Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1961 Jul 1;114(1):149–160. doi: 10.1084/jem.114.1.149

DEMONSTRATION OF THE SEQUENTIAL DEVELOPMENT OF VACCINIAL ANTIGENS AND VIRUS IN INFECTED CELLS: OBSERVATIONS WITH CYTOCHEMICAL AND DIFFERENTIAL FLUORESCENT PROCEDURES

Philip C Loh 1, John L Riggs 1
PMCID: PMC2137444  PMID: 13763191

Abstract

Virus-induced alterations in vaccinia virus-infected HeLa cells have been followed by immunofluorescent and cytochemical techniques. In a time sequence study, infected cells show an early increase in cytoplasmic RNA content, followed by appearance of centers of viral DNA synthesis in the cytoplasm. The centers of synthesis were detected at 4 hours post infection, with the acridine orange fluorochrome stain as compared to 6 hours with Feulgen and methyl green-pyronin stains. Marginal fragmentation of the inclusion bodies was seen at 8 to 10 hours post infection, and appears to coincide with the first increase in cell-associated virus. With the immunofluorescent technique, it was found that the LS antigen of the virus can be detected at about 4 hours post infection. This is followed at 5 to 6 hours post infection by the appearance of the NP antigen. Both antigens are found only in the cytoplasm, and precede the appearance of the infective particle. The HA antigen, a by-product of virus-cell interaction, is not seen until about 10 hours post infection; that is, several hours after the appearance of both the LS and NP antigens, and only after the appearance of mature virus. The successful application of the use of two immune sera, each labeled with a different fluorescent dye for the simultaneous visualization of two antigens within a cell, is reported. Using this technique, the sites of LS and NP antigen synthesis, were easily differentiated. The intimate mixing of the two antigens at a later stage appears to coincide with the fragmentation of the inclusion body and the first detectable increase in cell-associated virus. The evidence obtained strongly suggests that the typical inclusion body observed in vaccinia-infected cells is composed mainly of the NP antigen.

Full Text

The Full Text of this article is available as a PDF (778.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ARMSTRONG J. A., NIVEN J. S. Fluorescence microscopy in the study of nucleic acids; histochemical observations on cellular and virus nucleic acids. Nature. 1957 Dec 14;180(4598):1335–1336. doi: 10.1038/1801335a0. [DOI] [PubMed] [Google Scholar]
  2. BROWN A., MAYYASI S. A., OFFICER J. E. The toxic activity of vaccinia virus in tissue culture. J Infect Dis. 1959 Mar-Apr;104(2):193–202. doi: 10.1093/infdis/104.2.193. [DOI] [PubMed] [Google Scholar]
  3. CAIRNS J. The initiation of vaccinia infection. Virology. 1960 Jul;11:603–623. doi: 10.1016/0042-6822(60)90103-3. [DOI] [PubMed] [Google Scholar]
  4. COHN M. III. Techniques and analysis of the quantitative precipitin reaction. A. Reaction in liquid media. Methods Med Res. 1952;5:301–335. [PubMed] [Google Scholar]
  5. COONS A. H., KAPLAN M. H. Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med. 1950 Jan 1;91(1):1–13. doi: 10.1084/jem.91.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Craigie J., Wishart F. O. STUDIES ON THE SOLUBLE PRECIPITABLE SUBSTANCES OF VACCINIA : I. THE DISSOCIATION IN VITRO OF SOLUBLE PRECIPITABLE SUBSTANCES FROM ELEMENTARY BODIES OF VACCINIA. J Exp Med. 1936 Oct 31;64(5):803–817. doi: 10.1084/jem.64.5.803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Craigie J., Wishart F. O. STUDIES ON THE SOLUBLE PRECIPITABLE SUBSTANCES OF VACCINIA : II. THE SOLUBLE PRECIPITABLE SUBSTANCES OF DERMAL VACCINE. J Exp Med. 1936 Oct 31;64(5):819–830. doi: 10.1084/jem.64.5.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DEUTSCH H. F. II. Separation of antibody-active proteins from various animal sera by ethanol fractionation techniques. Methods Med Res. 1952;5:284–300. [PubMed] [Google Scholar]
  9. EAGLE H. The specific amino acid requirements of a human carcinoma cell (Stain HeLa) in tissue culture. J Exp Med. 1955 Jul 1;102(1):37–48. doi: 10.1084/jem.102.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. EPSTEIN M. A. An investigation into the purifying effect of a fluorocarbon on vaccinia virus. Br J Exp Pathol. 1958 Aug;39(4):436–446. [PMC free article] [PubMed] [Google Scholar]
  11. FLEWETT T. H. Intracellular growth of some viruses of the pox group; an electron microscopic study of infected chick chorionic cells. J Hyg (Lond) 1956 Sep;54(3):393–400. doi: 10.1017/s002217240004465x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GAYLORD W. H., Jr, MELNICK J. L. Intracellular forms of pox viruses as shown by the electron microscope (Vaccinia, Ectromelia, Molluscum Contagiosum). J Exp Med. 1953 Aug;98(2):157–172. doi: 10.1084/jem.98.2.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. JOKLIK W. K., RODRICK J. M. Biochemical studies on vaccinia virus in cultured cells. I. Incorporation of adenine-8-C14 into normal and infected cells. Virology. 1959 Nov;9:396–416. doi: 10.1016/0042-6822(59)90131-x. [DOI] [PubMed] [Google Scholar]
  14. MORGAN C., ELLISON S. A., ROSE H. M., MOORE D. H. Structure and development of viruses observed in the electron microscope. II. Vaccinia and fowl pox viruses. J Exp Med. 1954 Sep 1;100(3):301–310. doi: 10.1084/jem.100.3.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. NEWCOMER E. H. A new cytological and nistological fixing fluid. Science. 1953 Aug 7;118(3058):161–161. doi: 10.1126/science.118.3058.161. [DOI] [PubMed] [Google Scholar]
  16. NOYES W. F., WATSON B. K. Studies on the increase of vaccine virus in cultured human cells by means of the fluorescent antibody technique. J Exp Med. 1955 Sep 1;102(3):237–242. doi: 10.1084/jem.102.3.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. PETERS D. Morphology of resting vaccinia virus. Nature. 1956 Dec 29;178(4548):1453–1455. doi: 10.1038/1781453a0. [DOI] [PubMed] [Google Scholar]
  18. RIGGS J. L., LOH P. C., EVELAND W. C. A simple fractionation method for preparation of fluorescein-labeled gamma globulin. Proc Soc Exp Biol Med. 1960 Dec;105:655–658. doi: 10.3181/00379727-105-26207. [DOI] [PubMed] [Google Scholar]
  19. RIGGS J. L., SEIWALD R. J., BURCKHALTER J. H., DOWNS C. M., METCALF T. G. Isothiocyanate compounds as fluorescent labeling agents for immune serum. Am J Pathol. 1958 Nov-Dec;34(6):1081–1097. [PMC free article] [PubMed] [Google Scholar]
  20. Shedlovsky T., Smadel J. E. THE LS-ANTIGEN OF VACCINIA : II. ISOLATION OF A SINGLE SUBSTANCE CONTAINING BOTH L- AND S-ACTIVITY. J Exp Med. 1942 Jan 31;75(2):165–178. doi: 10.1084/jem.75.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smadel J. E., Hoagland C. L. ELEMENTARY BODIES OF VACCINIA. Bacteriol Rev. 1942 Jun;6(2):79–110. doi: 10.1128/br.6.2.79-110.1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. TAMM I., BABLANIAN R. On the role of ribonucleic acid in animal virus synthesis. II. Studies with ribonuclease. J Exp Med. 1960 Mar 1;111:351–368. doi: 10.1084/jem.111.3.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. TAMM I., NEMES M. M., OSTERHOUT S. On the role of ribonucleic acid in animal virus synthesis. I. Studies with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. J Exp Med. 1960 Mar 1;111:339–349. doi: 10.1084/jem.111.3.339. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES