EP3400007A1 - Virus de type orthomyxovirus du tilapia - Google Patents

Virus de type orthomyxovirus du tilapia

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Publication number
EP3400007A1
EP3400007A1 EP17735934.6A EP17735934A EP3400007A1 EP 3400007 A1 EP3400007 A1 EP 3400007A1 EP 17735934 A EP17735934 A EP 17735934A EP 3400007 A1 EP3400007 A1 EP 3400007A1
Authority
EP
European Patent Office
Prior art keywords
seq
tlv
nos
isolated
tilapia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP17735934.6A
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German (de)
English (en)
Other versions
EP3400007A4 (fr
Inventor
Arnon Dishon
Shlomit TAL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abic Biological Laboratories Ltd
Original Assignee
Kovax Ltd
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Filing date
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Publication of EP3400007A1 publication Critical patent/EP3400007A1/fr
Publication of EP3400007A4 publication Critical patent/EP3400007A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • 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.
  • 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.
  • 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.
  • TLV Tilapia Virus
  • 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.
  • 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.
  • TLV Tilapia virus
  • TLV Tilapia viruses
  • 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.
  • 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).
  • EM electron micrograph
  • FIG. 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.
  • FIG. 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).
  • 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).
  • Lane 21 -Healthy fish collected from the same farm.
  • 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, ⁇ - 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).
  • 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
  • SEQ ID Nos. 45 and 46 are respective forward and reverse primers specific to
  • SEQ ID Nos. 47 and 48 are respective forward and reverse primers specific to
  • 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.
  • Administration The introduction of a composition into a subject by a chosen route.
  • Administration of an active compound or composition 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.
  • PCR polymerase chain reaction
  • 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.
  • Animal Living multi-cellular vertebrate organisms, a category that includes for example, fish, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects, for example, humans, non-human primates, fish, dogs, cats, horses, and cows.
  • 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 RNA molecule. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • 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.
  • the base adenine (A) is complementary to thymidine (T) and uracil (U), while cytosine (C) is complementary to guanine (G).
  • T thymidine
  • U uracil
  • G guanine
  • the sequence 5'-ATCG-3' of one ssDNA molecule can bond to 3'-TAGC-5' of another ssDNA to form a dsDNA.
  • the sequence 5'-ATCG-3' is the reverse complement of 3'-TAGC-5'.
  • 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.
  • an agent such as a signal or particular nucleotide nucleic acid probe, amino acid, or protein, for example a TLV protein or nucleic acid
  • 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.
  • 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 S 35 and P 32 ), 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.
  • Orthomyxoviridae can have filamentous or spherical capsids. Their RNA genome is segmented and is transcribed and replicated by virus-encoded RNA dependent RNA polymerases.
  • 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 does not require absolute purity; rather, it is intended as a relative term.
  • 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.
  • pathogen a specific disease-causing agent
  • 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.
  • TLV Tilapia Virus
  • 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.
  • 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 nucleic acid further comprises a detectable label, such as a fluorescent label, radioactive label, or chemical label.
  • 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.
  • TLV Tilapia virus
  • 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.
  • kits for detecting a Tilapia virus (TLV) infection which contain at least one of the described isolated nucleic acids.
  • 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.
  • kits further include detectable label, which in particular embodiments can be associated with at least one of the isolated nucleic acids in the kit.
  • detectable label 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 are also described herein.
  • an isolated attenuated Tilapia virus 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.
  • the isolated attenuated TLV is passaged at least 20 times through tilapia cells.
  • the sublethal radiation dose is between 3.2-6.8 mJ/cm 2 , such as 6.8 mJ/cm 2 .
  • An exemplary isolated attenuated TLV provided herein was deposited with the CNCM (Pasteur Institute) as Accession No. CNCM 1-5075.
  • 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.
  • TLV Tilapia virus
  • such vaccination includes infecting a subject with the described isolated attenuated or inactivated TLVs.
  • vaccination includes administering to a subject an immunogenic composition comprising the described isolated attenuated or inactivated TLVs.
  • TLV 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • contig 4 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.
  • 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.
  • 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.
  • 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.
  • the primers can be 12-35 nt long, such as 15-25 nt, 18-30 nt and the like.
  • 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.
  • detectable label include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
  • the label is a radioactive label.
  • the label is a fluorescent label, such as any one of the fluorescent labels described herein.
  • 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.
  • the sample is isolated from an individual subject, such as a Tilapia fish.
  • the sample is collected from a population, such as a population of fish.
  • the sample is collected from an environment in which subjects live, such as fish pond and the like.
  • 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.
  • 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.
  • TLV has an RNA genome that does not pass through a
  • kits for detecting TLV in a sample include at least one of the TLV-specific probes and primers described above.
  • the kits include reagents for isolating viral RNA from a sample.
  • the kits include reagents and/or enzymes for reverse transcription and DNA amplification.
  • 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.
  • the supplied TLV-specific nucleic acids are pre-labeled with a detectable label.
  • a detectable label is included in the kit along with optional reagents and/or enzymes for associating the label with the nucleic acid.
  • 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.
  • 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
  • each passage is followed by exposure to a mutagenic condition prior to the next passage cycle.
  • a non-lethal (also described herein as "sublethal") mutagenic condition e.g. a condition wherein the virus may continue to infect cells
  • UV radiation such as 3.2-6.8 mJ/cm 2 , and more particularly 6.8 mJ/cm 2 .
  • 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/cm 2 , and then isolating an individual virus clone.
  • 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%-
  • 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.
  • 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.
  • an immunogenic composition e.g. a TLV vaccine
  • the pharmaceutically acceptable carrier can be water or a buffer, or additionally a composition stabilizer.
  • the vaccines are prepared as liquid solutions, emulsions or suspensions for injection.
  • delivery can be through immersion of fish in water.
  • 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.
  • the vaccine may be a lyophilized culture for reconstitution with a sterile diluent. For example 0.9% saline.
  • 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.
  • 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.
  • the described immunogenic composition additionally comprises at least one stabilizer to protect it from degradation, to enhance the shelf-life, or to improve freeze-drying efficiency.
  • 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 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.
  • 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
  • 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.
  • population vaccination methods can be used in various environments such as ponds, aquariums, natural habitat, fish farms and fresh water reservoirs.
  • 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 0.2 ⁇ 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.
  • CPE cytopathic effect
  • 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.
  • SEQ ID NO: 1 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).
  • SEQ ID NO: 2 The 520 amino acid conceptual translation of the sequence (in frame +2) is set out as SEQ ID NO: 2.
  • 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
  • the amino acid sequence set forth as SEQ ID NO: 2 resembles the PB1 RNA Polymerase of Orthomyxo virus.
  • 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.
  • This example demonstrates TLV detection in infected cultures or infected organs (liver, spleen, kidney, brain) derived from sick Tilapia fish:
  • 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.
  • 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.
  • FIG 7 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).
  • 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).
  • 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/cm 2 , virus clones were picked and propagated as a single viral clone.
  • 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).
  • 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.
  • 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.
  • TLV p20 (clone4) shows a favorable vaccination profile both for safety and efficacy parameters tested.
  • 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.

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Abstract

L'invention concerne l'isolation et la caractérisation d'un virus à ARN récemment découvert de poissons. L'invention concerne des procédés de détection du virus isolé, un vaccin à virus vivant atténué, ainsi qu'un vaccin à virus inactivé.
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