Eiko Matsuo, Kobe University

Eiko Matsuo1, Yoshitaka Harada1, Daisuke Motooka2, Takeshi Kobayashi3, Polly Roy4 1. Graduate School of Agricultural Science Kobe University, Kobe, Hyogo Japan 2. NGS Core Facility, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. 3. Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. 4. London School of Hygiene & Tropical Medicine, London, United Kingdom

Combatting and Exploiting dsRNA viruses

Genetic stability of exogenous genes is one important factor to develop RNA virus vector platforms. In Reoviridae, orbiviruses bear a unique protein, VP6, which could tolerate the partial substitution with exogenous genes in frame to generate viable tractable tools for transgenesis. Therefore, for the future application of orbiviruses, such as viral vectors, vaccine platforms and perhaps gene therapy, the instability of transgene, which was observed in our recent studies, is one of challenges to overcome. Here, using reverse genetics system for Ibaraki virus (IBAV), a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, an unessential region in VP6 gene (S9 segment) of IBAV were replaced with some transgenes and their genetical instability during replication was analyzed. The large and evolutionarily distant transgene destabilized the inserted S9 genome. In addition, most of the sequences trancated from S9-transgene segment included a part of transgenes and adjacent S9 sequence, indicating that replacement of an unessential region with transgenes could form unexpected pairs between transgene and S9 mRNA to distort its secondary structure. Similar to the previous study on rotavirus and mammalian orthoreovirus, codon-optimized transgenes, which possess EHDV-like codon usage biases, drastically stabilized the inserted S9 genome. Interestingly, secondary structure prediction of S9 mRNA revealed that the codon-optimized transgenes did not devastatingly distort the original S9 structure. Further study regarding destabilization mechanism in IBAV by transgene will be discussed.