WO2017118989A1

WO2017118989A1 - Orthomyxo-like virus of tilapia

Info

Publication number: WO2017118989A1
Application number: PCT/IL2017/050027
Authority: WO
Inventors: Arnon Dishon; Shlomit TAL
Original assignee: Kovax Ltd.
Priority date: 2016-01-10
Filing date: 2017-01-10
Publication date: 2017-07-13
Also published as: CO2018008288A2; EP3400007A1; CN108778324A; EP3400007A4; MX2018008529A; BR112018014042A2

Abstract

Described herein is the isolation and characterization of a newly-discovered RNA virus of fish. Methods for detection of the isolated virus, a live attenuated virus vaccine, and an inactivated virus vaccine are also described.

Description

ORTHOMYXO-LIKE VIRUS OF TILAPIA

CROSS REFERENCE TO RELATED APPLICATIONS

Benefit is claimed to U.S. Provisional Patent Application Nos 62/276,873, filed January 10, 2016, and 62/352,570, filed June 21, 2016, the contents of which are incorporated by reference herein in their entirety.

FIELD

This disclosure relates to isolation and characterization of a newly-discovered RNA virus of fish. Also disclosed are methods for detection of the isolated virus, a live attenuated virus vaccine, and an inactivated virus vaccine.

BACKGROUND

Tilapia is the common name for approximately hundred species of tropical chiclid fish which globally are the second most cultivated food fish following carp. Based on reports from the Food and Agriculture association of the United Nations (FAO), the Nile tilapia, Oreochromis niloticus, is the one of the major tilapia species cultivated for consumption, reaching a global production of 3.2 million tons (mt) in 2012 (FAO). Tilapia sp. is cultured in warm climate countries, with Israel being one of its most northern points of distribution.

According to the Israeli Ministry of Agriculture, 7,662 tons of Tilapia are consumed in Israel per year, accounting for 43% of the all fresh water fish consumption. Over the past two decades production of Tilapia fish in the Beit She'an valley has risen from 1,600 tons to 5,700 tons per annum.

In August of 2009, fish farmers reported sudden high mortality occurrences among Tilapia in the Beit She'an valley region. In the hybrid gray Tilapia; overall mortality rates reached 30%-40%. Damage to the Red Tilapia hybrid production was more dramatic, reaching losses of up to 100% in some of the fish ponds. Mortality was observed in juvenile and adult fish in both grow-out and reservoir ponds. In juvenile fish, mortality events were characterized by an initial loss of appetite, observance of weak fish at the edges of the pond, elevated presence of predatory birds, and ultimately fish mortality at varying rates. An elevated presence of known parasites was detected and treatment seemed to mitigate some of the loss. In larger fish, loss of appetite and lethargy is evident and acute high mortality rates over several days was followed by a recovery of the fish population. Diagnostic tests for presence of Nervous Necrosis Virus were negative. Over the summer of 2010, mortality reports of Tilapia began in June from both the Beit She'an valley and the Mediterranean coast areas. Predominant losses followed sex reversal and initial grow out. Testing at the Fish Health Center at Nir-David could not generate a disease model nor was it able to characterize an etiological agent. The unexplained epizootic events continued throughout the summers of 2011 and 2012. Initial statistical analysis of the mortality events in 2011 and 2012 did not reveal a significant correlation between the presence of parasites and mortality in the Tilapia farms. The identified parasites varied by species and therefore could not account for the epizootic events. Moreover, it is known that such parasites can be a secondary pathogen, and not the primary cause of mortality. No significant correlation was seen for presence of bacteria and mortality events. Additionally, the onset of disease and clinical signs were not characteristic of known viral pathogens of Tilapia.

A need therefore exists to determine the causative agent of the recent Tilapia epizootic events. SUMMARY

Provided herein is an isolated Tilapia Virus (TLV) deposited with the CNCM (Pasteur Institute) as accession number CNCM 1-4892.

Also provided herein are isolated orthomyxo-like viruses having a genome comprised of nucleic acid sequences at least 90% identical to each of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 22, and 24.

Further described herein are isolated nucleic acids comprising a nucleic acid at least 90% identical to a nucleic acid selected from the group consisting of SEQ ID NOs 25-48.

The present disclosure also describes methods for detecting a Tilapia virus (TLV) infection, which include isolating a sample from a subject suspected of being infected by TLV; generating a cDNA template from the sample; and amplifying a nucleic acid from the cDNA template using at least one oligonucleotide primer comprising a nucleic acid sequence at least 90% identical to at least one sequence selected from the group consisting of SEQ ID Nos: 25- 48, wherein amplification of a nucleic acid sequence with the primer indicates the presence of TLV in the sample.

Isolated attenuated and isolated inactivated Tilapia viruses (TLV) are also described herein. In particular embodiments, either of the isolated virus compositions also include an adjuvant.

Additionally provided are kits for detecting TLV, which include the described nucleic acids, and methods of vaccinating a subject against TLV through use of the isolated inactivated or attenuated viruses. The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an electron micrograph (EM) of infected tilapia cells at day 5 postinfection. Cells exhibit disintegration of cellular compartments and organelles. Cytoplasmic vacuoles contain an abundance of virus like structures varying in size and morphology (arrows, right panel).

Figure 2 shows non-infected (top panel) and infected (bottom panel) Tilapia fin cells

(TFC) maintained at 15°C-33°C. Infected cells at 15°C exhibit normal morphology with no apparent CPE. At 23°C-33°C cells exhibit cytoplasmic vacuolization and rounding of cells which eventually causes mortality of the cell culture.

Figure 3 is a phylogenic analysis of contig 4 of TLV resembling the PB1 RNA Polymerase of Orthomyxo virus. Using TREX-MAFFT sequence alignment software a Phylogenic tree was assembled, comparing known Orthomyxo virus PB1 amino acid sequences to that of TLV.

Figure 4 is a chart showing the generation of mutation in TLV through sub lethal irradiation. TLV was irradiated at increasing dosages of UV and the residual infectivity was determined by viral titration on 96 microwell plates, as shown in the chart. At the highest dosage where residual infectivity was maintained viral clones were picked and propagated.

Figure 5 is a chart showing the safety profile of two attenuated TLV clones compared to a placebo treated group.

Figure 6 is a chart showing the efficacy of vaccine prototypes when challenged with a lethal dose of wildtype TLV by IP injection.

Figure 7 is an electrophoresis gel showing detection of TLV using Conl9 primers, in infected cultures and demonstrating that TLV has an RNA genome that does not pass through a DNA stage during its propagation cycle. Specificity of Conl9 as a viral contig. Conl9 primers (lanes 1-5) or Endogenous tilapia control TL-NA primers (lane 6-10) were assayed using either DNA or RNA extracted from infected and non-infected TFC. RNA was used to generate total cDNA using Aurum Total RNA Mini kit (BIO-RAD Cat# 732-6820). Lane distribution as follows: MW: lOObp DNA ladder; 1: cDNA from infected TFC assayed with Conl9 primers; 2: cDNA from non-infected TFC assayed with Conl9 primers; 3: Conl9 Viral fragment cloned into a plasmid assayed with Conl9 primers; 4: DNA from infected TFC assayed with Conl9 primers; 5: DNA from non-infected TFC assayed with Con 19 primers; 6: cDNA from infected TFC assayed with TL-NA (endogenous control); 7: cDNA from non-infected TFC assayed with TL-NA (endogenous control); 8: DNA from infected TFC assayed with TL-NA (endogenous control); 9: DNA from non- infected TFC assayed with TL-NA (endogenous control); 10: Negative control: both primers sets tested in a reaction with no template.

Figure 8 is an electrophoresis gel showing use of Con4 as a diagnostic tool to identify

TLV from infected fish. TLV is readily identified in both experimentally infected fish as in field epizootics. RNA was extracted from fish brains and served as template for RT-PCR using Con4 primers (top pane) or a Tilapia endogenous marker (TL-NA) as control (bottom pane). Lanes 1-19: Naive Tilapia were injected with TLV and cohabitated with non-exposed fish. Over a 26 day observation period sick/dead fish were collected and kept at -80°C for further evaluation. Lane 20 - Sick fish sampled during an epizootic on a Tilapia farm in Israel. Lane 21 -Healthy fish collected from the same farm. MW - lOObp molecular marker, NC - negative control, water used as template, PC- positive control, RNA extracted from TLV infected TFC.

Figure 9 is a graph showing accumulated mortality of Tilapia fish challenged through IP injection with virulent TLV when vaccinated with inactivated TLV preparations as shown in the figure. Accumulated mortality of Tilapia fish challenged through IP injection with virulent TLV, 28 days post IP vaccination with: x - Medium from uninfected TFC cells, x - Naive fish, Δ - Formalin inactivated TLV in emulsion with Freund's incomplete adjuvant - IFA (1: 1), 0 - Formalin inactivated TLV, and O - emulsion containing Placebo - Medium from uninfected cells with Freund's incomplete adjuvant - IFA (1: 1).

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The nucleic and/or amino acid sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. As described herein:

SEQ ID NO: 1 is the sequence of Contig 4.

SEQ ID NO: 2 is the conceptual translation of Contig 4.

SEQ ID NO: 3 is the sequence of Contig 7.

SEQ ID NO: 4 is the conceptual translation of Contig 7.

SEQ ID NO: 5 is the sequence of Contig 5.

SEQ ID NO: 6 is the conceptual translation of Contig 5.

SEQ ID NO: 7 is the sequence of Contig 6.

SEQ ID NO: 8 is the conceptual translation of Contig 6.

SEQ ID NO: 9 is the sequence of Contig 8. SEQ ID NO: 10 is the conceptual translation of Contig 8.

SEQ ID NO: 11 is the sequence of Contig 14.

SEQ ID NO: 12 is the conceptual translation of Contig 14.

SEQ ID NO: 13 is the sequence of Contig 15.

SEQ ID NO: 14 is the conceptual translation of Contig 15.

SEQ ID NO: 15 is the sequence of Contig 12.

SEQ ID NO: 16 is the conceptual translation of Contig 12.

SEQ ID NO: 17 is the sequence of Contig 16.

SEQ ID NO: 18 is the conceptual translation of Contig 16

SEQ ID NO: 19 is the sequence of Contig 20.

SEQ ID NO: 20 is the conceptual phase 1 translation of Contig 20.

SEQ ID NO: 21 is the conceptual phase 2 translation of Contig 20.

SEQ ID NO: 22 is the sequence of Contig 21.

SEQ ID NO: 23 is the conceptual translation of Contig 21.

SEQ ID NO: 24 is the sequence of Contig 19.

SEQ ID NOs. 25 and 26 are respective forward and reverse primers specific to Contig 4.

SEQ ID NOs. 27 and 28 are respective forward and reverse primers specific to Contig 7.

SEQ ID NOs. 29 and 30 are respective forward and reverse primers specific to Contig 5.

SEQ ID NOs. 31 and 32 are respective forward and reverse primers specific to Contig 6.

SEQ ID NOs. 33 and 34 are respective forward and reverse primers specific to Contig 8.

SEQ ID NOs. 35 and 36 are respective forward and reverse primers specific to Contig 14.

SEQ ID NOs. 37 and 38 are respective forward and reverse primers specific to Contig 15.

SEQ ID NOs. 39 and 40 are respective forward and reverse primers specific to Contig 12.

SEQ ID NOs. 41 and 42 are respective forward and reverse primers specific to Contie 16. SEQ ID NOs. 43 and 44 are respective forward and reverse primers specific to

Contig 20.

SEQ ID NOs. 45 and 46 are respective forward and reverse primers specific to

Contig 21.

SEQ ID NOs. 47 and 48 are respective forward and reverse primers specific to

Contig 19.

SEQ ID NO: 49 is a mutated segment 4 sequence from an attenuated TLV.

SEQ ID NO: 50 is a mutated segment 5 sequence from an attenuated TLV.

DETAILED DESCRIPTION

I. Abbreviations

TFC tilapia fin cells

TLV tilapia virus

II. Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

In case of conflict, the present specification, including explanations of terms, will control. In addition, all the materials, methods, and examples are illustrative and not intended to be limiting.

Administration: The introduction of a composition into a subject by a chosen route. Administration of an active compound or composition, such as a TLV attenuated vaccine strain, can be by any route known to one of skill in the art. Administration can be local or systemic. Amplification: When used in reference to a nucleic acid, any technique that increases the number of copies of a nucleic acid molecule in a sample or specimen. An example of amplification is the polymerase chain reaction (PCR), in all of its currently practiced variations, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of in vitro amplification can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques. Non- limiting examples of PCR include RT-PCR and qPCR. Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP- A-320 308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Patent No. 6,025,134).

Animal: Living multi-cellular vertebrate organisms, a category that includes for example, fish, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term subject includes both human and veterinary subjects, for example, humans, non-human primates, fish, dogs, cats, horses, and cows. Similarly, the term "subject" includes both human and veterinary subjects, such as fish.

Attenuated virus: a virus that has been altered in order to reduce or eliminate its virulence. Attenuated viruses are "live", but have a reduced or eliminated ability to damage or kill host cells, and therefore cause a virus -associated pathology.

Biological Sample: Any sample that may be obtained directly or indirectly from an organism, including whole blood, plasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat, semen, vaginal secretion, sputum, fluid from ulcers and/or other surface eruptions, blisters, abscesses, tissues, cells (such as, fibroblasts, peripheral blood mononuclear cells, or muscle cells), organelles (such as mitochondria), organs, and/or extracts of tissues, cells (such as, fibroblasts, peripheral blood mononuclear cells, or muscle cells), organelles (such as mitochondria) or organs. A biological sample may also be a laboratory research sample such as a cell culture supernatant. The sample is collected or obtained using methods well known to those skilled in the art. In the current disclosure, "biological sample" is used interchangeably with "sample."

cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and transcriptional regulatory sequences. cDNA can also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding

RNA molecule. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

Complementary: A double- stranded DNA or RNA strand consists of two complementary strands of base pairs. Complementary binding occurs when the base of one nucleic acid molecule forms a hydrogen bond to the base of another nucleic acid molecule.

Normally, the base adenine (A) is complementary to thymidine (T) and uracil (U), while cytosine (C) is complementary to guanine (G). For example, the sequence 5'-ATCG-3' of one ssDNA molecule can bond to 3'-TAGC-5' of another ssDNA to form a dsDNA. In this example, the sequence 5'-ATCG-3' is the reverse complement of 3'-TAGC-5'. Similarly, cDNA produced from an ssRNA molecule will be the reverse complement of the RNA sequence.

Contacting: Placement in direct physical association. Includes both in solid and liquid form. Contacting can occur in vitro with isolated cells or in vivo by administering to a subject.

Detect: To determine if an agent (such as a signal or particular nucleotide nucleic acid probe, amino acid, or protein, for example a TLV protein or nucleic acid) is present or absent. In some examples, this can further include quantification.

Diagnosis: The process of identifying a disease, by its signs, symptoms, and results of various tests and methods, for example the methods disclosed herein.

Fluorophore: A chemical compound, which when excited by exposure to a particular stimulus, such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light).

Fluorophores are part of the larger class of luminescent compounds. Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminesce, but rather use a chemical source of energy. Therefore, the use of chemiluminescent molecules (such as aequorin) can eliminate the need for an external source of electromagnetic radiation, such as a laser. Examples of particular fluorophores that can be used in the probes and primers disclosed herein are provided in U.S. Patent No. 5,866,366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'- aminoethyl)aminonaphthalene-l- sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trif uoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3- (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6- carboxyf uorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; LightCycler Red 640; Cy5.5; and Cy56-carboxyfluorescein; 5- carboxyfluorescein (5-FAM); boron dipyrromethene difluoride (BODIPY); Ν,Ν,Ν',Ν'- tetramethyl-6-carboxyrhodamine (TAMRA); acridine, stilbene, -6-carboxy-fluorescein (HEX), TET (Tetramethyl fluorescein), 6-carboxy-X-rhodamine (ROX), Texas Red, 2',7'-dimethoxy- 4',5'-dichloro-6-carboxyfluorescein (JOE), Cy3, Cy5, VIC® (Applied Biosystems), LC Red 640, LC Red 705, Yakima yellow amongst others.

Inactivated virus: a virus that has been inactivated or "killed" in order to eliminate its virulence. Although not infectious, inactivated virus particles can provoke an immune response, and can form the basis of a vaccine. Non-limiting methods of viral inactivation include heat, UV exposure, or chemical means, such as exposure to formaldehyde (formalin). Isolated: A biological component (such as a nucleic acid molecule, protein or organelle) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include radioactive isotopes (such as S³⁵ and P³²), enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

Oligonucleotide: A plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 300 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 or 300 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases.

Orthomyxoviridae: An RNA virus family, which includes influenza virus.

Characterization and sequencing of the TLV virus described herein indicates it is an orthomyxovirus. Orthomyxoviridae can have filamentous or spherical capsids. Their RNA genome is segmented and is transcribed and replicated by virus-encoded RNA dependent RNA polymerases.

Probes and primers: Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this invention. A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

Primers are short nucleic acid molecules, preferably DNA oligonucleotides 10 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the PCR or other nucleic-acid amplification methods known in the art.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell.

Quantitative real time PCR: A method for detecting and measuring products generated during each cycle of a PCR, which products are proportionate to the amount of template nucleic acid present prior to the start of PCR. The information obtained, such as an amplification curve, can be used to quantitate the initial amounts of template nucleic acid sequence.

Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

Vaccine: a composition that induces a subject to acquire immunity protective against a specific disease-causing agent (pathogen), such as a virus or bacteria. Vaccines can include, inter alia, antigenic fragments of the pathogen, which in turn induce the immune response in the subject. Other vaccine examples include live attenuated or killed vaccines in which the pathogen itself is the basis for the vaccine. The vaccine described herein is a live attenuated TLV strain. Vaccination is the process of providing a vaccine, and thereby protection, to a subject against a pathogen. A vaccine is an exemplary type of immunogenic composition.

Virus: Microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so.

Virion: A complete viral particle including envelope, capsid, and nucleic acid elements. III. Overview of Several Embodiments

Described herein is an isolated Tilapia Virus (TLV) deposited with the CNCM (Pasteur Institute) as accession number CNCM 1-4892.

Also described herein are isolated orthomyxo-like viruses having a genome comprised of nucleic acid sequences at least 90% identical to each of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 22, and 24.

In particular embodiments, the nucleic acid further comprises a detectable label, such as a fluorescent label, radioactive label, or chemical label.

In particular embodiments of the detection methods described herein, the amplifying is carried out with at least one primer pair having sequences at least 90% to the primer pairs selected from the group consisting of SEQ ID NOs 25 and 26; SEQ ID NOs 27 and 28; SEQ ID NOs 29 and 30; SEQ ID NOs 31 and 32; SEQ ID NOs 33 and 34; SEQ ID NOs 35 and 36; SEQ ID NOs 37 and 38; SEQ ID NOs 39 and 40; SEQ ID NOs 41 and 42; SEQ ID NOs 43 and 44; SEQ ID NOs 45 and 46; and SEQ ID NOs 47 and 48.

The current disclosure also includes kits for detecting a Tilapia virus (TLV) infection, which contain at least one of the described isolated nucleic acids.

In particular embodiments, the kits contain at least one isolated nucleic acid which is at least one oligonucleotide primer pair having sequences at least 90% identical to the primer pairs selected from the group consisting of SEQ ID NOs 25 and 26; SEQ ID NOs 27 and 28; SEQ ID NOs 29 and 30; SEQ ID NOs 31 and 32; SEQ ID NOs 33 and 34; SEQ ID NOs 35 and 36; SEQ ID NOs 37 and 38; SEQ ID NOs 39 and 40; SEQ ID NOs 41 and 42; SEQ ID NOs 43 and 44; SEQ ID NOs 45 and 46; and SEQ ID NOs 47 and 48.

In particular embodiments, the kits further include detectable label, which in particular embodiments can be associated with at least one of the isolated nucleic acids in the kit. Isolated attenuated and isolated inactivated Tilapia viruses (TLV) are also described herein.

Additionally described herein is an isolated attenuated Tilapia virus (TLV) produced by the process of passaging the TLV through tilapia cells at least 10 times, irradiating the TLV at a sublethal radiation dose; and isolating an infectious attenuated clone. In particular embodiments, the isolated attenuated TLV is passaged at least 20 times through tilapia cells. In other particular embodiments, the sublethal radiation dose is between 3.2-6.8 mJ/cm², such as 6.8 mJ/cm². An exemplary isolated attenuated TLV provided herein was deposited with the CNCM (Pasteur Institute) as Accession No. CNCM 1-5075.

In particular embodiments, the isolated attenuated or inactivated TLV strains described herein are part of an immunogenic composition, such as a compositions that also include an adjuvant.

The present disclosure additionally describes methods for vaccinating a subject against a Tilapia virus (TLV) infection. In particular embodiments, such vaccination includes infecting a subject with the described isolated attenuated or inactivated TLVs. In other embodiments, vaccination includes administering to a subject an immunogenic composition comprising the described isolated attenuated or inactivated TLVs.

IV. Tilapia Virus and Methods of its Detection

Beginning in 2009, fish farmers in Northern Israel observed sickness and high mortality among the Tilapia in their farms. These events have continued over each successive summer. Described herein is the isolation and characterization of the causative agent of the sickness and mortality events, a newly discovered orthomyxo-like virus that is being called Tilapia Virus (TLV).

Primary characterization of TLV indicates a spherical, pleomorphic RNA virus, which propagates in a temperature dependent manner between 23°C-33°C. Virus has been grown to a high titer and deposited on June 15, 2014 at the CNCM (Pasteur Institute) under Accession No. CNCM 1-4892.

Genomic analysis reveals a segmented RNA genome with almost no homology to sequences in the GenBank database. BlastX analysis however showed that the TLV contig 4 (set our herein as SEQ NO: 1) includes a putative conserved domain of the Influenza RNA- dependent RNA polymerase subunit PB 1.

Contiguous (contig) sequences have been determined from TLV, set out herein as SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 22, and 24. In particular embodiments, the described TLV contains a genome having an identical sequence to those sequences set forth as SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 22, and 24. It is appreciated however that genomic variation from the described TLV sequences will exist in different isolated TLV. Accordingly, in particular embodiments, the isolated TLV described herein has a genome in which any one of the contigs can be less than 100% identical to the sequences set out as SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 22, and 24. Such sequences may be at least 99%, 95%, 90%, 85%, 80% or even less identical to one or more of the described contig sequences.

The isolated genomic sequences can be synthetically produced as described or as DNA (e.g. cDNA) or as shorter RNA or DNA fragments of the sequences described herein. Non- limited uses of such fragments include probes and primers for use in any method known to the art for detection of the virus in a sample. In particular embodiments, the fragments (which can be probes) can be least 99%, 95%, 90%, 85%, 80%, 75%, 70% or even less identical to the reverse complement of the contig sequence to be detected. Similarly, an isolated probe for use in detecting TLV can be 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, any percentage in between, or even less in length compared with the sequence to be detected. For example, contig 4 (SEQ ID NO: 1) is 1620 nucleotides (nt) long. A described isolated probe of contig 4 can therefore be identical to the sequence of SEQ ID NO: l, but can be significantly shorter in length, such as 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50 nt long, or any length in between. For example in other embodiments, an isolated nucleic acid that can be used as a probe for contig 4 (SEQ ID NO: 1) can be 35- 1620 nt long, such as 50- 1600 nt, 1000- 1600 nt, 500- 1500 nt, 750- 1200 nt, and any length in between. It will be understood that isolated nucleic acid fragments, including DNA or RNA fragments, of each of the contig sequences can similarly be used as a used as a probe sequence for the individual contig.

Similarly, the sequences set out as SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 22, and 24, or any shorter inclusive sequence can be amplified as described herein using primers developed from the described sequences. In particular embodiments, the primers can be 12-35 nt long, such as 15-25 nt, 18-30 nt and the like. Particular non-limiting examples of primers that can be used to amplify the described sequences include the oligonucleotide primers set forth herein as SEQ ID NOs 25-48, or primers at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even more identical to SEQ ID NOs 25-48.

The isolated nucleic acid sequences set out as SEQ ID NOs. 1, 3, 5, 7, 9, 1 1, 13, 15, 17, 19, 22, and 24, and fragments thereof (including probes and primers), can be covalently modified to include a detectable label as described herein. Particular non-limiting examples of such labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. In particular embodiments, the label is a radioactive label. In other embodiments, the label is a fluorescent label, such as any one of the fluorescent labels described herein.

Additionally described herein are methods of detecting TLV in a sample, such as a sample collected from a subject (e.g. a Tilapia fish). The methods include detecting a TLV nucleic acid in the sample, which indicates the presence of TLV in the sample.

In particular embodiments, the sample is isolated from an individual subject, such as a Tilapia fish. In other embodiments, the sample is collected from a population, such as a population of fish. In still other embodiments, the sample is collected from an environment in which subjects live, such as fish pond and the like. When the sample is a sample from one or more subjects, the presence of a TLV nucleic acid or protein in the sample indicates that the subject is infected by TLV.

Methods of nucleic acid isolation and their detection are well known in the art. In particular embodiments, the labeled probes or primers described herein can be used to detect TLV in a sample by standard hybridization techniques, including in an array, such as a microarray. Other methods of detection include any suitable method of nucleic acid amplification, such as PCR and its variations including RT-PCR and qPCR.

As an orthomyxo-like virus, TLV has an RNA genome that does not pass through a

DNA stage during its propagation cycle. Accordingly, when in DNA-form, the isolated nucleic acids described herein are synthetic. It will also be appreciated that methods of amplifying TLV nucleic acids by standard methods will include a reverse transcription step to produce cDNA as the basis for PCR methods and the like. Methods of reverse transcription and PCR are known in the art.

Also described herein are kits for detecting TLV in a sample. The described kits include at least one of the TLV-specific probes and primers described above. In particular embodiments, the kits include reagents for isolating viral RNA from a sample. In other embodiments, the kits include reagents and/or enzymes for reverse transcription and DNA amplification. In still other embodiments, the kits include at least one primer pair such as respective pairs from the oligonucleotides set forth herein as SEQ ID NOs 25-48, or primers at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even more identical to SEQ ID NOs 25-48. In particular embodiments, the supplied TLV-specific nucleic acids are pre-labeled with a detectable label. In other embodiments, a detectable label is included in the kit along with optional reagents and/or enzymes for associating the label with the nucleic acid.

V. Attenuated and Inactivated Tilapia Virus Vaccines

Further described herein are attenuated and inactivated TLV isolates, which can confer resistance to wildtype TLV infection.

The attenuated viruses provided herein are isolated and cloned following multiple passages through Tilapia cells, followed by exposure to a non-lethal mutagenic condition, such as ultraviolet radiation or chemical mutagen.

In particular embodiments, the attenuated TLV strain is produced by first passaging the virus, by methods known in the art (e.g. wherein each passage is a cycle of infection and isolation of virus resultant from the infection), at least 10 times through Tilapia cells, such as

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times through Tilapia cells. In particular examples, each passage is followed by exposure to a mutagenic condition prior to the next passage cycle. In other embodiments, exposure of the collected virus to a non-lethal (also described herein as "sublethal") mutagenic condition (e.g. a condition wherein the virus may continue to infect cells) occurs following the final passage. In some embodiments, the virus under attenuation is exposed to a sublethal dose of ultraviolet (UV) radiation, such as 3.2-6.8 mJ/cm², and more particularly 6.8 mJ/cm².

In a particular embodiment, an attenuated TLV strain is produced by passaging wildtype TLV through Tilapia Fin Cells for 20 passages, exposing the cells to a UV radiation dose of 6.8 mJ/cm², and then isolating an individual virus clone.

When provided as an immunogenic composition to a subject, such as in a vaccine, the described attenuated TLV strain can confer partial or complete resistance to TLV infection such that a population of vaccinated fish that is challenged by wildtype TLV will show at least a 60% survival rate, such as 60%-100% survival, such as 70%, 80%, 85%, 90%, 95%, 95%-

100%, or even up to approximately 100% survival.

A particular example of an attenuated TLV as described herein is the attenuated TLV that has been deposited with the CNCM (Pasteur Institute) on April 1, 2016 as Accession No. CNCM 1-5075.

Inactivated (killed) virus immunogenic compositions can be produced according to methods known in the art, including incubation with formalin as described herein.

In particular embodiments, the attenuated and/or inactivated TLV isolates is a component of an immunogenic composition (e.g. a TLV vaccine) that includes the attenuated and/or inactivated virus, a pharmaceutically acceptable carrier and optionally one or more adjuvants or other immune-stimulating agents.

In particular embodiments, the pharmaceutically acceptable carrier can be water or a buffer, or additionally a composition stabilizer.

In certain embodiments, the vaccines are prepared as liquid solutions, emulsions or suspensions for injection. In the example of a fish vaccine, delivery can be through immersion of fish in water. Accordingly, a liquid emulsion or emulsifiable concentrate can be prepared for addition to water where fish are held. Solid (e.g. powder) forms suitable for dissolution in, or suspension in, liquid vehicles or for mixing with solid food, prior to administration can also be prepared. In particular embodiments, the vaccine may be a lyophilized culture for reconstitution with a sterile diluent. For example 0.9% saline. Prior to drying, (e.g. lyophilization), a variety of ingredients may be added to the vaccine such as preservatives, antioxidants or reducing agents, a variety of excipients, etc. Such excipients may also be added to the dry virus after the drying step.

In particular embodiments, the immunogenic composition includes an adjuvant or other immune stimulant. Adjuvants are well known to the art. Particular non-limiting examples include muramyl dipeptides, avidine, aluminium hydroxide, aluminium phosphate, oils, oil emulsions, saponins, dextran sulphate, glucans, cytokines, block co-polymers, immuno stimulatory oligonucleotides and others known in the art may be admixed with the attenuated and/or inactivated TLV.

In other embodiments the described immunogenic composition (vaccine) additionally comprises at least one stabilizer to protect it from degradation, to enhance the shelf-life, or to improve freeze-drying efficiency. Non-limiting examples of stabilizers for use with the described vaccine include SPGA (Bovarnik et al., 1950, J. Bacteriology, vol. 59, p. 509), skimmed milk, gelatin, bovine serum albumin, carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, lactoses, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.

The described attenuated and/or inactivated TLV immunogenic composition (vaccine) can be used in methods of vaccination against TLV by infecting at least one subject with the attenuated TLV vaccine described herein and/or exposing the subject to the inactivated TLV.

In particular embodiments, the vaccine is administered to fish individually-orally, such as through their feed or by forced oral administration, or by injection, such as via the intramuscular or intraperitoneal route In alternative embodiments, the vaccine can be administered simultaneously to an entire fish population contained in a body of water by spraying, dissolving and/or immersing the vaccine in the water. Such population vaccination methods can be used in various environments such as ponds, aquariums, natural habitat, fish farms and fresh water reservoirs.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. EXAMPLES

Example 1: Isolation and Characterization of Tilapia Virus

A cell line was developed from Tilapia fins, and which was used to isolate an RNA virus from fish collected during the mortality events of 2012. The virus was temporarily designated Tilapia Virus - TLV. Virus was isolated as follows: Samples from frozen organs originated from sick Tilapia were thawed, pooled together, homogenized, filtered through a 0.2μιη filter and inoculated on naive Tilapia Fin Cells (TFC). TFC infected cultures were incubated at 28°C and monitored daily for cytopathic effect (CPE). Visible CPE was seen 4-7 days post inoculation and included clear plaque formation (Fig. 2). No CPE was evident in mock-infected TFC control cultures. Secondary inoculation of TFC naive cultures with supernatants from TFC cultures exhibiting CPE was repeated for over 20 passages with similar CPE results.

The Tilapia cell line propagates the isolated virus at high titers. Electron microscopy analysis of infected cell cultures shows virion structures within the cells (Figure 1). The TLV virus is spherical in shape, and shows variation in size and pleomorphic morphology. In infected cells virus particles appears in cytoplasmic vacuoles, and have an average diameter of ~65nm.

Primary characterization of the virus shows that viral propagation is temperature dependent and occurs at 23°C-33°C. The virus propagates in tilapia cell culture and causes cytopathic effect (CPE) at temperatures >24°C but not at 15°C (Figure 2). This observation is in accord with the field observations of disease occurrence. Throughout passages of the virus in cell culture we are able to maintain CPE and propagate the virus.

In order to fulfill Koch's postulates and establish that TLV is indeed the etiological agent of the disease a disease model was established in which tissue culture-isolated TLV was used to infect healthy naive Tilapia. The injected fish exhibited disease signs consistent with the field reports. Fish went off feed, showed lethargy, became emaciated and eventually died. Mortality ranged from 30%- 100%. Moreover, TLV was isolated and identified from the fish which died in the study, fulfilling Koch's postulates for TLV being the etiological agent.

Further characterization of the virus was accomplished through viral genomic sequencing. Sequencing of the virus revealed multiple sequences associated with infected cultures which did not overlap into a single sequence, suggesting a segmented genome. Of the twelve sequences determined and presented herein, two (Contigs 8 and 16) show homology to known sequences within Genbank (of Tilapia lake virus (Eyngor et al 2014; Genbank accession number KJ 605629, 1); the remaining sequences do not share homology with any other Genbank sequence. However, BlastX analysis identified a putative conserved domain in one of the viral fragments (contig 4) belonging to the Influenza RNA-dependent RNA polymerase subunit PB1. The full sequence (with U represented as T) is set out herein as SEQ ID NO: 1 (start and stop codons are predicted at nt 38 and 1595, respectively). The 520 amino acid conceptual translation of the sequence (in frame +2) is set out as SEQ ID NO: 2.

Other determined sequences, and putative translation products include: Contig 7 (SEQ ID NO: 3) and its frame 2 translation (SEQ ID NO: 4); Contig 5 (SEQ ID NO: 5), and its phase 6 translation (SEQ ID NO: 6); Contig 6 (SEQ ID NO: 7) and its phase 6 translation (SEQ ID NO: 8): Contig 8 (SEQ ID NO:9) and its phase 6 translation (SEQ ID NO: 10); Contig 14 (SEQ ID NO: 11) and its phase 5 translation (SEQ ID NO: 12); Contig 15 (SEQ ID NO: 13) and its phase 6 translation (SEQ ID NO: 14); Contig 12 (SEQ ID NO: 15) and its phase 5 translation (SEQ ID NO: 16); Contig 16 (SEQ ID NO: 17) and its phase 3 translation (SEQ ID NO: 18); Contig 20 (SEQ ID NO: 19), its phase 1 (SEQ ID NO: 20) and phase 2 (SEQ ID NO: 21) translations; Contig 21 (SEQ ID NO:22) and its phase 5 translation (SEQ ID NO: 23); and the nucleic acid sequence of Contig 19 (SEQ ID NO: 24).

The amino acid sequence set forth as SEQ ID NO: 2 resembles the PB1 RNA Polymerase of Orthomyxo virus. Using TREX-MAFFT sequence analysis software, a Phylogenic tree was assembled, comparing known Orthomyxo virus PB1 amino acid sequences (such as from influenza virus) to that of TLV. The results of this analysis are presented in Figure 3, which suggests that the isolated TLV may be an emerging pathogen in aquaculture of tilapia species and a new genus within the Orthomyxo viridae.

Example 2: Tilapia Virus Detection

This example demonstrates TLV detection in infected cultures or infected organs (liver, spleen, kidney, brain) derived from sick Tilapia fish:

A TLV infection is identified by detection of TLV RNA in an infected sample by PCR. Sample RNA is produced from viral-infected TFC or from organs derived from sick Tilapia fish, using Aurum Total RNA Mini kit according to the manufacturer's instructions (BIO-RAD Cat# 732-6820), and cDNA is generated from the isolated RNA using the Reverse Transcriptase Verso cDNA Kit according to manufacturer's instructions (Thermo scientific Cat# AB 1453/A). RT-PCR reactions were then carried out according to standard protocols using the produced cDNA as template, and with a set of primers for identifying Contig 4 (SEQ ID NOs 25-26)(as shown in Figure 8). Contig 4 contains a putative conserved domain belonging to the Influenza RNA-dependent RNA polymerase subunit PB 1.

The specific correlation between the 12 contigs identified are shown in Table 1. Table 1 is a summary comparison of the 16 observed TLV Contigs. Sucrose gradient purified TLV was sequenced using next generation sequencing technology. Genomic analysis revealed 16 distinct Contigs (at a range of 400-1620bp) which did not resemble known sequences appearing in the GenBank. To determine whether these sequences were in fact associated with TLV, specific primers were designed to amplify each of the sequences and RT-PCR was carried out using the primers sets on templates generated from infected and non-infected TFC. Of the 16 Contigs 12 were shown to be of viral origin, appearing only in infected cultures (see Table 1). The four Contigs which were positive for both infected and non-infected cultures are assumed to be unpublished endogenous Tilapia sequences (in bold).

Table 1 Summary of RT- PCR results of 16 Contigs assayed with specific primers designed for each assayed with templates from infected and non-infected TFC.

13 Con 78 254 + +

14 Con 86 423 + +

15 Con 6 175 - +

16 Con 19 150 - +

Detection of TLV is also demonstrated in Figure 7, which is an electrophoresis gel showing detection of TLV in infected cultures using Conl9 primers, and which demonstrates that TLV has an RNA genome that does not pass through a DNA stage during its propagation cycle. Conl9 was amplified in an RT-PCR reaction only when assayed on infected cultures following a reverse transcriptase reaction (Fig 7 lanel). Samples from non-infected cells and from DNA extracted from infected and non-infected cells assayed with Conl9 primers were negative (Fig 7 lanes 2, 4 and 5). Presence of genomic DNA and cDNA in the extractions tested was validated using primers amplifying a Tilapia endogenous gene (Fig 7 lanes 6-9). RNA was extracted from infected and non-infected TFC using Aurum Total RNA Mini kit (BIO-RAD Cat# 732-6820). Total cDNA was generated using Reverse Transcriptase Verso cDNA Kit (Thermo scientific Cat# AB 1453/A). DNA was produced from infected and non- infected TFC using DNeasy (QIAGEN Cat# 69504).

As indicated, in addition to showing the specific correlation of the contigs with TLV, Figure 7 also demonstrates that TLV possesses an RNA genome which does not pass through a DNA stage during its life cycle (compare lanes 5, 6, 8, and 9).

Example 3: Generation and Characterization of an Attenuated TLV Vaccine Strain

To generate an attenuated virus strain, a TLV isolate was continuously passaged on Tilapia fin cells for 20 passages, and subjected to UV irradiation at incremented intensities (Figure 4). At the sub lethal dosage of 6.8mJ/cm², virus clones were picked and propagated as a single viral clone.

To assay putative attenuated clones, Tilapia Fish were vaccinated with two cloned viruses and a placebo control through immersion for lhour. The vaccinated groups were: (1) Placebo control: medium devoid of viral antigen; (2) TLV plO (clone 6) - virus passaged 10 times in culture, UV irradiated and cloned; and (3) TLV p20 (clone 4) - TLVplO passaged 10 times more in culture UV irradiated and cloned. Fish were monitored for 35 days for mortality or other adverse events associated with disease (Figure 5).

Survivability in the placebo group was 100% with no abnormalities observed throughout the study period. The group exposed to TLV plO (clone 6) did show a mortality of approximately 20% associated with exposure to TLV virus. Fish vaccinated with TLV p20 (clone 4) did not exhibit any sign of disease and was comparable in feeding and behavior patterns with the placebo control group. TLV p20 (clone 4), also referred to as TLV clone 4, was deposited with the CNCM (Pasteur Institute) on April 1, 2016 as Accession No. CNCM I- 5075. Partial sequence characterization of this clone revealed deletions in at least segments 4 and 5 of the TLV genome (shown here as SEQ ID NOs 49 and 50, respectively). It will be appreciated that mutant- specific portions of these sequences can be used for detection of the attenuated strain, and distinguishing them from the wildtype strain.

In order to determine the effectiveness of the vaccines, fish surviving the safety portion were injected with a high titer of wildtype TLV virus that had been passaged 4 times in cultured Tilapia fin cells. A portion of the placebo group was not challenged with the wildtype virus to rule out any unassociated mortalities throughout the efficacy portion of the study.

The results of the efficacy challenge are shown in Figure 6. The challenged placebo group exhibited a mortality pattern which is evident of TLV infection. Fish became lethargic and went off feed, coloration of fish skin became dark and mortality began at day 12 post- exposure, peaking at 20 days, and reaching 100% mortality during the efficacy trial period. The unchallenged placebo group was uneventful and showed no sign of disease. Fish vaccinated with TLV plO (clone 6) exhibited a delayed onset of mortality only beginning at day 20 post-challenge, and peaking to 28% mortality. Fish vaccinated with TLV p20 (clone 4) showed no sign of disease and were comparable in behavior to the unchallenged placebo group. A mortality level of 2.9% (a single fish) was observed during the observation period.

These results indicates that TLV p20 (clone4) shows a favorable vaccination profile both for safety and efficacy parameters tested.

Example 4: Generation and Characterization of an Inactivated TLV Vaccine Strain

To generate the inactivated TLV vaccine strain, TLV is exposed to 0.2% formaldehyde and passaged until the exposed virus in non-infective. The ability of the isolated inactivated strains to confer resistance to TLV infection is then tested on live fish by injection with and without commercial adjuvants. Results are shown in Figure 9. As shown in the figure, mortality rate of Tilapia fish (lOgr) challenged by IP injection with TLV, reduced from 91.7% (11/12) mortality of the control group, to 33% (8/24, Relative Percent Survival = RPS = 63.4), when injected, 28 days prior to the challenge, with emulsion containing inactivated TLV (0.2% Formaldehyde) with Freund's incomplete adjuvant - IFA (1: 1). In contrast, no immune protection is gained when injecting Tilapia fish (lOgr) 28 days prior to the challenge with TLV, with either inactivated TLV (0.2% Formaldehyde) alone or with emulsion containing Placebo - Medium from uninfected cells with Freund's incomplete adjuvant - IFA (1: 1), 100% mortality (26/26 and 24/24 respectively).

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. An isolated attenuated Tilapia virus (TLV) produced by the process comprising: passaging the TLV through tilapia cells at least 10 times,

irradiating the TLV at a sublethal radiation dose; and

isolating an infectious attenuated clone.

2. The isolated attenuated TLV of claim 1, wherein the TLV is passaged at least 20 times through tilapia cells.

3. The isolated attenuated TLV of claim 1 or claim 2, wherein the sublethal radiation dose is a dose of ultraviolet radiation between 3.2-6.8 mJ/cm².

4. The isolated attenuated TLV of claim 3, wherein the sublethal radiation dose is 6.8 mJ/cm².

5. The isolated attenuated TLV of claim 1, comprising the attenuated TLV deposited with the CNCM (Pasteur Institute) as Accession No. CNCM 1-5075.

6. An immunogenic composition comprising the isolated attenuated TLV of any one of claims 1-5, and an adjuvant.

7. A method for vaccinating a subject against a Tilapia virus (TLV) infection comprising:

infecting a subject with the isolated attenuated TLVs of any one of claims 1-5 or administering to the subject the immunogenic composition of claim 6, thereby vaccinating the subject.

8. An isolated orthomyxo-like virus having a genome comprised of nucleic acid sequences at least 90% identical to each of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 22, and 24.

9. An isolated nucleic acid comprising a nucleic acid at least 90% identical to a nucleic acid selected from the group consisting of SEQ ID NOs 25-48.

10. The isolated nucleic acid of claim 9, further comprising a detectable label.

11. The isolated nucleic acid of claim 10, wherein the detectable label is a fluorescent label, radioactive label, or chemical label.

12. A method for detecting a Tilapia virus (TLV) infection, comprising:

isolating a sample from a subject suspected of being infected by TLV;

generating a cDNA template from the sample; and

amplifying a nucleic acid from the cD A template using at least one oligonucleotide primer comprising a nucleic acid sequence at least 90% identical to at least one sequence selected from the group consisting of SEQ ID Nos: 25-48, wherein amplification of a nucleic acid sequence with the primer indicates the presence of TLV in the sample.

13. The method of claim 12, wherein the amplifying is carried out with at least one primer pair having sequences at least 90% identical to the primer pairs selected from the group consisting of SEQ ID NOs 25 and 26; SEQ ID NOs 27 and 28; SEQ ID NOs 29 and 30; SEQ ID NOs 31 and 32; SEQ ID NOs 33 and 34; SEQ ID NOs 35 and 36; SEQ ID NOs 37 and 38; SEQ ID NOs 39 and 40; SEQ ID NOs 41 and 42; SEQ ID NOs 43 and 44; SEQ ID NOs 45 and 46; and SEQ ID NOs 47 and 48.

14. A kit for detecting a Tilapia virus (TLV) infection, comprising at least one isolated nucleic acid of claim 9.

15. The kit for detecting a Tilapia virus (TLV) infection of claim 14, wherein the at least one isolated nucleic acid is at least one oligonucleotide primer pair having sequences at least 90% identical to the primer pairs selected from the group consisting of SEQ ID NOs 25 and 26; SEQ ID NOs 27 and 28; SEQ ID NOs 29 and 30; SEQ ID NOs 31 and 32; SEQ ID NOs 33 and 34; SEQ ID NOs 35 and 36; SEQ ID NOs 37 and 38; SEQ ID NOs 39 and 40; SEQ ID NOs 41 and 42; SEQ ID NOs 43 and 44; SEQ ID NOs 45 and 46; and SEQ ID NOs 47 and 48.

16. The kit of claim 14 or claim 15, further comprising a detectable label.

17. An isolated inactivated Tilapia virus (TLV) produced by the process comprising: incubating TLV in formaldehyde; and

passaging the TLV through tilapia cells until the TLV is no longer infectious.

18. An immunogenic composition comprising the isolated inactivated TLV of claim 17, and an adjuvant.

19. A method for vaccinating a subject against a Tilapia virus (TLV) infection comprising:

administering to a subject a composition comprising the isolated inactivated TLV of claim 17 or the immunogenic composition of claim 18, thereby vaccinating the subject.