The use of fish in the study was approved by the National University of Malaysia (Approval no. UKM.PPI.AEC.800-4/3/1). Biological samples were collected from diseased Nile tilapia (Oreochromis niloticus) during unusual mortality events reported by farmers from May to December 2017 and July to November 2019. We collected a total of 85 moribund fish from 11 farms in 6 districts in 2017 and 69 moribund fish from 7 farms in 5 districts in 2019 (Table 1). Each fish was humanely killed using an overdose of clove oil (200 ppm). For PCR analysis, pieces of the liver, kidney, spleen and brain were collected as specimens from each fish immediately after killing and then pooled in RNA (Qiagen, Hilden, Germany) for preservation. In addition to the moribund fish farm samples, we also collected the same tissues from 114 clinically healthy tilapia brood fish from six tilapia hatcheries (H) in 2017 and 307 from 10 tilapia hatcheries in 2019. For histopathology, liver and brain specimens from individual fish were collected and preserved for 24–48 hr in neutral buffered formalin (10%), which was then replaced with 70% ethanol. 2.2 Total RNA isolation and RT-PCR diagnosis for TiLV Total RNA from the pooled samples of liver, kidney and spleen was extracted using TRIzol reagent (Invitrogen, Waltham, USA) based on (Eyngor et al., 2014; Tsofack et al., 2016). PCR mixtures and thermocycling conditions were performed according to Dong, Ataguba, et al. (2017). As a positive control, we used a recombinant plasmid containing a 415-bp fragment of the TiLV genome segment 3 (pGEM415_bp) (Dong, Siriroob, et al., 2017) and nuclease-free water as the negative control. Expected amplicon sizes from the first and nested amplification were 415 bp and 250 bp, respectively. Regarding samples collected in 2019, we used a newly published semi-nested RT-PCR targeting genome segment 1 of TiLV (Taengphu et al., 2020). This method is highly specific for TiLV and is reported to be 100 times more sensitive than previous protocols. The primers used were TiLV/nSeg1F: 5′-TCT GAT CTA TAG TGT CTG GGC C-3′; TiLV/nSeg1R: 5′-AGT CAT GCT CGC TTA CAT GGT-3′; and TiLV/nSeg1RN: 5′-CCA CTT GTG ACT CTG AAA CAG -3′. PCR mixtures and thermocycling conditions were carried out according to Taengphu et al. (2020). This time, for the positive control, we used a plasmid containing a 620-bp fragment of the partial TiLV genome segment 1 (Taengphu et al., 2020) and a nuclease-free water template as the negative control. Expected amplicon sizes from the first and nested amplification reactions were 620 and 274 bp, respectively. PCR products were electrophoresed in 1.5% agarose gel and stained with a SYBR Safe DNA Gel Stain before visualization under a gel documentation system (Maestrogen Inc, Model: SML01, Hsinchu, Taiwan). To confirm the specificity of our RT-PCR detection results, we sequenced representative amplicons from 11 samples that tested positive for TiLV by Sanger sequencing (Table 1). Sequence identity with the prototype strain of TiLV (KU751816) was determined by nucleotide blast (https://blast.ncbi.nlm.nih.gov). 2.3 | Sample selection for histopathology Representative samples of moribund fish (n = 6) that were collected from two affected farms in 2019 tested positive for TiLV by RT-PCR (Table 1). These were investigated histopathologically to confirm pathognomonic lesions of TiLV infection. The specimens were dehydrated, embedded in paraffin, sectioned at a thickness of 5 μm, stained with haematoxylin and eosin, and scrutinized underneath a light microscope. 2.4 | Amplification of 10 genomic segments of TiLV Three heavily infected samples (one collected in 2017 and two collected in 2019) were used for the recovery of the 10-genome segments of TiLV. Ten primer sets targeting putative open reading frames of 10 segments were used for RT-PCR amplification as previously described by Pulido, Chaparro, Dong and Senapin (2019). Amplified DNA products were gel-purified using the FavorPrep GEL/PCR Purification Kit (Favorgen, Pingtung, Taiwan). The purified DNA fragments were then ligated into the pGEM-T-easy vector (Promega, Madison, USA). The recombinant plasmids containing the target DNA fragment (verified by colony PCR using vector primers) were sequenced by Macrogen, South Korea, using T7 and SP6 primers. The obtained sequences were assembled, and the vector sequence was removed using the Geneious software (Biomatters, Inc, Auckland, New Zealand). The identity of nucleotide and amino acid sequences was determined by Blastn and Blastp, respectively, to the GenBank database. 2.5 | Phylogenetic analysis Previous studies suggested using genome segment 1 and concatenated 10 genome segments for phylogenetic analysis of TiLV (Chaput et al., 2020; Pulido et al., 2019; Taengphu et al., 2020). For this analysis, we used nine publicly available complete genomes of TiLV (GenBank) that originated from farmed tilapia in Israel (two), Ecuador (one), Peru (one), Thailand (two), the United States (two) and Bangladesh (one), as well as three newly sequenced genomes from Bangladesh (this study, Table S1). We created 12 concatenated genomes, each with 10 coding fragments and 9,052 bp long. Following multiple sequence alignments, using the MEGAX 10.1.7 program, we constructed a maximum-likelihood phylogenetic tree with the best DNA model TN93 + G and bootstrap of 1,000 replicates. Similarly, we also conducted a phylogenetic analysis on genome segment 1 sequences of 31 TiLV isolates (three from this study and 28 from GenBank) (Table S1) using the best DNA model K2 + G for this data set.