Microbiol. Immunol. Vol. 21 (9), 535-538, 1977
Electron
Microscopic Maturation
Observation of Group
of the
Budding
B Arboviruses
Akio OHYAMA,Tomiyoshi ITO, Eiki TANIMURA, Shiu-Chi HUANG, Jian-Yi HSUE,and Yoichi FURU Department of Microbiology, KansaiMedicalUniversity, Osaka (Receivedfor publication,December8, 1976) In the maturation process of group A arboviruses as generally known immature particles are enveloped while passing into the lumen of cytoplasmic vacuoles or while being extruded through the cell membrane (2). Differing from group A arboviruses, numerous efforts by many investigators (1, 3-5, 7, 8, 10-14) have failed to provide enough informations on the morphological maturation process of group B arboviruses. Our study has been focused on an electron microscopic insight into such maturation process of group B arboviruses by which the envelope is formed around the nucleocapsid. Three mouse-adapted virus strains, Japanese encephalitis virus (JEV) JaGAr-01, dengue virus (DV) type 4 H-241 (MB-2), and yellow fever virus (YFV) 17D, were used in this experiment. Vero cells were grown in Eagle's minimum essential medium added with calf serum in 10%, and the infected cells were maintained with a decreased concentration, 2%, of calf serum. Monolayered Vero cell cultures were inoculated with each virus suspension prepared by the following procedure; a 10% suckling mouse brain emulsion made with Dulbecco's phosphate-buffered saline (PBS) was centrifuged at 1,000 rpm for 5 min, and the supernatant was diluted to 1:100 with PBS. The infected Vero cells were frozen and thawed at harvest to use the supernatant as inocula for the next passage. The culture interval was 4 days for the first passage and 48 hr after the 2nd passage. Infected Vero cells at various stages were subjected to fixation with 2% glutaraldehyde and then with 1% OsO4, followed by dehydration with ethanol in serial concentrations. Peroxidase-labeled antibody and ferritin-labeled antibody, specific for each of the 3 virus strains used, were prepared for immuno-electron microscopy according to Nakane and Kawaoi (9) and Singer and Schick (15). The specific fluorescence for JEV was observed by the fluorescent antibody technique at the perinuclear region of cytoplasm of Vero cells, 15 hr after inoculation of virus. The development of specific immunofluorescence was slightly delayed in DV and YFV, when compared with that of JEV. The specific fluorescence for each virus extended gradually into the cytoplasmic area from the juxtanuclear to This report was presented at the 24th Annual Meeting of the Society of Japanese Virologists held at Nagoya, 30 October 1976. 535
536
A. OHYAMA
FT AL
2a Figs.
1 and
2
2b
NOTES
537
Fig. 3. Electron micrograph of YFV-infected Vero cell at 24 hr postinfection demonstrating budding particles from microvilli and cell membrane as well as immature particles (arrows).
peripheral region, concentrically or radially. The electron micrograph of ultrathin sections of Vero cells at 24 hr after inoculation with JEV showed a remarkable vacuole-like expansion of cisterna of Golgi apparatus and endoplasmic reticulum (ER). Some immature particles, 25 nm in diameter, were scattered or arranged in the cytoplasm, and others, heaping cell membrane, were found in contact with the inner surface of the cell membrane (Fig. 1). As particles were extruded, they were half-covered with the cell membrane, and then tailing of this membrane was obFig. 1. Electron micrograph of JEV-infected Vero cells at 24 hr postinfection demonstrating both budding virus particles from cell membrane and intracytoplasmic immature particles (arrows). Scales indicated in this and in the following represent 500 nm. Magnification is 1: 50,000 and insert is further enlargement (1: 100,000) of a part of every graph. Fig. 2. Electron micrographs of DV-infected Vero cells at 24 hr postinfection demonstrating budding virus particles from cell membrane and microvilli (a, b). Immature particles (arrows) are visible in cytoplasm (b).
538
A. OHYAMA
ET AL
served before complete pinching off of enveloped particles. Numerous electrondense mature particles with envelope, 45-50 nm in diameter, were found extracellularly or in the lumen of ER. All these findings were simultaneously observed in every specimen (Figs. 2 and 3). The identity of JEV, DV, and YFV grown in cultured Vero cells was repeatedly confirmed by neutralization tests both in vivoand in vitro, indirect fluorescent antibody staining, and immuno-electron microscopy against corresponding specific antibodies. Based on the electron microscopic observations here in described coupled with other reports (5, 6, 8, 13), we can conclude that group B arboviruses, as well as group A viruses, acquired their envelope from the cell membrane and other membranous structures, and mature virions were completed through budding. Our success in obtaining this definite picture of maturation of groupe B arboviruses may have resulted from the inoculation of an unusually high multiplicity of infection of these viruses. REFERENCES
1) 2)
3) 4) 5) 6) 7) 8) 9) 10) 11) 12)
13) 14) 15)
Higashi, N. 1964. Electron microscopic studies on the growth of Japanese encephalitis virus and PLT virus group. Prog. Virus Res. (The 4th Virus Symposium) : 167-184 (in Japanese). Higashi, N., Matsumoto, A., Tabata, K., and Nagatomo, Y. 1967. Electron microscope study of development of Chikungunya virus in green monkey kidney stable (Vero) cells. Virology 33: 55-69. Higashi, N. 1967. Electron microscopic study of Japanese encephalitis virus. Adv. Neurol. Sci. 11: 235-242 (in Japanese). Higashi, N. 1968. Maturation of arboviruses. Prog. Virus Res. (The 8th Virus Symposium) : 108 133 (in Japanese). Horzinek, M.C. 1973. The structure of Togaviruses. Prog. Med. Virol. 16: 109-156. Ko, K.K. 1976. Development of dengue virus type 4 in BHK cells. Biken J. 19: 43-51. Matsumura, T., Stollar, V., and Schlesinger, R.W. 1971. Studies on the nature of dengue viruses. V. Structure and development of dengue viruses. Virology 46: 344-355. Matsumura, T. 1975. Mechanism of the viral maturation in culture cells, especially in regard to Togaviruses. Tissue Culture 1: 168-175 (in Japanese). Nakane, P.K., and Kawaoi, A. 1974. Peroxidase-labeled antibody. A new method of conjugation. J. Histochem. Cytochem. 22: 1084-1091. Ohyama, A., Igarashi, A., Mantani, M., Naito, T., and Prakob, T. 1967. Morphological study of tissues infected with Dengue virus. Virus 17: 316-317 (in Japanese). Ohyama, A., Ito, T., Tanimura, E., Hsue, J-Y., Furu, Y., and Kitamura, S. 1972. Growth of Japanese encephalitis virus in tissue-cultured mosquito cells. Virus 22: 317 (in Japanese). Ohyama, A., Ito, T., Tanimura, E., Huang, S-C., Miyajima, N., Hotta, H., and Kitamura, S. 1972. Morphological study of tissue-cultured mosquito cells infected with Japanese encephalitis virus. Virus 22: 318 (in Japanese). Ohyama, A. 1976. Electron microscopic studies of group B arboviruses. Japan. J. Clin. Med. 34: 1641-1647 (in Japanese) . Ota, Z. 1965. Electron microscopic study of the development of Japanese B encephalitis virus in porcine kidney stable (PS) cells. Virology 25: 372-378. Singer, S. J., and Schick, A.F. 1961. The properties of specific stains for electron microscopy prepared by the conjugation of antibody molecules with ferritin. J. Biophys. Biochem. Cytol. 9: 519-537.
Requests for reprints should be addressed to Dr. Akio Ohyama, Department of Microbiology, Kansai Medical University, Moriguchi, Osaka 570, Japan.
Electron
Microscopic Maturation
Observation of Group
of the
Budding
B Arboviruses
Akio OHYAMA,Tomiyoshi ITO, Eiki TANIMURA, Shiu-Chi HUANG, Jian-Yi HSUE,and Yoichi FURU Department of Microbiology, KansaiMedicalUniversity, Osaka (Receivedfor publication,December8, 1976) In the maturation process of group A arboviruses as generally known immature particles are enveloped while passing into the lumen of cytoplasmic vacuoles or while being extruded through the cell membrane (2). Differing from group A arboviruses, numerous efforts by many investigators (1, 3-5, 7, 8, 10-14) have failed to provide enough informations on the morphological maturation process of group B arboviruses. Our study has been focused on an electron microscopic insight into such maturation process of group B arboviruses by which the envelope is formed around the nucleocapsid. Three mouse-adapted virus strains, Japanese encephalitis virus (JEV) JaGAr-01, dengue virus (DV) type 4 H-241 (MB-2), and yellow fever virus (YFV) 17D, were used in this experiment. Vero cells were grown in Eagle's minimum essential medium added with calf serum in 10%, and the infected cells were maintained with a decreased concentration, 2%, of calf serum. Monolayered Vero cell cultures were inoculated with each virus suspension prepared by the following procedure; a 10% suckling mouse brain emulsion made with Dulbecco's phosphate-buffered saline (PBS) was centrifuged at 1,000 rpm for 5 min, and the supernatant was diluted to 1:100 with PBS. The infected Vero cells were frozen and thawed at harvest to use the supernatant as inocula for the next passage. The culture interval was 4 days for the first passage and 48 hr after the 2nd passage. Infected Vero cells at various stages were subjected to fixation with 2% glutaraldehyde and then with 1% OsO4, followed by dehydration with ethanol in serial concentrations. Peroxidase-labeled antibody and ferritin-labeled antibody, specific for each of the 3 virus strains used, were prepared for immuno-electron microscopy according to Nakane and Kawaoi (9) and Singer and Schick (15). The specific fluorescence for JEV was observed by the fluorescent antibody technique at the perinuclear region of cytoplasm of Vero cells, 15 hr after inoculation of virus. The development of specific immunofluorescence was slightly delayed in DV and YFV, when compared with that of JEV. The specific fluorescence for each virus extended gradually into the cytoplasmic area from the juxtanuclear to This report was presented at the 24th Annual Meeting of the Society of Japanese Virologists held at Nagoya, 30 October 1976. 535
536
A. OHYAMA
FT AL
2a Figs.
1 and
2
2b
NOTES
537
Fig. 3. Electron micrograph of YFV-infected Vero cell at 24 hr postinfection demonstrating budding particles from microvilli and cell membrane as well as immature particles (arrows).
peripheral region, concentrically or radially. The electron micrograph of ultrathin sections of Vero cells at 24 hr after inoculation with JEV showed a remarkable vacuole-like expansion of cisterna of Golgi apparatus and endoplasmic reticulum (ER). Some immature particles, 25 nm in diameter, were scattered or arranged in the cytoplasm, and others, heaping cell membrane, were found in contact with the inner surface of the cell membrane (Fig. 1). As particles were extruded, they were half-covered with the cell membrane, and then tailing of this membrane was obFig. 1. Electron micrograph of JEV-infected Vero cells at 24 hr postinfection demonstrating both budding virus particles from cell membrane and intracytoplasmic immature particles (arrows). Scales indicated in this and in the following represent 500 nm. Magnification is 1: 50,000 and insert is further enlargement (1: 100,000) of a part of every graph. Fig. 2. Electron micrographs of DV-infected Vero cells at 24 hr postinfection demonstrating budding virus particles from cell membrane and microvilli (a, b). Immature particles (arrows) are visible in cytoplasm (b).
538
A. OHYAMA
ET AL
served before complete pinching off of enveloped particles. Numerous electrondense mature particles with envelope, 45-50 nm in diameter, were found extracellularly or in the lumen of ER. All these findings were simultaneously observed in every specimen (Figs. 2 and 3). The identity of JEV, DV, and YFV grown in cultured Vero cells was repeatedly confirmed by neutralization tests both in vivoand in vitro, indirect fluorescent antibody staining, and immuno-electron microscopy against corresponding specific antibodies. Based on the electron microscopic observations here in described coupled with other reports (5, 6, 8, 13), we can conclude that group B arboviruses, as well as group A viruses, acquired their envelope from the cell membrane and other membranous structures, and mature virions were completed through budding. Our success in obtaining this definite picture of maturation of groupe B arboviruses may have resulted from the inoculation of an unusually high multiplicity of infection of these viruses. REFERENCES
1) 2)
3) 4) 5) 6) 7) 8) 9) 10) 11) 12)
13) 14) 15)
Higashi, N. 1964. Electron microscopic studies on the growth of Japanese encephalitis virus and PLT virus group. Prog. Virus Res. (The 4th Virus Symposium) : 167-184 (in Japanese). Higashi, N., Matsumoto, A., Tabata, K., and Nagatomo, Y. 1967. Electron microscope study of development of Chikungunya virus in green monkey kidney stable (Vero) cells. Virology 33: 55-69. Higashi, N. 1967. Electron microscopic study of Japanese encephalitis virus. Adv. Neurol. Sci. 11: 235-242 (in Japanese). Higashi, N. 1968. Maturation of arboviruses. Prog. Virus Res. (The 8th Virus Symposium) : 108 133 (in Japanese). Horzinek, M.C. 1973. The structure of Togaviruses. Prog. Med. Virol. 16: 109-156. Ko, K.K. 1976. Development of dengue virus type 4 in BHK cells. Biken J. 19: 43-51. Matsumura, T., Stollar, V., and Schlesinger, R.W. 1971. Studies on the nature of dengue viruses. V. Structure and development of dengue viruses. Virology 46: 344-355. Matsumura, T. 1975. Mechanism of the viral maturation in culture cells, especially in regard to Togaviruses. Tissue Culture 1: 168-175 (in Japanese). Nakane, P.K., and Kawaoi, A. 1974. Peroxidase-labeled antibody. A new method of conjugation. J. Histochem. Cytochem. 22: 1084-1091. Ohyama, A., Igarashi, A., Mantani, M., Naito, T., and Prakob, T. 1967. Morphological study of tissues infected with Dengue virus. Virus 17: 316-317 (in Japanese). Ohyama, A., Ito, T., Tanimura, E., Hsue, J-Y., Furu, Y., and Kitamura, S. 1972. Growth of Japanese encephalitis virus in tissue-cultured mosquito cells. Virus 22: 317 (in Japanese). Ohyama, A., Ito, T., Tanimura, E., Huang, S-C., Miyajima, N., Hotta, H., and Kitamura, S. 1972. Morphological study of tissue-cultured mosquito cells infected with Japanese encephalitis virus. Virus 22: 318 (in Japanese). Ohyama, A. 1976. Electron microscopic studies of group B arboviruses. Japan. J. Clin. Med. 34: 1641-1647 (in Japanese) . Ota, Z. 1965. Electron microscopic study of the development of Japanese B encephalitis virus in porcine kidney stable (PS) cells. Virology 25: 372-378. Singer, S. J., and Schick, A.F. 1961. The properties of specific stains for electron microscopy prepared by the conjugation of antibody molecules with ferritin. J. Biophys. Biochem. Cytol. 9: 519-537.
Requests for reprints should be addressed to Dr. Akio Ohyama, Department of Microbiology, Kansai Medical University, Moriguchi, Osaka 570, Japan.
