Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

Discovery of an unrecognized nidovirus associated with granulomatous hepatitis in rainbow trout.

Rainbow trout (Oncorhynchus mykiss) is the principal species of inland-farmed fish in the Western hemisphere. Recently, we diagnosed in farmed rainbow trout a disease in which the hallmark is granulomatous-like hepatitis. No biotic agents could be isolated from lesions. Still, unbiased high-throughput sequencing and bioinformatics analyses revealed the presence of a novel piscine nidovirus that we named "Trout Granulomatous Virus" (TGV). TGV genome (28,767 nucleotides long) is predicted to encode non-structural (1a and 1 ab) and structural (S, M, and N) proteins that resemble proteins of other known piscine nidoviruses. High loads of TGV transcripts were detected by quantitative RT-PCR in diseased fish and visualized in hepatic granulomatous sites by fluorescence in situ hybridization. Transmission electron microscopy (TEM) revealed coronavirus-like particles in these lesions. Together, these analyses corroborated the association of TGV with the lesions. The identification and detection of TGV provide means to control TGV spread in trout populations.