A plasmid-based reverse genetics system for tilapia lake virus (TiLV)

Tilapia lake virus (TiLV) is a 10-segmented, single-stranded, negative-sense RNA virus first identified in 2014. It can cause up to 90 % mortality in tilapia, a species that is extensively cultured globally and serves as a crucial source of animal protein. However, scientific knowledge and research relating to TiLV is limited. The unavailability of a reverse genetics system (RGS) has made it challenging to elucidate the molecular mechanisms underlying TiLV replication and pathogenesis. We here report the establishment of an RGS and the successful rescue of recombinant tilapia lake viruses (TiLVs) from cloned cDNAs. Based on our discovery that TiLV can infect and replicate in Vero E6 cells, and through sequence analyses of the TiLV genome, we generated recombinant TiLVs. This was achieved by transfecting 10 plasmids containing the complete TiLV genome into Vero E6 cells that were co-cultured with fish-derived E11 cells. Sequence analysis of the genome of the rescued virus and comparison with parental TiLV genomic segments confirmed the successful rescue. We then used our system to generate reassortant viruses, as well as a HiBiT-tagged reporter TiLV. All TiLVs rescued using the RGS caused a clear cytopathic effect in E11 cells, which was indistinguishable from wild-type virus. Serial passage of the rescued viruses confirmed their genetic stability and demonstrated the robustness of the established RGS. This RGS will allow in-depth characterization of this emerged virus and facilitate the development of vaccines and antiviral therapeutics.

© 2024 Published by Elsevier B.V.