Engineering recombinant Orsay virus directly in the metazoan host Caenorhabditis elegans.

The recent identification of Orsay virus, the first virus that is capable of naturally infecting Caenorhabditis elegans, provides a unique opportunity to explore host-virus interaction studies in this invaluable model organism. A key feature of this system is the robust genetic tractability of the host, C. elegans, which would ideally be complemented by the ability to genetically manipulate Orsay virus in parallel. To this end, we developed a plasmid-based reverse genetics system for Orsay virus by creating transgenic C. elegans strains harboring Orsay virus cDNAs. Both wild-type and mutant Orsay viruses, including a FLAG epitope-tagged recombinant Orsay virus, were generated by use of the reverse genetics system. This is the first plasmid-based virus reverse genetics system in the metazoan C. elegans. The Orsay virus reverse genetics we established will serve as a fundamental tool in host-virus interaction studies in the model organism C. elegans. Importance: To date, Orsay virus is the first and the only identified virus capable of naturally infecting Caenorhabditis elegans. C. elegans is a simple multicellular model organism that mimics many fundamental features of human biology and has been used to define many biological properties conserved through evolution. Thus, the Orsay virus-C. elegans infection system provides a unique opportunity to study host-virus interactions. In order to take maximal advantage of this system, the ability to genetically engineer mutant forms of Orsay virus would be highly desirable. Most efforts to engineer viruses have been done with cultured cells. Here we describe the creation of mutant viruses directly in the multicellular organism C. elegans without the use of cell culture. We engineered a virus expressing a genetically tagged protein that could be detected in C. elegans. This provides proof of concept for modifying Orsay virus, which will greatly facilitate studies in this experimental system.