INTRODUCTION
Tilapia farming is one of the most important sectors in finfish species cultured worldwide and of major importance to global food security, with total global production estimated at more than 6 million tonnes in 2018 (Alathari et al. 2023).
METHODS AND MATERIALS
This study was performed on samples collected from seven farms in Lake Volta/Ghana, during an outbreak of ISKNV between October/2018- May/2022. Furthermore, water samples were collected to test the feasibility of this method in the field. Primers were designed with the PrimalScheme (v 1.3.2) to produce 2kb amplicons spanning the genome, for WGS of ISKNV from infected fish using a tiled PCR approach. Library preparation was conducted on the generated amplicons. Samples were multiplexed and sequenced using the MinION sequencer. A consensus genome was generated according to the ARTIC bioinformatics pipeline (
). Phylogenetic analysis of ISKNV within the Ghana outbreak of 2018–2022 was performed using Augur and visualised in Auspice. Finally, the number of ISKNV viral templates from 6 ng to 6 × 10−6 ng was measured using the ddPCR, to determine the minimal input for genome recovery of ISKNV using the tiled PCR protocol .
RESULTS AND DISCUSSION
In this study, we were able to recover up to ~96 % of the ISKNV genome, directly from fish samples collected from Ghana during the outbreak, using the tiled PCR protocol. Approximately 137 SNP s were detected, when compared with the original reference genome, and it has shown that changes were sufficient to detect a phylogeographic signal during this outbreak. Some of these non-synonymous mutations were associated with the MCP , ATPase gene and OFR022. Phylogenetic analysis of ISKNV within the Ghana outbreak of 2018–2022, performed using Augur and visualised in Auspice, showed the initial outbreaks in Lake Volta clustered into four distinct clades, and each clade had a mix of samples from different farms. To evaluate the optimal concentration of ISKNV needed for genome recovery using the tiled PCR method, we measured the number of ISKNV viral templates. The minimum requirement to recover 50% of an ISKNV genome was ~2410 viral templates. This work shows that PCR tiling approaches used successfully to track the evolutionary rate, signatures of host adaptation, and transmission patterns of RNA viruses (Quick et al. 2016) c an also be applied to monitor infections of large dsDNA fish viruses. Thus, this method can be utilised as a surveillance tool for other viral infections threatening the growth of the aquaculture industry.
FUTURE RESEARCH
Further studies will include determining the sensitivity of our developed protocol for different viruses affecting the growth of aquaculture. We have tested this method on Tilapia Lake Virus (TiLV ) samples- an RNA virus affecting tilapia in several continents. This work provides a platform from which it is feasible to replicate the Artic-Network “lab-in-a-suitcase” approach to disease tracking and management in aquaculture in remote and resource-limited locations. With appropriate training and guidance, this workflow represents a suitable framework for local authorities in lower- and middle-income countries to contain and track different viral diseases in their localities.
REFERENCES
J. Haydon, et al. 2019. “First Detection of Infectious Spleen and Kidney Necrosis Virus (ISKNV) Associated with Massive Mortalities in Farmed Tilapia in Africa.” .