Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8511, Japan.
Proc Natl Acad Sci U S A. 2021 Oct 19;118(42). doi: 10.1073/pnas.2105334118.
The family is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family , the genera , , , , and contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for viruses such as , , , and , which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus family ), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an -glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus but also the replication machinery of the family .
该科是一类无囊膜病毒，具有包含 9 到 12 个节段的双链 (ds) RNA 基因组。在该科中，属 、 、 、 和 包含具有 12 个节段 dsRNA 基因组的病毒种。用于生成重组传染性病毒的反向遗传学系统是研究病毒基因功能以及开发疫苗和治疗干预措施的有力工具。通常，这种方法已用于具有 10 或 11 个节段基因组的病毒，例如 、 、 和 。然而，对于具有包含 12 个节段基因组的病毒，尚未开发出反向遗传学系统。在此，我们描述了一种用于田尻 tick 病毒（TarTV）（属 科）的全质粒反向遗传学系统的开发，该病毒具有 12 个节段的基因组。通过将编码 TarTV 基因组的 12 个克隆互补 DNA 转染到表达 T7 RNA 聚合酶的幼仓鼠肾细胞中，生成了重组 TarTV。使用这项技术，我们生成了 VP12 突变病毒，并证明 VP12 是一种 -糖基化蛋白。我们还生成了一种表达 HiBiT 标记的 VP8 蛋白的报告病毒。该反向遗传学系统将增加我们对属生物学的理解，同时也增加我们对家族复制机制的理解。
