Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia

Abstract

Tilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5' and 3' termini, consistent with the genome organization found in other orthomyxoviruses. In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease.

Importance: The economic impact of worldwide trade in tilapia is estimated at $7.5 billion U.S. dollars (USD) annually. The infectious agent implicated in mass tilapia die-offs in two continents poses a threat to the global tilapia industry, which not only provides inexpensive dietary protein but also is a major employer in the developing world. Here we report characterization of the causative agent as a novel orthomyxo-like virus, tilapia lake virus (TiLV). We also describe complete genomic and protein sequences that will facilitate TiLV detection and containment and enable vaccine development.

FIG 1

Genomic characterization of the segments of TiLV isolated from tilapia in Israel. (A) TiLV segment 1 putative protein shows weak homology to motifs conserved in RNA-dependent polymerases. Sequence comparison of TiLV’s segment 1 predicted protein with motifs I to IV, conserved in polymerases of influenza virus strains C/JJ/50 (Inf C) (19), A/WSN/33 (Inf A) (34), and B/Lee/40 (Inf B) (35), vesicular stomatitis virus (VSV) (20), human immunodeficiency virus (HIV) (21), and poliovirus (Polio) (22). The relative motif positions are also shown. Invariant sequences in each motif are in boldface and underlined. TiLV sequences that show identity to one of the influenza virus sequences are highlighted in yellow. (B) Genomic segments of TiLV show conserved and homologous features at 5′ and 3′ termini.

FIG 2

Northern hybridization analysis indicates that TiLV is a segmented RNA virus. (A) Total RNA extracted from E-11 cells 6 days postinfection with TiLV from brains of tilapia in Israel (lanes 3, 6, and 9), from virions that were pelleted from the culture supernatant (lanes 4, 7, and 10), or from noninfected E-11 cells (lanes 2, 5, and 8). (B) Total RNA extracted from livers of tilapia in Ecuador (lanes 12 to 14). The extracts were hybridized to probe mixtures representing segments 1, 4, 7, and 10 (probe mixture 1 [lanes 2 to 4 and 12]), segments 2, 6, and 9 (probe mixture 2 [lanes 5 to 7 and 13]), or segments 3, 5, and 8 (probe mixture 3 [lanes 8 to 10 and 14]) to prevent signal overlap from segments of similar sizes. Influenza A virus RNA (A/Moscow/10/99) hybridized with three probes representing hemagglutinin (HA) (1,780 nt), NA (1450 nt), and matrix (1,002 nt) sequences served as size references (M [lanes 1 and 11]). Size markers appear on the left sides of the panels and segment numbers on the right.

FIG 3

Detection of TiLV RNA in brain and liver of infected tilapia and infected E-11 cells by in situ hybridization and image of dead tilapia in Israel. (A and B) Brain sections of infected Nile tilapia hybridized with Affymetrix Cy3-conjugated probes (red) of various polarities to TiLV segment 1 to detect genomic RNA (A) or mRNA (B). White arrowheads indicate hybridization signal. (C) Liver sections hybridized with Cy3-conjugated (red) Stellaris probes to segment 3 to detect mRNA. Nuclei are stained with DAPI (blue). (D) Liver section stained with hematoxylin and eosin reveals multinucleated giant cells (asterisk). (E) TiLV-infected E-11 cells hybridized with Quasar 670-conjugated (red) Stellaris probe to segment 3 to detect TiLV mRNA. Nuclei are stained with DAPI (blue). (F) Images of confocal sections of cells in panel E were reconstituted into a 3D image. (G) Dead tilapia at a fish farm in Israel.

FIG 4

TiLV deproteinized RNA is not infectious. Naive E-11 cell cultures were transfected with deproteinized RNA, extracted from cultured cells (“Cellular RNA” [A to C]) or pellets of culture supernatants (“Virion RNA” [D to F]), from TiLV-infected (“TiLV RNA” [A and D]) or NNV-infected (“NNV RNA” [B and E]) E-11 cells, or from naive E-11 cells (“E-11 RNA” [C and F]). Transfection with no RNA (“Mock” [G]) was also included. Bright-field images of transfected cultures were taken at 8 days posttransfection.