Rapid, seamless generation of recombinant poxviruses using host range and visual selection

Sameera Vipat, Greg Brennan, Chorong Park, Sherry L. Haller, Stefan Rothenburg

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV’s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3’ arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

Original languageEnglish (US)
Article numbere61049
Pages (from-to)1-7
Number of pages7
JournalJournal of Visualized Experiments
Issue number159
StatePublished - May 2020


  • E3L-mCherry
  • Host range
  • Immunology and Infection
  • Issue 159
  • Poxvirus
  • Protein kinase R
  • Recombination
  • Vaccinia virus

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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