Entirely plasmid-based reverse genetics system for rotaviruses

Significance

Rotaviruses (RVs) are a group of viruses that cause severe gastroenteritis in infants and young children. Until now, no strategy has been developed to generate infectious RVs entirely from cloned cDNAs. The absence of a reliable reverse genetics platform has been a major roadblock in the RV field, precluding numerous studies of RV replication and pathogenesis and hampering efforts to develop the next generation of RV vaccines. Here, we developed a plasmid-based reverse genetics system that is free from helper viruses and independent of any selection for RV. This technology will accelerate studies of RV pathobiology, allow rational design of RV vaccines, and yield RVs suitable for screening small molecules as potential antivirals.

Abstract

Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein–tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.