Isolation and Handling of Recombinant Vaccinia Viruses

  • Antonio Talavera
  • Javier M. Rodriguez
Part of the Methods in Molecular Biology book series (MIMB, volume 8)


In  Chapter 20, a general procedure was described for the construction of the intermediate vectors necessary for the insertion of foreign DNA sequences into the vaccinia virus (Vv) genome. The principles and basic methodology for the isolation of recombinant vaccinia viruses will be discussed in this chapter.


Vaccinia Virus Recombinant Virus Mycophenolic Acid Progeny Virus Recombinant Vaccinia Virus 
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  1. 1.
    Ensinger, M. J. (1982) Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR. J. Virol 43, 778–790.PubMedGoogle Scholar
  2. 2.
    Drillien, R., Spehner, D., and Kirn, A. (1982) Complementation and genetic linkage between vaccinia virus temperature-sensitive mutants. Virology 119, 372–381.PubMedCrossRefGoogle Scholar
  3. 3.
    Nakano, E., Panicah, D., and Paoletti, E. (1982) Molecular genetics of vaccinia virus Demonstration of marker rescue. Proc. Natl. Acad. Sa. USA 79, 1593–1596.CrossRefGoogle Scholar
  4. 4.
    Condit, R. C, Motyczka, A., and Spizz, G. (1983) Isolation, characterization, and physical mapping of temperature-sensitive mutants of vaccinia virus. Virology 128, 429–443.PubMedCrossRefGoogle Scholar
  5. 5.
    Drillien, R. and Spehner, D. (1983) Physical mapping of vaccinia virus temperaturesensitive mutations. Virology 131, 385–393.PubMedCrossRefGoogle Scholar
  6. 6.
    Tartaglia, J. and Paoletti, E. (1985) Physical mapping and DNA sequence analysis of the rifampicin resistance locus in vaccinia virus Virology 147, 394–404.PubMedCrossRefGoogle Scholar
  7. 7.
    Baldick, C J. and Moss, B. (1987) Resistance of vaccinia virus to rifampicin conferred by a single nucleotide substitution near the predicted NH2 terminus of a gene encoding an M 62,000 polypeptide. Virology 156, 138–145.PubMedCrossRefGoogle Scholar
  8. 8.
    Hruby, D. E. and Ball, L. A. (1982) Mapping and identification of the vaccinia virus thymidine kinase gene. J. Virol. 43, 403–409.PubMedGoogle Scholar
  9. 9.
    Jones, E. V. and Moss, B. (1984) Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA. J. Virol. 49, 72–77.PubMedGoogle Scholar
  10. 10.
    Panicali, D. and Paoletti, E. (1982) Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase from herpes simplex virus into the DNA of infectious vaccinia virus. Proc. Natl. Acad Sci. USA 79, 4927–4931.PubMedCrossRefGoogle Scholar
  11. 11.
    Mackett, M., Smith, G. I., and Moss, B. (1982) Vaccinia virus, a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA 79, 7415–7419.PubMedCrossRefGoogle Scholar
  12. 12.
    Mackett, M., Smith, G. I., and Moss, B. (1984) General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J. Virol 49, 857–864.PubMedGoogle Scholar
  13. 13.
    Chakrabaru, S., Brechling, K., and Moss, B. (1985) Vaccinia virus expression vector: Coexpression of β-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol. 5, 3403–3409.Google Scholar
  14. 14.
    Panicali, D., Grzelecki, A., and Huang, C. (1986) Vaccinia virus vectors utilizing the β-galactosidase assay for rapid selection of recombinant viruses and measurement of gene expression. Gene 47, 93–99.CrossRefGoogle Scholar
  15. 15.
    Rhim, J. S., Cho, H. Y, and Huebner, R.J. (1975) Nonproducer human cells induced by murine sarcoma virus. Int. J. Cancer 15, 23–29.PubMedCrossRefGoogle Scholar
  16. 16.
    Southern, E. (1975) Detection of specific sequences among DNA fragments seperated by gel electrophoresis. J. Mol. Biol. 98, 503–517.PubMedCrossRefGoogle Scholar
  17. 17.
    Joklik, W. K (1962) The preparation and characterization of highly purified radioactively labeled poxvirus. Btochim. Biophys. Acla 61, 292–302.Google Scholar
  18. 18.
    DeFilippes, F. M. (1976) Restriction enzyme digests of rapidly renaturing fragments of vaccinia virus DNA. J. Virol. 17, 227–238.Google Scholar
  19. 19.
    Plantrose, D. N., Nishimura, C, and Salzman, N. P. (1962) The purification of vaccinia virus from cell cultures. Virology 18, 294–301.CrossRefGoogle Scholar
  20. 20.
    Franke, C. A., Rice, C. M., Strauss, J. H., and Hruby, D. E. (1985) Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants. Mol. Cell. Biol. 5, 1918–1924.PubMedGoogle Scholar
  21. 21.
    Bertholet, C, Drillien, R., and Wittek, R. (1985) One hundred base pairs flanking sequence of a vaccinia virus late gene are sufficient to temporarily regulate transcription. Proc. Natl. Acad. Sci. USA 82, 2096–2100.PubMedCrossRefGoogle Scholar
  22. 22.
    Franke, C. A. and Hruby, D. E. (1988) Use of the gene encoding neomycin phosphotransferase II to convect linked markers into the vaccinia virus genome Nucleic Acids Res. 16, 1634.PubMedCrossRefGoogle Scholar
  23. 23.
    Boyle, D. B. and Coupar, B. E. H. (1988) A dominant selectable marker for the construction of recombinant poxviruses. Gene 65, 123–128.PubMedCrossRefGoogle Scholar
  24. 24.
    Falkner, F. G. and Moss, B. (1988) Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors J Virol 62, 1849–1854.PubMedGoogle Scholar
  25. 25.
    Patel, D. D., Ray, C. A., Drucker, R. P., and Pickup, D. J. (1988) A poxvirus-derived vector that directs high levels of expressed and cloned genes in mammalian cells. Proc. Natl. Acad Sci. USA 9931–9935.Google Scholar
  26. 26.
    Studier, F. W. and Moffat, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol Biol. 189, 113–130.PubMedCrossRefGoogle Scholar
  27. 27.
    Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83, 8122–8126.PubMedCrossRefGoogle Scholar
  28. 28.
    Fuerst, T. R., Earl, P. L., and Moss, B. (1987) Use of a hybrid vaccinia virus T7 RNA polymerase system for expression of target genes. Mol. Cell. Biol. 7, 2538–2544.PubMedGoogle Scholar
  29. 29.
    Fuerst, T. R. and Moss, B. (1989) Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 5′ untranslated leader. J. Mol. Biol. 206, 333–348.PubMedCrossRefGoogle Scholar
  30. 30.
    Lane, J. M., Ruben, F. L., Neff, J. M., and Millar, J. D. (1969) Complications in smallpox vaccination. 1968. National surveillance in the United States. N. Engl. J. Med. 21, 1201–1208.CrossRefGoogle Scholar
  31. 31.
    Shida, H., Hinuma, Y, Hatanaka, M., Morita, M., Kidokoro, M., Suzuki, K, Maruyama, T., Takahashi-Nishimari, F., Sugimotoo, M., Kitamura, A., Miyazama, T., and Hayami, M. (1988) Effects and virulences of recombinant vaccinia viruses derived form attenuated strains that express the human T-cell leukemia virus type I envelope gene. J. Virol. 62, 4474–4480.PubMedGoogle Scholar
  32. 32.
    Buller, R. M. L., Smith, G. L., Cremer, K., Notkins, A. L., and Moss, B. (1985) Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. Nature 317, 813–815PubMedCrossRefGoogle Scholar
  33. 33.
    Rodriguez, D., Rodriguez, J. R., Rodriguez J. F., Trauber, D., and Esteban, M. (1989) Highly attenuated vaccinia virus mutants for the generation of safe recombinant viruses. Proc. Natl. Acad. Sci. USA 86,1287–1291.PubMedCrossRefGoogle Scholar
  34. 34.
    Dallo, S. and Esteban, M. (1987) Isolation and characterization of attenuated mutants of vaccinia virus. Virology 159, 408–422.PubMedCrossRefGoogle Scholar
  35. 35.
    Dallo, S., Rodriguez J. F., and Esteban, M. (1987) A14 K envelope protein of vaccinia virus with an important role in virus-host cell interactions is altered during virus persistence and determines the plaque size phenotype of the virus. Virology 159, 423–432.PubMedCrossRefGoogle Scholar

Copyright information

© The Humana Press Inc., Clifton, NJ 1991

Authors and Affiliations

  • Antonio Talavera
    • 1
  • Javier M. Rodriguez
    • 2
  1. 1.Centro Nacional de BiotechnologiaMadridSpain
  2. 2.Centro de Biologia Molecular (CSIS-UAM)MadridSpain

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