A low fidelity virus shows increased recombination during the removal of an alphavirus reporter gene

Tiffany F. Kautz, Elizabeth Jaworski, Andrew Routh, Naomi L. Forrester

Research output: Contribution to journalArticlepeer-review


Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.

Original languageEnglish (US)
Article number660
Issue number6
StatePublished - Jun 2020


  • Alphavirus
  • Arbovirus
  • Fidelity
  • Recombination
  • Sequencing

ASJC Scopus subject areas

  • Infectious Diseases
  • Virology


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