A mix-and-match reverse genetics system for evaluating genetic determinants of orthobunyavirus neurological disease

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.