APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1976, p. 315-319 Copyright ©D 1976 American Society for Microbiology
Vol. 32, No. 3 Printed in U.S.A.
Inactivation of Semliki Forest Virus in Aerosols J. C.
JONG,l* M. HARMSEN, A. D. PLANTINGA,2 AND T. TROUWBORST3 Laboratory of Microbiology, State University, Utrecht, The Netherlands DE
Received for publication 24 February 1976
Purified Semliki forest virus in aerosols is inactivated rapidly at 40% and above 70% relative humidity. At all humidities tested the decay of virus infectivity runs parallel with the decrease in hemagglutination activity, whereas the biological integrity of the virus ribonucleic acid is preserved. Also, free infectious ribonucleic acid is stable after spraying at all relative humidities. Evidence is presented for the hypothesis that above 20% relative humidity, virus inactivation in aerosols is mainly due to surface-dependent factors, damaging the virus coat. MATERIALS AND METHODS Virus techniques. SFV strain SF/LS10/C1/A was obtained from C. J. Bradish, Porton, England. The virus was grown irk L cells in Eagle minimal essential medium without bicarbonate and with 0.01 M HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid). After 18 h at 37°C the infected cell culture was stored at 4°C. The supernatant was used for experiments. Plaque titration was performed on L cell monolayers with 48 h of incubation at 37°C. The crude suspension contained 6 x 108 plaqueforming units per ml. For purification, virus suspensions were centrifuged at low speed to remove cell debris and subsequently at 80,000 x g in a Spinco L50 ultracentrifuge at 4°C during 90 min. The sediment was suspended in 0.1 M NaCl with 0.001 M Na2HPO4, pH 7.6, using 0.005 times the original volume. The resulting suspension was subjected to gel exclusion chromatography over Sepharose 4B (Pharmacia, Uppsala). The purified preparation contained about 1010 plaque-fonning units per ml. The E260-E280 ratio was 1.47, indicating a nucleic acid content of 16%. Kaariainen et al. (10) reported a ratio of 1.3 to 1.4, corresponding to 11 to 13% nucleic acid. The purified virus was unstable. At 40C the titers of infectivity, iRNA and HA dropped by a factor of 2 per hour. Therefore, the preparations were titrated on these three parameters immediately before each experiment. iRNA. The virus suspension was supplied with sodium dodecyl sulfate (British Drug Houses, London) to 0.5% final concentration and added to an equal volume of phenol (Merck, p.a., redistilled and stored at -20°C under nitrogen), saturated with PBSE (phosphate-buffered saline, pH 7.2, with 0.01% ethylenediaminetetraacetic acid). This mixture was shaken vigorously by hand for 4 min at room temperature. The extraction was repeated with fresh phenol, with shaking for 2 min. twice ' Present address: National Institute of Public Health, After this, the water phase was shaken 5 times for Bilthoven, The Netherlands. 15 s with a double volume of ethyl ether (Merck, 2 Present address: Philips-Duphar, Weesp, The Netherp.a., distilled and washed with PBSE) each time. lands. 3Present address: Ministry of Health and Environmen- Finally, nitrogen gas or air was bubbled through the solution for 3 min at room temperature. Before tal Protection, Leidschendam, The Netherlands.
The inactivation of viruses in aerosols has been ascribed to desiccation or to processes located at the aerosol particle-air interface. The first mechanism is involved in the decay of the picornavirus causing encephalomyocarditis (EMC) in mice (5). The second factor prevails with bacteriophages Ti, T3, and MS2 (14-16). The localization of the damage to virions in aerosols has been studied with poliovirus and EMC virus. It has been demonstrated that, after destruction of the virus infectivity, the virus ribonucleic acid (RNA) retains its infectivity (3, 4). On the contrary, the hemagglutination activity (HA) of EMC virus in aerosols decreases parallel with the virus infectivity (3). The inactivation of lipid-containing viruses at spraying has not been investigated in these respects. We studied, therefore, the fate of Semliki forest virus (SFV) and its components in aerosols. SFV is a lipid-containing virus belonging to the alpha subgroup of the togaviruses. It shows HA and yields infectious RNA (iRNA) after extraction with phenol. Both properties are useful tools for localization of the damage at inactivation. Within 1 h after spraying from crude virus suspensions, virus decay is negligible (1). When working with purified preparations, however, considerable loss of infectivity has been observed. Addition of protein restored partly the aerosol stability of the virus (1), suggesting that surface-dependent processes could be responsible for the decay. This and other possibilities were tested in the present study, along with the fate of HA and RNA during inactivation.
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plating, 100 ,ug of diethylaminoethyl-dextran (Pharmacia, Uppsala, molecular weight 2 x 106) was added per ml. Dilutions were prepared in PBSE + diethylaminoethyl-dextran, and 2 x 107 L cells were suspended in 2-ml portions. After 2 min at 37°C the suspensions were added to preformed L-cell monolayers without removing the growth medium. After 1 h the plates were processed for plaque formation as with complete virus. The infectivity of the iRNA preparation was 0.01 to 0.1% of the original virus suspension and was completely abolished by incubation with ribonuclease (see below); this treatment did not influence the infectivity of whole virus for L cells. Ribonuclease treatment. Virus suspensions were supplied with 20 jig of ribonuclease (Boehringer, Mannheim) per ml. After 30 min at 37°C the virus was submitted to Sepharose 4B chromatography to remove the enzyme. In a control experiment SFViRNA was inactivated when added to the ribonuclease-containing virus suspension before, but not when added after chromatography. Hemagglutination. HA assays were performed with goose erythrocytes according to Clarke and Casals (2) in U-shaped disposable microtiter plates (Cooke Microtiter plates M24A, Dynatech, Billingshurst, Sussex, UK). HA patterns were read after 2 h at room temperature. HA titers of purified SFV suspensions were about 4 logs lower than infectivity titers and ranged around 106 units per ml. Experimental techniques. Aerosol apparatus and calculations have been described (6). The collection fluid was Dulbecco buffer (8) with 1% peptone. When the samples had to be assayed for iRNA, calcium and magnesium salts were omitted from the collection fluid. In the rotating bulb setup, a virus suspension is rotated in a large spherical flask to fonn a thin liquid film on the wall (17). Glycerol and OED were applied as before (5). Glycerol was mixed in various ratios with SFV in Dulbecco buffer. OED is a mixture of equal quantities of oxyethylene docosylether and oxyethylene octadecylether (11). Even in 10% OED the infectivity of SFV was, on standing for 1 h at room temperature, as stable as without the agent. On spraying, OED forms a film around each droplet (11). Graphs represent the results of at least two experiments.
RESULTS Inactivation of SFV in aerosols at different RHs. Maximal inactivation of purified SFV in aerosols occurred at 40% and above 70% relative humidity (RH) (Fig. 1). To test whether oxidation contributes to the decay, virus was sprayed in pure nitrogen at 20, 40, and 85% RH. No signiificant deviation from the aerosol experiments could be observed during 60 min after nebulization (not shown). Inactivation in glycerol solutions. In Fig. 1 the decrease of virus infectivity is plotted against the RH of the atmosphere that is in equilibrium with the glycerol-buffer mixture used. The aerosol data of the same virus preparation are included for comparison. It appears that the inactivation in aerosols at 10 to 20%
APPL. ENVIRON. MICROBIOL. log
Nt
No O_
-1
2.5 min 1.5 min in glycerol -2 \
m
\
n aer
\\ 0-5 min in aerosol
-3, \
\
/
K
1
/
o
230-35 min in aerosol
0/0 glycerol (w/w) -5 -
97 10
89 30
78 /. RH
50
64 70
35 90
FIG. 1. Inactivation of SFV in aerosols and in glycerol solutions. Purified SFV was sprayed and sampled from 0 to 5 min (0) and from 30 to 35 min (A). In other experiments, purified SFV was diluted in mixtures ofglycerol and phosphate-buffered saline in various ratios, corresponding to various relative humidities. Samples were taken from the solutions at 2.5 min (0) and at 32.5 min (A) after addition of virus.
RH roughly equals the decay in the corresponding glycerol solutions with 94 to 97% glycerol. At higher RH and lower glycerol concentration the two inactivation rates diverge. Sensitivity of SFV to surface exposure. The surface-active agent OED increased the stability of SFV in aerosols. The effect at 40% RH was less than at 85% RH (Fig. 2 and 3). In the rotating bulb system SFV was inactivated at the same rate as bacteriophage MS2 (Fig. 4). Phage MS2 is known to be vulnerable to surface-located forces (14). Fate of virus-RNA and HA at inactivation of SFV in aerosols. Purified virus preparations were nebulized at 10, 50, and 80% RH. Samples were assayed for infectious virus and for HA, and, after extraction with phenol, for iRNA. The result at 50% RH is shown in Fig. 5. Whole virus decay amounted to 2.7 logs in 60 min. The HA decreased parallel with the virus infectivity. The iRNA, on the contrary, was stable.
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INACTIVATION OF SEMLIKI FOREST VIRUS
317
these experiments. To destroy such RNA, the virus preparation was treated with ribonuclease before spraying. The resulting preparation was aerosolized at 15, 50 and 80% RH. Figure 6 shows the data of the 50% RH experiments. Again the virus RNA (iRNA-T) resisted spraying, whereas the virus infectivity (virus-T) was lowered by 2 logs. At 15 and 80% RH the same pattern was observed. At all these humidities inactivation proceeded at a reduced rate virus N when compared to the untreated virus. The reason is not known. o ~OEO10"A. \N~ W____¢f_ S\\ Stability of free SFV-iRNA in aerosols. Extracted SFV-iRNA was sprayed. Collection -x -_ _QOED 3*. \ fluids were titrated for iRNA. At 50% RH, free SFV-iRNA was completely stable (Fig. 5), in to whole virus. At 10 and 80% RH, no contrast OED */. inactivation of SFV-iRNA could be demonstrated either. NOED 0,5*/. N DISCUSSION SFV in crude suspension is stable in aerosols (1). To study the mechanism of aerosol inactiva4LO/. RH tion, protective materials have to be removed. The infectivity of the resulting purified virus FIG. 2. Influence of OED on aerosol inactivation suspension is unstable in aerosols (Fig. 1). Already 0 to 5 min after spraying, a factor of 30 or of SFV at 40% RH. more is lost. This can not be explained by the N
Nt lNo
FIG. 3. Influence of OED on aerosol inactivation of SFV at 85% RH.
15 45 60m;n 30 0 FIG. 4. Inactivation of SFiV and bacteriophage MS2 in a rotating bulb. A small volume of purified SFV together with phage MS2 was rotated slowly in a large glass bulb. A standing sample of the mixed
Similar data were obtained at 10 and 80% RH. The possible presence of SFV-iRNA outside the virions prevents a reliable interpretation of suspension served as a control.
318
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JONG ET AL.
Nt log
No
phage MS2, which is also sensitive to the rotat-
ing bulb treatment (Fig. 4), and to nebulization high RH (14). Conversely, EMC virus is stable in aerosols at high RH, as well as in the O _ iRNMA rotating bulb (5). The surface inactivation of a virus particle includes two steps (17): the sprayed iRNA and (i)(ii)penetration interface air-water by surdisruption into face tensions. Both phases presumably proceed -l ~ \ \ more readily with SFV as compared with EMC virus. The first process will be favored by the more hydrophobic nature of the lipid-containing surface of the SFV virion. The second step HA could be enhanced by the greater deformability -2 X \ of the lipoprotein membrane of SFV. The difference in behavior of EMC virus and phage MS2 is discussed earlier (5). Desiccation is prominent in aerosol decay of SFV at low RH. At 10 to 20% RH, the entire 3-_inactivation could be explained by this factor, because the loss of infectivity is the same as in glycerol solutions corresponding to these RH (Fig. 1). At 40% RH, dehydration probably still contributes to inactivation, as is suggested by 50 / RH the lower protective power of OED at 40% RH ,5 0 m, 45 0 15 30 60 min compared with 85% RH (Fig. 2 and 3). FIG. 5. Inactivation of SFV, SF V-HA, and SFVRNA in aerosols at 50% RH. After spraying of puri- lO N t fied SFV, aerosol samples were taken and titrated on No virus infectivity, on HA, and, after extraction with phenol, on iRNA (A). After spraying of free SFVat
iRNA, samples were assayed for iRNA (A).
&
RNA- U ~~~~~~~i
instability of the purified virus at standing, when only a factor of 2 is lost in 1 h. As shown in Fig., 1 SFV displays a region of high lability at high RH in aerosol, as has been -1l earlier described for SFV (1) and other lipidcontaining viruses: influenza virus (9), measles virus (7), Newcastle disease virus (13), respira\s v -T tory syncytial virus (12). With SFV another peak of inactivation was observed at 40% RH -2_ (Fig. 1). This peak is not reported by Benbough (1). It also occurs with respiratory syncytial virus (12). Oxygen is not involved in the aerosol inacti\ vation of SFV, the same decay being found in a 3 nitrogen atmosphere. The stability can be raised considerably by incorporating the surface-active agent OED in the spray medium (Fig. 2 and 3). This points at surface-located 500/o RH processes as the main mechanism of decay, as OED forms a layer at the particle-air interface, 0 30 15 45 60 min preventing the virus from entering this interFIG. 6. Aerosol inactivation of SFV and SFVface. Experiments in the rotating bulb system RNA at 50% RH. Purified SFV was nebulized before support this hypothesis. SFV in the thin film on (U) and after (T) treatment with ribonuclease to the wall of the bulb lost infectivity rapidly (Fig. remove extravirion iRNA. Samples were titrated on 4). whole virus (U: 0; T: 0) and, after extraction with This behavior is similar to that of bacterio- phenol, on iRNA (U: A; T: A).
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INACTIVATION OF SEMLIKI FOREST VIRUS
EMC virus is more vulnerable to desiccation than SFV. In concentrated glycerol solutions, as in aerosols with or without OED, infectivity titers may drop 5 logs in 32.5 min (5). The relative insensitivity of SFV to dehydration could be due to the lipids forning a barrier against the passage of water molecules. Perhaps this phenomenon is related to the fact that many respiratory viruses are provided with such a lipid membrane. This and the preceding study (5) show essentially different mechanisms for the aerosol inactivation of EMC virus at low RH on one hand and SFV at moderate and high RH on the other. Yet the consequences for the structural components are similar: destruction of the HA and conservation of the integrity of the RNA (Fig. 5 and 6). ACKNOWLEDGMENTS We wish to thank B. Khader Boutahar-Trouw for her excellent technical assistance and K. C. Winkler for helpful discussions. 1. 2.
3.
4.
LITERATURE CITED Benbough, J. E. 1969. The effect of relative humidity on the survival of airborne Semliki Forest virus. J. Gen. Virol. 4:473-477. Clarke, D. H., and J. Casals. 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7:561-573. de Jong, J. C. 1970. On the mechanism of the decay of poliomyelitis virus and encephalomyocarditis virus in aerosols, p. 210-211. In I. H. Silver (ed.), Aerobiology, Third International Symposium on Aerobiology, Brighton, 1969. Academic Press Inc., London. de Jong, J. C., M. Harmsen, and T. Trouwborst. 1973.
5. 6.
7.
8. 9.
10. 11. 12. 13. 14.
15. 16.
17.
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The infectivity of the nucleic acid of aerosol-inactivated poliovirus. J. Gen. Virol. 18:83-86. de Jong, J. C., M. Harmsen, and T. Trouwborst. 1975. Factors in the inactivation of encephalomyocarditis virus in aerosols. Infect. Immun. 12:29-35. de Jong, J. C., M. Harmsen, T. Trouwborst, and K. C. Winkler. 1974. Inactivation of encephalomyocarditis virus in aerosols: fate of virus protein and ribonucleic acid. Appl. Microbiol. 27:59-65. de Jong, J. G., and K. C. Winkler. 1964. Survival of measles virus in air. Nature (London) 201:1054-1055. Dulbecco, R., and M. Vogt. 1954. Plaque formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med. 99:167-182. Hemmes, J. H., K. C. Winkler, and S. M. Kool. 1960. Virus survival as a seasonal factor in influenza and poliomyelitis. Nature (London) 188:430-431. Kiarirainen, L., K. Simons, and C-H. von Bonsdorff. 1969. Studies in subviral components of Semliki forest virus. Ann. Med. Exp. Fenn. 47:235-248. Mihara, Y. 1966. Frost protection by fog droplets coated with monomolecular films. Nature (London) 212:602603. Rechsteiner, J., and K. C. Winkler. 1969. Inactivation of respiratory syncytial virus in aerosol. J. Gen. Virol. 5:405-410. Songer, J. R. 1967. Influence ofrelative humidity on the survival of some airborne viruses. Appl. Microbiol. 15:35-42. Trouwborst, T., and J. C. de Jong. 1973. Interaction of some factors in the mechanism of inactivation of bacteriophage MS2 in aerosols. Appl. Microbiol. 26:252257. Trouwborst, T., J. C. de Jong, and K. C. Winkler. 1972. Mechanism of inactivation in aerosols of bacteriophage Tl. J. Gen. Virol. 15:235-242. Trouwborst, T., and S. Kuyper. 1974. Inactivation of bacteriophage T3 in aerosols: effect of prehumidification on survival after spraying from solutions of salt, peptone, and saliva. Appl. Microbiol. 27:834-837. Trouwborst, T., S. Kuyper, J. C. de Jong, and A. D. Plantinga. 1974. Inactivation of some bacterial and animal viruses by exposure to liquid-air interfaces. J. Gen. Virol. 24:155-165.
Vol. 32, No. 3 Printed in U.S.A.
Inactivation of Semliki Forest Virus in Aerosols J. C.
JONG,l* M. HARMSEN, A. D. PLANTINGA,2 AND T. TROUWBORST3 Laboratory of Microbiology, State University, Utrecht, The Netherlands DE
Received for publication 24 February 1976
Purified Semliki forest virus in aerosols is inactivated rapidly at 40% and above 70% relative humidity. At all humidities tested the decay of virus infectivity runs parallel with the decrease in hemagglutination activity, whereas the biological integrity of the virus ribonucleic acid is preserved. Also, free infectious ribonucleic acid is stable after spraying at all relative humidities. Evidence is presented for the hypothesis that above 20% relative humidity, virus inactivation in aerosols is mainly due to surface-dependent factors, damaging the virus coat. MATERIALS AND METHODS Virus techniques. SFV strain SF/LS10/C1/A was obtained from C. J. Bradish, Porton, England. The virus was grown irk L cells in Eagle minimal essential medium without bicarbonate and with 0.01 M HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid). After 18 h at 37°C the infected cell culture was stored at 4°C. The supernatant was used for experiments. Plaque titration was performed on L cell monolayers with 48 h of incubation at 37°C. The crude suspension contained 6 x 108 plaqueforming units per ml. For purification, virus suspensions were centrifuged at low speed to remove cell debris and subsequently at 80,000 x g in a Spinco L50 ultracentrifuge at 4°C during 90 min. The sediment was suspended in 0.1 M NaCl with 0.001 M Na2HPO4, pH 7.6, using 0.005 times the original volume. The resulting suspension was subjected to gel exclusion chromatography over Sepharose 4B (Pharmacia, Uppsala). The purified preparation contained about 1010 plaque-fonning units per ml. The E260-E280 ratio was 1.47, indicating a nucleic acid content of 16%. Kaariainen et al. (10) reported a ratio of 1.3 to 1.4, corresponding to 11 to 13% nucleic acid. The purified virus was unstable. At 40C the titers of infectivity, iRNA and HA dropped by a factor of 2 per hour. Therefore, the preparations were titrated on these three parameters immediately before each experiment. iRNA. The virus suspension was supplied with sodium dodecyl sulfate (British Drug Houses, London) to 0.5% final concentration and added to an equal volume of phenol (Merck, p.a., redistilled and stored at -20°C under nitrogen), saturated with PBSE (phosphate-buffered saline, pH 7.2, with 0.01% ethylenediaminetetraacetic acid). This mixture was shaken vigorously by hand for 4 min at room temperature. The extraction was repeated with fresh phenol, with shaking for 2 min. twice ' Present address: National Institute of Public Health, After this, the water phase was shaken 5 times for Bilthoven, The Netherlands. 15 s with a double volume of ethyl ether (Merck, 2 Present address: Philips-Duphar, Weesp, The Netherp.a., distilled and washed with PBSE) each time. lands. 3Present address: Ministry of Health and Environmen- Finally, nitrogen gas or air was bubbled through the solution for 3 min at room temperature. Before tal Protection, Leidschendam, The Netherlands.
The inactivation of viruses in aerosols has been ascribed to desiccation or to processes located at the aerosol particle-air interface. The first mechanism is involved in the decay of the picornavirus causing encephalomyocarditis (EMC) in mice (5). The second factor prevails with bacteriophages Ti, T3, and MS2 (14-16). The localization of the damage to virions in aerosols has been studied with poliovirus and EMC virus. It has been demonstrated that, after destruction of the virus infectivity, the virus ribonucleic acid (RNA) retains its infectivity (3, 4). On the contrary, the hemagglutination activity (HA) of EMC virus in aerosols decreases parallel with the virus infectivity (3). The inactivation of lipid-containing viruses at spraying has not been investigated in these respects. We studied, therefore, the fate of Semliki forest virus (SFV) and its components in aerosols. SFV is a lipid-containing virus belonging to the alpha subgroup of the togaviruses. It shows HA and yields infectious RNA (iRNA) after extraction with phenol. Both properties are useful tools for localization of the damage at inactivation. Within 1 h after spraying from crude virus suspensions, virus decay is negligible (1). When working with purified preparations, however, considerable loss of infectivity has been observed. Addition of protein restored partly the aerosol stability of the virus (1), suggesting that surface-dependent processes could be responsible for the decay. This and other possibilities were tested in the present study, along with the fate of HA and RNA during inactivation.
315
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plating, 100 ,ug of diethylaminoethyl-dextran (Pharmacia, Uppsala, molecular weight 2 x 106) was added per ml. Dilutions were prepared in PBSE + diethylaminoethyl-dextran, and 2 x 107 L cells were suspended in 2-ml portions. After 2 min at 37°C the suspensions were added to preformed L-cell monolayers without removing the growth medium. After 1 h the plates were processed for plaque formation as with complete virus. The infectivity of the iRNA preparation was 0.01 to 0.1% of the original virus suspension and was completely abolished by incubation with ribonuclease (see below); this treatment did not influence the infectivity of whole virus for L cells. Ribonuclease treatment. Virus suspensions were supplied with 20 jig of ribonuclease (Boehringer, Mannheim) per ml. After 30 min at 37°C the virus was submitted to Sepharose 4B chromatography to remove the enzyme. In a control experiment SFViRNA was inactivated when added to the ribonuclease-containing virus suspension before, but not when added after chromatography. Hemagglutination. HA assays were performed with goose erythrocytes according to Clarke and Casals (2) in U-shaped disposable microtiter plates (Cooke Microtiter plates M24A, Dynatech, Billingshurst, Sussex, UK). HA patterns were read after 2 h at room temperature. HA titers of purified SFV suspensions were about 4 logs lower than infectivity titers and ranged around 106 units per ml. Experimental techniques. Aerosol apparatus and calculations have been described (6). The collection fluid was Dulbecco buffer (8) with 1% peptone. When the samples had to be assayed for iRNA, calcium and magnesium salts were omitted from the collection fluid. In the rotating bulb setup, a virus suspension is rotated in a large spherical flask to fonn a thin liquid film on the wall (17). Glycerol and OED were applied as before (5). Glycerol was mixed in various ratios with SFV in Dulbecco buffer. OED is a mixture of equal quantities of oxyethylene docosylether and oxyethylene octadecylether (11). Even in 10% OED the infectivity of SFV was, on standing for 1 h at room temperature, as stable as without the agent. On spraying, OED forms a film around each droplet (11). Graphs represent the results of at least two experiments.
RESULTS Inactivation of SFV in aerosols at different RHs. Maximal inactivation of purified SFV in aerosols occurred at 40% and above 70% relative humidity (RH) (Fig. 1). To test whether oxidation contributes to the decay, virus was sprayed in pure nitrogen at 20, 40, and 85% RH. No signiificant deviation from the aerosol experiments could be observed during 60 min after nebulization (not shown). Inactivation in glycerol solutions. In Fig. 1 the decrease of virus infectivity is plotted against the RH of the atmosphere that is in equilibrium with the glycerol-buffer mixture used. The aerosol data of the same virus preparation are included for comparison. It appears that the inactivation in aerosols at 10 to 20%
APPL. ENVIRON. MICROBIOL. log
Nt
No O_
-1
2.5 min 1.5 min in glycerol -2 \
m
\
n aer
\\ 0-5 min in aerosol
-3, \
\
/
K
1
/
o
230-35 min in aerosol
0/0 glycerol (w/w) -5 -
97 10
89 30
78 /. RH
50
64 70
35 90
FIG. 1. Inactivation of SFV in aerosols and in glycerol solutions. Purified SFV was sprayed and sampled from 0 to 5 min (0) and from 30 to 35 min (A). In other experiments, purified SFV was diluted in mixtures ofglycerol and phosphate-buffered saline in various ratios, corresponding to various relative humidities. Samples were taken from the solutions at 2.5 min (0) and at 32.5 min (A) after addition of virus.
RH roughly equals the decay in the corresponding glycerol solutions with 94 to 97% glycerol. At higher RH and lower glycerol concentration the two inactivation rates diverge. Sensitivity of SFV to surface exposure. The surface-active agent OED increased the stability of SFV in aerosols. The effect at 40% RH was less than at 85% RH (Fig. 2 and 3). In the rotating bulb system SFV was inactivated at the same rate as bacteriophage MS2 (Fig. 4). Phage MS2 is known to be vulnerable to surface-located forces (14). Fate of virus-RNA and HA at inactivation of SFV in aerosols. Purified virus preparations were nebulized at 10, 50, and 80% RH. Samples were assayed for infectious virus and for HA, and, after extraction with phenol, for iRNA. The result at 50% RH is shown in Fig. 5. Whole virus decay amounted to 2.7 logs in 60 min. The HA decreased parallel with the virus infectivity. The iRNA, on the contrary, was stable.
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these experiments. To destroy such RNA, the virus preparation was treated with ribonuclease before spraying. The resulting preparation was aerosolized at 15, 50 and 80% RH. Figure 6 shows the data of the 50% RH experiments. Again the virus RNA (iRNA-T) resisted spraying, whereas the virus infectivity (virus-T) was lowered by 2 logs. At 15 and 80% RH the same pattern was observed. At all these humidities inactivation proceeded at a reduced rate virus N when compared to the untreated virus. The reason is not known. o ~OEO10"A. \N~ W____¢f_ S\\ Stability of free SFV-iRNA in aerosols. Extracted SFV-iRNA was sprayed. Collection -x -_ _QOED 3*. \ fluids were titrated for iRNA. At 50% RH, free SFV-iRNA was completely stable (Fig. 5), in to whole virus. At 10 and 80% RH, no contrast OED */. inactivation of SFV-iRNA could be demonstrated either. NOED 0,5*/. N DISCUSSION SFV in crude suspension is stable in aerosols (1). To study the mechanism of aerosol inactiva4LO/. RH tion, protective materials have to be removed. The infectivity of the resulting purified virus FIG. 2. Influence of OED on aerosol inactivation suspension is unstable in aerosols (Fig. 1). Already 0 to 5 min after spraying, a factor of 30 or of SFV at 40% RH. more is lost. This can not be explained by the N
Nt lNo
FIG. 3. Influence of OED on aerosol inactivation of SFV at 85% RH.
15 45 60m;n 30 0 FIG. 4. Inactivation of SFiV and bacteriophage MS2 in a rotating bulb. A small volume of purified SFV together with phage MS2 was rotated slowly in a large glass bulb. A standing sample of the mixed
Similar data were obtained at 10 and 80% RH. The possible presence of SFV-iRNA outside the virions prevents a reliable interpretation of suspension served as a control.
318
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APPL. ENVIRON. MICROBIOL.
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Nt log
No
phage MS2, which is also sensitive to the rotat-
ing bulb treatment (Fig. 4), and to nebulization high RH (14). Conversely, EMC virus is stable in aerosols at high RH, as well as in the O _ iRNMA rotating bulb (5). The surface inactivation of a virus particle includes two steps (17): the sprayed iRNA and (i)(ii)penetration interface air-water by surdisruption into face tensions. Both phases presumably proceed -l ~ \ \ more readily with SFV as compared with EMC virus. The first process will be favored by the more hydrophobic nature of the lipid-containing surface of the SFV virion. The second step HA could be enhanced by the greater deformability -2 X \ of the lipoprotein membrane of SFV. The difference in behavior of EMC virus and phage MS2 is discussed earlier (5). Desiccation is prominent in aerosol decay of SFV at low RH. At 10 to 20% RH, the entire 3-_inactivation could be explained by this factor, because the loss of infectivity is the same as in glycerol solutions corresponding to these RH (Fig. 1). At 40% RH, dehydration probably still contributes to inactivation, as is suggested by 50 / RH the lower protective power of OED at 40% RH ,5 0 m, 45 0 15 30 60 min compared with 85% RH (Fig. 2 and 3). FIG. 5. Inactivation of SFV, SF V-HA, and SFVRNA in aerosols at 50% RH. After spraying of puri- lO N t fied SFV, aerosol samples were taken and titrated on No virus infectivity, on HA, and, after extraction with phenol, on iRNA (A). After spraying of free SFVat
iRNA, samples were assayed for iRNA (A).
&
RNA- U ~~~~~~~i
instability of the purified virus at standing, when only a factor of 2 is lost in 1 h. As shown in Fig., 1 SFV displays a region of high lability at high RH in aerosol, as has been -1l earlier described for SFV (1) and other lipidcontaining viruses: influenza virus (9), measles virus (7), Newcastle disease virus (13), respira\s v -T tory syncytial virus (12). With SFV another peak of inactivation was observed at 40% RH -2_ (Fig. 1). This peak is not reported by Benbough (1). It also occurs with respiratory syncytial virus (12). Oxygen is not involved in the aerosol inacti\ vation of SFV, the same decay being found in a 3 nitrogen atmosphere. The stability can be raised considerably by incorporating the surface-active agent OED in the spray medium (Fig. 2 and 3). This points at surface-located 500/o RH processes as the main mechanism of decay, as OED forms a layer at the particle-air interface, 0 30 15 45 60 min preventing the virus from entering this interFIG. 6. Aerosol inactivation of SFV and SFVface. Experiments in the rotating bulb system RNA at 50% RH. Purified SFV was nebulized before support this hypothesis. SFV in the thin film on (U) and after (T) treatment with ribonuclease to the wall of the bulb lost infectivity rapidly (Fig. remove extravirion iRNA. Samples were titrated on 4). whole virus (U: 0; T: 0) and, after extraction with This behavior is similar to that of bacterio- phenol, on iRNA (U: A; T: A).
VOL. 32, 1976
INACTIVATION OF SEMLIKI FOREST VIRUS
EMC virus is more vulnerable to desiccation than SFV. In concentrated glycerol solutions, as in aerosols with or without OED, infectivity titers may drop 5 logs in 32.5 min (5). The relative insensitivity of SFV to dehydration could be due to the lipids forning a barrier against the passage of water molecules. Perhaps this phenomenon is related to the fact that many respiratory viruses are provided with such a lipid membrane. This and the preceding study (5) show essentially different mechanisms for the aerosol inactivation of EMC virus at low RH on one hand and SFV at moderate and high RH on the other. Yet the consequences for the structural components are similar: destruction of the HA and conservation of the integrity of the RNA (Fig. 5 and 6). ACKNOWLEDGMENTS We wish to thank B. Khader Boutahar-Trouw for her excellent technical assistance and K. C. Winkler for helpful discussions. 1. 2.
3.
4.
LITERATURE CITED Benbough, J. E. 1969. The effect of relative humidity on the survival of airborne Semliki Forest virus. J. Gen. Virol. 4:473-477. Clarke, D. H., and J. Casals. 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7:561-573. de Jong, J. C. 1970. On the mechanism of the decay of poliomyelitis virus and encephalomyocarditis virus in aerosols, p. 210-211. In I. H. Silver (ed.), Aerobiology, Third International Symposium on Aerobiology, Brighton, 1969. Academic Press Inc., London. de Jong, J. C., M. Harmsen, and T. Trouwborst. 1973.
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The infectivity of the nucleic acid of aerosol-inactivated poliovirus. J. Gen. Virol. 18:83-86. de Jong, J. C., M. Harmsen, and T. Trouwborst. 1975. Factors in the inactivation of encephalomyocarditis virus in aerosols. Infect. Immun. 12:29-35. de Jong, J. C., M. Harmsen, T. Trouwborst, and K. C. Winkler. 1974. Inactivation of encephalomyocarditis virus in aerosols: fate of virus protein and ribonucleic acid. Appl. Microbiol. 27:59-65. de Jong, J. G., and K. C. Winkler. 1964. Survival of measles virus in air. Nature (London) 201:1054-1055. Dulbecco, R., and M. Vogt. 1954. Plaque formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med. 99:167-182. Hemmes, J. H., K. C. Winkler, and S. M. Kool. 1960. Virus survival as a seasonal factor in influenza and poliomyelitis. Nature (London) 188:430-431. Kiarirainen, L., K. Simons, and C-H. von Bonsdorff. 1969. Studies in subviral components of Semliki forest virus. Ann. Med. Exp. Fenn. 47:235-248. Mihara, Y. 1966. Frost protection by fog droplets coated with monomolecular films. Nature (London) 212:602603. Rechsteiner, J., and K. C. Winkler. 1969. Inactivation of respiratory syncytial virus in aerosol. J. Gen. Virol. 5:405-410. Songer, J. R. 1967. Influence ofrelative humidity on the survival of some airborne viruses. Appl. Microbiol. 15:35-42. Trouwborst, T., and J. C. de Jong. 1973. Interaction of some factors in the mechanism of inactivation of bacteriophage MS2 in aerosols. Appl. Microbiol. 26:252257. Trouwborst, T., J. C. de Jong, and K. C. Winkler. 1972. Mechanism of inactivation in aerosols of bacteriophage Tl. J. Gen. Virol. 15:235-242. Trouwborst, T., and S. Kuyper. 1974. Inactivation of bacteriophage T3 in aerosols: effect of prehumidification on survival after spraying from solutions of salt, peptone, and saliva. Appl. Microbiol. 27:834-837. Trouwborst, T., S. Kuyper, J. C. de Jong, and A. D. Plantinga. 1974. Inactivation of some bacterial and animal viruses by exposure to liquid-air interfaces. J. Gen. Virol. 24:155-165.
