Effect of Visible Light on Canine Distemper Virus'

JOURNAL OF BACTERIOLOGY, Feb., 1966 Copyright 1966 Amexican Society for Microbiology Vol. 91, No. 2 Printed in U.S.A. Effect of Visible Light on Canine Distemper Virus' GEORGE J. NEMO AND ERNEST C. CUTCHINS Department of Biology, The Catholic University ofamerica, Washington, D.C Received for publication 26 August 1965 ABsTRAcT NEMO, GEORGE J. (The Catholic University of America, Washington, D.C.), AND ERNEST C. CUTCHINS. Effect of visible light on canine distemper virus. J. Bacteriol. 91:798-802. 1966.-Canine distemper virus (CDV) was inactivated by visible light. The virus was light-sensitive in fluid suspension (in vitro) as well as during intracellular replication (in vivo). The addition of calf serum or glutathione reduced the extent of inactivation. CDV was less sensitive when suspended in distilled water or in the amino acid or Earle's salts components of the minimal essential medium of Eagle than when suspended in the vitamin component of the minimal essential medium of Eagle or in riboflavine (0.1 mg per liter). These findings indicate that, whereas some ingredient of the medium may enhance light sensitivity, its presence is not necessary for light inactivation of CDV. It is proposed that some substance derived from the host cell and intimately associated with the virus particle serves to render CDV light-sensitive. Raab in 1900 (12) was first to report the destructive effect of visible light on microorganisms as mediated through photosensitizing dyes. Numerous investigators extended these findings to include bacterial and animal viruses (3, 6, 9, 10, 14). The effect of visible light in the absence of dyes, however, is a phenomenon which has received little attention. Skinner and Bradish (13) found that the infectivity of various viruses, when exposed to daylight, was diminished. Lozovskaia (7) found that during and after the process of lyophilization, in the presence of light, the infectivity of measles virus and the PR8 strain of influenza virus was greatly diminished. Cutchins and Dayhuff (4) demonstrated that measles virus in suspension was readily inactivated by visible light, a finding subsequently confirmed by Wallis and Melnick (15). A virus closely related to measles virus in several ways is canine distemper virus (CDV). Numerous workers reported striking similarities between these two viruses, including ether sensitivity (8), cytopathic effect (11), serological overlap (16), and size and morphology of the virus particle (17). It was of interest, therefore, to determine the effect of visible light on CDV. MATERIALS AND MErHODS Virus. The Onderstepoort strain of CDV was employed throughout this study. Stock virus was I A portion of these data were presented before the Federated Societies for Experimental Biology, Atlantic City, N.J., 1965. prepared by infecting monolayers of chick embryo cell culture (CECC) in 8-oz (ca. 240-ml) bottles. The cells were washed twice in Hanks' balanced sal solution (HBSS). Seed virus (0.5 ml) was adsorbed to the cell sheet for 0.5 hr at 37 C, after which cells were fed the minimal essential medium of Eagle (MEME) (5) containing neither phenol red nor calf serum. Infected cultures were incubated at 37 C until CDV cytopathic effect was visible in approximately 5) to 75% of the cell sheet. At this point, the cultures were frozen and thawed once, clarified by centrifugation at 250 X g for 15 min, and stored at -65 C until needed. The ph of the harvest fluids was approximately 7.4 and was not adjusted. Method of irradiation. The method of illumination was the same as that employed by Cutchins and Dayhuff (4). Virus suspensions in amounts of 5.0 ml were irradiated, unless otherwise stated. Control tubes were treated similarly, except that these tubes were covered with several layers of aluminum foil. Plaque assay. Titrations for infectivity of irradiated samples were performed by use of the plaque-assay system in CECC of Bussell and Karzon (2). Virus dilutions in log10 steps were made in HBSS. Three cell cultures were inoculated with 0.5-mil volumes of selected dilutions. Plaques were counted on the 11th day. End points were calculated and expressed in plaque-forning units (PFU) per milliliter. RESULTS Inactivation ofcd V by artificial light. To determine whether visible light would inactivate CDV, an 8.0-ml sample of the stock virus was exposed to illumination for 150 min. Samples were taken at 30-min intervals and were assayed for infec- 798

VOL. 9 1, 1 966 PHOTOINACTIVATION OF CANINE DISTEMPER VIRUS 799 that oxidation plays an integral role in the inactivation process. Consequently, it was decided to 2 determine the effect of calf serum or glutathione Li. 2 0. on CDV inactivation. Virus suspensions containing 20%,;o calf serum or glutathione in an arbitrar- M 0 CD ily selected concentration of 5 mg/ml were irradiated for 120 and 180 min, respectively, along 0 -J with untreated virus. The ph of the glutathione In solution was adjusted to 7.0 before it was added to the virus suspension. The results of these studies are shown in Table (9 z 1. In the serum experiment, the sample containing no serum and the c- sample containing 20%7 calf <1 >Z serum had initial titers of 104 4and 104-0 PFU/ml, respectively. After 120 min of exposure, the suspension with calf serum contained considerable infectious 120 P.0 150 llo virus, whereas the titer of the suspension with no serum was equal to or less than 101-0 TIME OF EXPOSURE ( MI N) PFU/ml. It was subsequently found that the degree of protection was directly related to serum FIG. 1. Inactivationi of caninle distem per virus by concentration. ar-tificial liglht. In the glutathione experiment, the treated and untreated samples had initial titers of 103.8 and tivity. Infectivity titers of the various samples are 103 9 PFU/ml, respectively. After 180 min of shown in Fig. 1. No titer was obtaii ned for the exposure, the virus suspension to which glutathi- employed one had been added still contained 102 lpfu/ml, 90-min sample, because the dilutioms in anticipating the titer at this point Nwere higher whereas no virus was demonstrable in the un- the titer treated suspension. than the actual virus titer. At zero tinne, of the virus was 103 9 PFU/ml. After 30 min of Influence of components of the MEME on the exposure, the titer had fallen to 102 6 PFU/ml. inactivation of CDV by light. In a previous study Between 60 and 120 min, the titer had fallen from (4), it was found that measles virus suspended in 10 9 to 100.6 PFU/ml, respectively. No virus was distilled water was less sensitive to light than when detectable after 150 min of illuminatioin. The dark it was suspended in the harvest fluid (i.e., the control was sampled only at the end of the ex- spent culture fluid from cells fed the basal medium periment. A titer of 103 9 PFU/ml irldicated no of Eagle). It was of interest to pursue further the loss of infectivity in the absence of ligght. influence of the suspending menstruum on light Influence of calf serum or glutathiione on the sensitivity. Subsequently, it was found that, like light sensitivity of CDV. It was demonstrated by measles virus, CDV suspended in distilled water Cutchins and Dayhuff (4) that certain substances, was less sensitive to light. These findings suggested serum and glutathione, exerted a protlective effect the presence of a sensitizing substance in the har- that vest fluid, which was removed by washing. on the irradiated measles virus. The ifinding glutathione exerted a protective effect suggested MEME is a complex medium containing three TABLE 1. Inlfluenice of calf seruem or glutathionie oni the light selnsitivity of caninle distemper virus Calf serum expt Glutathione expt Exposure time No serum Calf serum (20%',) Untreated Glutathione (5 mg/ml) Light Dark Light Dark Light Dark Light Dark 0 4.4* t 4.0 3.9* t 3.8-60 2.9 3.6 1.9 2.6_ 120 <1.0 4.3 2.9 4.0 0.6 2.3 180 - -- < 1.0 3.9 2.1 3.8 * Logs(, PFU per milliliter. t Not done.

800 NEMO AND CUTCHINS J. BACTERIOL. TABLE 2. Influence of components of the minimal essential medium of Eagle (MEME) on the inactivation of canine distemper virus by light MEME Vitamin Riboflavine Amino acid Earle's salts Exposure time Inac- Inac- Inac- Inac- Inac- Light Dark tiva- Light Dark tiva- Light Dark tiva- Light Dark tivya- Light Dark tivation tion tion tion tion min % % % % % 0 4.3* -t 3.6 4.0 4.8 3.7 60 1.6 4.2 99.8 1.1 3.6 99.8 0.8 4.0 99.9 3.3 4.6 95.0 2.7 3.6 87.5 * Log10 PFU per milliliter. t Not done. major groups of compounds: vitamins, amino acids, and Earle's salts. Riboflavine, a vitamin thought to have photosensitizing capacity (4), is an essential component of MEME. The riboflavine content of the spent harvest fluid was determined by microbiological assay with Riboflavin Assay Medium (Difco), and the flavin content was found to be even greater than that of fresh MEME. Virus suspensions in the presence of 0.06% gelatin were sedimented in a Spinco model L ultracentrifuge at 80,000 X g for 2 hr. The virus pellets were then suspended in solutions of each of the three major components of MEME made to standard concentration with a ph of 7.2. Virus was also suspended in riboflavine (0.1 mg/liter). Virus suspended in fresh MEME served as a control. After 60 min of exposure, the virus suspended in MEME, vitamins, or riboflavine showed essentially the same degree of inactivation: 99.8, 99.8, and 99.9%, respectively (Table 2). The virus suspended in Earle's salts was only 87.5% inactivated, whereas the amino acid suspension showed 95.0% inactivation. In calculating the per cent inactivation, the titer of the dark control at the end of the exposure period was taken as 100% survival. Effect of I hr of illumination on CDV when exposed at various times during the replication cycle. With the finding that CDV was light-sensitive in vitro, it was of interest to determine whether the virus was light-sensitive in vivo, that is, during intracellular replication. CECC monolayers in 2-oz bottles were infected with approximately 100 PFU of virus. The bottles were filled with 60 ml of MEME without phenol red and were incubated at 35 C. Starting at the end of the adsorption period and at intervals up to 13 hr, duplicate cultures were illuminated for 1 hr, after which they were drained of fluid medium and overlaid with agar. The illumination apparatus consisted of two General Electric RFL 2 photoflood bulbs, each fastened to a separate ring stand and positioned side by side over the infected CECC monolayers. The heat generated by the bulbs decreased with use; this necessitated vertical repositioning of the bulbs to maintain constant temperature inside the bottles of cell cultures. The distance between the bulbs and the monolayers varied from a maximum of 28 cm, upon installation of a new bulb, to a minimum of 23 cm, when the bulb was discarded, because of loss of intensity. The illumination equipment was housed in a refrigerator (4 C), and a fan was used to increase air circulation. Filling the bottles aided in dissipation of heat and eliminated any air space inside the bottles in which heat could accumulate during the illuniination period. A 2-oz prescription bottle filled with MEME and containing a thermometer was irradiated along with the infected cultures; it served to monitor the temperature of the monolayers. Under these conditions, a temperature of 37 to 38 C could be maintained inside the bottles. As controls, duplicate companion cultures were transferred to a dark incubator (38.5 C) during the time the experimental cultures were being illuminated. The results of this study are seen in Table 3. With the time of inoculation taken as zero time, the first column shows the time in the replication cycle at which a set of cultures was illuminated. Each set of cultures received 1 hr of illumination. The PFU per cent survival in each case is based on the plaque count of the control cultures for that particular time interval. The data indicate that CDV is light-sensitive throughout its replication cycle, being most sensitive during the first 2 hr of the cycle. There is a gradually increasing resistance from the 2nd through the 11th hr. At the 13th hr, there is a sharp increase in sensitivity, which probably coincides with the release of new virus. Too much significance should not be attached to minor variations in the per cent survival of the virus, for several reasons. There is considerable variation in the thickness of glass of 2-oz bottles, which would contribute to variations in intensity of light reaching various portions of the

VOL. 91, 1966 PHOTOINACTIVATION OF CANINE DISTEMPER VIRUS TABLE 3. Effect of 1 hr of illumination on canine distemper virus at various times during one replication cycle Time after inoculation at * u which cultures were illuminated Plaque-forming unit for 1 hr per cent survival hr 0.5 20 1 36 2 46 2.5 60 3 50 4 76 5 70 6 70 9 54 11 79 13 48 cell monolayer. Although photoflood bulbs were replaced every 4 hr, their intensity gradually decreased with time. This gradual diminution of intensity would affect, to some extent, the amount of virus inactivated at a particular time. DISCUsSION The finding that CDV, like measles virus, was sensitive to light was not wholly unexpected in view of their other similarities. That the virus could be protected to some extent by the addition of serum is in agreement with the findings of others (4, 13). This protection may be the result of the screening afforded by the slight turbidity resulting from the addition of serum to the virus suspension. However, Cutchins and Dayhuff (4) were able to protect measles virus by use of a sucrose,gelatin stabilizer which did not absorb any wavelengths in the visible spectrum. Wallis and Melnick (15) emphasized the effect of ph on the photosensitivity of dye-treated viruses. It would not seem, however, that the increased survival observed when the virus was suspended in the amino acid or salts portion of MEME was due solely to the difference between the ph of these menstrua (ph 7.2) and that of the harvest fluid (ph 7.4). In photodynamic inactivation, dissolved molecular oxygen and a sensitizing dye are both essential requirements (9). In the photoinactivation of CDV, the protection afforded by the addition of glutathione suggests that oxygen is involved in its loss of infectivity in the presence of light. Should photodynamic inactivation and photoinactivation of viruses be similar phenomena, it then remains to demonstrate the presence and to determine the 801 source of the light-sensitizing substance involved in photoinactivation. The findings reported here show that CDV was most sensitive to light when suspended in whole MEME or the vitamin portion of this medium. Further, the virus was equally light-sensitive when suspended in a solution of riboflavine. This would indicate that riboflavine functioned as a photodynamic sensitizing dye to render the virus light-sensitive. In 1960, Schaffer (Federation Proc. 19:405) showed that proflavine, a compound very similar in structure to riboflavine, possessed photodynamic capacity. If this were the case, then virus suspended in a menstruum without vitamins should no longer be sensitive. However, when CDV was suspended in distilled water or the other components of MEME, although it was less sensitive than that suspended in the vitamin component, over 85 % of its infectivity was destroyed. Wallis and Melnick (15) reported that measles virus grown in a riboflavine-free system retained its natural photosensitive state. McCabe (M.S. thesis, Catholic Univ. America, Washington, D.C., 1964) found vaccinia virus, whether derived from the chorioallantoic membrane or CECC, was light-sensitive. These findings indicate that, although the nutrients of a tissue culture system may contain a sensitizing substance, its presence is not necessary for light sensitivity. If photosensitivity is due to the presence of some sensitizing substance, that substance probably is derived from the intracellular environment of the host cell. In a study undertaken to determine the extent of light sensitivity, viruses representing the presently accepted groups of animal viruses (1) were examined (Cutchins, unpublished data). With certain exceptions, light sensitivity could be correlated with size and complexity of structure. Generally, the larger viruses possessing an outer envelope (1) were found to be light-sensitive. From the results of that study, it was postulated that, during the formation of the virus particle, some substance derived from the intracellular milieu becomes intimately associated with the mature particle, rendering it light-sensitive. This substance may be some host-cell flavin. CDV possesses the size and complexity (17) to have this sensitizing substance associated with the virus particle. The possibility remains, however, that light sensitivity is a unique and intrinsic characteristic of certain viruses. Until the sensitivity of rigidly pure suspensions of a virus or its nucleic acid are determined, this question cannot be finally answered. However, the practical importance of light sensitivity cannot be ignored in all operations involving sensitive viruses.

802 NEMO AND CUTCHINS J. BACTERIOL. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI-05113-01 from the National In stitute of Allergy and Infectious Diseases. We thank R. H. Bussell and D. T. Karzon of the University of Buffalo School of Medicine, Buffalo, N.Y., for supplying the Onderstepoort strain of CDV. LITERATURE CITED 1. ANDREWES, C. 1964. Viruses of vertebrates. The Williams & Wilkins Co., Baltimore. 2. BUSSELL, R. H., AND D. T. KARZON. 1962. Canine distemper virus in chick embryo cell culture. Plaque assay, growth, and stability. Virology 18:589-600. 3. CROWTHER, D., AND J. L. MELNICK. 1961. The incorporation of neutral red and acridine orange into developing poliovirus particles making them photosensitive. Virology 14: 11-21. 4. CUTCHINS, E. C., AND T. R. DAYHUFF. 1962. Photoinactivation of measles virus. Virology 17:420-425. 5. EAGLE, H. 1959. Amino acid metabolism in mammalian cell cultures. Science 130:432-437. 6. HIATT, C. W., E. KAUFMAN, J. J. HELPRIN, AND S. BARON. 1960. Inactivation of viruses by the photodynamic action of toluidine blue. J. Immunol. 84:480-484. 7. LoZOVSKAIA, L. S. 1959. Destructive action of light on measles and influenza viruses during desiccation and subsequent storage in vacuo. Vopr. Virusol. 4:56-59. 8. PALM, C. R., AND F. L. BLACK. 1961. A comparison of canine distemper and measles viruses. Proc. Soc. Exptl. Biol. Med. 107:588-590. 9. PERDRAU, J. R., AND C. TODD. 1933. Photodynamic action of methylene blue on bacteriophage. Proc. Roy. Soc. London Ser. B 112: 277-287. 10. PERDRAU, J. R., AND C. TODD. 1933. Photodynamic action of methylene blue on certain viruses. Proc. Roy. Soc. London Ser. B 112: 288-298. 11. PEREIRA, H. G. 1961. The cytopathic effect of animal viruses. Advan. Virus Res. 8:245-285. 12. RAAB, 0. 1900. Ueber die wirkung fluorescierender stoffe auf infusorien. Z. Biol. 39:524-546. 13. SKINNER, H. H., AND C. J. BRADISH. 1954. Exposure to light as a source of error in the estimation of the infectivity of virus suspensions. J. Gen. Microbiol. 10:377-397. 14. WALLIS, C., AND J. L. MELNICK. 1963. Photodynamic inactivation of poliovirus. Virology 21:332-341. 15. WALLIS, C., AND J. L. MELNICK. 1964. Irreversible photosensitization of viruses. Virology 23: 520-527. 16. WARREN, J. 1960. The relationships of the viruses of measles, canine distemper, and rinderpest. Advan. Virus Res. 7:27-60. 17. WATERSON, A. P. 1962. Two kinds of myxovirus. Nature 193:1163-1164. Downloaded from http://jb.asm.org/ on May 19, 2018 by guest