Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1979 May;9(5):609–614. doi: 10.1128/jcm.9.5.609-614.1979

Development of dengue virus plaques under serum-free overlay medium.

B Malewicz, H M Jenkin
PMCID: PMC275358  PMID: 39085

Abstract

An improved plaque assay for dengue virus was developed utilizing baby hamster kidney (BHK-21) cells initially grown in shaker culture. Different media preparations were tested for uniform and fast formation of BHK-21 cell sheets. Several overlay formulas were tested to develop a rapid plaque assay in 6- and 24-well plastic plates. The best results were obtained utilizing Eagle minimal essential medium (pH 7.2 to 7.4) supplemented with 1 mg of NaHCO3 per ml and 5% newborn calf serum for the formation of cell monolayers after 8 to 24 h of incubation at 37 degrees C. Serum-free Eagle minimal essential medium supplemented with 1% methylcellulose and buffered with 10 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (pH 7.4 to 7.6) was used as an overlay medium. This system allowed for plaque formation after 3 days of incubation of dengue type 2 virus and after 4 days for dengue type 1 and 4 viruses.

Full text

PDF
611

Images in this article

611Image
on p.611

Selected References

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

  1. Bentley J., Wickham E. A. A simple plaque assay for influenza A 2 viruses and its application to antiviral screening. Arch Gesamte Virusforsch. 1971;33(3):234–241. doi: 10.1007/BF01254679. [DOI] [PubMed] [Google Scholar]
  2. Clarke E. J., Jr, Suitor E. C., Jr, Jenkin H. M. A serologic survey for arbovirus antibodies in inhabitants of a southwest Taiwan village. Trop Geogr Med. 1967 Dec;19(4):326–332. [PubMed] [Google Scholar]
  3. EAGLE H. Amino acid metabolism in mammalian cell cultures. Science. 1959 Aug 21;130(3373):432–437. doi: 10.1126/science.130.3373.432. [DOI] [PubMed] [Google Scholar]
  4. Eylar O. R., Wisseman C. L., Jr Thermal inactivation of type 1 Dengue virus strains. Acta Virol. 1975 Apr;19(2):167–168. [PubMed] [Google Scholar]
  5. Fujita N., Karabatsos N., Shope R. E. Effects of protamine sulfate on dengue type 1 viral activities. Microbiol Immunol. 1978;22(6):315–323. doi: 10.1111/j.1348-0421.1978.tb00376.x. [DOI] [PubMed] [Google Scholar]
  6. Fujita N., Tamura M., Hotta S. Dengue virus plaque formation on microplate cultures and its application to virus neutralization (38564). Proc Soc Exp Biol Med. 1975 Feb;148(2):472–475. doi: 10.3181/00379727-148-38564. [DOI] [PubMed] [Google Scholar]
  7. Guskey L. E., Jenkin H. M. Adaptation of BHK-21 cells to growth in shaker culture and subsequent challenge by Japanese encephalitis virus. Appl Microbiol. 1975 Sep;30(3):433–438. doi: 10.1128/am.30.3.433-438.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. HOLLAND J. J., McLAREN L. C. Improved method for staining cell monolayers for virus plaque counts. J Bacteriol. 1959 Oct;78:596–597. doi: 10.1128/jb.78.4.596-597.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hotta S., Fujita N., Maruyama T. Research on dengue in tissue culture. I. Plaque formation in an established monkey kidney cell line culture. Kobe J Med Sci. 1966 Sep;12(3):179–187. [PubMed] [Google Scholar]
  10. Igarashi A., Mantani M. Rapid titration of dengue virus type 4 infectivity by counting fluorescent foci. Biken J. 1974 Sep;17(3):87–93. [PubMed] [Google Scholar]
  11. Jenkin H. M., Anderson L. E. The effect of oleic acid on the growth of monkey kidney cells (LLC-MK2). Exp Cell Res. 1970 Jan;59(1):6–10. doi: 10.1016/0014-4827(70)90616-6. [DOI] [PubMed] [Google Scholar]
  12. MAGEE W. E., SHEEK M. R., SAGIK B. P. Methods of harvesting mammalian cells grown in tissue culture. Proc Soc Exp Biol Med. 1958 Nov;99(2):390–392. doi: 10.3181/00379727-99-24360. [DOI] [PubMed] [Google Scholar]
  13. Nash D. R., Halstead S. B., Stenhouse A. C., McCue C. Nonspecific factors in monkey tissues and serum causing inhibition of plaque formation and hemagglutination by dengue viruses. Infect Immun. 1971 Feb;3(2):193–199. doi: 10.1128/iai.3.2.193-199.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Okuno Y., Sasao F., Fukunaga T., Fukai K. An application of PAP (peroxidase-anti-peroxidase) staining technique for the rapid titration of dengue virus type 4 infectivity. Biken J. 1977 Mar;20(1):29–33. [PubMed] [Google Scholar]
  15. Ong S. B. Plaque formation of dengue viruses in LLC-MK 2 cell cultures. Southeast Asian J Trop Med Public Health. 1971 Jun;2(2):147–150. [PubMed] [Google Scholar]
  16. Russell P. K., Nisalak A., Sukhavachana P., Vivona S. A plaque reduction test for dengue virus neutralizing antibodies. J Immunol. 1967 Aug;99(2):285–290. [PubMed] [Google Scholar]
  17. SCHULZE I. T., SCHLESINGER R. W. Plaque assay of dengue and other group B arthropod-borne viruses under methyl cellulose overlay media. Virology. 1963 Jan;19:40–48. doi: 10.1016/0042-6822(63)90022-9. [DOI] [PubMed] [Google Scholar]
  18. Shiraki K., Hotta S. Dengue-1 virus permissiveness of established cell line cultures. Microbiol Immunol. 1977;21(7):411–415. doi: 10.1111/j.1348-0421.1977.tb00305.x. [DOI] [PubMed] [Google Scholar]
  19. Sinarachatanant P., Olson L. C. Replication of dengue virus type 2 in Aedes albopictus cell culture. J Virol. 1973 Aug;12(2):275–283. doi: 10.1128/jvi.12.2.275-283.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stim T. B. Dengue virus plaque development in simian cell systems. I. Factors influencing virus adsorption and variables in the agar overlay medium. Appl Microbiol. 1970 May;19(5):751–756. doi: 10.1128/am.19.5.751-756.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stim T. B. Dengue virus plaque development in simian cell systems. II. Agar variables and effect of chemical additives. Appl Microbiol. 1970 May;19(5):757–757. doi: 10.1128/am.19.5.757-762.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. WAYMOUTH C. Rapid proliferation of sublines of NCTC clone 929 (strain L) mouse cells in a simple chemically defined medium (MB 752/1). J Natl Cancer Inst. 1959 May;22(5):1003–1017. doi: 10.1093/jnci/22.5.1003. [DOI] [PubMed] [Google Scholar]
  23. Wallis C., Ver B., Melnick J. L. The role of serum and fetuin in the growth of monkey kidney cells in culture. Exp Cell Res. 1969 Dec;58(2):271–282. doi: 10.1016/0014-4827(69)90505-9. [DOI] [PubMed] [Google Scholar]
  24. Yuill T. M., Sukhavachana P., Nisalak A., Russell P. K. Dengue-virus recovery by direct and delayed plaques in LLC-MK2 cells. Am J Trop Med Hyg. 1968 May;17(3):441–448. doi: 10.4269/ajtmh.1968.17.441. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES