INFECTION AND IMMUNITY, May 1976, p. 1454-1458 Copyright C 1976 American Society for Microbiology
Vol. 13, No. 5 Printed in U.S.A.
Use of the Hemadsorption Phenomenon for Determining Virus and Neutralizing Antibody Titers of Rabies N. MINAMOTO,* K. KURATA, I. KAIZUKA, AND H. SAZAWA National Veterinary Assay Laboratory, Kokubunji, Tokyo, 185 Japan Received for publication 20 November 1975
Chicken embryo cells infected with the HEP Flury strain of rabies virus adapted to tissue culture produced a hemadsorption (HAD) phenomenon by using goose erythrocytes. The optimal conditions for HAD included the incubation of cell cultures at 37 C for 3 days after virus inoculation, the use of a 0.4% suspension of goose erythrocytes in phosphate buffer adjusted at pH 6.2, and adsorptioh of erythrocytes at 4 C. This phenomenon was inhibited with antirabies serum. Virus titer obtained with the HAD technique was almost the same as with the fluorescent antibody technique or the intracerebral inoculation of suckling mice. Results of the neutralization test by using the HAD technique could be easily determined 3 days after inoculation of chicken embryo cells with the mixture of 100 mean tissue culture infective doses of virus and diluted serum. The neutralizing antibody titers coincided with those obtained in mice.
Although several methods have been used for determining rabies virus or antibodies, each method has a few inherent disadvantages. For example, they need large numbers of animals (1), more than a 5-day interim between inoculation and final results (1, 2, 11, 16), and an accurate procedure (11, 16). Some methods indicate sporadic results (5) and negative or very low titer for evaluation of immune effectiveness (9). For the improvement of techniques to measure virus and antibody, we investigated the hemadsorption (HAD) phenomenon by which virus could be detected more rapidly and in the presence of less hemagglutinin than in the hemagglutination (HA) and hemagglutination inhibition (HI) techniques. Recently, the HAD phenomenon has been attempted primarily with street virus adapted to hamster kidney cells by Selimov and Ilyasova (12). However, detailed conditions of the reaction or practical methods employing the HAD phenomenon for the titration and serum neutralization (SN) test of rabies virus have not been reported. In this paper, we describe the HAD phenomenon produced in a system of the HEP Flury strain of fixed rabies virus adapted to tissue culture (HF-TC strain) and chicken embryo (CE) cells, which have been generally used in many laboratories, and show that this phenomenon now can be used for determining virus and SN antibody titers of rabies with reproducible results. MATERIALS AND METHODS Virus strain. The principal yirus used was the HF-TC strain of rabies virus obtained from A. Kondo, National Institute of Health, Tokyo, Japan,
which had been adapted to 7-day CE cells as described earlier (5), and had been purified by cloning three times after the 100th passage by A. Kondo. HF-TC strain is a large-plaque variant. The stock virus (103rd to 106th passage level), which was prepared as described previously (9), was used. Titration of virus by HAD. Serial 10-fold diluted virus (0.05 ml) was inoculated into four tube monolayers. After 1 h at 37 C, the inoculum was removed and each tube was washed twice with phosphatebuffered saline (PBS) (3). Then, to each tube was added 0.5 ml of maintenance medium (medium 199 with 0.4% sodium bicarbonate and 0.11% bovine albumin [fraction V]). After incubation of the cell monolayers at 37 C for 3 days, the maintenance medium was removed, and each tube was washed once with PBS. Then, 0.2 ml of a 0.4% suspension of adult goose erythrocytes in phosphate buffer, pH 6.2 (0.15 M NaCl, 0.056 M Na2HPO4, and 0.144 M NaH2PO4, pH 6.2), was added to each tube. Goose blood was preserved in equal part of Alsever solution and was stored at 4 C. Erythrocytes were used for each experiment after being washed three times with saline. The tubes were placed in a horizontal position at approximately 4 C for 20 min and returned to upright position at room temperature for an additional time, about 10 min. Then, foci of HAD on infected cell monolayers were investigated by microscope under low magnification (x 100). If foci of HAD were observed on cell monolayers, the test tube was read as viral positive (Fig. 1). Based on the above results, mean tissue culture infective dose (TCID50) was calculated by the method of Reed and Muench (10). SN test by HAD. Anti-rabies sera were obtained from guinea pigs administered several or only one injection (6) and were diluted 10-fold. An equal volume of virus suspension containing about 100 TCID,,) per 0.05 ml was added to each diluted serum. After incubation in a water bath at 37 C for 1 h, the
1454
VOL. 13, 1976
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USE OF THE HEMADSORPTION PHENOMENON
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FIG. 1. HAD induced by the HF-TC strain of rabies virus. (a) HAD reaction by goose erythrocytes in CE cell monolayers at 3 days after virus inoculation (grade of HAD: + ++ - + + + +); (b) focus of HAD (+ + +); (c) HAD-negative CE cell monolayers. (Magnification xl00). mixtures were inoculated onto four cell monolayers in 0.05-ml amounts. Adsorption of the mixtures was allowed to take place at 37 C for 1 h, and the cell monolayers were carefully washed twice with phosphate buffer. The reading of results was performed by means of HAD at 3 days after incubation. SN titer of serum tested was calculated by the method of
Reed and Muench (10) from the highest dilution of serum that inhibited HAD completely.
RESULTS . .
Conditions of erythrocytes, pH, temperature, and observational days for the HAD phe-
1456
MINAMOTO ET AL.
nomenon. CE cell monolayers infected with the HF-TC strain induced the HAD phenomenon by erythrocytes of hamsters, guinea pigs, rabbits, fowl, geese, pigs, sheep, and horses. However, a 0.4% goose erythrocyte suspension showed the most clear and sensitive HAD phenomenon. Consequently, the following experiments were done with goose erythrocytes. Virus suspensions diluted 10-fold serially from 10-' to 10-7 were inoculated into CE cell cultures in amounts of 0.05 ml. The optimal conditions for HAD were established by investigating the effect of each condition on virus titer determined by HAD. The influence of various pH values of phosphate buffer to suspend erythrocytes on HAD was investigated. The highest virus titer was obtained at pH 6.2 (Table 1), but the HAD phenomenon was not observed with a suspension of erythrocytes in saline adjusted to various pH values (5.8 to 7.0). Next, we tested the effect of various temperatures on the adsorption of erythrocytes. The maximum titer (107 TCID5o/ml) was obtained by incubation at 4 C. Almost the same titer was observed at room temperature, but the HAD pattern was not satisfactorily developed. At 37 C, the HAD phenomenon was not observed. In the various adsorption times of erythrocytes, the HAD pattern was fully developed at 20 min, but an adsorption time less than 15 min was not enough to develop the HAD pattern. The time course of HAD appearance after inoculation of approximately 50,000 TCID;,,/ml of virus was investigated. HAD was first recognized (+ + +) 1 day after inoculation, and it became satisfactory after 3 days (+ + + +++ +). Then, virus titration of infected cell culture fluid which was harvested for 3 days after inoculation was performed to find the optimal observation period in days so as to determine the virus titer by means of HAD. Virus titer reached a peak at 3 days, and thereafter the same titer was maintained until 5 days (Table 2). Virus titers of the same material determined by the fluorescent antibody (FA) technique and in suckling mice inoculation were 104 TCID.5/ml and 10-' mean lethal doses/ml, respectively. Accordingly, the following experiments using HAD were performed at pH 6.2 and 4 C, with 0.4% goose erythrocytes, at 3 days after virus inoculation. Under these conditions, a positive reaction easily indicated a recognizable characteristic pattern so that it was not necessary to remove the unadsorbed erythrocytes, because almost all unadsorbed erythrocytes were precipitated to the bottom of the tube by returning it to an upright position. On the other hand, if HAD foci were washed with phosphate buffer or an-
INFECT. IMMUN. TABLE 1. Effect of various pH of erythrocyte suspension on the rabies virus titers determined by HAD pHa
Virus titerb
5.8 6.0 6.2 6.4
7.4 7.5 7.8 4.7 3.2
6.6
Saline 8 (5.8 7.0) -1. a Goose erythrocytes were suspended in phosphate buffer adjusted to various pH values. b Titers determined by using HAD as indicator instead of CPE at 3 days after virus inoculation, mean of two titrations (log TCID50/milliliter). -
TABLE 2. Titration by HAD of HF-TC strain in CE cell monolayers Days after inoculation
Dilution of virusa 1
2
3
4
5
4/4b
Control
1/4 0/4 0/4 0/4 0/4
4/4 4/4 4/4 3/4 1/4 0/4
4/4 4/4 4/4 4/4 1/4 0/4
4/4 4/4 4/4 4/4 1/4 0/4
4/4 4/4 4/4 4/4 1/4 0/4
Virus titer
50
7.8
8.0
8.0
8.0
10-3 10-4 10-; 10-6 10-'
(log TCID501/Ml)
a Cell monolayers were inoculated with 0.05 ml of diluted virus. b Number of HAD positive tubes/number of tested tubes.
other solution (higher pH than 6.2), the foci became slightly indistinct. Specificity of the HAD phenomenon. The HAD phenomenon was inhibited specifically by adding rabies immune serum to infected cell monolayers and was not inhibited by normal guinea pig serum, Japanese encephalitis, hemagglutinating encephalomyelitis, parainfluenza 1 and 3, Newcastle disease, equine influenza, vesicular stomatitis, infectious canine hepatitis virus, and Mycoplasma (M. synoviae, M. pneumoniae, and M. gallisepticum) immune sera. SN test in immune sera. The HAD phenomenon was applied to rabies SN test as indicator of virus growth instead of cytopathic effect. (CPE). Twenty-four SN tests were performed by using two guinea pig sera at different stage of immunization and a hyperimmune serum of guinea pig in order to investigate whether reproducible results could be obtained in rabies SN titers. Titers of three sera by SN test in mice were 30, 74, and 14,111, respectively. All
VOL. 13, 1976
USE OF THE HEMADSORPTION PHENOMENON
1457
tests indicated reproducible results, but the differences in each SN titer were less than twofold 80 except two (tests 2 and 5, Table 3). Comparison of SN titers determined by 40 HAD and mice. Ten guinea pigs were vaccinated subcutaneously with 4 ml of inactivated 2: vaccine. Then blood samples were collected by 20 intracardial puncture for titration of SN anti4.4 bodies at intervals of a week. The geometrical V. 10 means of SN titers determined by HAD and mice are shown in Fig. 2. SN antibodies measured by both methods became detectable 7 days 0 2 3 4 5 after vaccination, and with a striking degree of Time in weeks after vaccination parallelism both titers increased until 21 days. The comparative SN titers of 60 sera deterFIG. 2. Responses of SN antibody in guinea pigs mined by both tests are shown in Fig. 3. The immunized with the vaccine. (a) SN titers detertiters determined by HAD were about 1.8 times mined by HAD; (b) SN titers determined by mice; (c) (varying from 0.4 to 6.3) higher than the mouse geometric mean SN titer of 10 guinea pig sera. SN titers. The correlation coefficient for the results of both methods was +0.95. -4
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DISCUSSION It is already known that the HAD phenomenon occurs on tissue culture cells infected with myxovirus (13, 15), paramyxovirus (14), and coronavirus (8), which are hemagglutinating viruses. The HAD phenomenon was provided for a practical approach to diagnostic and fundamental studies of these agents. Because HAD depends on direct visualization under low magnification (x 100), it is detectable in the presence of minimal hemagglutinin associated with tissue culture cells, resulting in more sensitivity than with procedures based on the development of CPE or usual HA by virus suspension. In the experiments described in this paper, we demonstrate that the HF-TC strain which TABLE 3. Reproducibility of SN antibody titer determined by HAD Serum No. of test la 2b 3i
1 102 45 10,240 2 20 128 14,488 91 3 23 10,240 4 128 7,362 25 81 5 20 10,240 6 23 161 12,190 7 128 23 12,093 NTv 8 128 14,488 Mean ± SID 26 ± 9 118 ± 26 11,056 ± 2,982 a Guinea pig serum 2 weeks after single injection of vaccine. b Guinea pig serum 5 weeks after single injection of vaccine. " Hyperimmune serum of guinea pig. d NT, Not tested. ' SD, Standard deviation.
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FIG. 3. Relationship between SN antibody titers determined by HAD and mice; (a) SN titer determined by HAD; (b) SN titer determined by mice.
grew in CE cell monolayers and exhibited HA activity regularly induced the HAD phenomenon with several species of erythrocytes. The optimal conditions of the HAD phenomenon were almost the same as with the HA test (4, 7) with regard to low temperature, pH of 6.2, and the use of 0.4% goose erythrocytes. In addition, from the fact that goose erythrocytes fully adsorbed on infected cell monolayers were released by washing with phosphate buffer (higher pH than 6.2) or saline, it was suggested that the mechanism of the HAD phenomenon was identified with that of the HA reaction, because rabies virus adsorbed to goose erythrocytes could be eluted again from the agglutin-
1458
MINAMOTO ET AL.
ated erythrocytes by different temperature or pH in the HA test. But the optimal pH of our HAD differed from the first report (12) in that the Mochalin strain of rabies street virus adapted to hamster kidney cells induced the HAD phenomenon on cell cultures with several erythrocytes suspended in saline (pH 7.0) at 9 to 10 days after virus infection. The reason for this difference remains unknown. In the future, more detailed experiments are necessary to elucidate whether other rabies virus strains adapted to tissue culture monolayers require different conditions in HAD. Under the conditions mentioned above, the final results for virus titration using HAD techniques were obtained at 3 days after virus inoculation, and this method was so sensitive that virus titer coincided with those determined by the FA technique and suckling mice. Recently, a rapid method for determining the results of the rabies SN test was obtained in 1 day by using the FA technique (14). As a simple method, without special equipment, we usually employ the HI test for evaluating immune effectiveness, but this method is not applicable to rabies virus. HI antibody in animals immunized with rabies vaccine was negative or had a very low titer (9). This simple method by HAD was originally developed for use in applying the SN test to the measurement of rabies antibody. These results clearly demonstrate that SN antibody titers determined by using HAD as indicator instead of CPE coincided with those determined by mice, and this method obtained reproducible titers excluding some undefined factors such as the HI test (4, 9). This method makes it possible to determine rabies antibody titers on many sera without special equipment, and only 3 days after the mixtures of virus and serum are inoculated to the CE cell monolayers. ACKNOWLEDGMENT We are grateful to Y. Fujisaki, National Institute of Animal Health, Kodaira, Tokyo, Japan, for his review of this manuscript.
INFECT. IMMUN. LITERATURE CITED 1. Atanasiu, P. 1973. Quantitative assay and potency test of antirabies serum and immunoglobulin, p. 314-318. In M. M. Kaplan and H. Koprowski (ed.), Laboratory techniques in rabies. World Health Organization, Geneva, Switzerland. 2. Debbie, J. G., J. A. Andrulonis, and M. K. Abelseth. 1972. Rabies antibody determination by immunofluorescence in tissue culture, Infect. Immun. 5:902-904. 3. Dulbecco, R., and M. Vogt. 1954. Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99:167-182. 4. Halonen, P. E., F. A. Murphy, B. N. Fields, and D. R. Reese. 1968. Hemagglutinin of rabies and some other bullet shaped viruses. Proc. Soc. Exp. Biol. Med. 127:1037-1042. 5. Kondo, A. 1963. Growth characteristics of rabies virus in primary chick embryo cells. Virology 27:199-204. 6. Kurata, K., N. Minamoto, S., Ohta, and H. Sazawa. 1972. Agar gel diffusion test as a simple diagnostic method of rabies. Annu. Rep. Natl. Vet. Assay Lab. no. 9, p. 3-10. 7. Kuwert, E., T. J. Wiktor, F. Sokol, and H. Koprowski. 1968. Hemagglutination by rabies virus. J. Virol. 2:1381-1392. 8. Mengeling, W. L. 1972. Hemadsorption plaque assays for hemagglutinating encephalomyelitis virus. Am. J. Vet. Res. 33:2075-2080. 9. Minamoto, N., S. Ohta, and K. Kurata. 1974. Hemagglutination inhibition test for rabies antibody in immunized guinea pigs. Annu. Rep. Natl. Vet Assay Lab. no. 11, p. 9-13. 10. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27:493-497. 11. Sedwick, W. D., and T. J. Wiktor. 1967. Reproducible plaquing system for rabies, lymphocytic choriomeningititis, and other ribonucleic acid viruses in BHK21/13S agarose suspension. J. Virol. 1:1224-1226. 12. Selimov, M. A., and R. Sh. IIyasova. 1968. The hemadsorption phenomenon produced by rabies street virus adapted to tissue culture. Vopr. Virusol. 13: 76-80. 13. Shelokov, A., J. E. Vogel, and L. Chi. 1958. Hemadsorption (adsorption-hemagglutination) test for viral agents in tissue culture with special reference to influenza. Proc. Soc. Exp. Biol. Med. 97:802-809. 14. Smith, J. S., P. A. Yager, and G. M. Baer. 1973. A rapid reproducible test for determining rabies neutralizing antibody. Bull. W.H.O. 48:535-541. 15. Vogel, J. E., and A. Shelokov. 1957. Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture. Science 126:358-359. 16. Yoshino, K., and T. Morishima. 1971. An improvement in the plaque assay of rabies virus in chick embryo cells Arch. Gesamte Virusforsch. 34:40-50.
Vol. 13, No. 5 Printed in U.S.A.
Use of the Hemadsorption Phenomenon for Determining Virus and Neutralizing Antibody Titers of Rabies N. MINAMOTO,* K. KURATA, I. KAIZUKA, AND H. SAZAWA National Veterinary Assay Laboratory, Kokubunji, Tokyo, 185 Japan Received for publication 20 November 1975
Chicken embryo cells infected with the HEP Flury strain of rabies virus adapted to tissue culture produced a hemadsorption (HAD) phenomenon by using goose erythrocytes. The optimal conditions for HAD included the incubation of cell cultures at 37 C for 3 days after virus inoculation, the use of a 0.4% suspension of goose erythrocytes in phosphate buffer adjusted at pH 6.2, and adsorptioh of erythrocytes at 4 C. This phenomenon was inhibited with antirabies serum. Virus titer obtained with the HAD technique was almost the same as with the fluorescent antibody technique or the intracerebral inoculation of suckling mice. Results of the neutralization test by using the HAD technique could be easily determined 3 days after inoculation of chicken embryo cells with the mixture of 100 mean tissue culture infective doses of virus and diluted serum. The neutralizing antibody titers coincided with those obtained in mice.
Although several methods have been used for determining rabies virus or antibodies, each method has a few inherent disadvantages. For example, they need large numbers of animals (1), more than a 5-day interim between inoculation and final results (1, 2, 11, 16), and an accurate procedure (11, 16). Some methods indicate sporadic results (5) and negative or very low titer for evaluation of immune effectiveness (9). For the improvement of techniques to measure virus and antibody, we investigated the hemadsorption (HAD) phenomenon by which virus could be detected more rapidly and in the presence of less hemagglutinin than in the hemagglutination (HA) and hemagglutination inhibition (HI) techniques. Recently, the HAD phenomenon has been attempted primarily with street virus adapted to hamster kidney cells by Selimov and Ilyasova (12). However, detailed conditions of the reaction or practical methods employing the HAD phenomenon for the titration and serum neutralization (SN) test of rabies virus have not been reported. In this paper, we describe the HAD phenomenon produced in a system of the HEP Flury strain of fixed rabies virus adapted to tissue culture (HF-TC strain) and chicken embryo (CE) cells, which have been generally used in many laboratories, and show that this phenomenon now can be used for determining virus and SN antibody titers of rabies with reproducible results. MATERIALS AND METHODS Virus strain. The principal yirus used was the HF-TC strain of rabies virus obtained from A. Kondo, National Institute of Health, Tokyo, Japan,
which had been adapted to 7-day CE cells as described earlier (5), and had been purified by cloning three times after the 100th passage by A. Kondo. HF-TC strain is a large-plaque variant. The stock virus (103rd to 106th passage level), which was prepared as described previously (9), was used. Titration of virus by HAD. Serial 10-fold diluted virus (0.05 ml) was inoculated into four tube monolayers. After 1 h at 37 C, the inoculum was removed and each tube was washed twice with phosphatebuffered saline (PBS) (3). Then, to each tube was added 0.5 ml of maintenance medium (medium 199 with 0.4% sodium bicarbonate and 0.11% bovine albumin [fraction V]). After incubation of the cell monolayers at 37 C for 3 days, the maintenance medium was removed, and each tube was washed once with PBS. Then, 0.2 ml of a 0.4% suspension of adult goose erythrocytes in phosphate buffer, pH 6.2 (0.15 M NaCl, 0.056 M Na2HPO4, and 0.144 M NaH2PO4, pH 6.2), was added to each tube. Goose blood was preserved in equal part of Alsever solution and was stored at 4 C. Erythrocytes were used for each experiment after being washed three times with saline. The tubes were placed in a horizontal position at approximately 4 C for 20 min and returned to upright position at room temperature for an additional time, about 10 min. Then, foci of HAD on infected cell monolayers were investigated by microscope under low magnification (x 100). If foci of HAD were observed on cell monolayers, the test tube was read as viral positive (Fig. 1). Based on the above results, mean tissue culture infective dose (TCID50) was calculated by the method of Reed and Muench (10). SN test by HAD. Anti-rabies sera were obtained from guinea pigs administered several or only one injection (6) and were diluted 10-fold. An equal volume of virus suspension containing about 100 TCID,,) per 0.05 ml was added to each diluted serum. After incubation in a water bath at 37 C for 1 h, the
1454
VOL. 13, 1976
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USE OF THE HEMADSORPTION PHENOMENON
1455
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FIG. 1. HAD induced by the HF-TC strain of rabies virus. (a) HAD reaction by goose erythrocytes in CE cell monolayers at 3 days after virus inoculation (grade of HAD: + ++ - + + + +); (b) focus of HAD (+ + +); (c) HAD-negative CE cell monolayers. (Magnification xl00). mixtures were inoculated onto four cell monolayers in 0.05-ml amounts. Adsorption of the mixtures was allowed to take place at 37 C for 1 h, and the cell monolayers were carefully washed twice with phosphate buffer. The reading of results was performed by means of HAD at 3 days after incubation. SN titer of serum tested was calculated by the method of
Reed and Muench (10) from the highest dilution of serum that inhibited HAD completely.
RESULTS . .
Conditions of erythrocytes, pH, temperature, and observational days for the HAD phe-
1456
MINAMOTO ET AL.
nomenon. CE cell monolayers infected with the HF-TC strain induced the HAD phenomenon by erythrocytes of hamsters, guinea pigs, rabbits, fowl, geese, pigs, sheep, and horses. However, a 0.4% goose erythrocyte suspension showed the most clear and sensitive HAD phenomenon. Consequently, the following experiments were done with goose erythrocytes. Virus suspensions diluted 10-fold serially from 10-' to 10-7 were inoculated into CE cell cultures in amounts of 0.05 ml. The optimal conditions for HAD were established by investigating the effect of each condition on virus titer determined by HAD. The influence of various pH values of phosphate buffer to suspend erythrocytes on HAD was investigated. The highest virus titer was obtained at pH 6.2 (Table 1), but the HAD phenomenon was not observed with a suspension of erythrocytes in saline adjusted to various pH values (5.8 to 7.0). Next, we tested the effect of various temperatures on the adsorption of erythrocytes. The maximum titer (107 TCID5o/ml) was obtained by incubation at 4 C. Almost the same titer was observed at room temperature, but the HAD pattern was not satisfactorily developed. At 37 C, the HAD phenomenon was not observed. In the various adsorption times of erythrocytes, the HAD pattern was fully developed at 20 min, but an adsorption time less than 15 min was not enough to develop the HAD pattern. The time course of HAD appearance after inoculation of approximately 50,000 TCID;,,/ml of virus was investigated. HAD was first recognized (+ + +) 1 day after inoculation, and it became satisfactory after 3 days (+ + + +++ +). Then, virus titration of infected cell culture fluid which was harvested for 3 days after inoculation was performed to find the optimal observation period in days so as to determine the virus titer by means of HAD. Virus titer reached a peak at 3 days, and thereafter the same titer was maintained until 5 days (Table 2). Virus titers of the same material determined by the fluorescent antibody (FA) technique and in suckling mice inoculation were 104 TCID.5/ml and 10-' mean lethal doses/ml, respectively. Accordingly, the following experiments using HAD were performed at pH 6.2 and 4 C, with 0.4% goose erythrocytes, at 3 days after virus inoculation. Under these conditions, a positive reaction easily indicated a recognizable characteristic pattern so that it was not necessary to remove the unadsorbed erythrocytes, because almost all unadsorbed erythrocytes were precipitated to the bottom of the tube by returning it to an upright position. On the other hand, if HAD foci were washed with phosphate buffer or an-
INFECT. IMMUN. TABLE 1. Effect of various pH of erythrocyte suspension on the rabies virus titers determined by HAD pHa
Virus titerb
5.8 6.0 6.2 6.4
7.4 7.5 7.8 4.7 3.2
6.6
Saline 8 (5.8 7.0) -1. a Goose erythrocytes were suspended in phosphate buffer adjusted to various pH values. b Titers determined by using HAD as indicator instead of CPE at 3 days after virus inoculation, mean of two titrations (log TCID50/milliliter). -
TABLE 2. Titration by HAD of HF-TC strain in CE cell monolayers Days after inoculation
Dilution of virusa 1
2
3
4
5
4/4b
Control
1/4 0/4 0/4 0/4 0/4
4/4 4/4 4/4 3/4 1/4 0/4
4/4 4/4 4/4 4/4 1/4 0/4
4/4 4/4 4/4 4/4 1/4 0/4
4/4 4/4 4/4 4/4 1/4 0/4
Virus titer
50
7.8
8.0
8.0
8.0
10-3 10-4 10-; 10-6 10-'
(log TCID501/Ml)
a Cell monolayers were inoculated with 0.05 ml of diluted virus. b Number of HAD positive tubes/number of tested tubes.
other solution (higher pH than 6.2), the foci became slightly indistinct. Specificity of the HAD phenomenon. The HAD phenomenon was inhibited specifically by adding rabies immune serum to infected cell monolayers and was not inhibited by normal guinea pig serum, Japanese encephalitis, hemagglutinating encephalomyelitis, parainfluenza 1 and 3, Newcastle disease, equine influenza, vesicular stomatitis, infectious canine hepatitis virus, and Mycoplasma (M. synoviae, M. pneumoniae, and M. gallisepticum) immune sera. SN test in immune sera. The HAD phenomenon was applied to rabies SN test as indicator of virus growth instead of cytopathic effect. (CPE). Twenty-four SN tests were performed by using two guinea pig sera at different stage of immunization and a hyperimmune serum of guinea pig in order to investigate whether reproducible results could be obtained in rabies SN titers. Titers of three sera by SN test in mice were 30, 74, and 14,111, respectively. All
VOL. 13, 1976
USE OF THE HEMADSORPTION PHENOMENON
1457
tests indicated reproducible results, but the differences in each SN titer were less than twofold 80 except two (tests 2 and 5, Table 3). Comparison of SN titers determined by 40 HAD and mice. Ten guinea pigs were vaccinated subcutaneously with 4 ml of inactivated 2: vaccine. Then blood samples were collected by 20 intracardial puncture for titration of SN anti4.4 bodies at intervals of a week. The geometrical V. 10 means of SN titers determined by HAD and mice are shown in Fig. 2. SN antibodies measured by both methods became detectable 7 days 0 2 3 4 5 after vaccination, and with a striking degree of Time in weeks after vaccination parallelism both titers increased until 21 days. The comparative SN titers of 60 sera deterFIG. 2. Responses of SN antibody in guinea pigs mined by both tests are shown in Fig. 3. The immunized with the vaccine. (a) SN titers detertiters determined by HAD were about 1.8 times mined by HAD; (b) SN titers determined by mice; (c) (varying from 0.4 to 6.3) higher than the mouse geometric mean SN titer of 10 guinea pig sera. SN titers. The correlation coefficient for the results of both methods was +0.95. -4
V}
i
320 r
DISCUSSION It is already known that the HAD phenomenon occurs on tissue culture cells infected with myxovirus (13, 15), paramyxovirus (14), and coronavirus (8), which are hemagglutinating viruses. The HAD phenomenon was provided for a practical approach to diagnostic and fundamental studies of these agents. Because HAD depends on direct visualization under low magnification (x 100), it is detectable in the presence of minimal hemagglutinin associated with tissue culture cells, resulting in more sensitivity than with procedures based on the development of CPE or usual HA by virus suspension. In the experiments described in this paper, we demonstrate that the HF-TC strain which TABLE 3. Reproducibility of SN antibody titer determined by HAD Serum No. of test la 2b 3i
1 102 45 10,240 2 20 128 14,488 91 3 23 10,240 4 128 7,362 25 81 5 20 10,240 6 23 161 12,190 7 128 23 12,093 NTv 8 128 14,488 Mean ± SID 26 ± 9 118 ± 26 11,056 ± 2,982 a Guinea pig serum 2 weeks after single injection of vaccine. b Guinea pig serum 5 weeks after single injection of vaccine. " Hyperimmune serum of guinea pig. d NT, Not tested. ' SD, Standard deviation.
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-
*:
: *
*o < 10
00 *-
Si . < 10
10
20 M
40 -
SN
80
160
titerb
FIG. 3. Relationship between SN antibody titers determined by HAD and mice; (a) SN titer determined by HAD; (b) SN titer determined by mice.
grew in CE cell monolayers and exhibited HA activity regularly induced the HAD phenomenon with several species of erythrocytes. The optimal conditions of the HAD phenomenon were almost the same as with the HA test (4, 7) with regard to low temperature, pH of 6.2, and the use of 0.4% goose erythrocytes. In addition, from the fact that goose erythrocytes fully adsorbed on infected cell monolayers were released by washing with phosphate buffer (higher pH than 6.2) or saline, it was suggested that the mechanism of the HAD phenomenon was identified with that of the HA reaction, because rabies virus adsorbed to goose erythrocytes could be eluted again from the agglutin-
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MINAMOTO ET AL.
ated erythrocytes by different temperature or pH in the HA test. But the optimal pH of our HAD differed from the first report (12) in that the Mochalin strain of rabies street virus adapted to hamster kidney cells induced the HAD phenomenon on cell cultures with several erythrocytes suspended in saline (pH 7.0) at 9 to 10 days after virus infection. The reason for this difference remains unknown. In the future, more detailed experiments are necessary to elucidate whether other rabies virus strains adapted to tissue culture monolayers require different conditions in HAD. Under the conditions mentioned above, the final results for virus titration using HAD techniques were obtained at 3 days after virus inoculation, and this method was so sensitive that virus titer coincided with those determined by the FA technique and suckling mice. Recently, a rapid method for determining the results of the rabies SN test was obtained in 1 day by using the FA technique (14). As a simple method, without special equipment, we usually employ the HI test for evaluating immune effectiveness, but this method is not applicable to rabies virus. HI antibody in animals immunized with rabies vaccine was negative or had a very low titer (9). This simple method by HAD was originally developed for use in applying the SN test to the measurement of rabies antibody. These results clearly demonstrate that SN antibody titers determined by using HAD as indicator instead of CPE coincided with those determined by mice, and this method obtained reproducible titers excluding some undefined factors such as the HI test (4, 9). This method makes it possible to determine rabies antibody titers on many sera without special equipment, and only 3 days after the mixtures of virus and serum are inoculated to the CE cell monolayers. ACKNOWLEDGMENT We are grateful to Y. Fujisaki, National Institute of Animal Health, Kodaira, Tokyo, Japan, for his review of this manuscript.
INFECT. IMMUN. LITERATURE CITED 1. Atanasiu, P. 1973. Quantitative assay and potency test of antirabies serum and immunoglobulin, p. 314-318. In M. M. Kaplan and H. Koprowski (ed.), Laboratory techniques in rabies. World Health Organization, Geneva, Switzerland. 2. Debbie, J. G., J. A. Andrulonis, and M. K. Abelseth. 1972. Rabies antibody determination by immunofluorescence in tissue culture, Infect. Immun. 5:902-904. 3. Dulbecco, R., and M. Vogt. 1954. Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99:167-182. 4. Halonen, P. E., F. A. Murphy, B. N. Fields, and D. R. Reese. 1968. Hemagglutinin of rabies and some other bullet shaped viruses. Proc. Soc. Exp. Biol. Med. 127:1037-1042. 5. Kondo, A. 1963. Growth characteristics of rabies virus in primary chick embryo cells. Virology 27:199-204. 6. Kurata, K., N. Minamoto, S., Ohta, and H. Sazawa. 1972. Agar gel diffusion test as a simple diagnostic method of rabies. Annu. Rep. Natl. Vet. Assay Lab. no. 9, p. 3-10. 7. Kuwert, E., T. J. Wiktor, F. Sokol, and H. Koprowski. 1968. Hemagglutination by rabies virus. J. Virol. 2:1381-1392. 8. Mengeling, W. L. 1972. Hemadsorption plaque assays for hemagglutinating encephalomyelitis virus. Am. J. Vet. Res. 33:2075-2080. 9. Minamoto, N., S. Ohta, and K. Kurata. 1974. Hemagglutination inhibition test for rabies antibody in immunized guinea pigs. Annu. Rep. Natl. Vet Assay Lab. no. 11, p. 9-13. 10. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27:493-497. 11. Sedwick, W. D., and T. J. Wiktor. 1967. Reproducible plaquing system for rabies, lymphocytic choriomeningititis, and other ribonucleic acid viruses in BHK21/13S agarose suspension. J. Virol. 1:1224-1226. 12. Selimov, M. A., and R. Sh. IIyasova. 1968. The hemadsorption phenomenon produced by rabies street virus adapted to tissue culture. Vopr. Virusol. 13: 76-80. 13. Shelokov, A., J. E. Vogel, and L. Chi. 1958. Hemadsorption (adsorption-hemagglutination) test for viral agents in tissue culture with special reference to influenza. Proc. Soc. Exp. Biol. Med. 97:802-809. 14. Smith, J. S., P. A. Yager, and G. M. Baer. 1973. A rapid reproducible test for determining rabies neutralizing antibody. Bull. W.H.O. 48:535-541. 15. Vogel, J. E., and A. Shelokov. 1957. Adsorption-hemagglutination test for influenza virus in monkey kidney tissue culture. Science 126:358-359. 16. Yoshino, K., and T. Morishima. 1971. An improvement in the plaque assay of rabies virus in chick embryo cells Arch. Gesamte Virusforsch. 34:40-50.
