Miettinen Heini M, Abbott Matthew J, Evans Alyssa B
Department of Microbiology and Cell Biology, Montana State University, Bozeman, Montana, United States of America.
PLoS One. 2025 Apr 28;20(4):e0315694. doi: 10.1371/journal.pone.0315694. eCollection 2025.
The encephalitic orthobunyaviruses have tri-segmented, negative sense RNA genomes and can cause severe neurological disease in humans, including La Crosse virus (LACV), which is the leading cause of pediatric arboviral encephalitis in the United States. However, little is known about the genetic factors that drive neuropathogenesis. Reverse genetics systems (RGS) are valuable tools for studying viral genetics and pathogenesis. Plasmid-based cDNA reverse genetics systems are available for LACV, however the plasmid backbones are medium-copy number and have a propensity for recombination. We therefore generated a plasmid-based cDNA reverse genetics system for LACV utilizing a more stable and high-copy number plasmid backbone. Additionally, we created the first full reverse genetics systems for two closely related orthobunyaviruses, Jamestown Canyon virus (JCV), and Inkoo virus (INKV), which have differing reported disease incidences in humans and differing neuropathogenic phenotypes in mice compared to LACV. We compared wild type (wt) viruses with RGS-derived wt viruses in human neuronal cells and in mice, and found that RGS-derived wt viruses maintained the replication and neuropathogenic phenotypes of their wt counterpart. Additionally, we demonstrated that reverse genetics plasmids from different parental viruses can be readily mixed-and-matched to generate reassortant viruses. This system provides a valuable genetic tool utilizing viruses with differing neuropathogenic phenotypes to investigate the genetic determinants of orthobunyavirus neuropathogenesis.
脑炎正布尼亚病毒具有三分节段的负链RNA基因组，可导致人类严重的神经系统疾病，包括拉科罗斯病毒（LACV），它是美国儿童虫媒病毒性脑炎的主要病因。然而，对于驱动神经发病机制的遗传因素知之甚少。反向遗传学系统（RGS）是研究病毒遗传学和发病机制的宝贵工具。基于质粒的cDNA反向遗传学系统可用于LACV，然而质粒骨架是中拷贝数的，并且有重组倾向。因此，我们利用更稳定和高拷贝数的质粒骨架构建了基于质粒的LACV cDNA反向遗传学系统。此外，我们为两种密切相关的正布尼亚病毒——詹姆斯敦峡谷病毒（JCV）和因库奥病毒（INKV）创建了首个完整的反向遗传学系统，与LACV相比，它们在人类中的报告发病率不同，在小鼠中的神经致病表型也不同。我们在人类神经元细胞和小鼠中比较了野生型（wt）病毒与RGS衍生的wt病毒，发现RGS衍生的wt病毒保持了其wt对应物的复制和神经致病表型。此外，我们证明了来自不同亲本病毒的反向遗传性质粒可以很容易地混合搭配以产生重配病毒。该系统提供了一种有价值的遗传工具，利用具有不同神经致病表型的病毒来研究正布尼亚病毒神经发病机制的遗传决定因素。
