Publication Database

Development of a reverse genetics system for iridoviruses based on TAR cloning in Saccharomyces cerevisiae, including the establishment of a novel method for genomic DNA rescue using a heterologous fish helper virus (2025)

Art

Hochschulschrift

Autor

Vladimirova, Daria (WE 5)

Quelle

Berlin, 2025 — 118 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/50002

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

A reverse genetics system (RGS) for iridovirus frog virus 3 (FV3, Ranavirus rana1), one of the key members in Megaviricetes class, was successfully developed using transformation- associated recombination (TAR) cloning in yeast Saccharomyces cerevisiae. This method enabled the construction of BAC-YAC constructs containing the full-length FV3 genome, from which infectious virus could be rescued. One of the constructed clones - FV3-S3Ar - retained key phenotypic and functional characteristics of the parental FV3, demonstrating the integrity of the construct. To evaluate the established RGS applicability, a recombinant virus was generated by replacing ORF64R with an enhanced green fluorescent protein (EGFP), constructing a reporter virus FV3-Δ64R-EGFP. The resulting reporter virus showed stable replication and persistent EGFP expression over several passages. This confirmed the platform’s suitability for targeted gene manipulation and the viability of the system. An alternative rescue method using the heterologous fish ranavirus Largemouth bass virus virus (LMBV, Ranavirus micropterus1) as a helper virus was developed and validated. This approach proved effective for virus rescue and may provide a safer and more straightforward option compared to the use of a traditional UV-irradiated helper virus. Overall, this system offers a reliable and flexible tool for the genetic mutagenesis of FV3 and paves the way for future investigations into viral gene function, pathogenicity, and potential vaccine development.