Archives of Virology

Archives of Virology 61, 313--319 (1979)

© by Springer-Vertag 1979

Rhinoviruses: Kinetics of Ultraviolet Inactivation and Effects of UV and Heat on Immunogenicity By J. H. HUGHES, M. MITCItELL, and V. V. HAMPARIAN Departments of Medical Microbiology and Pediatrics, The Ohio State University, Columbus, Ohio, and Children's Hospital Research Foundation, Columbus, Ohio, U.S.A. With 2 Figures Accepted April 2, 1979

Summary The kinetics of ultraviolet inactivation for two h u m a n rhinoviruses and poliovirus were compared. No major differences in the rates of ultraviolet inactivation were detectable. All viral preparations inactivated b y ultraviolet irradiation induced neutralizing antibody in guinea pigs. In contrast, when guinea pigs were immunized with a heat inactivated rhinovirns preparation, little or no neutralizing antibody was elicited. Immune electron microscopy of the heated rhinovirus preparations revealed the presence of particles resembling empty capsids. These results suggest that rhinoviruses and enteroviruses are affected in a similar manner when subjected to ultraviolet or heat inactivation.

Introduction Ultraviolet spectra of absorption and kinetics of inactivation have been established for many animal DNA and I~NA viruses (2, i0, 23, 24, 28, 29). It has been clearly shown that the viral nucleic acid is the major site affected by ultraviolet irradiation at a wavelength of 260 nanometers. Several studies involving the effect of ultraviolet irradiation on poliovirus have been published (9, 18, 19, 25). With ultraviolet irradiation as the modifying agent, infectivity is lost prior to changes in antigenic specificity. Whether similar conditions exist for rhinoviruses is not known. The alteration of picornavirns antigens after exposure to heat has been reported for coxsackieviruses (27) and for polioviruses (I, 5, 6, 13, 18, 19, 22, 25, 26). This phenomenon has been reported for human rhinoviruses (5, 7, 20) with heat treated antigens used in neutralization, immunodiffusion, complement fixation tests and in a vaccine. The present study was done to determine the ultraviolet in-

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314

J, H. I~UGI~ES, M. MITCI~ELL, and V. V. HASIPAIClA~-:

a c t i v a t i o n k i n e t i c s of t w o h u m a n r h i n o v i r u s e s . U l t r a v i o l e t i n a c t i v a t i o n k i n e t i c s of p o l i o v i r u s t y p e 2 was i n c l u d e d for c o m p a r a t i v e p u r p o s e s . W e also e x a m i n e d t h e e f f e c t of u l t r a v i o l e t i r r a d i a t i o n a n d h e a t o n t h e i m m u n o g e n i e i t y of h u m a n rhinoviruses.

Materials and Methods Cell Culture, Viruses, and Serology The I l e L a cells used were o b t a i n e d from Microbiological Associates, Bethesda, Maryland. I n this laboratory, the cells were carried in Eagle's m i n i m u m essential m e d i u m w i t h Earle's balanced salt solution s u p p l e m e n t e d w i t h 10 percent fetal calf serum and 0.15 percent sodium bicarbonate. Penicillin, streptomycin, p o l y m y x i n B, and chlortetracyeline, at 100 units, 100, 10, and 50 ~g/ml respectively, were incorporated into t h e medium. F o r m a i n t e n a n c e purposes, t l e L a cells were grown in the same medium, b u t fetal calf s e r u m was reduced to 5 pereent, and sodium bicarbonate was increased to 0.30 pereent. The viruses used in this study were poliovirus t y p e 2 Sabin strain and h u m a n rhinovirus t y p e s 17 and 40. Viral preparations, after growth in t~eLa cells, were partia.lly purified by a single fluorocarbon extraction. A n t i s e r a preparation, s e r u m n e u t r a l i z a t i o n tests and i m m u n e electron microscopy were p e r f o r m e d as previously described (3, 11, 12). Ultraviolet Inactivation U l t r a v i o l e t irradiation of viruses was p e r f o r m e d on a rocker platforra ( D r u m m o n d Scientific Co., Broomall, Pennsylvania). The distance of the rocker p l a t f o r m from the s t a t i o n a r y t a m p was 18.8 era. A General Electric 15 w a t t Germicidal bulb ( G I 5 T 8 ) was used as an ultraviolet light source. Measurements of the energy o u t p u t were 25.8 × 104 ¢o 32.0 × t04 ergs/see/em 2 a t 1 m e t e r a.I~d were taken before and after each e x p e r i m e n t using a Westinghouse Sterilamp m e t e r ® (Westinghouse Electric Corporalion, Bloomfield, N e w Jersey). F o r kinetics of viral inactivation, a t : l 0 dilution of poliovirus t y p e 2 and rhinovirus t y p e s 17 and 40 was m a d e in 5 ml a m o u n t s in serum free m e d i u m and distributed into 10 × 100 m m glass petri dishes. A single petri dish was placed u n c o v e r e d in t h e center of a rocking p l a t f o r m and virus exposed for various lengths of t i m e to ultraviolet irradiation. F o r control purposes, an aliquot of each viral p r e p a r a t i o n was t r e a t e d similarly b u t w i t h o u t exposure to ultraviolet irradiation, l~esidual i n f e e t i v i t y after irradiation was m e a s u r e d b y plaque assay (¢). Heat Inactivation Ten ml ampules were filled w i t h 8 ml of rhinovirus t y p e 17 and flame sealed. A f t e r equilibration on ice, all ampules were i m m e r s e d in a 56 ° C w a t e r b a t h for t6 minutes. After h e a t inactivation, the ampules were r e m o v e d from the w a t e r b a t h and placed at 4 ° C for one-half hour before being frozen at - - 8 0 ° C. Controls were h a n d l e d similarly b u t were n o t h e a t inactivated. H e a t t r e a t e d preparations of rhinovirus t y p e 17 were e x a m i n e d by i m m u n e electron microscopy ( I E N ) for the presence of D- and C-antigenic particles. The 1)-particles are n o t p e n e t r a t e d w i t h stain, while C-partieles are p e n e t r a t e d by stain (14).

Results and Discussion T h e k i n e t i c s of u l t r a v i o l e t i n a c t i v a t i o n of r h i n o v i r u s t y p e s 17 a n d 40 a n d p o l i o v i r u s t y p e 2 a r e p r e s e n t e d i n F i g u r e 1. T h e i n a c t i v a t i o n c u r v e s w e r e p l o t t e d u s i n g t h e b e s t fit line, w h i c h was d e t e r m i n e d b y u s i n g t h e l e a s t s q u a r e s m e t h o d . T h e i n a c t i v a t i o n r a t e s for all v i r u s e s w e r e similar. H a l f - l i f e t i m e s for r h i n o v i r u s t y p e s 17 a n d 40 w e r e a p p r o x i m a t e l y 3 a n d 5 seconds r e s p e c t i v e l y . T h e half-life for p o l i o v i r u s t y p e 2 w a s a p p r o x i m a t e l y 4.5 seconds. All half-life t i m e s w e r e

Ultraviolet and Heat Effects on lghinoviruses

315

determined from the best fit, inactivation plots. W h e n tested undiluted, infectivity for all viruses was gone before 90 seconds of exposure to ultraviolet irradiation. The slopes of the inactivation plots were --0.078 for rhinovirus t y p e 17, --0.087 for rhinovirus t y p e 4~0, a,n d - - 0 . 0 7 3 for poliovirus t y p e 2. Zero-order correlation coefficients for a,ll viral inactivation curves were within 1.0 percent. Control viral preparations which were rocked w i t h o u t ultraviolet irradiation, did not lose infectivity. 8 ¸

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Fig. 1. The kinetics of ultraviolet inactivated human rhinoviruse types 17, 40 and poliovirus type 2 W h e n viral preparations were treated with ultraviolet irradiation for 7 rainutes and used as antigens for evoking neutralizing antibody, there was less t h a n a tenfold reduction in the geometric m e a n serum neutralizing a n t i b o d y titers when compared to controls (Table 1). The ultraviolet inactivation time for these experiments (7 minutes) is estimated from Figure 1 to be an average of about 6 times t h a t

316

J . H . HUGHES, IVi. MITCHELL,and V. V.

HAMPAI~IAN

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needed to destroy all infectivity. The reduction in geometric mean titers between ultraviolet treated and untreated preparations was 2.1-fold for rhinovirus type 17, 5.7-fold for rhinovirus type 40, and 6.4-fold for poliovirus type 2. Although not shown, when similar preparations were irradiated for 13 mim~tes and used as antigen, significant amounts of neutralizing antibody were produced for all viruses. KATAGIRI et al. (16) also found that almost all poliovirus type 1 treated with ultraviolet, light for 10 minutes retained N (D) antigenicity. The production of neutralizing antibody with ultraviolet treated rhinovirus preparations demonstrates that infectivity is not required for immunogenieity. Thus, for picornaviruses, infectivity does not appear to be required for production of neutralizing antibody when viral preparations are inactivated with ultraviolet irradiation. Table 1. Serum neutralizing antibody titers induced by native, ultraviolet and heat treated picornavirus antigens Virus

Treatment ~

GMT b

Nmnber of animals

l~hinovirus type 17 ghinovirus type 17

None Ultraviolet

2793 1302

6 7

Rhinovirus type 40 I~hinovirus type 40

None Ultraviolet

2988 516

6 8

Poliovirus type 2 Poliovirus type 2 II,hinovirus type 17 Rhinovirus type 17

None Ultraviolet None Heat

4006 618 1875 12

4 5 5 5

Ultraviolet treatment was for 7 minutes and heat treatment for 16 minutes at 56° C as described in the text b Geometric Mean Titer. Antisera were tested against 10--300 TCIDao of virus When rhinovirus type 17 was he,ted at 56 ° C, the titer dropped from 7.0 to 3.5 TCID50/ml. A native (unheated) preparation elicited high levels of neutralizing antibody, yielding a geometric mean titer of 1875 (Table 1). On. the other hand, a heated preparation induced little if any neutralizing antibody. The geometric mean titer obtMned after immmdzation with the heat treated preparation was t2. This was greater than a 150-fold reduction in titer. Since there was a dramatic loss of ability to induce neutralizing antibody by the heated but not the ultraviolet irradiated preparations, we decided to examine the heated preparation by IEM for the presence of C (stain penetrated) and D (not stain penetrated) viral particles. Figure 2 shows the results obtained with native and heat treated weparations for rhinovirus type 17. The native (control) preparation consisted of apparently normal particles, while the heated preparation showed mostly particles filled with stain. The particles penetrated with stain probably have C antigenieity, since they are agglutinated b y a dilution (1:100) o f human serum devoid of neutralizing activity (12). The human serum used did not agglutinate native viral antigen when tested by IEM. Morphological, structural and antigenic studies involving the degradation of either potiovirus or rhinovirus by heat (1, 5, 6, 8, 15, 20, 21, 30) have revealed

Ultraviolet and Heat Effects on ]ghinoviruses

317

the production of empty viral capsids and other products such as fibrillar structures, 135 S infectious particles, B particles, and 80 S ribonucleoprotein complexes. The viral products produced are dependent u r o n the time and temperature of inactivation. The heated rhinovirus preparations generated during this study and observed by IElV[ consisted of empty ealosid-like material. No fibrillar, ribonucleoprotein-like structures or full parbicles were apparent. Using poliox, irus heated at 55 ° C for 30 minutes, HIssMA et al. (8) and KATAGIRI and tIINVMA (17) found infectivity was reduced to less than 0.01 percent of

Fig. 2. Immune electron microscopy of heat treated rhinovirus type 17 preparations. A Native lghinovirus type 17 preparation without amtiserum. B Heated tihinovirus type 17 preparation with human serum diluted 1:100. Magnification 300,000 ×, size bar is 50 nanometers

318

J. ~ . HUGHES, M. MITCHELL, and V. V. H~5~.~I~X~N:

u n h e a t e d virus, e m p t y capsids were formed a n d no N (D) a n t i g e n i c i t y was detectable. W h e n poliovirus preparations were heated for only one m i n u t e at 55 ° C, i n f e c t i v i t y was reduced to less t h a n 0.0001 percent of control samples a n d the stain p e n e t r a t e d particles generated were aggregated b y a n t i s e r u m with C-antigen specificity (6). Dooe~T~ et al. (7) observed t h a t when a rhi~ovirus vaccine was heated for 30 m i n u t e s at 56 ° C, it was no longer antigenic. DINNOCK (5) showed t h a t rhinovirus preparations heated for 30 m i n u t e s at 5 5 ° C failed to combine with n e u t r a l i z i n g a n t i b o d y . Similarly, when our r h i n o v i r u s preparations were heated ~t 56 ° C for 16 minutes, most of the particles were p e n e t r a t e d with stain, infectivity wa.s reduced to less t h a n 0.001 percent of controls, a n d little or no neutrMizing a n t i b o d y was evoked in guinea pigs. This is i n contra.st to our ultraviolet t r e a t e d viral p r e p a r a t i o n s t h a t completely lost i n f e c t i v i t y w i t h i n a few m i n u t e s of t r e a t m e n t b u t still elicited n e u t r a l i z i n g a n t i b o d y . These results suggest t h a t h u m a n rhinoviruses react i n a m a n n e r similar to enteroviruses when subjected to either ultraviolet or heat i n a c t i v a t i o n .

Aeknowledfl ements The authors wish to recognize the capable technical assist, ante of Ms. K. Fowler.

References 1. B~EISrDL, M.: The structure of heated potiovirus particles. J. gem Virol. l i , 147 to 156 (197t). 2. CA~E~O~', K. R. : Ultraviolet irradiation of herpes simplex virus : action spectrmn for tile survival of infectivity in relation to the small-plaque effect. J. gen. Virol. lg, 5 t - - 5 4 (1973). 3. CONANT, 1%. ~{., HASIPArtLd.N, V. V. : /~hinoviruses: basis for a numbering system. 1. I-IeLa cells for propagation and serologic procedures. J. Immunot. t00, 1 0 7 - - i t 3 (t968). 4. CONA~T, R. M., S o ~ s o ~ , N. L., HA~_r~A~I~?¢, V. V.: Plaque Formation by I%hinoviruses. Proc. Soc. exp. Biol. Med. 128, 51--56 (19(18). 5. DIM~oeK, N. J. : Differences between the thermal inactivation of pieornaviruses at "high" and "low" temperatures. Virology 31, 338--353 (1967). 6. DI~Moe~, N. J., HA~nis, W. J.: Heterogeneity of apparently empty poliovirus particles stained with phosphotungstic acid after heating of the virus at "low" temperature. Virology 31[, 715--719 (1967). 7. DOGGETT, J. E., B¥~OE, M. L., TYRaEL~, D. A. J. : Some attempts to produce an experimental vaccine with rhinoviruses. Brit. med. J. 1, 34--36 (1963). 8. HINUMA, Y., NATAGII~I, S., FUI~UDA, M., FUKUSHI, K., WATANABE, Y. : Kinetic studies on the thermal degradation of purified poliovirus. Biken J. 8, 143 153 (1965). 9. ~ I ~ u ~ A , Y., KA'rAGI~, S., A I ~ w A , S. : I m m u n e response to H particles of poliovirus. Virology 4@, 773--776 (i~970). 10. ~[OLLAENDEI¢, A., 0LIP~AN'T, g. ~¥. : The inactivating effect of monochromatic ultraviolet radiation on influenza virus. J. Bacteriot. 48, 447--454 (1944). 11. I-IVCH~S, J. H., C~ES~A, S., LIx, N., CONANT, ]~. M., HASiPARIAN, V. V.: Acid la.bility of rhinoviruses: loss of C and D antigeniciW after treatment at pI~ 3.0. J. Immunol. 112, 9t9--925 (1974). 12. I-IUGHES, J. ~J[., GIgAU, J. M., HILTY, M. D., CHEMA, S., OTTOLENGIII, A. C., H~P~aIAN, V. V. : Picornaviruses: rapid differentiation and identification by immune electron microscopy and immunodiffusion. J. reed. Mierob. 10, 203--212 (1977).

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and

Heat

Effects on Rhinoviruses

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13. HtJ~M~ELEI~, K., HA~IPAt~IAN,V. V. : Studies on the complement-fixing antigens of poliomyelitis. I. Demonstration of type and group specific antigens in native and heated viral preparations. J. Immunol. 81, 499--505 (1958). 14. HVMNELEt~, K., ANDERSON, T. t~., BROWN, t~. A. : Identification of poliovirus particles of different antigenicity by specific agglutination as seen in the electron microscope. Virology 16, 84--90 (1962). t5. JORDAN, L., MA¥O~, H. D.: Studies on the degradation of poliovirus by heat. Mierobios 9, 51--60 (1971). 16. I~TAGI~I, S., I : h N ~ A , Y., ISHIDA, N. : Biophysical properties of polio~drus particles irradiated with ultraviolet light. Virology 32, 337--343 (1967). 17. KATAGIRI, S., 11INUMA, Y. : Complement-fixing antibodies produced in guinea pigs immunized with purified N and I t antigens of poliovirus. Tohoku J. exp. Med. 88, 153--160 (1966). 18. LE B o u v I ~ , G. L. : The modification of poliovirus antigens by heat and ultraviolet light. Lancet 2, 1013----1016 (1955). 19. LE BGUV~E]~, G. L. : The D -->C change in poliovirus particles. Brit. J. exp. Pathol. 40, 605----620 (1959). 20. LO~BEIm-HoLM, K., YI~, F. H . : Antigenic determinants of infective and inactivated human rhinovirus type 2. J. Virol. 12, 114--123 (1973). 21. MeGRNC~O~, S., ?¢IAYO]:~,H. D. : Internal components released from rhinovirus and poliovirus by heat. J. gen. Virol. 10, 203- -207 (1971). 22. POWELL, t-L 5I., CVJ~BERTSON, C. G. : Short t e r m heat stability of poliomyelitis vaccine. £roc. Soc. exp. Biol. Med. 89, 490--492 (1955). 23. P~AV~I~,A. M. : The physical state of viral nucleic acid and the sensitivity of viruses to ultraviolet light. Biophys. J. 5, 257--273 (1965). 24. I~Ivn~s, T. M., GATES, F. L. : Ultra-violet light and vaccine virus. II. The effect of monochromatic ultra-violet light upon vaccine virus. J. exp. lVied. 47, 45--49 (1928). 25. ROlZ~ASr, B., MAVEn, M. M., ROANE, P. R., JR. : ImmunochemicM studies of poliovirus. IV. Alteration of the immunologic specificity of purified poliomyelitis virus by heat and ultraviolet light. J. Immunol. 82, 19--25 (1959). 26. ScEs~I])~, N. J., L E ~ E ' r T ~ , E. H. : Modification of the homotypic specificity of poliomyelitis complement-fixing antigens by heat. J. exp. Ned. 104, 99--120 (1956). 27. ScI~ID~, N. J., D E ~ I S , J., F ~ o ~ 5 ~ : N , L. H., LE~rNET~E, E. It.." Serologic reactivity of certain antigens obtained by fractionation of coxsackie viruses in cesium chloride density gradients. J. Immunol. ~0, 654--662 (1963). 28. S ~ , E. H°, B ~ ) s o x , It. S. : A comparison of ultraviolet action spectra for vaccinia virus and T 2 bacteriophage. J. gen. Virol. 18, 55--60 (1973). 29. SwRv~, E., GAffES, F. L., M~'~Z~H¥, J. B.: Properties of the causative agent of a chicken tumor. II. The inactivation of the tumor-producing agent by monochromatic ultra-violet light. J. exp. Med. 55, 441--444 (1932). 30. VAN ELSEX, A. A., BOEYE, A., T~.uc~¥, H. : Formation of fibrillar structures from poliovirus by alkaline disruption and other treatments. Virol. 36, 511--514 (1968). Author's address: J. I~I. H u e ~ s , Ph. D., D e p a r t m e n t of Medical l~{icrobiology, 5072 Graves I-~all, College of Medicine, The Ohio State University, 333~¥. 10th Avenue, Columbus, OH432t0, U.S.A. I~eceived December 5, 1978