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Comparative titration of Chilo iridescent virus in vivo and in vitro
JOURNAL
OF INVERTEBRATE
PATHOLOGY
Comparative
32, 394-395
(1978)
Titration of Chile Iridescent In Vivo and In Vitro
For the titration of infectivity of insect iridoviruses, an in vivo assay (M. Ohba, Sci. Bull. Fat. Agr., Kyushu Univ. 30, 5165, 1975) using the dilution end point technique has been employed; this technique has also been utilized for in vitro assays (A. H. McIntosh and M. Kimura, Zntervirology 4,257-267,1974; S. R. Webb, J. D. Paschke, G. W. Wagner, and W. R. Campbell, J. Znvertebr. Pathol. 26, 205212, 1975; D. C. Kelly, J. Znvertebr. Pathol. 27, 415-418, 1976; R. M. Elliott, T. Lescott, and D. C. Kelly, Virology 81, 309316, 1977). Recently a plaque assay technique for an iridovirus from Simulium sp. (B. S. Batson, M. R. L. Johnston, M. K. A. Arnold, and D. C. Kelly, J. Znvertebr. Pathol. 27, 133-135, 1976) has been developed (D. A. Brown, T. Lescott, K. A. Hat-rap, and D. C. Kelly, J. Gen. Virol. 38, 175-178, 1977). In the present experiment, sensitivity of an in vitro assay using Antheraea eucalypti cultured cells (T. D. C. Grace, Nature (London) 195,788789, 1962) was compared with that of in vivo assay using the greater wax moth, Galleria mellonella, larvae for the titration of Chilo iridescent virus (CIV). Antheraea cells were supplied by.Dr. J. L. Vaughn and maintained at 25-26°C in Grace’s medium (Gibco) supplemented with 5% silkworm (Bombyx mori) larval hemolymph and 5% fetal calf serum. Silkworm hemolymph was heat-treated at 60°C for 5 min. After preculture of 3 days, Antheraea cells were harvested by centrifugation at 500 rpm for 5 min and resuspended in a small amount of Grace’s medium. CIV from infected Galleria larvae was propagated once in Antheraea cells. Infected culture was then centrifuged at 1000 rpm for 10 min, and the supernatant 0022-2011/78/0323-0394$01.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
394
Virus
fluid was used as the inoculum in the present experiment. Virus inoculation was performed by mixing 1.5 ml of cell suspension containing 1.5 x 10’ cells with 0.5 ml of virus suspension ( 106.45TCID50). After incubation of the mixture at 26°C for 1 hr with intermittent shaking, cells were sedimented by centrifugation and washed once in 10 ml of Grace’s medium. Cells were then seeded in 10 ml of medium at the concentration of 5 x lo5 cells/ml and incubated at 25-26°C. At intervals of 24 hr, 0.3-ml aliquots of the infected culture were removed and diluted fivefold with Grace’s medium. Diluted cultures were centrifuged at 500 r-pm for 5 min, and sedimented cells were smeared on glass slides, airdried, fixed in methanol for l-3 min, and stained with Giemsa solution for the estimation of the proportion of CIV-infected cells. The supematant was stored at -20°C until the titration of virus infectivity. For the virus titration, serial IO-fold dilution of the supematant with Grace’s medium was performed and 0.1 ml of dilution was introduced into a Leighton culture tube. In each Leighton tube, l-3 x lo5 cells were seeded in 0.9 ml of the medium and cultured at 25-26°C for l-2 days prior to virus inoculation. Virus titration in the Galleria larvae (150-200 mg) was also performed at 2526°C in parallel with the in vivo titration. Each Galleria larva was injected hemocoelitally with 5 ~1 of virus suspension. In vivo and in vitro infections were determined by the formation of cytoplasmic inclusions 7 days after virus inoculation as described previously (M. Ohba, Sci. Bull. Fat. Agr., Kyushu Univ. 30, 51-65, 1975). Infectivity titers were estimated by the method of L. J. Reed and H. Muench (Amer. J. Hyg. 27,
395
NOTES &Or
0
24
48
72
Hours
88
944
after
imculaticm
182
FIG. 1. The proportion
of inclusion-forming Anrheraea eucnlypti cells during the course of CIV infection. At each sampling time, cells were collected and stained with Giemsa solution. Approximately 150 cells were observed to estimate the percentage of CIV-infected cells.
024487288l2Ol44 Hours after
imoulation
FIG.
2. Multiplication of CIV in Antheraea cells. Infectivity of the released virus in the supematant fluid was titrated in both Antheraea cell culture and Galleria mellonella larvae simultaneously.
eucalypti
leria larvae
493-497, 1938) and expressed as negative log tissue culture IDSo (TCID,& and Galleria intrahemocoelic IDSo (GIHID,,). The results are shown in Figures 1 and 2. Although the multiplicity of infection was relatively low, the release of the progeny virus apparently occurred within 24 hr after virus inoculation. The infectivity titer of the released virus almost reached its maximum 3 days after virus inoculation, and at the same time the proportion of the infected cells reached its maximum of approximately 84%. Cells remaining uninfected with CIV seemed to be resistant to CIV infection. Such cells could be maintained, and a carrier culture similar to a cell line persistently infected with CIV reported by J. Mitsuhashi (Nature (London) 215, 863864, 1967) was established in another experiment (M. Ohba and K. Aizawa, unpubl.). The results obtained here reveal that Gal-
are two- to sevenfold more sensitive to CIV infection than Antheraea cells suggesting that an in vivo assay system is superior to an in vitro system from the point of view of sensitivity. Furthermore, it is difficult to titrate the infectivity of melanized material because of the toxic effect of melanization on cultured cells, while there is no such a problem in an in vivo assay. Of course, these problems in cell culture do not prohibit the general application of insect cell culture to invertebrate virology. KEY WORDS: Chilo iridescent virus; Antheraea eucalypti cell culture; Galleria mellonella ; comparative infectivity titration; dilution end point technique. MICHIO OHBA KEIO AIZAWA Institute of Biological Control Faculty of Agriculture Kyushu University Fukuoka 812, Japan Received March 8* 1978
OF INVERTEBRATE
PATHOLOGY
Comparative
32, 394-395
(1978)
Titration of Chile Iridescent In Vivo and In Vitro
For the titration of infectivity of insect iridoviruses, an in vivo assay (M. Ohba, Sci. Bull. Fat. Agr., Kyushu Univ. 30, 5165, 1975) using the dilution end point technique has been employed; this technique has also been utilized for in vitro assays (A. H. McIntosh and M. Kimura, Zntervirology 4,257-267,1974; S. R. Webb, J. D. Paschke, G. W. Wagner, and W. R. Campbell, J. Znvertebr. Pathol. 26, 205212, 1975; D. C. Kelly, J. Znvertebr. Pathol. 27, 415-418, 1976; R. M. Elliott, T. Lescott, and D. C. Kelly, Virology 81, 309316, 1977). Recently a plaque assay technique for an iridovirus from Simulium sp. (B. S. Batson, M. R. L. Johnston, M. K. A. Arnold, and D. C. Kelly, J. Znvertebr. Pathol. 27, 133-135, 1976) has been developed (D. A. Brown, T. Lescott, K. A. Hat-rap, and D. C. Kelly, J. Gen. Virol. 38, 175-178, 1977). In the present experiment, sensitivity of an in vitro assay using Antheraea eucalypti cultured cells (T. D. C. Grace, Nature (London) 195,788789, 1962) was compared with that of in vivo assay using the greater wax moth, Galleria mellonella, larvae for the titration of Chilo iridescent virus (CIV). Antheraea cells were supplied by.Dr. J. L. Vaughn and maintained at 25-26°C in Grace’s medium (Gibco) supplemented with 5% silkworm (Bombyx mori) larval hemolymph and 5% fetal calf serum. Silkworm hemolymph was heat-treated at 60°C for 5 min. After preculture of 3 days, Antheraea cells were harvested by centrifugation at 500 rpm for 5 min and resuspended in a small amount of Grace’s medium. CIV from infected Galleria larvae was propagated once in Antheraea cells. Infected culture was then centrifuged at 1000 rpm for 10 min, and the supernatant 0022-2011/78/0323-0394$01.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
394
Virus
fluid was used as the inoculum in the present experiment. Virus inoculation was performed by mixing 1.5 ml of cell suspension containing 1.5 x 10’ cells with 0.5 ml of virus suspension ( 106.45TCID50). After incubation of the mixture at 26°C for 1 hr with intermittent shaking, cells were sedimented by centrifugation and washed once in 10 ml of Grace’s medium. Cells were then seeded in 10 ml of medium at the concentration of 5 x lo5 cells/ml and incubated at 25-26°C. At intervals of 24 hr, 0.3-ml aliquots of the infected culture were removed and diluted fivefold with Grace’s medium. Diluted cultures were centrifuged at 500 r-pm for 5 min, and sedimented cells were smeared on glass slides, airdried, fixed in methanol for l-3 min, and stained with Giemsa solution for the estimation of the proportion of CIV-infected cells. The supematant was stored at -20°C until the titration of virus infectivity. For the virus titration, serial IO-fold dilution of the supematant with Grace’s medium was performed and 0.1 ml of dilution was introduced into a Leighton culture tube. In each Leighton tube, l-3 x lo5 cells were seeded in 0.9 ml of the medium and cultured at 25-26°C for l-2 days prior to virus inoculation. Virus titration in the Galleria larvae (150-200 mg) was also performed at 2526°C in parallel with the in vivo titration. Each Galleria larva was injected hemocoelitally with 5 ~1 of virus suspension. In vivo and in vitro infections were determined by the formation of cytoplasmic inclusions 7 days after virus inoculation as described previously (M. Ohba, Sci. Bull. Fat. Agr., Kyushu Univ. 30, 51-65, 1975). Infectivity titers were estimated by the method of L. J. Reed and H. Muench (Amer. J. Hyg. 27,
395
NOTES &Or
0
24
48
72
Hours
88
944
after
imculaticm
182
FIG. 1. The proportion
of inclusion-forming Anrheraea eucnlypti cells during the course of CIV infection. At each sampling time, cells were collected and stained with Giemsa solution. Approximately 150 cells were observed to estimate the percentage of CIV-infected cells.
024487288l2Ol44 Hours after
imoulation
FIG.
2. Multiplication of CIV in Antheraea cells. Infectivity of the released virus in the supematant fluid was titrated in both Antheraea cell culture and Galleria mellonella larvae simultaneously.
eucalypti
leria larvae
493-497, 1938) and expressed as negative log tissue culture IDSo (TCID,& and Galleria intrahemocoelic IDSo (GIHID,,). The results are shown in Figures 1 and 2. Although the multiplicity of infection was relatively low, the release of the progeny virus apparently occurred within 24 hr after virus inoculation. The infectivity titer of the released virus almost reached its maximum 3 days after virus inoculation, and at the same time the proportion of the infected cells reached its maximum of approximately 84%. Cells remaining uninfected with CIV seemed to be resistant to CIV infection. Such cells could be maintained, and a carrier culture similar to a cell line persistently infected with CIV reported by J. Mitsuhashi (Nature (London) 215, 863864, 1967) was established in another experiment (M. Ohba and K. Aizawa, unpubl.). The results obtained here reveal that Gal-
are two- to sevenfold more sensitive to CIV infection than Antheraea cells suggesting that an in vivo assay system is superior to an in vitro system from the point of view of sensitivity. Furthermore, it is difficult to titrate the infectivity of melanized material because of the toxic effect of melanization on cultured cells, while there is no such a problem in an in vivo assay. Of course, these problems in cell culture do not prohibit the general application of insect cell culture to invertebrate virology. KEY WORDS: Chilo iridescent virus; Antheraea eucalypti cell culture; Galleria mellonella ; comparative infectivity titration; dilution end point technique. MICHIO OHBA KEIO AIZAWA Institute of Biological Control Faculty of Agriculture Kyushu University Fukuoka 812, Japan Received March 8* 1978