Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus

Arch Virol. 2020 May;165(5):1079-1087. doi: 10.1007/s00705-020-04583-w. Epub 2020 Mar 6.

Abstract

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

MeSH terms

  • Animals
  • Cell Line
  • DNA, Complementary / genetics
  • Hemorrhagic Disease Virus, Epizootic / genetics*
  • Hemorrhagic Disease Virus, Epizootic / growth & development
  • Mesocricetus
  • Plasmids
  • RNA, Viral / genetics
  • Recombination, Genetic
  • Reoviridae Infections / virology
  • Reverse Genetics / methods*
  • Virology / methods*

Substances

  • DNA, Complementary
  • RNA, Viral