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

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Authors

Guo, Yunze
Pretorius, Jakobus Maree
Xu, Qingyuan
Wu, Donglai
Bu, Zhigao
Theron, Jacques
Sun, Encheng

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Publisher

Springer

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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Keywords

Epizootic hemorrhagic disease virus (EHDV), Reverse genetics system (RGS), Viral RNA transcripts, DNA-based reverse genetics systems

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Citation

Guo, Y., Pretorius, J.M., Xu, Q. et al. Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus. Archives of Virology 165, 1079–1087 (2020). https://doi.org/10.1007/s00705-020-04583-w.