he M-E components. The results of this experiment are shown in Table 2. When either lactalbumin hydrolyzate, phenol red, glucose, or monkey kidney cell extract was
WALLIS
336
108
AND MELNICK
Id
107
105
IO’
CONCENTRATION
OF VIRUS (PFWml)
EXPOSED
IN
TO LIGHT
IO-‘M
NEUTRAL
RED
FIG. 2. Effect of tissue culture constituents on photosensitivity of type 1 poliovirus. Virus harvest deriving from MK cells maintained in lactalbumin hydrolyzate (M-E) medium and Earle’s salt solution (ESS) were diluted to contain IO*, IO’, lo’, lo’, and lo4 PFU/ml in IO-’ M neutral red in phosphate buffer at pH 8. A dialyzed aliquot of the M-E harvest and a sample of the ESS harvest passed through a Dowex anion exchange column were also diluted as described above. All samples were exposed to light for 1 hour. Control samples with dye in the dark showed no loss in infectivity.
mixed with the virus before addition of neutral red, photosensitization was partially or complet,ely prevented. However, if neutral red were added to the virus first, the virus sample was completely inactivated when exposed to light, regardless of what was added later. Effect of Different Concentrations of Neutral Red on Pho toinactiva tion of Poliovirus Resin-purified virus was diluted to contain lo5 PFU/ml in different concentrations of neutral red in phosphate buffer at, pH 8.0. Samples were exposed to light for 60
minutes. The results of this experiment are shown in Fig. 3. No photosensitization occurred in virus samples containing 10-4.g M neutral red or less. At 10e4.” M, the titer was decreased by 1.5 log, and at 10-4.3 M, by 3.7 log. At 1O-4 M, complete loss of infectivity occurred. A repeat experiment showed essentially the same first-order inactivation kinetics. Effect of Time on Photoinactivation of Type 1 Poliovirus The resin-purified virus was diluted as described above in lo-” M neutral red at pH 8.0. Samples were then exposed to light
INACTIVATION
OF POLIOVIRUS TABLE
337
BY LIGHT
2
EFFECT OF INDIVIDUAL COMPONENTS OF M-E MEDIUM AND OF MONKEY EXTRACT ON PHOTOSBNSITIVITY OF TYPE 1 POLIOVIRUS”
KIDNEY
CELL
LogiD virus titer (PFU/ml) Components added to virus samples
Component added before neutral red*
Component added after neutral red=
Exposed to light”
Held in dark
Exposed to lighta
Held in dark
0.0 5.0 1.5
4.8 4.9 5.1
0.0 0.0 0.0
4.9 5.2 5.2
3.5 0.0 0.0
5.0 4.8 5.0
0.0 0.0 0.0
4.8 5.1 5.1
3.0 5.0
5.3 4.9
0.0 0.0
5.0 5.0
Phosphate buffer, pH 8.0 0.5% Lactalbumin hydrolyzate 100 Units penicillin + IO0 pg streptomycin/ ml O.O02$7oPhenol red 0.22% NaHC03 Earle’s salt solution without glucose and phenol red 0.1% Glucose Soluble fraction of ruptured kidney cellsd
(1Dialyzed virus was diluted to contain lo5 PFU/ml in pH 8 buffer. b One part of component at the concentration described above was mixed with 8 parts of virus (106 PFU/ml in buffer) and incubated at 4°C for 15 minutes. Then, 1 part of 1O-3 M neutral red was added to 9 parts of the virus-component mixture. Samples were stored at 4” for 15 minutes, and then exposed to light for 1 hour. c One part of IO-3iM neutra1 red was mixed with 8 parts of virus (105PFU/ml in buffer) and incubated at 4” for 15 minutes. Then, 1 part of the indicated M-E component or cell fraction was added to 9 parts of the virus-dye mixture. Samples were stored at 4” for 15 minutes, and then exposed to light for 1 hour. d Normal MK monolayers in bottles containing about lo6 cells were washed repeatedly with buffer and then changed to 2 ml of buffer (pH 8.0). Cultures were frozen and thawed 3 times and cell debris for virus samples. sedimented at 2000 rpm. The supernatant fluid was used as an additive 6 All samples exposed to light were at pH 8.
for 0, 5, 15, 30, and 60 minutes. The results of this experiment, plotted in Fig. 4, again show that virus was inactivated exponentially as a first-order reaction. Eject
of Distance of Light Source on Photoinactivation Resin-purified virus was diluted to contain lo5 PFU/ml in 10h4 M neutral red at pH 8.0. Samples were exposed to light for 60 minutes at distances of 2, 5, and 10 inches. The results of this experiment are shown in Table 3. After exposure at a distance of 2 inches, no infectivity was detectable; at 5 inches, 2.2 log titer was lost; and at 10 inches only 0.7 log loss of infectivity titer occurred. Efiect of Incubation of Virus-Dye Mixtures on Photosensitivity of Type I Poliovirus The experiments described above were accomplished without incubation of virus-
dye mixtures before light exposure (unless specified). An experiment was performed to determine whether more efficient photosensitization resulted by prior holding of the virus-dye mixtures at 4” or at 37”. Type 1 virus (resin-purified) was diMed to contain lo5 PFU/ml in 10W4 M neutral red at pH 8.0. Samples were placed at 4” and 37” in the dark for 0 and 4 hours. Samples were then exposed to light for 30 minutes. No significant difference in degree of inactivation was observed between virus-dye samples exposed to light without prior incubation and those held at 4” and 37” for 4 hours before exposure to light (loss of 3.5 -C 0.5 log titer). Effect of Other Photodynamic Type I Poliovirus
Dyes
on
Resin-purified virus was diluted to contain lo5 PFU/ml in lOA M of (a) neutral red, (b) acridine orange, (c) methylene
WALLIS
338
A_\D MELNICK
l EXR I 0 EXP. 2
5’
10-4.6
10-4.3
MOLARITY
NEUTRAL
10-4.9
FINAL
10-4.0
RED
3. Effect of different concentrations of neutral red on photosensitivity of type 1 poliovirus. Virus was purified by passage through an anion exchange column and diluted to contain 10’ PFU/ml in different concentrations of neutral red at pH 8. Samples were exposed FIG.
to light for 1 hour. blue, (d) o-toluidine blue, and (e) proflavine at pH 8.0. Samples were exposed to light for 1 hour. The results of this experiment are shown in Table 4. All the dyes used, except acridine orange, completely sensitized poliovirus to light. In the case of acridine orange, only 1.5 log virus titer was lost. Effect of Neutral Red on Photosensitivity of Other Polioviruses Types 1, 2, and 3 poliovirus (attenuated and virulent) in 0. l-ml volumes were seeded onto drained monolayer cultures ( lo6 cells per bottle) so that the input of virus was 2-4 PFU/cell. After 2 hours’ adsorption at
0 MINUTES
15
30
EXPOSED
TO LIGHT
60
FIG. 4. Effect of different exposure times on photosensitivity of type 1 poliovirus. Purified virus was diluted to contain loj PFU/ml in IO-* neutral red at pH 8. Samples were exposed to light for times indicated.
INACTIVATION TABLE 3 EFFECT OF DISTANCE OF LIGHT SOURCE ON PHOTOSENSITIVITY OF TYPE 1 POLIOVIRUB
Distance from light source (inches)
Log10 virus titer (PFU/ml)
2 5 10
0.0 3.0 4.5
Control
5.2
in dark
a Purified virus was diluted to contain lo5 PFU/ ml in 10s4 M neutral red at pH 8.0, and samples were exposed to light for 1 hour at distances shown.
TABLE THE
4
EFFECT OF OTHER PHOTODYNAMIC DYES ON TYPE 1 POLIOVIRUS”
Log10 virus titer (PFU/ml) Dye
Neutral red Acridine orange Methylene blue Toluidine blue Proflavine None
339
OF POLIOVIRUS BY LIGHT
Exposed to light
Held in dark
0.0 3.5 0.0 0.0 0.0 5.0
5.2 5.0 5.0 5.0 5.1 5.3
a Type 1 (resin-purified) Mahoney virus was diluted to contain lo5 PFU/ml in 10e4 M dye. Samples were exposed to light for 1 hour. Control virus samples in dye were wrapped in aluminum foil to keep them in the dark.
37”, cultures were washed with M-E medium and changed to 4 ml of M-E medium. All cultures manifested marked cytopathic changes between 8 and 16 hours and were frozen. Harvests were thawed and centrifuged at 2000 rpm for 10 minutes, and supernatants were collected. Each virus was then diluted lO,OOO-fold in 10W4 M neutral red or dye-free buffer at pH 8.0 to reduce the concentration of tissue culture ingredients. Samples were then exposed to light for 1 hour. The results of this experiment are shown in Table 5. All six viruses were significantly inactivated.
Photosensitivity of Poliovirus That of Vaccinia Virus
Compared to
Hiatt et al. (1960) showed that vaccinia virus, unlike enteroviruses, could be readily inactivated in toluidine blue in the presence of light. In view of our finding that purified mature poliovirus can also be made photosensitive, we investigated the reversibility of the binding of dye to virus. For this, cation exchange columns were used, as they readily adsorb basic dyes. A number of experiments were performed, all of which yielded similar findings. One such experiment is reported in detail below. Type 1 poliovirus and vaccinia virus were each diluted to contain lo5 PFU/ml in 10W5 M toluidine blue and in dye-free phosphate buffer at, pH 8. Samples were incubated at 4” for 1 hour, and then exposed to light at 2532” for 1 hour. A duplicate series of samples stored in the dark at 4” for 1 hour were passed through cation exchange colTABLE EFFECT OF NEUTRAL TYPES 1, 2, AND
5 RED AND LIGHT 3 POLIOVIRUSES”
ON
-
1Log10 virus titer (PFU ml) Virus strain
II 1
Ekpt. no.
neutral
reC
-7
Dye-free
d:k
EX- I Held Nosed to d:k ight
2
3.5 3.8
3.6 3.6
3.4 3.7
EX1wsed to 1light
Held
-
Attenuated Type 1, LSc
1
Type
2, P712
3
3.6
3.5
3.3
Type
3, Leon
4 5
3.0 2.9
3.1 3.5
3.0 3.2
1, Mahoney
6 7
Type
2, MEFl
8
3.8
3.8
Type
3, Saukett
Virulent Type
9 -
3.8 -
a M-E virus harvests were diluted lO,OOO-fold in 1OW M neutral red or in dye-free buffer at pH 8. Samples were exposed to light for 1 hour. Dark controls w-ere wrapped in aluminum foil.
340
WALLIS VACCINIA
Toluidine
AND
POLIOVIRUS TYPE 1
VIRUS
blue
Li~L$i~;:
:;;gfifip;:
Li.jy.
Li&rk 4.5
4.3
Cation
Cation
cation
Cation
4.5
MELNICK
Lifirk 4.6
3.8
Ligfirk
FIG. 5. Reversibility of binding of dye by poliovirus and vaccinia virus. Results pressed as log titers (PFU/ml). The details of the experiment are given in the text.
umns to yield water-clear filtrates. They were then exposed to light for 1 hour. Proper dark controls were employed. The results of this experiment are shown in Fig. 5. The vaccinia virus sample in toluidine blue kept in the dark, the sample in dyefree buffer exposed to light, and the sample in dye-free buffer kept in the dark, all had similar titers (10j.2, 105.2, and 105.3 PFU/ ml, respectively). This indicated that the dye at the level used was not toxic to the virus if light were excluded and that dyefree virus exposed to light was not inactivated. However, the sample of vaccinia virus in toluidine blue exposed to light was completely inactivated. The sample of vaccinia virus in toluidine blue that had been passed through t.he cation exchange column yielded a dye-free filtrate. When exposed to light, this sample had the same titer as the corresponding sample kept in the dark (104.5 PFU/ml). Since the virus was no longer sensitive to light when the dye was removed, vaccinia virus exposed to t,oluidine blue did not take up the dye irreversibly. Vaccinia virus in dye-free buffer had similar titers when exposed to light or when kept in the dark. Poliovirus in toluidine blue in the dark, or in dye-free buffer in the dark or exposed to light, yielded similar titers (104.s, 105.0, and 105.1, respectively). Thus, as with vac-
5.3
5.1
4.0
are ex-
cinia, the dye at the level used was not toxic to poliovirus unless exposed to light. The sample of poliovirus in toluidine blue that was passed through the cation exchange column yielded a clear filtrate that was no longer photosensitive. The titer of the dark control was 104.0 PFU/ml, that of the sample exposed to light, 103,s. Thus, it is apparent that toluidine blue may also be reversibly bound to poliovirus. However only 10% of free virus was recovered in the filtrate, and 90% remained on the column bound to the dye. In contrast, when poliovirus in dye-free buffer at pH 8 was passed through the exchange column, no detectable adsorption of virus to the column occurred. Thus the poliovirus-dye complex seems t.o be more firmly bound than t’he vaccinia virus-dye complex. DISCUSSION
Earlier studies (Hiatt, 1960; Hiatt et al., 1960; Opton and Green, 1960; LoGrippo and Basinski, 1960; Crowther and Melnick, 1961) showed that mature polioviruses grown in the absence of photosensitive dyes could not be inactivated in dyes when exposed to light. Experiments described in this report show that intact polioviruses can be photodynamically inactivated under the proper conditions. We have shown that pH 8.0 is critical for photosensitization of poliovirus and that organic components
INACTIVATION
OF POLIOVIRUS
such as those present in tissue culture constituents (both dialyzable and nondialyzable) prevent photosensitivity. Phosphate buffer was the diluent of choice for the virus-dye mixtures, since organic buffers prevented photoinactivation. Most attempts to inactivate mature poliovirus in the presence of a photosensitive dye have been made with crude virus harvests. However, Schaffer (1962) used purified virus grown in the absence of dye and still failed, because the importance of pH control was not known at the time. Also, as he observed, the high concentration of proflavine used (100 pg/ml) might actually have prevented the light from penetrating. In his experiments, added proflavine at this concentrat,ion actually protected virus grown in the presence of the dye from phot.oinactivation. The earlier results on photoresistance of poliovirus and photosensitivity of larger RNA and DNA viruses in the presence of dye has led to speculation that permeable pores may be present in DNA viruses but not in polio and other enteroviruses (Hiatt, 1960; Mayor et al., 1963). However, in view of our present results showing that polioviruses under proper conditions can be rendered as photosensitive as vaccinia virus, the mechanism by which dyes are bound to and dissociated from viruses will have to be investigated further. ADDENDUM Since this manuscript was submitted, we have tested 10 echoviruses and 5 coxsackieviruses (4 group B strains, and 1 group A strain) for photodynamic inactivation in the presence of toluidine blue or neutral red. The results were similar to those reported above for the polioviruses.
341
BY LIGHT REFERENCES
CROWTHER,D., and MELNICK, J. L. (1961). The incorporation of neutral red and acridine orange into developing poliovirus particles making them photosensitive. Virology 14, 11-21. HIATT, C. W. (1960). Photodynamic inactivation of viruses. Trans. N.Y. Acad. Sci. 23, 66-78. HIATT, C. W., KAUFMAN, E., HELPRIN, J. L., and BARON,S. (1960). Inactivation of viruses by photodynamic action of toluidine blue. J. Immunol.
84,48&484. LEDINKO, N. (1958). Production complement-fixing cells treated with
poliovirus proflavine.
of noninfectious particles in HeLa Virology 6, 512-
524. LOGRIPPO,G., and BASINSKI, D. H. (1960). The value of acridine orange for in plasma and whole blood.
sterilizing viruses Federation Proc.
19,56.
D., and DIWAN, A. R. (1961). Studies on the acridine orange staining of two purified RNA viruses: poliovirus and tobacco mosaic virus. Virology 14, 74-82. MAI’OR, H. D., JAMISON,R. M., and DIWAN, A. R. (1963). Studies on viral replication using acridine orange as a vital dye. Submitted to Virology. OPTON,E. M., and GREEN,R. H. (1960). Effects of neutral red and light on polioviruses. Federation MAYOR, H.
Proc.19,408. SCHAFFER,F. L. (1962). Binding
of proflavine by and photoinactivation of poliovirus propagated in the presence of the dye. Virology 18, 41%
425. TOMITA, Y., and PRINCE, A. M.
(1963). Photodynamic inactivation of arbor viruses by neutral red and visible light. Proc. Sot. Ezptl. Biol. Med.
112,887-890. WALLIS, C., and MELNICK, J. L. (1962a). Suppression of adventitious agents in monkey kidney cultures. Texas Rept. Biol. Med. 20, 465-475. WALLIS, C., and MELNICK, J. L. (1962b). Magnesium chloride enhancement of cell susceptibility to poliovirus. Virology 16, 122-132.
21,
332-341 (1963)
Photodynamic CRAIG Department of Virology
Inactivation
WALLIS
JOSEPH
.4ND
and Epide,miology, Houston, Accepted
Baylor Texas
of Poliovirus’ L. MELNICK University
College of Medicine,
July 8,196s
Poliovirus, as usually found in fluids of infected cultures, cannot be inactivated photodynamically in the presence of heterotricyclic dyes. However, if the mature virus is freed of extraneous organic material, dyes can attach to the virus and render it light-sensitive. Optimal conditions for photosensitivity of poliovirus were found to be pH 8.0 in phosphate buffer in lo-’ M dye (neutral red, toluidine blue, or proflavine). Organic buffers (Tris and glycine) prevent photosensitization of poliovirus. When the pH is lowered, the photosensitive virus-dye complex appears to dissociate and the free virus again becomes light-resistant. A cation exchange column adsorbs most of the virusdye complex. That part of the virus which dissociates from the dye on the column is no longer photosensitive. The attachment of dye to vaccinia virus is also reversible, and virus resistant to light can be obtained by chromatographic adsorption of dye from the virus-dye complex. INTRODUCTION
the present study we have found that under certain conditions dye may be bound reversibly to mature virus, making the virus photosensitive.
Several investigators have reported that polioviruses and other enteroviruses are resistant to inactivation by heterotricyclic dyes in the presence of white light (Ledinko, 1958; Hiatt et al., 1960; Opton and Green, and Basinski, 1960; 1960 ; LoGrippo Crowther and Melnick, 1961; Schaffer, 1962). However, if poliovirus is grown in the presence of such dyes, the dye is incorporated into the virus during replication; upon exposure to light, loss of infectivity results (Crowther and Melnick, 1961; Mayor and Diwan, 1961; Schaffer, 1962). On the other hand, several viruses, even
MATERIALS
Monkey kidney (MK) cells. Kidneys from immature rhesus monkeys were trypsinized and grown in M-H la&albumin medium containing 0.2 mM AIC& to suppress adventitious agents and then maintained in M-E medium (Wallis and Melnick, 1962a). Viruses. Virulent strains of poliovirus were plaque-purified lines of type 1, Mahoney; type 2, MEFl; and type 3, P24. Attenuated strains were Sabin’s plaque-purified lines as used in the oral poliovaccine (type 1, LSc; type 2, P712; and type 3, Leon). The WR strain of vaccinia virus was used. All viruses were grown in MK cells maintained in M-E medium, unless otherwise specified in the detailed description of experiments. Virus assays. All virus titrations were
when grown in the absence of dyes, are readily sensitized to photoinactivation. These include members of the adeno-, arbo-, papova-, pox-, and reovirus groups (Hiatt, 1960; Tomita and Prince, 1963). In l Aided tion, and stitute of Institutes Service.
AND METHODS
by a grant from The National Foundaby grant AI-05382 from the National InAllergy and Infectious Diseases, National of Health, United States Public Health 332
INACTIVATION
OF POLIOVIRUS
carried out by determination of plaqueforming units (PFU) using bottle cultures. For poliovirus, MgClz at a concentration of 25 mM was incorporated into the overlay medium to expedite plaque counting (Wallis and Melnick, 196213). All titers are given in logarithms to the base 10. Exposure to light. Virus samples were dispensed into new 13 x loo-mm lime glass tubes having an 0.8-mm wall. Samples were exposed 2 inches from 2 “daylight” type fluorescent lighting tubes (General Electric, Cool White, 15 watts each). At this distance, the temperature of samples increased from an initial temperature of 25” to 30” within 5-10 minutes, but never exceeded 35” even after exposures of over an hour. Up to 20 virus samples could be exposed simultaneously to uniform lighting in a single experiment. Tubes were placed in a rack and backed with aluminum foil for maximum reflection. Virus controls, in the presence or absence of dye, were wrapped in foil to keep them dark and were placed in the rack in locations similar to those of the samples being exposed to light. All virus samples were held in the dark as much as possible when light was not purposely being applied. Thus, experiments were performed and virus was titrated in darkened rooms to minimize the effect of extraneous light. Buffers. Sorensen’s pH 8.0 buffer (0.005 M NaH2P04 and 0.095 M Na2HP04) was used throughout these studies. Other buffers used were glycine (0.05 M) adjusted to pH 4-6 with HCl and to pH 8-9 with NaOH ; phosphate buffer (0.1 M) for pH 6-8; and Tris buffer for pH 8-10. Dyes. These were certified grade (Wational Aniline). They were dissolved in distilled water to make a lop2 M stock (calculated on the basis of an 84% dye content) and sterilized by boiling for 10 minutes. In the alkaline range, neutral red formed a precipitate at concentrations of 1O-3 M. Therefore all dye stocks were diluted to lop3 M in distilled water, and then diluted further in buffer at the pH required for the test. No precipitates formed under these conditions. Exchange resins. Dowex l-X8 (Cl-),
BY LIGHT
333
200400 mesh. This anion exchange resin was prepared by washing repeatedly with phosphate buffer at pH 8 and then was autoclaved. Upon cooling, the resin was washed further with sterile pH 8 buffer until the filtrate obtained was at pH 8. The columns of cation exchange resin, Dowex 50 W-X4 (H+), 50-100 mesh, were prepared in the same way. Excess fluid was removed from columns by positive nitrogen pressure, and virus samples were then passed through the column. RESULTS
The Effect of pH on Photosensitivity Mature Type 1 Poliovirus
of
The earlier experiments on polioviruses were repeated and confirmed-tissue culture fluids containing poliovirus could not be photosensitized. However, when the virus fluids were first dialyzed at 4” for 24 hours against distilled water, and the solution was made alkaline, the virus could be made photosensitive. The details of such experiments are given below. Type 1 (Mahoney) virus having a titer of 108.0 PFU/ml, was dialyzed and diluted lOOO-fold to contain 105.0 PFU/ml in 1O-4 M neutral red from pH 4 to 9. A duplicate series of samples was made in buffers without dye. Samples were exposed to light for 1 hour. The results of two experiments are plotted in Fig. 1. The dark control samples containing virus-dye mixtures and the dyefree samples exposed to light failed to manifest any detectable loss of infectivity and are not plotted in this figure. The virus-dye mixtures exposed to light at pH 4.0-6.0 showed no significant loss of infectivity. However, in 0.1 M phosphate buffer at pH 7.0 the infectivity titer decreased by 1.2 log; at pH 7.5 by 3.0 log; and at pH 8.0, complete inactivation occurred. In the organic buffers (Tris and glycine) at pH 8.0 or higher, only a slight drop in titer occurred (0.1-0.4 log). Thus the organic buffers seemed to prevent the attachment of neutral red to virus, or its penetration into the virus particle. As shown in Fig. 1, pH 8 was optimum for photosensitivity. To determine whether
334
WALLIS
AND
MELNICK
0 EXP. I o EXP. 2
5 1 4
I
I 5
I
I
6
7
, 6
PH FIG. 1. Effect of pH on photodynamic inactivation of type 1 poliovirus. Virus was dialyzed and then diluted to contain 10’ PFU/ml in 10m4M neutral red at different pH levels. Samples were exposed to white light for 1 hour at 2532°C. Control samples in neutral red kept in the dark and samples in dye-free buffers, exposed to light, showed no detectable loss of infectivity and are not plotted.
the virus could be liberated from the dye by changing pH, the following experiment was performed: dialyzed type 1 virus (lo8 PFU/ml) was diluted loo-fold in lop4 neutral red at (A) pH 6.0 and (B) 8.0. Samples were held at 4” for 15 minutes and then samples A and B were each further diluted tenfold in 10e4 M neutral red at (C) pH 6.0 and (D) pH 8.0. All four samples (A + C) (B + C) (A -+ D) and (B + D) were then immediat,ely exposed to light for 1 hour. The results are shown in Table 1. From these results it appears that the virusdye complex formed at pH 8 is readily dissociable on lowering the pH to 6. The virusdye mixture formed and exposed to light at pH 8 was completely inactivated, while an aliquot of this virus-dye mixture shifted to pH 6.0 and then exposed to light did not lose titer. The virus-dye mixture initially
TABLE
1
EFFECT OF PH ON PHOTOSENSITIVITY OF TYPE 1 POLIOVIRUSQ Initial pH adjustment after dialysis of virus
pH during exposure to light
Loglo virus titer (PFU/ml) Exposed to light
Held in dark
PH 6 (-4
G (Cl 8 CD)
4.5 0.0
4.9 4.8
PH 8 03)
6 (Cl 8 CD)
4.6 0.0
4.8 5.0
a Dialyzed Mahoney virus (lo8 PFU/ml) was diluted loo-fold in 10e4 M neutral red at pH 6.0 and at pH 8.0. These samples were held at 4°C for 15 minutes, then were diluted tenfold in 1OW M neutral red at pH (C) 6.0 and (D) 8.0, and immediately exposed to light for 1 hour.
INACTIVATION
OF POLIOVIRUS
at pH 6 remained photoresistant when held at this pH but became extremely photosensitive when the pH was raised to 8. Effect of Tissue Culture Constituents Photoinactivation of Poliovirus
on
Significant photosensitization as described above occurred only if the virus harvests were first dialyzed and then diluted at least loo-fold in the dye using phosphate buffer at pH 8. This indicated that tissue culture harvests contained both dialyzable and nondialyzable constituents that interfered with the attachment of dye to the virus particle. The effect of extraneous material present in the harvest was investigated as follows: Cultures were inoculated with type 1 Mahoney virus at an input of 5-10 PFU per cell. After an adsorption period of 2 hours at 37”, the cells were washed with buffered saline, and then half were changed with M-E lactalbumin hydrolyzate medium and half were changed with Earle’s salt solution (ESS) to reduce the organic constituents in the harvest. After 7 hours’ additional incubation at 37”, all cultures manifested extensive cytopathic changes. They were harvested by freezing and thawing, and the suspension was clarified by centrifugation at 2000 rpm for 15 minutes. A sample of the M-E harvest was dialyzed overnight at 4” against distilled water. A sample of the ESS harvest was passed through an anion exchange column. The virus titer of the M-E harvest was 1O8.5PFU/ml; the M-E dialyzed sample, 1O8.3; the ESS harvest, 10s,4; and the filtrate of the ESS harvest that had been passed through the resin column was 108.2. Each of the samples described was diluted to contain 10s, 107, 106, 105, and lo4 PFU/ml in 1O-4 M neutral red at pH 8.0 and in dye-free buffer at pH 8. Samples were then exposed to light for 1 hour and duplicates were held in the dark. The results are plotted in Fig. 2. Control samples held in the dark and dye-free samples exposed to light showed no loss of infectivity and are not plotted. The sample derived from the M-E harvest showed significant photosensitivity
BY LIGHT
335
only after the virus had been first diluted lOOO-fold. At this dilution, lo5 PFU/ml were present and there was a 2.5 log loss in titer after exposure to light. In the sample diluted lO,OOO-fold, complete photosensitization occurred. On the other hand, the dialyzed M-E harvest could be inactivated after being diluted only lOO-fold in neutral red (2.2 log loss in titer) and complete photosensitization (5 log loss) of the virus was accomplished with the virus diluted lOOOfold. As shown in Fig. 2, the harvest made in salt solution (ESS) had an inactivation curve similar to that of the dialyzed M-E harvest, which suggests that the amino acids and other dialyzable ingredients of the M-E medium interfere with photoinactivation. The anion resin exchange was able to adsorb interfering substances present in the ESS harvests, and yielded the most sensitive virus. When lo* PFU/ml of resinpurified virus was mixed with dye and exposed to light, 1.7 log reduction in titer was achieved. With a tenfold dilution of the virus (to contain lo7 PFU/ml in dye), 4 log reduction in titer resulted, and upon a loo-fold dilution, complete inactivation occurred (6.0 log loss). A series of experiments was carried out to determine which ingredients of the M-E medium prevented photosensitization. The details of a typical experiment follow. Mahoney virus purified by passage through the anion resin was diluted to contain lo5 PFU/ml in pH 8.0 buffer, and 2.4-ml samples were dispensed. One part (0.3 ml) of the various components that constitute M-E medium (as listed in Table 2) was added to 8 parts (2.4 ml) of virus sample, and stored at 4” for 15 minutes. Then 1 part (0.3 ml) of low3 M neutral red was added to each sample, so that the final concentration of dye was 10v4 M. All tubes were further incubated at 4” for 15 minutes and then exposed to light for 1 hour. A duplicat,e series of samples of virus was treated wit’h neutral red before the addition of each of t,he M-E components. The results of this experiment are shown in Table 2. When either lactalbumin hydrolyzate, phenol red, glucose, or monkey kidney cell extract was
WALLIS
336
108
AND MELNICK
Id
107
105
IO’
CONCENTRATION
OF VIRUS (PFWml)
EXPOSED
IN
TO LIGHT
IO-‘M
NEUTRAL
RED
FIG. 2. Effect of tissue culture constituents on photosensitivity of type 1 poliovirus. Virus harvest deriving from MK cells maintained in lactalbumin hydrolyzate (M-E) medium and Earle’s salt solution (ESS) were diluted to contain IO*, IO’, lo’, lo’, and lo4 PFU/ml in IO-’ M neutral red in phosphate buffer at pH 8. A dialyzed aliquot of the M-E harvest and a sample of the ESS harvest passed through a Dowex anion exchange column were also diluted as described above. All samples were exposed to light for 1 hour. Control samples with dye in the dark showed no loss in infectivity.
mixed with the virus before addition of neutral red, photosensitization was partially or complet,ely prevented. However, if neutral red were added to the virus first, the virus sample was completely inactivated when exposed to light, regardless of what was added later. Effect of Different Concentrations of Neutral Red on Pho toinactiva tion of Poliovirus Resin-purified virus was diluted to contain lo5 PFU/ml in different concentrations of neutral red in phosphate buffer at, pH 8.0. Samples were exposed to light for 60
minutes. The results of this experiment are shown in Fig. 3. No photosensitization occurred in virus samples containing 10-4.g M neutral red or less. At 10e4.” M, the titer was decreased by 1.5 log, and at 10-4.3 M, by 3.7 log. At 1O-4 M, complete loss of infectivity occurred. A repeat experiment showed essentially the same first-order inactivation kinetics. Effect of Time on Photoinactivation of Type 1 Poliovirus The resin-purified virus was diluted as described above in lo-” M neutral red at pH 8.0. Samples were then exposed to light
INACTIVATION
OF POLIOVIRUS TABLE
337
BY LIGHT
2
EFFECT OF INDIVIDUAL COMPONENTS OF M-E MEDIUM AND OF MONKEY EXTRACT ON PHOTOSBNSITIVITY OF TYPE 1 POLIOVIRUS”
KIDNEY
CELL
LogiD virus titer (PFU/ml) Components added to virus samples
Component added before neutral red*
Component added after neutral red=
Exposed to light”
Held in dark
Exposed to lighta
Held in dark
0.0 5.0 1.5
4.8 4.9 5.1
0.0 0.0 0.0
4.9 5.2 5.2
3.5 0.0 0.0
5.0 4.8 5.0
0.0 0.0 0.0
4.8 5.1 5.1
3.0 5.0
5.3 4.9
0.0 0.0
5.0 5.0
Phosphate buffer, pH 8.0 0.5% Lactalbumin hydrolyzate 100 Units penicillin + IO0 pg streptomycin/ ml O.O02$7oPhenol red 0.22% NaHC03 Earle’s salt solution without glucose and phenol red 0.1% Glucose Soluble fraction of ruptured kidney cellsd
(1Dialyzed virus was diluted to contain lo5 PFU/ml in pH 8 buffer. b One part of component at the concentration described above was mixed with 8 parts of virus (106 PFU/ml in buffer) and incubated at 4°C for 15 minutes. Then, 1 part of 1O-3 M neutral red was added to 9 parts of the virus-component mixture. Samples were stored at 4” for 15 minutes, and then exposed to light for 1 hour. c One part of IO-3iM neutra1 red was mixed with 8 parts of virus (105PFU/ml in buffer) and incubated at 4” for 15 minutes. Then, 1 part of the indicated M-E component or cell fraction was added to 9 parts of the virus-dye mixture. Samples were stored at 4” for 15 minutes, and then exposed to light for 1 hour. d Normal MK monolayers in bottles containing about lo6 cells were washed repeatedly with buffer and then changed to 2 ml of buffer (pH 8.0). Cultures were frozen and thawed 3 times and cell debris for virus samples. sedimented at 2000 rpm. The supernatant fluid was used as an additive 6 All samples exposed to light were at pH 8.
for 0, 5, 15, 30, and 60 minutes. The results of this experiment, plotted in Fig. 4, again show that virus was inactivated exponentially as a first-order reaction. Eject
of Distance of Light Source on Photoinactivation Resin-purified virus was diluted to contain lo5 PFU/ml in 10h4 M neutral red at pH 8.0. Samples were exposed to light for 60 minutes at distances of 2, 5, and 10 inches. The results of this experiment are shown in Table 3. After exposure at a distance of 2 inches, no infectivity was detectable; at 5 inches, 2.2 log titer was lost; and at 10 inches only 0.7 log loss of infectivity titer occurred. Efiect of Incubation of Virus-Dye Mixtures on Photosensitivity of Type I Poliovirus The experiments described above were accomplished without incubation of virus-
dye mixtures before light exposure (unless specified). An experiment was performed to determine whether more efficient photosensitization resulted by prior holding of the virus-dye mixtures at 4” or at 37”. Type 1 virus (resin-purified) was diMed to contain lo5 PFU/ml in 10W4 M neutral red at pH 8.0. Samples were placed at 4” and 37” in the dark for 0 and 4 hours. Samples were then exposed to light for 30 minutes. No significant difference in degree of inactivation was observed between virus-dye samples exposed to light without prior incubation and those held at 4” and 37” for 4 hours before exposure to light (loss of 3.5 -C 0.5 log titer). Effect of Other Photodynamic Type I Poliovirus
Dyes
on
Resin-purified virus was diluted to contain lo5 PFU/ml in lOA M of (a) neutral red, (b) acridine orange, (c) methylene
WALLIS
338
A_\D MELNICK
l EXR I 0 EXP. 2
5’
10-4.6
10-4.3
MOLARITY
NEUTRAL
10-4.9
FINAL
10-4.0
RED
3. Effect of different concentrations of neutral red on photosensitivity of type 1 poliovirus. Virus was purified by passage through an anion exchange column and diluted to contain 10’ PFU/ml in different concentrations of neutral red at pH 8. Samples were exposed FIG.
to light for 1 hour. blue, (d) o-toluidine blue, and (e) proflavine at pH 8.0. Samples were exposed to light for 1 hour. The results of this experiment are shown in Table 4. All the dyes used, except acridine orange, completely sensitized poliovirus to light. In the case of acridine orange, only 1.5 log virus titer was lost. Effect of Neutral Red on Photosensitivity of Other Polioviruses Types 1, 2, and 3 poliovirus (attenuated and virulent) in 0. l-ml volumes were seeded onto drained monolayer cultures ( lo6 cells per bottle) so that the input of virus was 2-4 PFU/cell. After 2 hours’ adsorption at
0 MINUTES
15
30
EXPOSED
TO LIGHT
60
FIG. 4. Effect of different exposure times on photosensitivity of type 1 poliovirus. Purified virus was diluted to contain loj PFU/ml in IO-* neutral red at pH 8. Samples were exposed to light for times indicated.
INACTIVATION TABLE 3 EFFECT OF DISTANCE OF LIGHT SOURCE ON PHOTOSENSITIVITY OF TYPE 1 POLIOVIRUB
Distance from light source (inches)
Log10 virus titer (PFU/ml)
2 5 10
0.0 3.0 4.5
Control
5.2
in dark
a Purified virus was diluted to contain lo5 PFU/ ml in 10s4 M neutral red at pH 8.0, and samples were exposed to light for 1 hour at distances shown.
TABLE THE
4
EFFECT OF OTHER PHOTODYNAMIC DYES ON TYPE 1 POLIOVIRUS”
Log10 virus titer (PFU/ml) Dye
Neutral red Acridine orange Methylene blue Toluidine blue Proflavine None
339
OF POLIOVIRUS BY LIGHT
Exposed to light
Held in dark
0.0 3.5 0.0 0.0 0.0 5.0
5.2 5.0 5.0 5.0 5.1 5.3
a Type 1 (resin-purified) Mahoney virus was diluted to contain lo5 PFU/ml in 10e4 M dye. Samples were exposed to light for 1 hour. Control virus samples in dye were wrapped in aluminum foil to keep them in the dark.
37”, cultures were washed with M-E medium and changed to 4 ml of M-E medium. All cultures manifested marked cytopathic changes between 8 and 16 hours and were frozen. Harvests were thawed and centrifuged at 2000 rpm for 10 minutes, and supernatants were collected. Each virus was then diluted lO,OOO-fold in 10W4 M neutral red or dye-free buffer at pH 8.0 to reduce the concentration of tissue culture ingredients. Samples were then exposed to light for 1 hour. The results of this experiment are shown in Table 5. All six viruses were significantly inactivated.
Photosensitivity of Poliovirus That of Vaccinia Virus
Compared to
Hiatt et al. (1960) showed that vaccinia virus, unlike enteroviruses, could be readily inactivated in toluidine blue in the presence of light. In view of our finding that purified mature poliovirus can also be made photosensitive, we investigated the reversibility of the binding of dye to virus. For this, cation exchange columns were used, as they readily adsorb basic dyes. A number of experiments were performed, all of which yielded similar findings. One such experiment is reported in detail below. Type 1 poliovirus and vaccinia virus were each diluted to contain lo5 PFU/ml in 10W5 M toluidine blue and in dye-free phosphate buffer at, pH 8. Samples were incubated at 4” for 1 hour, and then exposed to light at 2532” for 1 hour. A duplicate series of samples stored in the dark at 4” for 1 hour were passed through cation exchange colTABLE EFFECT OF NEUTRAL TYPES 1, 2, AND
5 RED AND LIGHT 3 POLIOVIRUSES”
ON
-
1Log10 virus titer (PFU ml) Virus strain
II 1
Ekpt. no.
neutral
reC
-7
Dye-free
d:k
EX- I Held Nosed to d:k ight
2
3.5 3.8
3.6 3.6
3.4 3.7
EX1wsed to 1light
Held
-
Attenuated Type 1, LSc
1
Type
2, P712
3
3.6
3.5
3.3
Type
3, Leon
4 5
3.0 2.9
3.1 3.5
3.0 3.2
1, Mahoney
6 7
Type
2, MEFl
8
3.8
3.8
Type
3, Saukett
Virulent Type
9 -
3.8 -
a M-E virus harvests were diluted lO,OOO-fold in 1OW M neutral red or in dye-free buffer at pH 8. Samples were exposed to light for 1 hour. Dark controls w-ere wrapped in aluminum foil.
340
WALLIS VACCINIA
Toluidine
AND
POLIOVIRUS TYPE 1
VIRUS
blue
Li~L$i~;:
:;;gfifip;:
Li.jy.
Li&rk 4.5
4.3
Cation
Cation
cation
Cation
4.5
MELNICK
Lifirk 4.6
3.8
Ligfirk
FIG. 5. Reversibility of binding of dye by poliovirus and vaccinia virus. Results pressed as log titers (PFU/ml). The details of the experiment are given in the text.
umns to yield water-clear filtrates. They were then exposed to light for 1 hour. Proper dark controls were employed. The results of this experiment are shown in Fig. 5. The vaccinia virus sample in toluidine blue kept in the dark, the sample in dyefree buffer exposed to light, and the sample in dye-free buffer kept in the dark, all had similar titers (10j.2, 105.2, and 105.3 PFU/ ml, respectively). This indicated that the dye at the level used was not toxic to the virus if light were excluded and that dyefree virus exposed to light was not inactivated. However, the sample of vaccinia virus in toluidine blue exposed to light was completely inactivated. The sample of vaccinia virus in toluidine blue that had been passed through t.he cation exchange column yielded a dye-free filtrate. When exposed to light, this sample had the same titer as the corresponding sample kept in the dark (104.5 PFU/ml). Since the virus was no longer sensitive to light when the dye was removed, vaccinia virus exposed to t,oluidine blue did not take up the dye irreversibly. Vaccinia virus in dye-free buffer had similar titers when exposed to light or when kept in the dark. Poliovirus in toluidine blue in the dark, or in dye-free buffer in the dark or exposed to light, yielded similar titers (104.s, 105.0, and 105.1, respectively). Thus, as with vac-
5.3
5.1
4.0
are ex-
cinia, the dye at the level used was not toxic to poliovirus unless exposed to light. The sample of poliovirus in toluidine blue that was passed through the cation exchange column yielded a clear filtrate that was no longer photosensitive. The titer of the dark control was 104.0 PFU/ml, that of the sample exposed to light, 103,s. Thus, it is apparent that toluidine blue may also be reversibly bound to poliovirus. However only 10% of free virus was recovered in the filtrate, and 90% remained on the column bound to the dye. In contrast, when poliovirus in dye-free buffer at pH 8 was passed through the exchange column, no detectable adsorption of virus to the column occurred. Thus the poliovirus-dye complex seems t.o be more firmly bound than t’he vaccinia virus-dye complex. DISCUSSION
Earlier studies (Hiatt, 1960; Hiatt et al., 1960; Opton and Green, 1960; LoGrippo and Basinski, 1960; Crowther and Melnick, 1961) showed that mature polioviruses grown in the absence of photosensitive dyes could not be inactivated in dyes when exposed to light. Experiments described in this report show that intact polioviruses can be photodynamically inactivated under the proper conditions. We have shown that pH 8.0 is critical for photosensitization of poliovirus and that organic components
INACTIVATION
OF POLIOVIRUS
such as those present in tissue culture constituents (both dialyzable and nondialyzable) prevent photosensitivity. Phosphate buffer was the diluent of choice for the virus-dye mixtures, since organic buffers prevented photoinactivation. Most attempts to inactivate mature poliovirus in the presence of a photosensitive dye have been made with crude virus harvests. However, Schaffer (1962) used purified virus grown in the absence of dye and still failed, because the importance of pH control was not known at the time. Also, as he observed, the high concentration of proflavine used (100 pg/ml) might actually have prevented the light from penetrating. In his experiments, added proflavine at this concentrat,ion actually protected virus grown in the presence of the dye from phot.oinactivation. The earlier results on photoresistance of poliovirus and photosensitivity of larger RNA and DNA viruses in the presence of dye has led to speculation that permeable pores may be present in DNA viruses but not in polio and other enteroviruses (Hiatt, 1960; Mayor et al., 1963). However, in view of our present results showing that polioviruses under proper conditions can be rendered as photosensitive as vaccinia virus, the mechanism by which dyes are bound to and dissociated from viruses will have to be investigated further. ADDENDUM Since this manuscript was submitted, we have tested 10 echoviruses and 5 coxsackieviruses (4 group B strains, and 1 group A strain) for photodynamic inactivation in the presence of toluidine blue or neutral red. The results were similar to those reported above for the polioviruses.
341
BY LIGHT REFERENCES
CROWTHER,D., and MELNICK, J. L. (1961). The incorporation of neutral red and acridine orange into developing poliovirus particles making them photosensitive. Virology 14, 11-21. HIATT, C. W. (1960). Photodynamic inactivation of viruses. Trans. N.Y. Acad. Sci. 23, 66-78. HIATT, C. W., KAUFMAN, E., HELPRIN, J. L., and BARON,S. (1960). Inactivation of viruses by photodynamic action of toluidine blue. J. Immunol.
84,48&484. LEDINKO, N. (1958). Production complement-fixing cells treated with
poliovirus proflavine.
of noninfectious particles in HeLa Virology 6, 512-
524. LOGRIPPO,G., and BASINSKI, D. H. (1960). The value of acridine orange for in plasma and whole blood.
sterilizing viruses Federation Proc.
19,56.
D., and DIWAN, A. R. (1961). Studies on the acridine orange staining of two purified RNA viruses: poliovirus and tobacco mosaic virus. Virology 14, 74-82. MAI’OR, H. D., JAMISON,R. M., and DIWAN, A. R. (1963). Studies on viral replication using acridine orange as a vital dye. Submitted to Virology. OPTON,E. M., and GREEN,R. H. (1960). Effects of neutral red and light on polioviruses. Federation MAYOR, H.
Proc.19,408. SCHAFFER,F. L. (1962). Binding
of proflavine by and photoinactivation of poliovirus propagated in the presence of the dye. Virology 18, 41%
425. TOMITA, Y., and PRINCE, A. M.
(1963). Photodynamic inactivation of arbor viruses by neutral red and visible light. Proc. Sot. Ezptl. Biol. Med.
112,887-890. WALLIS, C., and MELNICK, J. L. (1962a). Suppression of adventitious agents in monkey kidney cultures. Texas Rept. Biol. Med. 20, 465-475. WALLIS, C., and MELNICK, J. L. (1962b). Magnesium chloride enhancement of cell susceptibility to poliovirus. Virology 16, 122-132.