Abstract
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, posing a significant threat to the global tilapia industry. Currently, there is no cure for TiLV infection, nor is there a commercially available vaccine. Since the first identification of TiLV in 2014, a decade has passed without substantial progress in TiLV research. A major bottleneck in TiLV research is the lack of a reverse genetics system (RGS), which has hindered efforts to elucidate the molecular mechanisms underlying TiLV replication and pathogenesis. Therefore, the primary objective of this study is to develop an RGS for TiLV.Firstly, we identified that TiLV can infect and replicate in Vero E6 cells after susceptibility testing of various cell lines, thereby expanding the available systems for studying TiLV to a well-characterized cell line. Additionally, we investigated the effect of mammalian type I interferon (IFN) on TiLV infection in this cell culture system. Our results demonstrated that mammalian type I IFN exhibited robust antiviral activity against TiLV. Importantly, a recombinant fish type I IFN produced in mammalian cells also showed potent antiviral activity against TiLV in fish-derived E11 cells.
Secondly, based on the above discovery and through comprehensive sequence analyses of the TiLV genome, we successfully generated recombinant TiLV. This was achieved by transfecting 10 synthetic plasmids containing the complete TiLV reference genome (GCF_001630085.1, TiLV isolate Til-4-2011) into Vero E6 cells that were co-cultured with E11 cells. Sequence analysis of the genome of the rescued virus, compared with the TiLV reference genome, confirmed the successful rescue. In addition, we established a reliable method for sequencing the complete genome of TiLV, assembling the complete genome of a wild-type TiLV strain originating from Israel (named TiLV-Israel-HK). The new recombinant TiLV (TiLV-Israel-HK) was also successfully recovered using this RGS. Furthermore, we generated reassortant viruses using this system, providing the first experimental evidence of reassortment in TiLV.
Thirdly, we recovered a HiBiT-tagged reporter TiLV, which provides a powerful tool for monitoring virus replication both in vitro and in vivo. Notably, all TiLVs rescued using our RGS caused a clear cytopathic effect in E11 cells, which was indistinguishable from that caused by the wild-type TiLV.
In conclusion, we first identified that the fish virus, TiLV, can infect and replicate in mammalian cells, specifically the Vero E6 cells. Furthermore, we demonstrated the protective effect of type I IFN against TiLV infection, which may point to a mechanism of TiLV pathogenesis and aid in the development of vaccines and antiviral therapies against this important fish pathogen. Most importantly, we established the first RGS for TiLV, which will significantly expedite studies into the molecular mechanisms underlying TiLV replication and pathogenesis, and facilitate the development of novel vaccines and antiviral therapeutics. In the course of establishing the RGS, we also developed a robust method for sequencing the complete genome of TiLV. Additionally, we identified the consensus polyadenylation signal sequence in the TiLV genomic segments.
| Date of Award | 22 Jan 2025 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Akos KENEZ (Supervisor) & Klaus OSTERRIEDER (External Co-Supervisor) |