Molecular Detection of Tilapia Lake Virus (TiLV) in Farmed Mono-sex Nile Tilapia (Tilapia niloticus) in Bangladesh

Abstract

Background and Objectives: Tilapia lake virus (TiLV) has been marked as an emerging infectious agent that causes mass die-offs in farmed mono-sex Nile tilapia (Tilapia niloticus) in Bangladesh, indicates rapid diagnostic assay. This study was aimed to develop molecular detection method to confirm the TiLV in Tilapia niloticus and construct a genetic baseline to control this disease. Materials and Methods: The research aims to the detection of TiLV followed by complementary techniques of PCR based approaches such as reverse-transcription polymerase chain reaction (RT-PCR) and RT-quantitative (q) PCR using SYBR Green I dye. The RNA quantification, followed by a PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR Green I dye. Results: This research reported a novel RNA virus allowing its clinical signs lethargy, skin erosion, exophthalmia, detached scales and 15-82% morbidity rate. The RT-PCR amplified a 491 bp fragment from segment 3 in both cases. The PCR amplification efficiency of 98.5% over a wide linear range of 2.98×101 to 2.98×1010 TiLV copies, while the NTC (no template control) produced no fluorescence and therefore no amplification. The sequence of amplified PCR products received 100, 98 and 97% identity. The phylogenetic relationship of 17 TiLV sequences was chosen to compare with GeneBank resulting a common ancestor while closely related with Columbia, India, Malaysia and Thailand. The highest pair-wise alignment score was received 90.20 for MH338228.1 (Columbia), 85.57 for MF582636.1 (India), 85.30 for MH213048.1 (Malaysia) and 86.93 for MH213039.1 (Thailand) using the sequence of TiLV segments of one TiLV-positive strain. Conclusion: The mono-sex Nile tilapia was infected with common fish pathogens, such as Aeromonas and Streptococcus. This newly developed SYBR Green-based RT-qPCR assay can be as an essential tool for TiLV diagnostics and should help to control the dissemination of this virus worldwide.