Development of reverse genetics for Ibaraki virus to produce viable VP6-tagged IBAV

• Published in: FEBS open bio Vol. 5; no. 1; pp. 445 - 453
• Main Authors: Matsuo, Eiko, Saeki, Keiichi, Roy, Polly, Kawano, Junichi
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01-01-2015; John Wiley & Sons, Inc; Elsevier; Wiley
• Subjects: Cattle industry; Disease; Enzymes; Epizootic hemorrhagic disease virus; Genomes; Hemorrhage; Hemorrhagic disease; Ibaraki virus; Infections; Livestock; Localization; Molecular modelling; Mortality; Polyethylene glycol; Primary replication; Proteins; Replication; Reverse genetics system; Viable VP6-tagged virus; Viruses; VP6; VP6 protein; EHD; Reverse genetics system; Epizootic hemorrhagic disease virus; RG; EHDV; Viable VP6-tagged virus; IBAV; Primary replication; Ibaraki virus; VP6; VIB; RG, reverse genetics; VIB, virus inclusion body; EHDV, epizootic hemorrhagic disease virus; IBAV, Ibaraki virus; EHD, epizootic hemorrhagic disease
• ISSN: 2211-5463; 2211-5463
• DOI: 10.1016/j.fob.2015.05.006

•A reverse genetics system for Ibaraki virus (IBAV) was developed.•The RG system was used to produce viable VP6-tagged IBAV.•A region of VP6 (aa 34–82) is not required for IBAV replication in tissue culture.•The insertion of tags into the nonessential VP6 region did not disrupt replication.•IBAV VP6 quickly assembled into puncta in the cytosol of infected cells.
Ibaraki virus (IBAV) is a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, which belongs to the Orbivirus genus of the Reoviridae family. Although EHDV, including IBAV, represents an ongoing threat to livestock in the world, molecular mechanisms of EHDV replication and pathogenesis have been unclear. The reverse genetics (RG) system is one of the strong tools to understand molecular mechanisms of virus replication. Here, we developed a RG system for IBAV to identify the nonessential region of a minor structural protein, VP6, by generating VP6-truncated IBAV. Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV. We demonstrated that all VP6-tagged IBAV could replicate in BHK cells in the absence of any helper VP6 protein. Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV. Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).