A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV).

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.