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

Archives of Virology
Yunze GuoEncheng Sun

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.

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Citations

Jan 14, 2021·Virus Research·Yuta Kanai, Takeshi Kobayashi
Sep 30, 2021·Annual Review of Virology·Yuta Kanai, Takeshi Kobayashi

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Methods Mentioned

BETA
genetic modification
dissection
transfection
PCR
transmission electron microscopy
electrophoresis
electron microscopy

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