Rotaviruses, a genetically diverse and rapidly evolving group of segmented double-stranded RNA viruses, are a major cause of severe life-threatening gastroenteritis in infants and young. Progress towards understanding rotavirus biology and pathogenesis, and the potential development of more effective next generation rotavirus vaccines, is hampered by the lack of a fully recombinant reverse genetics (RG) system for the virus. Without this fundamental tool, many studies are not possible including those intended to understand the role of viral proteins in virulence and spread in the infected animal, identify and characterize gene-specific packaging (assortment) signals in viral RNAs, and analyze virus replication and assembly by live-cell imaging of infected cells expressing fluorescently-tagged viral proteins. The consequence for vaccine development and evaluation is perhaps even more profound, as an RG system is critical for generating live-virus vaccine candidates through directed attenuation of viral virulence determinants, for modifying antigenic epitopes in older vaccine virus strains to better match those of contemporary viruses, and for generating vaccine candidates through directed reassortment of the genomes of human and animal virus strains. Moreover, an effective RG system is essential for pursing attempts to develop rotaviruses as plug-and-play expression vectors of antigens of other enteric/mucosal pathogens (e.g., norovirus). In previous work, we established a highly efficient single gene RG system for rotavirus and showed that foreign sequences can be inserted into, and stably maintained by, the rotavirus genome. Investigators working on other viruses that, like rotaviruses, belong to the Reoviridae family, have developed fully recombinant RG systems using T7 transcription vectors to drive the expression of complete genome sets of viral +RNAs in cells expressing T7 RNA polymerase. Through the introduction of 2A stop-start translational elements, these RG systems have been used to generate autonomously replicating recombinant Reoviridae that express foreign antigens and fluorescent proteins. In this R03 grant application, we propose to (i) establish a fully recombinant plasmid based RG system for rotavirus using T7 expression vectors and (ii) develop recombinant rotaviruses as plug-and-play expression vectors using 2A elements to drive synthesis of foreign proteins. Achievement of these aims will revolutionize the nature of studies that can be pursued for a virus that kills >200,000 children each year.
Rotavirus, a genetically diverse and rapidly evolving pathogen, is a major cause of life-threatening diarrheal disease in infants and young children throughout the world. Despite its importance, many aspects of the biology and pathogenesis of rotavirus remain poorly understood largely because of the lack of a robust reverse genetics system to dissect the properties and function of the virus?s genes. In this proposal, we will pursue development of a rotavirus reverse genetics systems that not only will allow study of the virus but also enable the construction of more effective next generation rotavirus vaccines.
| Borodavka, Alexander; Desselberger, Ulrich; Patton, John T (2018) Genome packaging in multi-segmented dsRNA viruses: distinct mechanisms with similar outcomes. Curr Opin Virol 33:106-112 |
