Engineering iridoviruses: development of reverse genetics and virus rescue systems.

Iridoviruses are a family of large DNA viruses that infect insects and poikilotherm vertebrates, including amphibians, reptiles, and fish. Notably, members of the genus cause mass mortality in fish and amphibians, threatening aquaculture and contributing to global amphibian decline. Despite their omnipresence and impact, key aspects of iridovirus biology remain unknown, largely due to the absence of reverse genetics systems. In this study, we developed, characterized, and utilized a reverse genetics system for frog virus 3 (FV3, ), one of the most widely studied iridoviruses. The rescued virus exhibits growth and phenotypic properties identical to those of the parental virus isolate. Furthermore, we established an alternative approach of virus reconstitution from genomic DNA, utilizing a heterologous iridovirus as a helper. This novel approach enables rapid and facile rescue of modified viruses from naked DNA. The reverse genetics and rescue systems described in this study will advance iridovirus research by facilitating efficient genetic modification of the virus genome in yeast or bacteria. This could clear the path to elucidating functions of virus genes and allow a much more detailed understanding of iridovirus biology. Moreover, owing to the promiscuous nature of FV3 with its ability to infect hosts from different classes of animals, the FV3 system has the potential to serve as a platform for the development of modified live vaccines for a variety of fish and amphibian species.IMPORTANCEIridoviruses pose a substantial threat to aquaculture and global amphibian populations, yet research has been hindered by the lack of a reverse genetics system. In this study, we describe the development of the first such system for this virus family. We constructed a synthetic clone of frog virus 3 (FV3) that can be propagated and genetically manipulated in both yeast and bacteria, yielding a virus that has biological properties identical to the parental virus isolate. Furthermore, we developed a novel helper virus-based system for the rescue of FV3 from purified DNA. This system provides an essential tool for advancing our understanding of iridovirus biology and serves as a platform for the development of modified live virus vaccines.