JEREMIAH T. SIKI,1'2* MARY LOU BERNINGER,1 ALFONSO TORRES,1 JAMESA. HOUSE,' .... Indeed, Sullivan et al. (15, 16), using viruses ... f. 0. 1. 2. 3. 4. 5. 6. 0. 1. 2. 3. 4. 5. 6. 0. 1. 2. 3. 4. 5. 8. Irradiation Level (Mrads). FIG. 2. Titration of ...
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1993, P. 428432
Vol. 31, No. 2
0095-1137/93/020428-05$02.00/0 Copyright © 1993, American Society for Microbiology
Effect of Gamma Irradiation on Reactivity of Rinderpest Virus Antigen with Bovine Immune Serum in Enzyme-Linked Immunosorbent Assays and Virus Neutralization and Indirect Fluorescent-Antibody Tests JEREMIAH T. SIKI,1'2* MARY LOU BERNINGER,1 ALFONSO TORRES,1 JAMES A. CHARLES A. MEBUS,1
AND
HOUSE,'
EDWARD J. DUBOVI2
Foreign Animal Disease Diagnostic Laboratory, USDA/APHIS/VS/NVSL, P. O. Box 848, Greenport, New York 11944, 1* and Diagnostic Laboratory, NYS College of Veterinary Medicine, Cornell University, P.O. Box 786, Ithaca, New York 148512 Received 8 October 1992/Accepted 20 November 1992
Gamma irradiation effectively inactivated gradient-purified rinderpest virus. Irmdiated antigen and sera remained functional in enzyme-linked immunosorbent assays, virus neutralization tests, and indirect fluorescent-antibody tests. Irradiation, however, led to a dose-dependent decrease in reactivity, particularly significant (P < 0.05) when both reagents were irradiated. To avoid false-positive reactions, only one reagent (serum or antigen) may be irradiated.
Gamma irradiation (GI) is used to ensure innocuity of a variety of biological products that may contain live viruses. It has been shown to be an effective means for inactivating virus contaminants in sewage (18), tissue culture media (7, 15, 17), serum (9), and animal tissues (10, 16). Irradiated products may be useful as diagnostic reagents in laboratories that lack adequate biocontainment facilities. However, GI may lead to some loss in immunoreactivity either directly or indirectly through the formation of free radicals and peroxides. A few reports have described the effect of GI on viral antigens for use in various diagnostic tests, including direct immunofluorescence (10), indirect immunofluorescence and reverse passive hemagglutination (7), and Western blotting (immunoblotting) (1). The immunogenicity of an irradiated bluetongue virus vaccine has also been studied (3, 5). All those reports involved antigen-antibody reactions in which only the antigen was irradiated. There is no general agreement regarding the effect of GI on immunoreactivity. Here, we report the results of studies on the reactivity of gammairradiated rinderpest virus (RPV) and bovine sera in three commonly used serological tests-virus neutralization (VN), enzyme-linked immunosorbent assay (ELISA), and indirect fluorescent-antibody test (IFAT). Rinderpest, an economically important viral disease of cattle, sheep, goats, and wild ruminants, is presently limited to some areas of Africa and Asia where biocontainment facilities are rare. Diagnostic workers in those regions may need inactivated RPV antigen. Laboratories in disease-free areas may also need to possess some diagnostic capability. Our experiments were designed to include test conditions likely to be encountered in a laboratory using RPV antigens and sera under minimal biocontainment. The RBOK vaccine strain of RPV was grown in African green monkey kidney (Vero) cells and gradient purified as described elsewhere (4, 19). Briefly, RPV was concentrated from clarified infected cell culture supernatants by pelleting at 125,000 x g for 1 h. Purification was then carried out by *
successively centrifuging the resuspended pellet through a 20 to 60% step sucrose gradient for 1 h and then through a 15 to 45% continuous potassium tartrate gradient for 4 h. At each step, the virus was collected by pelleting at 125,000 x g for 1 h. The final pellet was resuspended in sterile phosphate-buffered saline (PBS) and stored at -70°C until used. Two other members of the Morbillivirus genus, the Edmonston strain of measles virus (MV) (provided by Max J. G. Appel) and the attenuated Nigeria 75/1 isolate of peste des petits ruminants virus (PPRV) (6), were used to find out whether irradiated RPV sera remained cross-reactive. Both viruses were grown in Vero cells and gradient purified as described for RPV. Three reference bovine serum specimens from our laboratory stocks were used: a control negative serum specimen (VN titer < 1 log2), a weakly positive (WP) serum specimen (VN titer = 8 log2) from a steer experimentally infected with the Kabete 0 strain of RPV, and a strongly positive (SP) serum specimen (VN titer = 13 log2) from a steer hyperimmunized with the Pendik strain of RPV. The sera were reconstituted from lyophilized stocks and stored at -70°C until used. Seven 1.5-ml aliquots of purified virus were made. An equal number of 1.5-ml aliquots of each serum were also prepared in screw-cap glass vials. One set of serum and virus, nonirradiated controls, were stored at -70°C; the six other sets were packed in cotton, hermetically sealed in metal cans, and irradiated while at -70°C with 1, 2, 3, 4, 5, and 6 megarads of gamma radiation (1 megarad = 10 kGy). The irradiator was a 50Co Gammacell 220 (Nordion International, Inc., Kanata, Ontario, Canada). The tissue culture infectivity of pre- and postirradiation RPV samples was assayed in Vero cells with 10-fold serial dilutions. The 50% endpoints were calculated by the Reed and Muench method (12). The initial RPV preparation had a titer of 6.5 log10 50% tissue culture infective doses per ml. All six levels of irradiation reduced the titer to < 1.0 log1o 50% tissue culture infective dose per ml. Virus-neutralizing antibodies in sera were titrated by a modification of the micromethod (13). Titrations were done in Vero cells with approximately 100 50% tissue culture infective doses of virus (RPV, MV, or
Corresponding author. 428
VOL. 31, 1993
NOTES
12
3
5
Vr6
Mrad
Mrad Mrad
10
E1z C\le
0
4
2
RPV
PPRV
Virus
Neutralized
FIG. 1. Neutralizing titers of NS and SP RPV IS against three morbilliviruses. Titers are expressed as log2 of the reciprocal of the highest dilution of serum that neutralized virus-specific cytopathic effects in two of two wells. ND, not done.
PPRV)
per well. Figure 1 presents the VN titers, expressed of the reciprocal of the highest dilution of serum that neutralized virus-specific cytopathic effects in two of two wells, for the SP serum. Similar results were obtained for the WP serum (data not shown) except that the titers were 4 log2 to 5 log2 lower. The negative serum did not neutralize any of the viruses. High doses of gamma radiation led to a reduction in neutralizing titers in both the homologous (RPV) and heterologous (MV and PPRV) systems (Fig. 1). The change in titer could not be assessed statistically for lack of sufficient replicates. Indirect ELISA was performed using a modification of established procedures (2, 8). Three experiments were done to investigate the effect of irradiation on the ELISA reactivity of RPV antigen and sera. In the first experiment, each antigen was titrated in eight replicates against a constant 1:20 dilution of nonirradiated sera (Fig. 2). In the second experiment, duplicate serial dilutions of sera were titrated against a previously determined (data not shown) optimal 1:125 dilution of antigen (Fig. 3). Finally, with sera and antigen at routine ELISA dilutions (1:20 and 1:125, respectively), all four combinations of irradiated and nonirradiated antigen and sera were tested: normal antigen (NAg)-normal serum (NS), NAg-irradiated serum (IS), irradiated antigen (IAg) NS, and IAg-IS (Fig. 4). Only one level of irradiation was used for each reagent, corresponding to the dose normally used in our laboratory to inactivate reagents being imported as log2
429
into the United States: 3 megarads for sera and 6 megarads for virus. Twelve replicates were used for each antigenserum combination, and the mean optical density (OD) values for appropriate pairs were compared by the least significant difference method after an analysis of variance for a nested design was performed (14). Figures 2 and 3 present the effects of GI on antigen and serum ELISA reactivities, respectively. The results indicate that high doses of GI resulted in a reduction in sensitivity. That decrease was particularly important for WP serum, which became negative at a 1:64 dilution for all six levels of irradiation (Fig. 3). Figure 4 presents mean ODs (n = 12) for various combinations of antigens and sera. Statistical analysis of those data for SP serum showed a significant (P < 0.05) decrease in sensitivity only when both reagents were irradiated. For WP serum, the decrease in sensitivity was statistically significant (P < 0.05) whether one or both reagents were irradiated. When 3-megarad-irradiated sera were tested against nonirradiated MV and PPRV antigens, the pattern of reactivity was similar to that observed for the NS-NAg and IS-NAg RPV pairs in Fig. 4, except the signal strength was generally 15 to 20% lower than corresponding values for RPV, as should be expected for heterologous reactions (data not shown). In comparison to the signal given by normal reagents (Fig. 4), the mean declines in OD values for the LAg-IS pairs were 28 and 51% for SP and WP sera, respectively. It should be emphasized, however, that GI even at high doses did not affect the detectability of antibody when reagents were used at routine dilutions (1:125 for antigen and 1:20 for serum) (see cutoff points, Fig. 2, 3, and 4). Furthermore, irradiation had no effect on specificity, as negative serum tested negative throughout. NS and IS (0, 3, and 6 megarads) diluted 1:50 in PBS were tested by IFAT on acetone-fixed RPV-, PPRV-, and MVinfected or mock-infected Vero cells, as previously described for MV (11). The test was read by using a blinded code to prevent interpretation bias. No staining was observed with the negative serum or on mock-infected cells. For both SP and WP sera, irradiation resulted in a dosedependent reduction in sensitivity for each of the three viruses. It was still possible, however, to clearly distinguish between WP and negative sera even when the highest dose (6 megarads) of irradiation was used. The effects observed on IAg and IS tested by ELISA, VN test, and IFAT were attributable to GI since the integrity of the reagents was otherwise preserved by keeping them cold (-70°C) during irradiation. Our results indicate that 1 megarad was sufficient to inactivate infectivity of the RPV antigen preparation for tissue culture. Other studies, involving different viruses, have shown a more gradual dose-dependent inactivation process (9, 10, 17, 18). The difference with our results may be explained more by the environment of the virus particles than by the type of virus. Indeed, Sullivan et al. (15, 16), using viruses from various families, have reported that the ease of inactivation of viruses depends on suspension media, with viruses suspended in culture media or in tissues requiring higher irradiation levels than the same viruses suspended in distilled water. They proposed that the presence of free radical scavengers (various proteins) in menstrua may reduce susceptibility of virus to GI. The absence of proteins in our virus preparation, other than constituents of the virus itself, may thus explain its higher susceptibility to GI inactivation. GI led to a dose-dependent reduction in antiserum activity as measured by VN test, ELISA, and IFAT. IAg and irradiated antibody, however, retained a satisfactory level of
430
J. CLIN. MICROBIOL.
NOTES a) SP serum 1.2
b) WP serum
0
0
c) Neg serum
-
1.0
0
Antigen dilutions: o 1/62.5 * 1/125*
j-
*~~~~~~~~
-
0
v
1/250 1/500
o
1/1000
v
0
* 1/2000
*10 8)* 0
0
Ce
0
0.6 -
P--
C
0
x=G9|
-_
1
2
3
4
5
6
Irradiation Level (Mrad) FIG. 3. Titration of NS and IS against NAg at 1/125 dilution. Symbols are similar to those in the legend to Fig. 2. Notice the prozone effect of SP serum at a high concentration (1/4 dilution).
NOTES
VOL. 31, 1993
431
2.0
T
NS/NAg NS/IAg IS/NAg IS/IAg
1.8 -
1--l-
1.6 -
0 C) LO ,-.4
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0 1.0 (3)
-
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Serum Identification FIG. 4. ELISA reactivities of various combinations of antigens and sera. Mean ODs plus standard errors of the means (n = 12) are shown. Antigens were treated with 6 megarads and sera were treated with 3 megarads, the normal dosages administered to reagents being imported into the United States. Neg, negative.
live virus antigens and to treat sera of exotic origin for use in ELISA, IFAT, and VN test in minimal biocontainment laboratories. For diagnostic purposes, however, only one irradiated reagent (serum or virus) may be used in a test without running the risk of getting false-negative reactions. We wish to acknowledge Barry Latney for assistance in irradiating the reagents used in this study.
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5. Campbell, C. H. 1985. Immunogenicity of bluetongue virus inactivated by gamma irradiation. Vaccine 3:401-406. 6. Diallo, A., W. P. Taylor, P.-C. Lefevre, and A. Provost. 1989. Att6nuation d'une souche de virus de la peste des petits ruminants: candidat pour un vaccin homologue vivant. Rev. Elev. Med. Vet. Pays Trop. 42:311-319. 7. Elliott, L. H., J. B. McCormick, and K. M. Johnson. 1982. Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation. J. Clin. Microbiol. 16:704-708. 8. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immunosorbent assay, ELISA-III. Quantitation of specific antibodies by enzyme-labeled antiimmunoglobulin in antigen-coated tubes. J. Immunol. 109:129-135. 9. House, C., J. A. House, and R. J. Yedloutschnig. 1990. Inactivation of viral agents in bovine serum by gamma irradiation. Can. J. Microbiol. 36:737-740. 10. McVicar, J. W., C. A. Mebus, A. Brynjolfsson, and J. S. Walker. 1982. Inactivation of African swine fever virus in tissues by gamma radiation. Am. J. Vet. Res. 43:318-319. 11. Norrby, E., S.-N. Chen, T. Togashi, H. Sheshberadaran, and K. P. Johnson. 1982. Five measles antigens demonstrated by use of mouse hybridoma antibodies in productively infected tissue culture cells. Arch. Virol. 71:1-11. 12. Reed, L. J., and H. Muench. 1937. A simple method of estimating fifty percent endpoints. Am. J. Hygiene 27:493-497.
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NOTES
13. Rossiter, P. B., D. M. Jessett, and W. P. Taylor. 1985. Microneutralisation systems for use with different strains of peste des petits ruminants virus and rinderpest virus. Trop. Anim. Health Prod. 17:75-81. 14. Snedecor, G. W., and W. G. Cochran. 1989. Statistical methods, 8th ed. Iowa State University Press, Ames. 15. Sullivan, R., A. C. Fassolitis, E. P. Larkin, R. B. Bead, Jr., and J. T. Peeler. 1971. Inactivation of thirty viruses by gamma radiation. Appl. Microbiol. 22:61-65. 16. Sullivan, R., P. V. Scarpino, A. C. Fassolitis, E. P. Larkin, and J. T. Peeler. 1973. Gamma radiation inactivation of coxsackie-
J. CLIN. MICROBIOL.
virus B-2. Appl. Microbiol. 26:14-17. 17. Thomas, F. C., A. G. Davies, G. C. Dulac, N. G. Willis, G. Papp Vid, and A. Girard. 1981. Gamma ray inactivation of some animal viruses. Can. J. Comp. Med. 45:397-399. 18. Thomas, F. C., T. Ouwerkerk, and P. McKercher. 1982. Inactivation by gamma irradiation of animal viruses in simulated laboratory effluent. Appl. Environ. Microbiol. 43:1051-1056. 19. Underwood, B., and F. Brown. 1974. Physico-chemical characterisation of rinderpest virus. Med. Microbiol. Immunol. 160: 125-132.
