CN108778324A

CN108778324A - The just glutinous sample virus of Tilapia mossambica

Info

Publication number: CN108778324A
Application number: CN201780015550.2A
Authority: CN
Inventors: A·迪逊; S·塔尔
Original assignee: Co Of Co
Current assignee: Yabek Biological Laboratory Co., Ltd.
Priority date: 2016-01-10
Filing date: 2017-01-10
Publication date: 2018-11-09
Also published as: WO2017118989A1; EP3400007A4; MX2018008529A; CO2018008288A2; EP3400007A1; BR112018014042A2

Abstract

This document describes the separation of the RNA virus of newfound fish and characterizations.Also describe virus, the method for attenuated live virus vaccines and the viral vaccine of inactivation for detecting separation.

Description

The just glutinous sample virus of Tilapia mossambica

Cross reference to related applications

It is required that the U.S. Provisional Patent Application No. 62/276,873 submitted on January 10th, 2016 and on June 21st, 2016 The equity of the U.S. Provisional Patent Application No. 62/352,570 of submission, content is by quoting being integrally incorporated herein with them.

Technical field

This disclosure relates to the separation of the RNA virus of newfound fish and characterizations.It also discloses for detecting separation The method of virus, attenuated live virus vaccines and the viral vaccine of inactivation.

Background technology

Tilapia mossambica is the adopted name of nearly hundred kinds of tropical cichlids, is second at most foster after carp in the whole world The food fish grown.Based on the report from United Nations Food and Agriculture Organization (FAO), bolti-Tilapia mossambica (Oreochromis niloticus)-is main Tilapia mossambica kind one of of the cultivation for consumption, and global yield reaches within 2012 To 3,200,000 tons (mt) (FAO).Tilapia mossambica species temperature climate country cultivation, Israel be its most northern distributed point it One.

According to State of Israel-Ministry of Agriculture, 7,662 tons of Tilapia mossambicas are consumed every year in Israel, account for whole fresh-water fishes consumption 43%.In past 20 years, Tilapia mossambica rises to 5,700 tons in the yield of shellfish spy Xie Anshan paddy from annual 1,600 ton.

There is high mortality suddenly in the Tilapia mossambica of in August, 2009, family report shellfish spy's Xie Anshan Canyon of breeding fish.In hybridization ash In Tilapia mossambica, general mortality rate reaches 30%-40%.The damage of red tilapia hybridization production is even more serious, is reached in some fish ponds To up to 100% loss.The death of juvenile fish and adult fish is all observed in growing pond and reservoir pond.In juvenile fish, dead thing Part is characterized in that initially losing the appetite, and observes that weak fish, the presence of grazing eclipse birds increase at pond edge, and not synchronized The final fish death rate of rate.It detects that the presence of known parasite increases, and handles and seem to alleviate some losses.Larger Fish in, loss of appetite and drowsiness apparent, and the acute high mortality in several days then restores fish population.Nerve necrosis The existing diagnostic test of virus is feminine gender.

Summer in 2010, the death rate of Tilapia mossambica was reported in June from shellfish spy's Xie Anshan paddy and Mediterranean littoral two Person starts.Main loss is formed along with sex reversal and initially.Test in the fish health center of Nir-David not will produce disease Disease model can not characterize pathogen.The epidemic disease event of unknown cause continued entire summer in 2011 and 2012.? The initial statistical analysis of death incident in 2011 and 2012 does not disclose the death rate in the presence and Tilapia mossambica farm of parasite Significant correlation.The parasite of identification changes with species, and therefore may be without method interpretation epidemic disease event.Furthermore it is known that This parasite may be secondary pathogen, and not the main reason for the death rate.The presence of bacterium and death incident do not have Significant correlation.In addition, the breaking-out of disease and clinical symptoms are not the features of known Tilapia mossambica viral pathogen.

Therefore, exist for the pathogen of the nearest Tilapia mossambica epidemic disease event of determination and need.

Invention content

There is provided herein the Rofe fish diseases for the separation that the registration number of CNCM (Institute Pasteur) preservation is CNCM I-4892 Malicious (TLV).

The just glutinous sample virus of separation is also provided herein, have by with SEQ ID NOs.1,3,5,7,9,11,13,15, 17, the genome of each of 19,22 and 24 at least 90% same nucleic acid sequence compositions.

The nucleic acid of separation described further herein comprising with the nucleic acid at least 90% selected from SEQ ID NOs 25-48 Same nucleic acid.

This disclosure further describes for detecting Rofe fish virus (TLV) infective method comprising feels from suspecting The subject for having contaminated TLV detaches sample；CDNA templates are generated from sample；It is come from using at least one Oligonucleolide primers to expand The nucleic acid of cDNA templates, at least one Oligonucleolide primers include with selected from SEQ ID Nos：At least one sequence of 25-48 is extremely The amplification of few 90% same nucleic acid sequence, more control sequences primer indicates that there are TLV in sample.

There is also described herein the inactivation Rofe fish virus (TLV) of the attenuation of separation and separation.In a specific embodiment, divide From any one of virus composition further include adjuvant.

Be additionally provided with the kit for detecting TLV comprising the nucleic acid of description, and by using separation inactivation or Attenuated virus makes the method that subject is directed to TLV vaccine inoculations.

By reference to the described in detail below of attached drawing progress, foregoing end other objects, feature and advantage will become more to show And it is clear to.

Description of the drawings

Fig. 1 is the electron micrograph (EM) in the Rofe fry cell of the postoperative infection of infection 5 days.Cell presents cellular regions The disintegration of room and organelle.Cytoplasm vacuole includes the size abundant virus-like structure (right figure arrow) different with form.

Fig. 2 shows (figure below) Rofe fin cell for maintaining the (above) being uninfected by 15 DEG C -33 DEG C and infection (TFC).Infection cell shows normal morphology at 15 DEG C, without apparent CPE.At 23 DEG C -33 DEG C, cell shows cytoplasm sky Alveolation and cell rounding finally cause the death of cell culture.

Fig. 3 is analogous to the Phylogenetic Analysis of the contig 4 of the TLV of the PB1RNA polymerases of orthomyxovirus.It uses TREX-MAFFT sequence alignment program package systems development tree, by the sequence of known orthomyxovirus PB1 amino acid sequences and TLV Compare.

Fig. 4 is to show the chart for generating mutation in TLV by sub- Lethal irradiation.TLV with increased UV doses, and And remaining infectivity is measured by the titration of virus on 96 microwell plates, as shown in chart.Maintaining residual infectivity Maximum dose level under, select and propagative viruses clone.

Fig. 5 is the chart of the security situation for the TLV clones for showing two attenuations compared with placebo treatment group.

Fig. 6 is to show to inject when facing the wild type TLV of lethal dose by IP, the chart of the effect of vaccine prototype.

Fig. 7 is running gel, is shown in the culture of infection and uses Con19 primer detection TLV, and shows that TLV has Have during its breeding cycle not by the rna gene group in DNA stages.Use the DNA extracted from TFC that is infection and being uninfected by Or RNA measures Con19 and compares TL-NA primer (swimming lanes as viral contig Con19 primers (swimming lane 1-5) or endogenous Tilapia mossambica Specificity 6-10).Using Aurum Total RNA Mini kits (BIO-RAD Cat#732-6820), RNA is used for Generate total cDNA.Swimming lane distribution is as follows：MW：100bp DNA ladders；1：It is measured come the TFC's of self-infection with Con19 primers cDNA；2：It is measured from the cDNA for being uninfected by TFC with Con19 primers；3：Con19 viral fragments are cloned into be surveyed with Con19 primers In fixed plasmid；4：It is measured come the DNA of the TFC of self-infection with Con19 primers；5：It is measured with Con19 primers from being uninfected by The DNA of TFC；6：With TL-NA measurement come the cDNA (endogenous control) of the TFC of self-infection；7：It is measured with TL-NA from being uninfected by The cDNA (endogenous control) of TFC；8：With TL-NA measurement come the DNA (endogenous control) of the TFC of self-infection；9：With TL-NA measurement come From the DNA (endogenous control) for the TFC being uninfected by；10：Negative control：Two primer sets are tested in the reaction of not template.

Fig. 8 is running gel, and display identifies the TLV for the fish for carrying out self-infection using Con4 as diagnostic tool.TLV exists Identification is easy in the fish of experimental infection and field epidemic disease.RNA is extracted from fish brain, and is used as template, for using Con4 primers The RT-PCR of (above) or Tilapia mossambica endogenous marker object (TL-NA) (figure below) as a contrast.Swimming lane 1-19：Natural Tilapia mossambica is used TLV is injected and is inhabited jointly with unexposed fish.Within 26 day observation period, collects sick fish/dead fish and is maintained at -80 DEG C, For further assessing.The sick fish that swimming lane 20-samples during the epidemic disease of the Tilapia mossambica farm of Israel.Swimming lane 21-from The healthy fish that identical farm collects.MW -100bp molecular marked compounds, NC-negative control, water are used as template, and the PC- positives are right According to from the TFC extractions RNA of infection TLV.

Fig. 9 be when being inoculated with the TLV preparations of inactivation as shown, display by the hypertoxic TLV of IP injections by The figure of the cumulative mortality of the Tilapia mossambica of challenge.After IP vaccine inoculations 28 days, it is challenged by the TLV of IP injection severe toxicity Tilapia mossambica cumulative mortality, wherein：Culture mediums of the x- from the TFC cells being uninfected by, the natural fishes of x-, good fortune in Δ-lotion The TLV and incomplete Freund's adjuvant-IFA (1 of your Malin's inactivation:1), the TLV and Ο-of ◇-Formalin inactivation include placebo Culture medium of the lotion-from the cell being uninfected by and incomplete Freund's adjuvant-IFA (1:1).

The brief description of the sequence of description

Using the standard letter abbreviation of nucleotide base and the three-letter codes of amino acid show provided herein nucleic acid and/ Or amino acid sequence, as defined in 37C.F.R.1.822.A chain of each nucleic acid sequence is only shown, but any is carried And the chain showed is understood to include complementary strand.As described herein：

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

SEQ ID NO：2 be the theoretical translation of contig 4.

SEQ ID NO：3 be the sequence of contig 7.

SEQ ID NO：4 be the theoretical translation of contig 7.

SEQ ID NO：5 be the sequence of contig 5.

SEQ ID NO：6 be the theoretical translation of contig 5.

SEQ ID NO：7 be the sequence of contig 6.

SEQ ID NO：8 be the theoretical translation of contig 6.

SEQ ID NO：9 be the sequence of contig 8.

SEQ ID NO：10 be the theoretical translation of contig 8.

SEQ ID NO：11 be the sequence of contig 14.

SEQ ID NO：12 be the theoretical translation of contig 14.

SEQ ID NO：13 be the sequence of contig 15.

SEQ ID NO：14 be the theoretical translation of contig 15.

SEQ ID NO：15 be the sequence of contig 12.

SEQ ID NO：16 be the theoretical translation of contig 12.

SEQ ID NO：17 be the sequence of contig 16.

SEQ ID NO：18 be the theoretical translation of contig 16

SEQ ID NO：19 be the sequence of contig 20.

SEQ ID NO：20 be the translation of theory stage 1 of contig 20.

SEQ ID NO：21 be the translation of theory stage 2 of contig 20.

SEQ ID NO：22 be the sequence of contig 21.

SEQ ID NO：23 be the theoretical translation of contig 21.

SEQ ID NO：24 be the sequence of contig 19.

SEQ ID NOs.25 and 26 are the forward and reverse primer to 4 specificity of contig respectively.

SEQ ID NOs.27 and 28 are the forward and reverse primer to 7 specificity of contig respectively.

SEQ ID NOs.29 and 30 are the forward and reverse primer to 5 specificity of contig respectively.

SEQ ID NOs.31 and 32 are the forward and reverse primer to 6 specificity of contig respectively.

SEQ ID NOs.33 and 34 are the forward and reverse primer to 8 specificity of contig respectively.

SEQ ID NOs.35 and 36 are the forward and reverse primer to 14 specificity of contig respectively.

SEQ ID NOs.37 and 38 are the forward and reverse primer to 15 specificity of contig respectively.

SEQ ID NOs.39 and 40 are the forward and reverse primer to 12 specificity of contig respectively.

SEQ ID NOs.41 and 42 are the forward and reverse primer to 16 specificity of contig respectively.

SEQ ID NOs.43 and 44 are the forward and reverse primer to 20 specificity of contig respectively.

SEQ ID NOs.45 and 46 are the forward and reverse primer to 21 specificity of contig respectively.

SEQ ID NOs.47 and 48 are the forward and reverse primer to 19 specificity of contig respectively.

SEQ ID NO：49 be 4 sequence of mutation section from attenuation TLV.

SEQ ID NO：50 be 5 sequence of mutation section from attenuation TLV.

Specific implementation mode

I. it abridges

TFC Rofe fin cells

TLV Rofe fish virus

II. term

Unless otherwise stated, all technical and scientific terms used herein has common with present disclosure fields The identical meaning that technical staff is generally understood.Unless context clearly indicates in other ways, singular references " one (a) ", " one (an) " and " being somebody's turn to do (the) " includes plural number instruction.Similarly, unless context clearly indicates in other ways, word " or (or) " it is intended to include " and (and) ".It will further be understood that providing all base sizes or amino acid size of nucleic acid or polypeptide It is approximate with all molecular weight or molecular mass values, and provides for describing.Although in the practice or survey of present disclosure Can be used in examination and similar or equivalent method and material those of is described herein, but be described below suitable method and Material.Term " including (comprise) " is meant " including (include) ".Abbreviation " such as (e.g.) " is originated from Latin language for example (exempli gratia), and herein for indicating non-limiting examples.Therefore, abbreviation " such as (e.g.) " and term " such as (for example) " is synonymous.

In the case of a conflict, subject to the present specification, include the explanation of term.In addition, all materials, method and reality Example is schematical and is not intended to restrictive.

Using：Composition is introduced into subject by the approach of selection.Well known by persons skilled in the art can be passed through What approach applies reactive compound or composition, such as TLV attenuated vaccine bacterial strains.Using can be local or whole body.

Amplification：When referring to nucleic acid in use, referring to any technology for the copy number for increasing the nucleic acid molecules in sample or sample. The example of amplification is polymerase chain reaction (PCR), with its all variant put into practice at present, wherein in allowing primer and sample The biological sample collected from subject is set to be contacted with a pair of of Oligonucleolide primers under conditions of nucleic acid-templated hybridization.Primer is suitable Under conditions of extend, from template dissociate, and then re-annealing, extension and dissociation with the copy number of amplification of nucleic acid.It can use Standard technique, by electrophoresis, restriction endonuclease cut mode, oligonucleotide hybridization or connection, and/or nucleic acid sequencing come table Levy the product of amplification in vitro.The non-limiting examples of PCR include RT-PCR and qPCR.Other examples of Amplification Technologies include Strand displacement amplification (see U.S. Patent number 5,744,311)；Without transcription isothermal duplication (see U.S. Patent number 6,033,881)；It repairs Chain reaction amplification (see WO 90/01069)；Ligase chain reaction expands (see EP-A-320 308)；Gap filling ligase chain is anti- It should expand (see U.S. Patent number 5,427,930)；The ligase detection of coupling and PCR (see U.S. Patent number 6,027,889)；With NASBA^TMRNA is without transcription amplification (see U.S. Patent number 6,025,134).

Animal：Many cells vertebrate organism living, classification include such as fish, mammal and birds.Term is fed Newborn animal includes both people and non-human mammal.Similarly, term subject includes both people and veterinary subject, such as People, non-human primates, fish, dog, cat, horse and cow.Similarly, term " subject " includes both people and veterinary subject, than Such as fish.

Attenuated virus：The virus having changed, to reduce or eliminate its virulence.Attenuated virus is " work ", still With the destruction that reduces or eliminates or the ability of host cell is killed, and is therefore caused and viral associated pathology.

Biological sample：Any sample that can be directly or indirectly obtained from organism, including whole blood, blood plasma, serum, tear Liquid, mucus, saliva, urine, hydrothorax, liquor pleurae, gastric juice, sweat, sperm, vaginal fluid, phlegm, from ulcer and/or other The liquid of surface eruption, blister, abscess, tissue, cell are (for example, fibroblast, peripheral blood mononuclear cells or muscle are thin Born of the same parents), organelle (such as mitochondria), organ, and/or tissue extract, cell be (for example, fibroblast, peripheral blood mononuclear are thin Born of the same parents or muscle cell), organelle (such as mitochondria) or organ.Biological sample can also be that laboratory research sample is such as thin Born of the same parents' culture supernatant.Sample is collected or obtained using method well known to those skilled in the art.In this disclosure, " biology Sample " is used interchangeably with " sample ".

CDNA (complementary DNA)：Lack the DNA fragmentation of internal non-coding section (introne) and transcription regulating nucleotide sequence.cDNA Can also include non-translational region (UTR), the translation control being responsible in corresponding RNA molecule.In the lab by from cell The reverse transcription of the mRNA of extraction synthesizes cDNA.

It is complementary：Double-stranded DNA or RNA chains are made of two complementary strands of base-pair.When a nucleic acid molecules base with it is another When the base of one nucleic acid molecules forms hydrogen bond, complementary combination occurs.In general, bases adenine (A) and thymidine (T) and Uracil (U) is complementary, and cytimidine (C) is complementary with guanine (G).For example, the sequence 5'-ATCG-3' of a ssDNA molecule can To be bound to the 3'-TAGC-5' of another ssDNA to form dsDNA.In this example, sequence 5'-ATCG-3' is 3'-TAGC- The reverse mutual complement of 5'.Similarly, the cDNA generated by ssRNA molecules by be RNA sequence reverse mutual complement.

Contact：It is placed as direct physical interconnection.Including solid and liquid form.Contact can utilize the cell of separation in body Outer generation is occurred in vivo by being applied to subject.

Detection：Determination existence or non-existence reagent (such as signal or specific nucleotide nucleic acid probe, amino acid or albumen Matter, such as TLV protein or nucleic acid).In some instances, this may further include quantization.

Diagnosis：Disease is identified by its sign, symptom and the result of various tests and method such as method disclosed herein Process.

Fluorogen：A kind of chemical compound, when the light for such as limiting wavelength by being exposed to particular stimulation is excited, Emit light (fluorescing), such as with different wave length (such as light of more long wavelength).

Fluorogen is a part for the luminophor compared with major class.Luminophor includes chemiluminescent molecule, no It needs the light of specific wavelength to shine, but uses chemical energy source.Therefore, chemiluminescent molecule (such as water wood shine egg Use in vain) can no longer need external source of electromagnetic radiation, such as laser.In the U.S. Patent number 5,866 of Nazarenko etc., It is provided in 366 and can be used for the example that the specific fluorescent of probe and primer disclosed herein is rolled into a ball, such as 4- acetylaminohydroxyphenylarsonic acids 4'- different Thiocyano- talan -2,2' disulfonic acid, acridine and derivative such as acridine and acridine isothiocyanates, 5- (2'- amino second Base) amino naphthalenes -1- sulfonic acid (EDANS), -3,5 disulfonic acid of 4- amino-N- [3- vinylsulfonyls) phenyl] naphthalimide Ester (fluorescein VS), N- (4- anilino- -1- naphthalenes) maleimide, anthranilamide, bright orange, cumarin and derivative Such as cumarin, 7- amino -4- methylcoumarins (AMC, coumarin 1 20), 7- amino -4- trifluoromethyl coumaran (coumarans 151)；Phloxin (cyanosine)；4', 6- diamidino -2-phenylindone (DAPI)；5', 5 "-dibromo pyrogallols-sulfonephthalein (bromopyrogallol red)；7- diethylaminos -3- (4'- phenyl isothiocyanates base) -4- methylcoumarins；Diethylenetriamines five Acetic acid esters；4,4'- diisothiocyanic acids dihydro-stilbene -2,2'- disulfonic acid；Two sulphurs of 4,4'- diisothiocyanic acid base talan -2,2'- Acid；5- [dimethylamino] naphthalene -1- sulfonic acid chlorides (DNS, dansyl Cl)；The different sulphur cyanogen of 4- dimethyl aminophenylazo phenyl -4'- Acid esters (DABITC)；Eosin and derivative such as eosin and eosin isothiocyanates；Erythrosine and derivative such as Erythrosin B and Erythrosine isothiocyanates；Ethidium；Fluorescein and derivative such as 5-carboxyfluorescein (FAM), 5- (4,6- dichlorotriazines- 2- yls) Aminofluorescein (DTAF), bis- chloro- 6- Fluoresceincarboxylic acids (JOE) of 2'7'- dimethoxy-4 's ' 5'-, fluorescein, fluorescein Isothiocyanates (FITC) and QFITC (XRITC)；Fluorescamine；IR144；IR1446；Peacock green isothiocyanates；4- methyl umbrellas Shape ketone；O-cresolphthalein；Nitrotyrosine；Pararosaniline；It is phenol red；B- phycoerythrin；O-phthalaldehyde；Pyrene and derivative are such as Pyrene, butyric acid pyrene and succinimido 1- butyric acid pyrenes；Reactive Red 4 (Cibacron^TMBrilliant Red 3B-A)；Rhodamine and Derivative such as 6- carboxy-X-rhodamines (ROX), 6- carboxyrhodamines (R6G), Sulforhodamine B sulfonic acid chloride, rhodamine (Rhod), rhodamine B, Rhodamine 123, rhodamine X isothiocyanates, Sulforhodamine B, Sulforhodamine 101 and sulphonyl sieve The sulfonyl chloride derivatives (texas Red) of pellet bright 101；N, N, N', N'- tetramethyl -6- carboxyrhodamines (TAMRA)；Tetramethyl Rhodamine；Tetramethylrhodamine isothiocyanates (TRITC)；Riboflavin；Rosolic acid and terbium chelate derivative； LightCycler red 640；Cy5.5；With Cy56- Fluoresceincarboxylic acids；5-carboxyfluorescein (5-FAM)；Dipyrromethene difluoro Change boron (BODIPY)；N, N, N', N'- tetramethyl -6- carboxyrhodamines (TAMRA)；Acridine, stilbene, -6- carboxy-fluoresceins (HEX), TET (tetramethyl fluorescein), 6- carboxy-X-rhodamines (ROX), texas Red, 2', 7'- dimethoxy-4 's ', 5'- Two chloro- 6- Fluoresceincarboxylic acids (JOE), Cy3, Cy5,(Applied Biosystems), LC are red 640, LC is red 705, Yakima Huangs etc..

The virus of inactivation：It has been inactivated or " kill " is to eliminate the virus of its virulence.Although without infectiousness, The virion of inactivation can excite immune response, and can form the basis of vaccine.The non-limiting method of inactivation of virus Including heating, UV exposures or chemical mode, for example it is exposed to formaldehyde (formalin).

Separation：The biological components substantially with other biological components isolated or purifieds in the cell of organism (such as nucleic acid molecules, protein or organelle), the component is naturally occurring in the organism, other biological components i.e. its Its chromosome and exchromosomal DNA and RNA, protein and organelle.Separated nucleic acid and protein include passing through standard The nucleic acid and protein of purification process purifying.The term further includes the nucleic acid and egg by recombinantly expressing preparation in host cell White matter and chemically synthesized nucleic acid.

Label：Another molecule is directly or indirectly conjugated in favor of detecting detectable compound or the combination of the molecule Object.The specific non-limiting examples of label include radioactive isotope (such as S³⁵And P³²), zymolyte, confactor, ligand, Chemiluminescence or fluorescer, haptens and enzyme.

Oligonucleotides：By the nucleotide for multiple connections that natural phosphodiester is keyed, length is at about 6 and about 300 Between nucleosides.Oligonucleotide analogs refer to plays similar effect but the part with non-naturally-occurring part with oligonucleotides.Tool The oligonucleotides and oligonucleotide analogs of body may include the linear order that length is up to about 300 nucleotide, and for example, at least 6 The sequence (such as DNA or RNA) of a base, for example, at least 8,10,15,20,25,30,35,40,45,50,100 or even 200 Or 300 bases are long, or from about 6 to about 50 bases, for example, about 10-25 base, such as 12,15 or 20 bases.

Orthomyxovirus section：RNA virus family comprising influenza virus.The characterization of TLV viruses described herein and sequencing refer to Show that it is orthomyxovirus.Orthomyxovirus section can have Filamentous or spherical capsid.Their rna gene group is compiled by dependovirus The RNA polymerase of the RNA of code is divided and is transcribed and replicates.

Probe and primer：Nucleic acid probe and primer can be easily prepared based on the nucleic acid molecules provided in the present invention.It visits Needle includes the nucleic acid for the separation for being connected to detectable label or reporter molecule.Typical label includes radioactive isotope, enzyme Substrate, confactor, ligand, chemiluminescence or fluorescer, haptens and enzyme.

Primer is short nucleic acid molecules, and preferably length is the DNA oligonucleotides of 10 nucleotide or more.Primer can be with It is annealed to form the hybrid between primer and target dna chain by the target dna chain of nucleic acid hybridization and complementation, and then drawn Object is by archaeal dna polymerase along target dna chain extension.Primer pair can be used for the amplification of nucleic acid sequence, for example, by PCR or originally Other nucleic acid amplification methods known to field.

Purifying：Term purification does not require absolute purity；But it is intended as relative terms.Thus, for example, purifying Protein formulation be it is mentioned that the protein protein formulation purer than the protein in its natural surroundings in the cell.

Quantitative real-time PCR：Method for detecting and measuring the product generated during each of PCR is recycled, the production Product to before starting PCR the amount of existing template nucleic acid it is proportional.The information of acquisition, such as amplification curve can be used for quantifying The primary quantity of template nucleic acid sequence.

Sequence identity：Similitude between two nucleic acid sequences or two amino acid sequences, according to the phase between sequence It is expressed like property, also referred to as sequence identity.Sequence identity is generally according to percentage identity (or similitude or homology) To weigh；Percentage is higher, and two sequences are more similar.

Vaccine：Subject is induced to obtain the group for specific pathogenic agent (pathogen) the such as immunoprotection of virus or bacterium Close object.Vaccine especially may include the antigen fragment of pathogen, induce the immune response in subject again.Other vaccine examples Including attenuated live vaccine or vaccine is killed, wherein pathogen itself is the basis of vaccine.Vaccine described herein is attenuated live TLV Bacterial strain.Vaccine inoculation is to provide vaccine to protect subject to support antiviral process.Vaccine is immunogenic composition Exemplary types.

Virus：In the microcosmic infectious organisms of living cells internal reproduction.Virus is substantially by being surrounded by protein coat The core composition of single nucleic acid, and with only in the ability of living cells internal reproduction." virus replication " is by occurring at least The generation of the other virus of one viral lifecycle.Virus may destroy the normal function of host cell, cause cell table It is now the mode determined by virus.For example, when the cell being uninfected by is not done that usually, virus infection can cause cell to produce Raw cell factor, or in response to cell factor.

Virion：Complete virion includes coating, capsid and nucleic acid element.

III. the general introduction of several embodiments

This document describes the Rofe fish diseases for the separation that the registration number of CNCM (Institute Pasteur) preservation is CNCM I-4892 Malicious (TLV).

There is also described herein the just glutinous sample virus of separation, have by with SEQ ID NOs.1,3,5,7,9,11,13,15, 17, the genome of each of 19,22 and 24 at least 90% same nucleic acid sequence compositions.

In a specific embodiment, nucleic acid further comprises detectable label, for example, fluorescent marker, radioactive label or Chemical labeling.

In the specific implementation mode of detection method described herein, expanded using at least one primer pair, at least One primer pair have with selected from least 90% same sequence of following primer pair：SEQ IDNOs 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；43 Hes of SEQ ID NOs 44；SEQ ID NOs45 and 46；With SEQ ID NOs 47 and 48.

Present disclosure further includes for detecting Rofe fish virus (TLV) infective kit, and it includes at least one The nucleic acid of the separation of description.

In a specific embodiment, kit includes the nucleic acid of at least one separation, for drawing with selected from following At least one Oligonucleolide primers pair of the object sequence same at least 90%：SEQ ID NOs25 and 26；SEQ ID NOs 27 With 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 NOs39 and 40；SEQ ID NOs 41 and 42；SEQ ID NOs 43 and 44；SEQ ID NOs 45 and 46；With SEQ ID NOs 47 and 48.

In a specific embodiment, kit further comprises detectable label, in a specific embodiment can be with It is associated with the nucleic acid that at least one of kit detaches.

There is also described herein the Rofe fish virus (TLV) of the attenuation of separation and the inactivation of separation.

The attenuation Rofe fish virus (TLV) of the separation generated by following methods further described herein：Pass through Tilapia mossambica Cell passes on TLV at least 10 times, irradiates TLV with sub- lethal radiation dose；And detach the attenuated clones body of infection.Specific real It applies in mode, so that the attenuation TLV of separation is passed at least 20 times by Rofe fry cell.In other specific implementation modes, Asia causes Dead dose of radiation is in 3.2-6.8mJ/cm²Between, such as 6.8mJ/cm².The attenuation TLV of exemplary separation provided herein by CNCM (Institute Pasteur) preservation, registration number are CNCM I-5075.

In a specific embodiment, the attenuation of separation described herein or the TLV bacterial strains of inactivation are immunogenic compositions A part, for example further include the composition of adjuvant.

In addition present disclosure describes the method to subject's vaccine inoculation for Rofe fish virus (TLV) infection.? In specific implementation mode, this vaccine inoculation includes that subject is made to infect the attenuation of the separation described or the TLV of inactivation.Other In embodiment, vaccine inoculation includes that the immunogenicity of the attenuation for the separation for including description or the TLV of inactivation is applied to subject Composition.

IV. Rofe fish virus and its detection method

Since 2009, the family of breeding fish of northern Israel observed that the disease of Tilapia mossambica and height are dead in its farm Rate.These events are continuing in each continuous summer.This document describes the separation of disease and the pathogen of death incident and Characterization, pathogen are to be referred to as the newfound just glutinous sample virus of Rofe fish virus (TLV).

Primary characterization instruction spherical shape, the pleomorphism RNA virus of TLV, it is numerous with temperature-dependent manner between 23 DEG C -33 DEG C It grows.Virus has grown to high titre, and is deposited in CNCM (Baths on June 15th, 2014 with registration number CNCM I-4892 Moral research institute).

Genome analysis discloses the rna gene group of segmentation, with the sequence in GenBank databases almost without homologous Property.However, BlastX analysis shows that TLV contigs 4 (it is described herein as be SEQ NO：1) include that influenza RNA Dependent RNA is poly- The presumption conserved domain of synthase subunits PB1.

Continuous (contig) sequence is determined from TLV, it is described herein as SEQ ID NOs.1,3,5,7,9,11, 13,15,17,19,22 and 24.

In a specific embodiment, the TLV of description include have be illustrated as SEQ ID NOs.1,3,5,7,9,11,13, 15,17,19,22 have mutually homotactic genome with those of 24 sequences.However, it is recognized that carrying out the TLV sequences of self-described Genome variation will be present in the TLV of different separation.Therefore, in a specific embodiment, separation described herein There is TLV such genome, wherein any of contig it is same in being stated as SEQ ID can be less than 100% The sequence of NOs.1,3,5,7,9,11,13,15,17,19,22 and 24.This sequence can in the Contig of description One or more at least 99%, 95%, 90%, 85%, 80% is even less same.

The genome sequence of separation can be synthetically produced or as described as DNA (such as cDNA) or as being described herein Sequence relatively short rna or DNA fragmentation be synthetically produced.The non-limiting purposes of this segment includes being used for use known in the art In the probe and primer of any method for detecting the virus in sample.In a specific embodiment, segment (it can be probe) It can be with the reverse complementary sequence at least 99%, 95%, 90%, 85%, 80%, 75%, 70% of Contig to be detected Or it is even less same.Similarly, compared with sequence to be detected, the length of the probe of the separation for detecting TLV can be 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, therebetween any hundred Divide ratio, or even smaller.For example, (the SEQ ID NO of contig 4：1) a length of 1620 nucleotide (nt).Therefore the overlapping of description The probe of the separation of group 4 can be with SEQ ID NO:1 sequence is same, but length can be significantly shorter, such as a length of 1600,1500,1400,1300,1200,1100,1000,900,800,700,600,500,400,300,200,100,50nt, Or any length therebetween.Such as in other embodiments, it can be used as (the SEQ ID NO of contig 4：1) separation of probe Nucleic acid can be with a length of 35-1620nt, such as 50-1600nt, 1000-1600nt, 500-1500nt, 750-1200nt and its Between any length.It should be appreciated that the nucleic acid fragment of the separation of each Contig, including DNA or RNA segments, it can be with class As be used as the probe sequence of single contig.

Similarly, using the primer developed from the sequence of description, be stated as SEQ ID NOs.1,3,5,7,9,11,13, 15,17,19,22 and 24 sequence or any shorter inclusive sequence, can be amplified as described herein.Specific In embodiment, primer can be with a length of 12-35nt, such as 15-25nt, 18-30nt etc..It can be used for expanding drawing for the sequence of description The specific non-limiting examples of object include be illustrated as the Oligonucleolide primers of SEQ ID NOs 25-48 herein, or with SEQ ID NOs 25-48 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even more are same Primer.

It is stated as the nucleic acid sequence of the separation of SEQ ID NOs.1,3,5,7,9,11,13,15,17,19,22 and 24, and Its segment (including probe and primer) can include detectable label as described herein by covalent modification.This mark The specific non-limiting examples of note include radioactive isotope, zymolyte, confactor, ligand, chemiluminescence or fluorescer, half Antigen and enzyme.In a specific embodiment, label is radioactive label.In other embodiments, label is fluorescent marker, than Any one of fluorescent marker as described herein.

Detection sample further described herein is such as from the side of the TLV in the sample that subject (such as Tilapia mossambica) collects Method.This method includes the TLV nucleic acid detected in sample, indicates that there are TLV in sample.

In a specific embodiment, sample is detached from individual subjects such as Tilapia mossambica.In other embodiments, from kind The population of group such as fish collects sample.In still other embodiment, from receipts such as the environment such as fishponds that wherein subject lives Collect sample.When sample is the sample from one or more subjects, in sample TLV nucleic acid or protein presence instruction by Examination person has infected TLV.

Nucleic acid detaches and its detection method is well known in the art.In a specific embodiment, by standard hybridisation methods, Including array, such as microarray, label probe or primer described herein can be used for detecting the TLV in sample.Other detection sides Method includes any suitable nucleic acid amplification method, such as PCR and its modification, including RT-PCR and qPCR.

As just glutinous sample virus, TLV has does not pass through the rna gene group in DNA stages during its breeding cycle.Therefore, When in DNA form, the nucleic acid of separation described herein is synthesis., it will also be appreciated that expanding TLV cores by standard method Acid method by including reverse transcription step to generate cDNA as the basis of PCR method etc..The method of reverse transcription and PCR are abilities Known to domain.

There is also described herein the kits for detecting the TLV in sample.The kit of description includes above-mentioned TLV- special Property at least one of probe and primer.In a specific embodiment, kit includes for the examination from sample isolated viral RNA Agent.In other embodiments, kit includes reagent and/or the enzyme for reverse transcription and DNA cloning.In still other embodiment party In formula, kit includes at least one primer pair, such as from the oligonucleotides for being illustrated as SEQ ID NOs 25-48 herein It is each right, or with SEQ IDNOs 25-48 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even more same primer.In a specific embodiment, the TLV specific nucleic acids provided are pre- with detectable label First mark.In other embodiments, it is detectable label together with for will mark and the associated optional reagent of nucleic acid and/or Enzyme is included in kit.

V. the Tilapia mossambica viral vaccine for being attenuated and inactivating

The TLV isolates of described further herein attenuation and inactivation can assign the resistance to wild type TLV infection.

After by the passage repeatedly of Rofe fry cell, attenuated virus provided herein is detached and cloned, is subsequently exposed to Nonlethal mutagenic condition, such as ultraviolet radioactive or chemical mutagen.

In a specific embodiment, by methods known in the art (such as wherein every time passage be infection and by infecting The period of the separation of the virus of generation), lacked 10 times by making virus be passaged to by Rofe fry cell first, for example pass through Rofe Fry cell 10,11,12,13,14,15,16,17,18,19,20 or more times, generates the TLV bacterial strains of attenuation.In specific example In, passage is after being exposed to mutagenic condition every time, followed by next passage period.In other embodiments, final After passage, make the virus of collection be exposed to nonlethal (being also described as " sub- lethal ") mutagenic condition (such as its Middle virus can continue the condition of infection cell).In some embodiments, the virus under attenuation is made to be exposed to ultraviolet (UV) The sublethal dose of radiation, such as 3.2-6.8mJ/cm², and more specifically 6.8mJ/cm²。

In a specific embodiment, wild type TLV is passed on by 20 generations by Rofe fin cell, cell is made to be exposed to 6.8mJ/cm²UV dose of radiations, and then detach precursor virus clone, generate the TLV bacterial strains of attenuation.

When being provided to subject as immunogenic composition, such as in vaccine, the attenuation TLV bacterial strains of description can To assign the resistance partially or completely infected TLV so that will be shown by the population of the fish of the vaccine inoculation of wild type TLV attacks Show at least 60% survival rate, such as 60%-100% survival rates, for example, 70%, 80%, 85%, 90%, 95%, 95%- 100% or even as high as about 100% survival rate.

The specific example of attenuation TLV is such attenuation TLV as described herein, in preservation on April 1 in 2016 At CNCM (Institute Pasteur), registration number is CNCM I-5075.

(kill) viral immunogenic compositions of inactivation can be generated according to methods known in the art, including the use of Formalin as described herein is incubated.

In a specific embodiment, the TLV isolates for being attenuated and/or inactivating are immunogenic composition (such as TLV epidemic diseases Seedling) component comprising attenuation and/or inactivation virus, pharmaceutically acceptable carrier and optionally one or more adjuvants Or other immunostimulant.

In a specific embodiment, pharmaceutically acceptable carrier can be water or buffer solution, or additionally composition is steady Determine agent.

In some embodiments, vaccine is prepared as liquid solution, lotion or suspension for injection.In fish vaccine In example, it can be delivered by the way that fish to be immersed in the water.Therefore, liquid emulsion or emulsifiable concentrate can be prepared to add Into the water for accommodating fish.Before administration, it can also prepare suitable for being dissolved or suspended in liquid-carrier or being suitable for eating with solid Solid (such as powder) form of object mixing.In a specific embodiment, vaccine can be for reconstructing with sterile diluent It is freeze-dried culture.Such as 0.9% physiological saline.Before drying (such as freeze-drying), it can be added to vaccine various Ingredient, such as preservative, antioxidant or reducing agent, various excipient etc..This excipient can also be after the drying step It is added to dry virus.

In a specific embodiment, immunogenic composition includes adjuvant or other immunostimulant.Adjuvant is this field It is well known.Specific non-limiting examples include muramyl dipeptide, avidin, aluminium hydroxide, aluminum phosphate, oil, oil breast Agent, saponin(e, dextran sulfate, glucan, cell factor, block copolymer, immunostimulatory oligonucleotide and can be with attenuation And/or the other materials known in the art of the TLV mixing of inactivation.

In other embodiments, the immunogenic composition (vaccine) of description also comprise at least one stabilizer to prevent Only it is degraded, and enhances the shelf-life, or improves freeze-drying efficiency.With the non-limiting examples for the stabilizer that the vaccine of description is used together Including SPGA (Bovarnik etc., volume 1950, J.Bacteriology, 59, page 509), skim milk, gelatin, cow's serum Albumin, carbohydrate such as D-sorbite, mannitol, trehalose, starch, sucrose, glucan or glucose, lactose, egg White matter such as albumin or casein or its catabolite and buffer solution, such as alkali metal phosphate.

By making an at least subject infect attenuation TLV vaccines described herein and/or subject is made to be exposed to inactivation TLV, the attenuation of description and/or the TLV immunogenic compositions (vaccine) of inactivation can be used for the vaccine inoculation side for TLV Method.

In a specific embodiment, vaccine is individually administered orally to fish, such as by its feed or by forcing to take orally Using, or by injection, such as via path in intramuscular or peritonaeum.

In alternative embodiments, by the way that vaccine is sprayed, dissolve and/or is immersed in the water, vaccine can be applied simultaneously With the entire fish population being extremely included in water body.This population vaccine inoculation method can be used for various environment, such as pond, Aquarium, natural habitat, fish farm and fresh water reservoir.

Following embodiments are provided to illustrate certain specific features and/or embodiment.These embodiments are not necessarily to be construed as Present disclosure is limited to the specific features or embodiment of description.

Embodiment

Embodiment 1：The separation of Rofe fish virus and characterization

Cell line is developed from Rofe fin, and it is used to detach RNA from the fish collected during death incident in 2012 Virus.Virus is temporarily designated as Rofe fish virus-TLV.Following isolated viral：By the freezing organ from illness Tilapia mossambica Sample thaws, and concentrates in together, homogenizes, be filtered through 0.2 μm of filter, and is seeded on natural Rofe fin cell (TFC). The TFC cultures infected are incubated at 28 DEG C, and monitoring cytopathic effect (CPE) daily.It can after being inoculated with 4-7 days See CPE, and includes clearly Plaque Formation (Fig. 2).CPE is unconspicuous in the TFC control cultures of simulated infection.With Supernatant from the TFC cultures for showing CPE was repeated more than for 20 generations to the natural culture secondary inoculations of TFC, had similar CPE results.

Tilapia mossambica cell line is with the virus of high titre breeding separation.The electron-microscopic analysis of the cell culture of infection is aobvious Intracellular Vims particle structures (Fig. 1) are shown.The shape of TLV viruses is spherical shape, and shows size and polymorphic form Variation.In infected cell, virion appears in cytoplasm vacuole, and the average diameter with~65nm.

The primary characterization of virus shows that virus breeding is temperature dependency and is happened at 23 DEG C -33 DEG C.Virus exists In Rofe fish cell culture breeding and>Cause cytopathic effect (CPE) (figure at a temperature of 24 DEG C rather than 15 DEG C 2).This observation result is consistent with the pathogenetic field observation result of disease.In entire viral passages in cell culture, I Be able to maintain that CPE and propagative viruses.

In order to realize Koch's it is assumed that and determine TLV be strictly disease pathogen, establish disease model, wherein The TLV of tissue culture separation be used to infect the natural Tilapia mossambica of health.The fish of injection shows the disease consistent with account on the spot Sick body is levied.Fish is lost the appetite, and display is drowsiness, becomes thin and weak and final death.Death rate range is from 30%-100%.Moreover, TLV is detached and is identified from fish dead in research, and the TLV for realizing Koch is the hypothesis of pathogen.

The further characterization for completing virus is sequenced by viral genome.The sequencing of virus discloses the culture with infection Associated multiple sequences, are not overlapped into single sequence, show the genome of segmentation.In 12 for measuring and presenting herein In sequence, two (contig 8 and 16) display and Genbank (Tilapia mossambica lake virus (Eyngor et al 2014；Genbank Registration number KJ605629.1) interior known array homology；Remaining sequence is not shared with any other Genbank sequences homologous Property.However, BlastX analyses are in the viral fragment (contig 4) for belonging to influenza RNA Dependent RNA polymerase subunits PB1 One in identify the conserved domain of presumption.Complete sequence (wherein U is expressed as T) is described herein as SEQ ID NO：1 (starting and terminator codon are predicted at nt 38 and 1595 respectively).520 amino acid theories of sequence (meeting frame+2) are turned over It translates and is stated as SEQ ID NO：2.

The sequence of other measurement and the translated product of presumption include：Contig 7(SEQ ID NO：3) it is translated with its frame 2 (SEQ ID NO：4)；(the SEQ ID NO of contig 5：5) and its 6th section is translated (SEQ ID NO：6)；(the SEQ ID of contig 6 NO：7) and its 6th section is translated (SEQ ID NO：8)：(the SEQ ID NO of contig 8:9) and its 6th section is translated (SEQ ID NO： 10)；(the SEQ ID NO of contig 14:11) and its 5th section is translated (SEQ ID NO：12)；(the SEQ ID NO of contig 15:13) With its 6th section translation (SEQ ID NO：14)；(the SEQ ID NO of contig 12:15) and its 5th section is translated (SEQ ID NO： 16)；(the SEQ ID NO of contig 16:17) and its 3rd section is translated (SEQ ID NO：18)；(the SEQ ID NO of contig 20:19), Its paragraph 1 (SEQ ID NO：And the 2nd section of (SEQ ID NO 20)：21) it translates；(the SEQ ID NO of contig 21:22) and its 5th Section translation (SEQ ID NO：23)；With (the SEQ ID NO of contig 19：24) nucleic acid sequence.

It is illustrated as SEQ ID NO：2 amino acid sequence is similar to the PB1RNA polymerases of orthomyxovirus.Use TREX- MAFFT sequence analysis softwares, compared with known orthomyxovirus PB1 amino acid sequences (for example coming from influenza virus), system hair Educate the phylogenetic evolution tree that chadogram is similar to TLV.The analysis result is presented in figure 3, this shows that the TLV of separation can be The new category in new emerging pathogens and orthomyxovirus section in the aquaculture of Tilapia mossambica kind.

Embodiment 2：Tilapia mossambica viral diagnosis

The embodiment shows in the culture of the infection from illness Tilapia mossambica or the organ (liver,spleen,kidney, brain) of infection TLV detection：

The TLV RNA in infection sample, which are detected, by PCR identifies that TLV is infectious.(BIO- according to the manufacturer's instructions RAD Cat#732-6820), using Aurum Total RNA Mini kits, from the TFC of virus infection or from from illness The organ of Tilapia mossambica generates sample RNA, and (Thermo scientific Cat#AB 1453/ according to the manufacturer's instructions A), using reverse transcriptase Verso cDN kits, cDNA is generated from the RNA of separation.Then according to standard scheme, generation is used CDNA as template, and utilize for identify contig 4 (SEQ ID NOs 25-26) one group of primer carry out RT-PCR It reacts (as shown in Figure 8).Contig 4 includes the conserved domain of presumption, and it is sub- to belong to influenza RNA Dependent RNA polymerase Unit PB1.

The specific correlation between 12 contigs of identification has been displayed in Table 1.Table 1 is the TLV weights of 16 observations The summary of folded group is compared.Sucrose gradient purified TLV is sequenced using next-generation sequencing technologies.Genome analysis shows 16 not Same contig (in the range of 400-1620bp), with the known array dissmilarity occurred in GenBank.In order to determine this Whether a little sequences are actually associated with TLV, and design specific primer is to expand each sequence, and use is not felt by infection and The primer sets in template that the TFC of dye is generated carry out RT-PCR.It is viral source that be shown in has 12 in 16 contigs, only It appears in the culture of infection and (is shown in Table 1).It is considered as to four contigs that the culture for infecting and being uninfected by is positive Unpub endogenous Tilapia mossambica sequence (runic).

The summary of the RT-PCR results for 16 contigs that the specific primer of table 1 measures, specific primer design is every It is a to be measured for the template of self-infection and the TFC being uninfected by.

The detection of TLV is confirmed in the figure 7, is detected in the culture of infection using 19 primer of contig for display The running gel of TLV, and this shows that TLV has during its breeding cycle not by the rna gene group in DNA stages.Overlapping Group 19 is only expanding (Fig. 7 swimming lanes 1) when being measured on the culture of infection after reverse transcriptase reaction in RT-PCR reactions.With The sample that 19 primer of contig measures the DNA from the cell being uninfected by and from the cell extraction for infecting and being uninfected by is feminine gender (Fig. 7 swimming lanes 2,4 and 5).Use genomic DNA and cDNA in the extract of the primer validation test of amplification Tilapia mossambica endogenous gene Presence (Fig. 7 swimming lane 6-9).Using Aurum Total RNA Mini kits (BIO-RADCat#732-6820) from infection RNA is extracted with the TFC being uninfected by.Use reverse transcriptase Verso cDN kits (Thermo scientific Cat#AB 1453/A) generate total cDNA.Using DNeasy (QIAGEN Cat#69504) DNA is generated from the TFC for infecting and being uninfected by.

As indicated, other than display contig is with the specific correlation of TLV, Fig. 7 also shows that TLV has in its life Do not pass through the rna gene group in DNA stages (comparing swimming lane 5,6,8 and 9) during the life period.

Embodiment 3：It is attenuated generation and the characterization of TLV vaccine strains

In order to generate attenuated virus strains, TLV isolates were constantly passed on for 20 generations on Rofe fin cell, and to pass The intensity experience UV irradiations (Fig. 4) of increasing.In 6.8mJ/cm²Sublethal dose under, to be selected and breed be single to virus clone Virus clone.

In order to test the attenuated clones of presumption, by impregnating 1 hour, sieve is inoculated with two kinds of clonal virus and placebo Non- fish.The group of inoculation is：(1) placebo：There is no the culture medium of viral antigen；(2) TLV p10 (clone 6)-exist virus It is passed on 10 times in culture, UV is irradiated and cloned；(3) TLVp20 (clone 4)-passes on TLVp10 10 times in culture, UV irradiates and clone.Monitor fish the death rate or with the relevant other adverse events of disease, continue 35 days (Fig. 5).

The survival rate of placebo is 100%, and exception is not observed during entire research.It is exposed to TLVp10 (clones 6) group shows and is exposed to relevant about 20% death rate of TLV viruses really.Do not have with the fish of TLVp20 (clone 4) inoculation Show any disease sign, and on the feed with it is suitable with placebo group in behavior pattern.TLV p20 (clone 4), also referred to as For TLV clones 4, CNCM (Institute Pasteur) is deposited on April 1st, 2016, registration number is CNCM I-5075.The clone Partial sequence characterization disclose the missings of at least section 4 and 5 of TLV genomes and (be respectively indicated as SEQ IDNOs 49 herein With 50).It recognizes, the mutant specific parts of these sequences can be used for detecting attenuated strain, and by they and wild-type bacteria Strain distinguishes.

In order to determine the effect of vaccine, the fish to be survived in security with the wild type TLV virus injections of high titre is wild Type TLV viruses pass on 4 times in the Rofe fin cell of culture.A part for placebo is not attacked by wild-type virus It hits, to exclude any incoherent death rate in the entire effect part of research.

The result of effect attack is shown in figure 6.Placebo under attack show be obviously TLV infection dead mould Formula.Fish becomes lethargic sleep and loss of appetite, the colour-darkening of fish-skin, and starts death within 12 days after exposure, reaches peak at 20 days Value, and reach 100% death rate during potency test.The placebo for being not affected by attack is without incident, and is shown Showing does not have disease sign.Show the death rate that delay starts with the fish of TLV p10 (clone 6) inoculation, opens within 20 days only after attack Begin, and peak value reaches 28% death rate.Disease sign is not shown with the fish of TLV p20 (clone 4) inoculation, and is expert at It is upper suitable with placebo that is being not affected by attack.The mortality level of 2.9% (single fish) is observed during observation.

These are the result shows that TLV p20 (clone 4) are shown for the safety of test and the advantageous inoculation of efficacy parameter Vaccine overview.

Embodiment 4：The generation of the TLV vaccine strains of inactivation and characterization

In order to generate the TLV vaccine strains of inactivation, TLV is exposed to 0.2% formaldehyde and is passed on until exposed virus It is non-infectious.Then by injecting and not injecting business adjuvant, the inactivated strain that separation is tested on live fish is assigned to TLV The ability of the resistance of infection.As a result it shows in fig.9.As shown in the figure, when 28 days before attack inject comprising inactivation TLV (0.2% formaldehyde) and incomplete Freund's adjuvant-IFA (1:1) when lotion, pass through the Tilapia mossambica that IP injects TLV attacks The death rate of (10gr) drop to 33% from the death rate of control group 91.7% (11/12) (8/24, relative percentage survival= RPS=63.4).On the contrary, individually injecting TLV (0.2% first inactivated to Tilapia mossambica (10gr) when 28 days before being attacked with TLV Aldehyde) or inject comprising placebo culture medium and incomplete Freund's adjuvant-IFA (1 from the cell being uninfected by:1) when lotion, There is no immunoprotection, the death rate is 100% (being respectively 26/26 and 24/24).

In view of many possible embodiments that can apply the open principle invented, it should be appreciated that the implementation of explaination Mode is only the preferred embodiment of the present invention, and is not construed as limiting the scope of the invention.But the scope of the present invention is by weighing Profit requires to be limited.Therefore, the whole inventions fallen in these scope and spirit of the claims are claimed in we.

Sequence table

<110>Kovacs company

<120>The just glutinous sample virus of Tilapia mossambica

<130> 3099/1.2

<150> US 62/276,873

<151> 2016-01-10

<150> US 62/352,570

<151> 2016-06-21

<160> 50

<170> PatentIn version 3.5

<210> 1

<211> 1620

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<213>Tilapia mossambica lake virus

<400> 1

ctcttaatta cgcactatta ctgtactacc ataaggtatg tgggcatttc aagaaggagt 60

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agagtcaata gacagagcgt ctgaaatcat gacaggaaaa tcgtacaatg ctgtccacac 180

tggggactta agtaagctgc ctaatcaggg agaaagccca ctgaggatag ttgattccga 240

cctctactca gagaggagtt gctgttgggt tatagagaag gagggcagaa ttgtgtgcaa 300

aagtaccacg ctcacccgcg gtatgacgag cctgttgaac acaacaaagt gtagttctcc 360

atctgagctc atatgtaagg ttttgacagt ggaatcccta tctgaaaaga taggtgatac 420

gagcgtcgag gagttacttt ctcatggcag gtactttaag tgcgcacttc gcgaccaaga 480

gaggggtaaa cccaagagca gagctatctt tctgtcacat cctttcttca ggttgctttc 540

ctctgtagta gagacgcacg ctagatctgt gctgtcaaag gtctcagcag tgtacaccgc 600

tactgctagt gcagaacaac gggctatgat ggccgcacag gttgtagagt caagaaaaca 660

tgttcttaat ggcgactgta ctaagtataa tgaggcaatc gacgcagaca cactgctaaa 720

agtgtgggat gcaataggca tggggtcaat tggagtcatg ctcgcttaca tggtgcgcag 780

gaaatgcgtt ctcattaaag acactctagt agagtgtcca ggaggtatgt tgatggggat 840

gtttaacgca actgccacct tggcactgca agggacgact gacagattcc tgtctttcag 900

cgacgacttt ataacatcgt ttaactcgcc tgctgaatta cgcgagatag aggatctgct 960

tttcgtaagc tgtcataact tgtcgctaaa gaagagttac atttcagttg cctcactgga 1020

aataaactcg tgtaccctca ctagggacgg tgacctagcc acagggttag gttgtactgc 1080

tggtgtcccc ttcagggggc cacttgtgac tctgaaacag actgcagcta tgttatctgg 1140

cgctgttgac tcaggagtta tgccattcca ctcagcagaa cgtctgttcc agataaagca 1200

gcaggaatgt gcctataggt ataacaaccc cacttacaca acgaggaatg aggacttcct 1260

ccccacatgc ctgggaggga agactgtaat tagctttcaa tctctactga cttgggattg 1320

ccacccattt tggtaccaag tgcaccctga tggcccagac actatagatc agaaagtcct 1380

gtctgtcctt gcctcaaaga ctcgcagaag gagaacccga ctagaggctc tctcagactt 1440

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agctaggcag gctcacttga agtccttggg cttggaacaa cccacaaact ttaactatgc 1560

tatttataaa gcagtccagc ccaccgctgg gtgctaagta actatatagg cgaatgagag 1620

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Met Trp Ala Phe Gln Glu Gly Val Cys Lys Gly Asn Leu Leu Ser Gly

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Pro Thr Ser Met Lys Ala Pro Asp Ser Ala Ala Arg Glu Ser Ile Asp

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Arg Ala Ser Glu Ile Met Thr Gly Lys Ser Tyr Asn Ala Val His Thr

35 40 45

Gly Asp Leu Ser Lys Leu Pro Asn Gln Gly Glu Ser Pro Leu Arg Ile

50 55 60

Val Asp Ser Asp Leu Tyr Ser Glu Arg Ser Cys Cys Trp Val Ile Glu

65 70 75 80

Lys Glu Gly Arg Ile Val Cys Lys Ser Thr Thr Leu Thr Arg Gly Met

85 90 95

Thr Ser Leu Leu Asn Thr Thr Lys Cys Ser Ser Pro Ser Glu Leu Ile

100 105 110

Cys Lys Val Leu Thr Val Glu Ser Leu Ser Glu Lys Ile Gly Asp Thr

115 120 125

Ser Val Glu Glu Leu Leu Ser His Gly Arg Tyr Phe Lys Cys Ala Leu

130 135 140

Arg Asp Gln Glu Arg Gly Lys Pro Lys Ser Arg Ala Ile Phe Leu Ser

145 150 155 160

His Pro Phe Phe Arg Leu Leu Ser Ser Val Val Glu Thr His Ala Arg

165 170 175

Ser Val Leu Ser Lys Val Ser Ala Val Tyr Thr Ala Thr Ala Ser Ala

180 185 190

Glu Gln Arg Ala Met Met Ala Ala Gln Val Val Glu Ser Arg Lys His

195 200 205

Val Leu Asn Gly Asp Cys Thr Lys Tyr Asn Glu Ala Ile Asp Ala Asp

210 215 220

Thr Leu Leu Lys Val Trp Asp Ala Ile Gly Met Gly Ser Ile Gly Val

225 230 235 240

Met Leu Ala Tyr Met Val Arg Arg Lys Cys Val Leu Ile Lys Asp Thr

245 250 255

Leu Val Glu Cys Pro Gly Gly Met Leu Met Gly Met Phe Asn Ala Thr

260 265 270

Ala Thr Leu Ala Leu Gln Gly Thr Thr Asp Arg Phe Leu Ser Phe Ser

275 280 285

Asp Asp Phe Ile Thr Ser Phe Asn Ser Pro Ala Glu Leu Arg Glu Ile

290 295 300

Glu Asp Leu Leu Phe Val Ser Cys His Asn Leu Ser Leu Lys Lys Ser

305 310 315 320

Tyr Ile Ser Val Ala Ser Leu Glu Ile Asn Ser Cys Thr Leu Thr Arg

325 330 335

Asp Gly Asp Leu Ala Thr Gly Leu Gly Cys Thr Ala Gly Val Pro Phe

340 345 350

Arg Gly Pro Leu Val Thr Leu Lys Gln Thr Ala Ala Met Leu Ser Gly

355 360 365

Ala Val Asp Ser Gly Val Met Pro Phe His Ser Ala Glu Arg Leu Phe

370 375 380

Gln Ile Lys Gln Gln Glu Cys Ala Tyr Arg Tyr Asn Asn Pro Thr Tyr

385 390 395 400

Thr Thr Arg Asn Glu Asp Phe Leu Pro Thr Cys Leu Gly Gly Lys Thr

405 410 415

Val Ile Ser Phe Gln Ser Leu Leu Thr Trp Asp Cys His Pro Phe Trp

420 425 430

Tyr Gln Val His Pro Asp Gly Pro Asp Thr Ile Asp Gln Lys Val Leu

435 440 445

Ser Val Leu Ala Ser Lys Thr Arg Arg Arg Arg Thr Arg Leu Glu Ala

450 455 460

Leu Ser Asp Leu Asp Pro Leu Val Pro His Arg Leu Leu Val Ser Glu

465 470 475 480

Ser Asp Val Ser Lys Ile Arg Ala Ala Arg Gln Ala His Leu Lys Ser

485 490 495

Leu Gly Leu Glu Gln Pro Thr Asn Phe Asn Tyr Ala Ile Tyr Lys Ala

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Val Gln Pro Thr Ala Gly Cys

515

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<213>Tilapia mossambica lake virus

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actctctatt accaaataca tttacttctg aaaaatgagt cagtttgaga aatcattcaa 60

gggcagaact gaggtcacaa taaccgaata tcgctctcat actgtcaaag atgtgcacag 120

aagcttactt acggctgaca aatctctaag gaagtcattc tgctttagga acgccctaaa 180

ccagttcttg gataaagatt tgcctctttt gcccattcgg ccaaaattag agtccagggt 240

tgctgtgaaa aagtctaagc tgaggagtca gctgtcgttc agacccggtt tgactcagga 300

ggaagcaatt gatctttaca acaagggcta tgatggtgac agcgtctcag gtgccttaca 360

agacagggta gtcaatgagc ctgtagctta ctcgagtgca gataatgaca aatttcacag 420

gggcttggcg gctctagggt acactttggc tgatagagca tttgatacgt gcgaatccgg 480

cttcgtgaga gcaattccta ctactccatg tgggttcata tgttgtgggc caggttcttt 540

caaagattca cttggatttg tgataaaaat cggcgaattc tggcacatgt atgacgggtt 600

ccaacacttc gtcgctgtcg aagatgctaa gttcttagca agtaagtctc cttcgttttg 660

gttggcaaaa cgtcttgcaa agaggctgaa tctggtccca aaagaggatc catctgtagc 720

agcagctgag tgcccttgta aaaaagtgtg ggaagctagt tttgctaggg cacctactgc 780

actagatcca tttggaggca gggccttctg cgaccagggt tgggtgtacc acagggacgt 840

agggtatgca acagctaacc acatatcaca ggagacactt tttcaacagg cgctttcagt 900

gaggaacctt ggaccgcaag gtagtgcaaa tgtctcaggc tcaatacata ccgccctgga 960

caggctcaga gcagcgtaca gtaggggaac acccgcctct agatctatac ttcaagggct 1020

tgcgaatctc atcacacctg taggtgaaaa ctttgaatgt gatctcgaca agaggaagct 1080

caatataaag gcattacgtt ctcccgagag gtacattacg atagagggcc tggttgtaaa 1140

cctggacgat gtggttagag ggttctacct tgacaaggcg aaagtcactg ttctctcgag 1200

atcaaagtgg atgggttacg aggacttgcc tcag 1234

<210> 4

<211> 400

<212> PRT

<213>Tilapia mossambica lake virus

<400> 4

Met Ser Gln Phe Glu Lys Ser Phe Lys Gly Arg Thr Glu Val Thr Ile

1 5 10 15

Thr Glu Tyr Arg Ser His Thr Val Lys Asp Val His Arg Ser Leu Leu

20 25 30

Thr Ala Asp Lys Ser Leu Arg Lys Ser Phe Cys Phe Arg Asn Ala Leu

35 40 45

Asn Gln Phe Leu Asp Lys Asp Leu Pro Leu Leu Pro Ile Arg Pro Lys

50 55 60

Leu Glu Ser Arg Val Ala Val Lys Lys Ser Lys Leu Arg Ser Gln Leu

65 70 75 80

Ser Phe Arg Pro Gly Leu Thr Gln Glu Glu Ala Ile Asp Leu Tyr Asn

85 90 95

Lys Gly Tyr Asp Gly Asp Ser Val Ser Gly Ala Leu Gln Asp Arg Val

100 105 110

Val Asn Glu Pro Val Ala Tyr Ser Ser Ala Asp Asn Asp Lys Phe His

115 120 125

Arg Gly Leu Ala Ala Leu Gly Tyr Thr Leu Ala Asp Arg Ala Phe Asp

130 135 140

Thr Cys Glu Ser Gly Phe Val Arg Ala Ile Pro Thr Thr Pro Cys Gly

145 150 155 160

Phe Ile Cys Cys Gly Pro Gly Ser Phe Lys Asp Ser Leu Gly Phe Val

165 170 175

Ile Lys Ile Gly Glu Phe Trp His Met Tyr Asp Gly Phe Gln His Phe

180 185 190

Val Ala Val Glu Asp Ala Lys Phe Leu Ala Ser Lys Ser Pro Ser Phe

195 200 205

Trp Leu Ala Lys Arg Leu Ala Lys Arg Leu Asn Leu Val Pro Lys Glu

210 215 220

Asp Pro Ser Val Ala Ala Ala Glu Cys Pro Cys Lys Lys Val Trp Glu

225 230 235 240

Ala Ser Phe Ala Arg Ala Pro Thr Ala Leu Asp Pro Phe Gly Gly Arg

245 250 255

Ala Phe Cys Asp Gln Gly Trp Val Tyr His Arg Asp Val Gly Tyr Ala

260 265 270

Thr Ala Asn His Ile Ser Gln Glu Thr Leu Phe Gln Gln Ala Leu Ser

275 280 285

Val Arg Asn Leu Gly Pro Gln Gly Ser Ala Asn Val Ser Gly Ser Ile

290 295 300

His Thr Ala Leu Asp Arg Leu Arg Ala Ala Tyr Ser Arg Gly Thr Pro

305 310 315 320

Ala Ser Arg Ser Ile Leu Gln Gly Leu Ala Asn Leu Ile Thr Pro Val

325 330 335

Gly Glu Asn Phe Glu Cys Asp Leu Asp Lys Arg Lys Leu Asn Ile Lys

340 345 350

Ala Leu Arg Ser Pro Glu Arg Tyr Ile Thr Ile Glu Gly Leu Val Val

355 360 365

Asn Leu Asp Asp Val Val Arg Gly Phe Tyr Leu Asp Lys Ala Lys Val

370 375 380

Thr Val Leu Ser Arg Ser Lys Trp Met Gly Tyr Glu Asp Leu Pro Gln

385 390 395 400

<210> 5

<211> 1228

<212> DNA

<213>Tilapia mossambica lake virus

<400> 5

ccaattaccg tctaagaatt taccagtttt tgatgacttg ttgaagaata tatagaaaac 60

ttgggctcag aggggccctt ggcagcctgt gcagctttcc gcgcatgggt ggcctatttc 120

gttgcctatc ttccaacagc tcctgctgtc tctttgaggc agtgatacag actcttccca 180

cttctcttac attttatgac accgtcctgt ttctgaatgg caatgcactc ttccagcaga 240

tttgtattcc ctgtcccctc aatcctggag tcggcaattg ctgagagccc gtggctagcc 300

accaccagga agcagcttcc tgtagcctgc agaggtagac caacaacaac accaatactc 360

ccgtccccct gcgaggggct ccaacactca gcaaggacac cgtcgcaaag gccccaagca 420

attgaggcaa gcaactccct gagggtgacc tggtcgccta tctgcagcct gtggccaatg 480

atcagaaacc tcggaggcgg gattatagca gtctgattca actttttgta gttctttcct 540

ctttctcctg ctgctgcttt ctttgcaaaa tagccaccaa ccagccctgg gcaagcacga 600

taggaatccc cactcaggta tgggaaccac tgagcatcct ccaaagtctg aatcctctcg 660

tcgagagcaa ccaaactgac gtacctagcc ttccagacct caatgcgaac acacactctc 720

aatagagagt tatggtcctt catcatttga atcatagctg cgtcccctga ggcttttatc 780

accttcatct ttgagcccag tgcaggcaca caggttgcgc agtattcagt ccatctgctg 840

gttaatataa ggctccttcc gaccctcatc cctgcttctg gcgccttcct tgatttctta 900

gcgttctccc tctctctctt cttttcactg gctctcagaa ccttagcctc ctgaccatcg 960

agggagtgag tgtgcatttt aaagaggaat gcagcagcat cagatatcac actgttgtcc 1020

tccctgttaa aggaacttcc aaacacatga ccacaagctt cattgaaaac ctcaaggttt 1080

cttgggagtt cagcttgtgc ctgcgaagta ctgggtgaac tttcatccat tgctacctct 1140

ggtgcaacag tgctggcagc tgccatacta gtctttgtag ttctcaccat tttcagtttg 1200

ttaagttagc tattctgggt aataggag 1228

<210> 6

<211> 354

<212> PRT

<213>Tilapia mossambica lake virus

<400> 6

Met Val Arg Thr Thr Lys Thr Ser Met Ala Ala Ala Ser Thr Val Ala

1 5 10 15

Pro Glu Val Ala Met Asp Glu Ser Ser Pro Ser Thr Ser Gln Ala Gln

20 25 30

Ala Glu Leu Pro Arg Asn Leu Glu Val Phe Asn Glu Ala Cys Gly His

35 40 45

Val Phe Gly Ser Ser Phe Asn Arg Glu Asp Asn Ser Val Ile Ser Asp

50 55 60

Ala Ala Ala Phe Leu Phe Lys Met His Thr His Ser Leu Asp Gly Gln

65 70 75 80

Glu Ala Lys Val Leu Arg Ala Ser Glu Lys Lys Arg Glu Arg Glu Asn

85 90 95

Ala Lys Lys Ser Arg Lys Ala Pro Glu Ala Gly Met Arg Val Gly Arg

100 105 110

Ser Leu Ile Leu Thr Ser Arg Trp Thr Glu Tyr Cys Ala Thr Cys Val

115 120 125

Pro Ala Leu Gly Ser Lys Met Lys Val Ile Lys Ala Ser Gly Asp Ala

130 135 140

Ala Met Ile Gln Met Met Lys Asp His Asn Ser Leu Leu Arg Val Cys

145 150 155 160

Val Arg Ile Glu Val Trp Lys Ala Arg Tyr Val Ser Leu Val Ala Leu

165 170 175

Asp Glu Arg Ile Gln Thr Leu Glu Asp Ala Gln Trp Phe Pro Tyr Leu

180 185 190

Ser Gly Asp Ser Tyr Arg Ala Cys Pro Gly Leu Val Gly Gly Tyr Phe

195 200 205

Ala Lys Lys Ala Ala Ala Gly Glu Arg Gly Lys Asn Tyr Lys Lys Leu

210 215 220

Asn Gln Thr Ala Ile Ile Pro Pro Pro Arg Phe Leu Ile Ile Gly His

225 230 235 240

Arg Leu Gln Ile Gly Asp Gln Val Thr Leu Arg Glu Leu Leu Ala Ser

245 250 255

Ile Ala Trp Gly Leu Cys Asp Gly Val Leu Ala Glu Cys Trp Ser Pro

260 265 270

Ser Gln Gly Asp Gly Ser Ile Gly Val Val Val Gly Leu Pro Leu Gln

275 280 285

Ala Thr Gly Ser Cys Phe Leu Val Val Ala Ser His Gly Leu Ser Ala

290 295 300

Ile Ala Asp Ser Arg Ile Glu Gly Thr Gly Asn Thr Asn Leu Leu Glu

305 310 315 320

Glu Cys Ile Ala Ile Gln Lys Gln Asp Gly Val Ile Lys Cys Lys Arg

325 330 335

Ser Gly Lys Ser Leu Tyr His Cys Leu Lys Glu Thr Ala Gly Ala Val

340 345 350

Gly Arg

<210> 7

<211> 1023

<212> DNA

<213>Tilapia mossambica lake virus

<400> 7

tctcaatcaa gcacttaaaa ctgtacctgg gcatttcgcc cacacgatag gacatatagt 60

gtcacatgta tttattgatt ttacagcagg atcttactgt cactggtggt gatatcgaaa 120

gatcaattac aggattcaat ctacgcattt ttgtatagct aagaaaagta cgagcaagaa 180

gataagcatt tgacttctta taagcaactt catcctgcat cgcggaccac tcgctgatga 240

gctggtgacg ctttatttcg gctggtagta ggacttccac tgcggaaaag tggcacttgt 300

atggggagtg attctcctcc agtgactcaa ctaataccca acactgtttt gtgctattgt 360

ctctgcagct ccacttgtat tcagttctga agccgtttgg cgtcatatat gttctatcta 420

tagtacgctt acagtttgta caaaggccac gcgacattag catacaggtg ctaaagatgg 480

gcctcctctt cgaaaggcac agcgatgtct caaacagttt agagcttatt tttatagtac 540

gtattttacc tcggcccggt attccccggc tctccaatat cttgtcagtt acaggctcca 600

gttcttttct cgatattccc gggcacaaaa caaatcctcc aagcataaag tttagcgtcg 660

ggttacatcg tttctcgaca aagttgcagt tcagatgatg gagttcccct tttgatccct 720

ccaaatcata catgtagtat ggagatccac ctgagtctat gacttcgttg ttaccatcaa 780

gtcccagaac cactttctgt actgctggat ctagataatc actactcgaa tatgaacctt 840

caacagaata tttgtccccg tcaataacta cgcttgacgt gtagtttgat ctcccgtaaa 900

accgccacat gttgtccacg cattgatcgc ttccgctaaa gagtgttgaa aacaaagtaa 960

gggttcttat caccctaagc caactcattg gacaatcctg tagataaaaa tgcatgcgag 1020

agg 1023

<210> 8

<211> 317

<212> PRT

<213>Tilapia mossambica lake virus

<400> 8

Met His Phe Tyr Leu Gln Asp Cys Pro Met Ser Trp Leu Arg Val Ile

1 5 10 15

Arg Thr Leu Thr Leu Phe Ser Thr Leu Phe Ser Gly Ser Asp Gln Cys

20 25 30

Val Asp Asn Met Trp Arg Phe Tyr Gly Arg Ser Asn Tyr Thr Ser Ser

35 40 45

Val Val Ile Asp Gly Asp Lys Tyr Ser Val Glu Gly Ser Tyr Ser Ser

50 55 60

Ser Asp Tyr Leu Asp Pro Ala Val Gln Lys Val Val Leu Gly Leu Asp

65 70 75 80

Gly Asn Asn Glu Val Ile Asp Ser Gly Gly Ser Pro Tyr Tyr Met Tyr

85 90 95

Asp Leu Glu Gly Ser Lys Gly Glu Leu His His Leu Asn Cys Asn Phe

100 105 110

Val Glu Lys Arg Cys Asn Pro Thr Leu Asn Phe Met Leu Gly Gly Phe

115 120 125

Val Leu Cys Pro Gly Ile Ser Arg Lys Glu Leu Glu Pro Val Thr Asp

130 135 140

Lys Ile Leu Glu Ser Arg Gly Ile Pro Gly Arg Gly Lys Ile Arg Thr

145 150 155 160

Ile Lys Ile Ser Ser Lys Leu Phe Glu Thr Ser Leu Cys Leu Ser Lys

165 170 175

Arg Arg Pro Ile Phe Ser Thr Cys Met Leu Met Ser Arg Gly Leu Cys

180 185 190

Thr Asn Cys Lys Arg Thr Ile Asp Arg Thr Tyr Met Thr Pro Asn Gly

195 200 205

Phe Arg Thr Glu Tyr Lys Trp Ser Cys Arg Asp Asn Ser Thr Lys Gln

210 215 220

Cys Trp Val Leu Val Glu Ser Leu Glu Glu Asn His Ser Pro Tyr Lys

225 230 235 240

Cys His Phe Ser Ala Val Glu Val Leu Leu Pro Ala Glu Ile Lys Arg

245 250 255

His Gln Leu Ile Ser Glu Trp Ser Ala Met Gln Asp Glu Val Ala Tyr

260 265 270

Lys Lys Ser Asn Ala Tyr Leu Leu Ala Arg Thr Phe Leu Ser Tyr Thr

275 280 285

Lys Met Arg Arg Leu Asn Pro Val Ile Asp Leu Ser Ile Ser Pro Pro

290 295 300

Val Thr Val Arg Ser Cys Cys Lys Ile Asn Lys Tyr Met

305 310 315

<210> 9

<211> 926

<212> DNA

<213>Tilapia mossambica lake virus

<400> 9

cataatcctc tattagaacg tcgtaacctt tagcgaaagc gtcgaaagcg atcatctcgc 60

aaatgggtgt actgtcatcc gcaatcttac tgcacaaagt gaataataaa gtgagcttaa 120

gggtattgta ccccttatct cagaagccag ctggtagcct ttcctgaact cgtctttaca 180

gacgctaagt gctagccggt gcctattaaa atgttgagcc actgcttggc taacccttgt 240

agagtcgagg cattccagaa gtaagatgac gtcccatctt gtctcaagac cactagctct 300

gtccagatca cccttcctac ttatgggagg tagttccaac atatccagct tgtaaatttc 360

tcgggtactc acaaagtctt gctcccctat attagccgat tgcttgggat ctaggtgcat 420

cacatgcaca gctgccctgt acccgtcaaa ctcaaaatcg tgttcacagc caggtttact 480

tagacctaca actaagtggt gagtggaggc ggttggtctc cttttactgt gctttccaga 540

gtcgcgcatg actggtacag ctagtatgct ggtattatcg ctatgcagta ctttccctgc 600

ctgagttgtg cttctagcaa tcaacatcaa aagctcacga gcaagtgggg cactagccgg 660

tagaggtaat atcttctgtg tagcaggctt atgagaagca actgtatacc tttgtatcca 720

ccctccattg cggaactcaa attctctatc acgtgcgtac tcgttcagta taagttctct 780

tgcctcctgg tcaagaccac attcctcacc gcaggcgagg aactttgagc actcgaagaa 840

tccatattgc ctctttagct cagctgtctc cttggatatg tccgcaagtc tgggtggtgc 900

cacccactcg atacgaggct tcgggc 926

<210> 10

<211> 290

<212> PRT

<213>Tilapia mossambica lake virus

<400> 10

Pro Lys Pro Arg Ile Glu Trp Val Ala Pro Pro Arg Leu Ala Asp Ile

1 5 10 15

Ser Lys Glu Thr Ala Glu Leu Lys Arg Gln Tyr Gly Phe Phe Glu Cys

20 25 30

Ser Lys Phe Leu Ala Cys Gly Glu Glu Cys Gly Leu Asp Gln Glu Ala

35 40 45

Arg Glu Leu Ile Leu Asn Glu Tyr Ala Arg Asp Arg Glu Phe Glu Phe

50 55 60

Arg Asn Gly Gly Trp Ile Gln Arg Tyr Thr Val Ala Ser His Lys Pro

65 70 75 80

Ala Thr Gln Lys Ile Leu Pro Leu Pro Ala Ser Ala Pro Leu Ala Arg

85 90 95

Glu Leu Leu Met Leu Ile Ala Arg Ser Thr Thr Gln Ala Gly Lys Val

100 105 110

Leu His Ser Asp Asn Thr Ser Ile Leu Ala Val Pro Val Met Arg Asp

115 120 125

Ser Gly Lys His Ser Lys Arg Arg Pro Thr Ala Ser Thr His His Leu

130 135 140

Val Val Gly Leu Ser Lys Pro Gly Cys Glu His Asp Phe Glu Phe Asp

145 150 155 160

Gly Tyr Arg Ala Ala Val His Val Met His Leu Asp Pro Lys Gln Ser

165 170 175

Ala Asn Ile Gly Glu Gln Asp Phe Val Ser Thr Arg Glu Ile Tyr Lys

180 185 190

Leu Asp Met Leu Glu Leu Pro Pro Ile Ser Arg Lys Gly Asp Leu Asp

195 200 205

Arg Ala Ser Gly Leu Glu Thr Arg Trp Asp Val Ile Leu Leu Leu Glu

210 215 220

Cys Leu Asp Ser Thr Arg Val Ser Gln Ala Val Ala Gln His Phe Asn

225 230 235 240

Arg His Arg Leu Ala Leu Ser Val Cys Lys Asp Glu Phe Arg Lys Gly

245 250 255

Tyr Gln Leu Ala Ser Glu Ile Arg Gly Thr Ile Pro Leu Ser Ser Leu

260 265 270

Tyr Tyr Ser Leu Cys Ala Val Arg Leu Arg Met Thr Val His Pro Phe

275 280 285

Ala Arg

290

<210> 11

<211> 897

<212> DNA

<213>Tilapia mossambica lake virus

<400> 11

ctgagcagcg agagaaccat cagcgaaagc accaggtaat agacaaactt atatttctct 60

agcatcccaa acactattgc aaggatagct tccccaaagg ctgcaaattg ctccttgaat 120

ttgcctatgt tatcagattt acagacgagg gatttaatcg agcaaaactc tcctagcgtc 180

ataccggtga cttcccgtgt caaagcttct aaatactccc aaatctcaga aacatctgct 240

tgagctgcat ctgctaggcc gatgacccca gcatagttgt aaacacccat gccaattgct 300

actgagctag caagtaaaca cagtataatg aggcctgcta ctacaaacga cttcggctgc 360

cttgaggcca gcatggtctt gtagtcgtcc acagtgggtc tggtacagtc acactgacct 420

ggcagtacgg tggaatacac ctccaaatct ctgcactcgc tactgtggct gcacctggaa 480

acgttgtata gaacaacccc actatcttcg tcaatcctaa cttcagtagc tttccaatca 540

cctcttcccc ctgctttggg aagtttgtac ccgagggctg ggaagctgtt aggtactgca 600

tgtaacccag tgcagaacga cccacccgag gatggggtta caatgtagtg cacccccata 660

gtgcaggagc aactctccat cctgaatttg gatgcaaatc cacgccttat ttcactttct 720

atctccctac tttggtaggt attgtttaga tatgattgta gtcgttttat ccttaaactt 780

tcatcacttg ctagtgcgca aactaggcag cttatcagga ccagcgcctg catagctatt 840

ggagtctgag ataagagaaa catttggctc catcctgaat ttggatgcaa atccacg 897

<210> 12

<211> 298

<212> PRT

<213>Tilapia mossambica lake virus

<400> 12

Val Asp Leu His Pro Asn Ser Gly Trp Ser Gln Met Phe Leu Leu Ser

1 5 10 15

Gln Thr Pro Ile Ala Met Gln Ala Leu Val Leu Ile Ser Cys Leu Val

20 25 30

Cys Ala Leu Ala Ser Asp Glu Ser Leu Arg Ile Lys Arg Leu Gln Ser

35 40 45

Tyr Leu Asn Asn Thr Tyr Gln Ser Arg Glu Ile Glu Ser Glu Ile Arg

50 55 60

Arg Gly Phe Ala Ser Lys Phe Arg Met Glu Ser Cys Ser Cys Thr Met

65 70 75 80

Gly Val His Tyr Ile Val Thr Pro Ser Ser Gly Gly Ser Phe Cys Thr

85 90 95

Gly Leu His Ala Val Pro Asn Ser Phe Pro Ala Leu Gly Tyr Lys Leu

100 105 110

Pro Lys Ala Gly Gly Arg Gly Asp Trp Lys Ala Thr Glu Val Arg Ile

115 120 125

Asp Glu Asp Ser Gly Val Val Leu Tyr Asn Val Ser Arg Cys Ser His

130 135 140

Ser Ser Glu Cys Arg Asp Leu Glu Val Tyr Ser Thr Val Leu Pro Gly

145 150 155 160

Gln Cys Asp Cys Thr Arg Pro Thr Val Asp Asp Tyr Lys Thr Met Leu

165 170 175

Ala Ser Arg Gln Pro Lys Ser Phe Val Val Ala Gly Leu Ile Ile Leu

180 185 190

Cys Leu Leu Ala Ser Ser Val Ala Ile Gly Met Gly Val Tyr Asn Tyr

195 200 205

Ala Gly Val Ile Gly Leu Ala Asp Ala Ala Gln Ala Asp Val Ser Glu

210 215 220

Ile Trp Glu Tyr Leu Glu Ala Leu Thr Arg Glu Val Thr Gly Met Thr

225 230 235 240

Leu Gly Glu Phe Cys Ser Ile Lys Ser Leu Val Cys Lys Ser Asp Asn

245 250 255

Ile Gly Lys Phe Lys Glu Gln Phe Ala Ala Phe Gly Glu Ala Ile Leu

260 265 270

Ala Ile Val Phe Gly Met Leu Glu Lys Tyr Lys Phe Val Tyr Tyr Leu

275 280 285

Val Leu Ser Leu Met Val Leu Ser Leu Leu

290 295

<210> 13

<211> 756

<212> DNA

<213>Tilapia mossambica lake virus

<400> 13

ctcatgctac catccttagt gaacggtact aagtaccata agaagctttc agaccaatta 60

tccctgcttt caaaagtaat tagttaactt agaaaggcct ccccaacaag acaacgaaac 120

agctgggtaa ttagagttca aatgtgatcc ctttagggat tggcactaac ccaactctgt 180

ggtaggaaca ccacgattca ttgacatcag ttgcaccctt acttatagcc tctaggtatg 240

tggcaaccct gctcaactca gtcagtggtc gagtgctggt caccagcgct gatctcttgg 300

tccagtgtgg aaactttgga gatggcgcac agagccagta ctttgcttcc cttctagtgt 360

agaaggtagt tgaacagaga tgcatgtccc ctttgactag ataggctcct ttcgggcctc 420

ttactgctgt accctcctgg gcaagtaccc ttctgactgg taatccgggg ctcacttcat 480

acttccattt caaaggtacc ttttctccac catcggttgg gtgactcgtc aatacaatcc 540

aggttgtgcc atttgtattg ggtgtaaacc cagttgcaca aaagaaccat acgttcgagg 600

caccagagta atgctctagg agcagtcgat ggaacagccc agtctgactt ccgctgtcct 660

tggggagggt gcttttgcgc gtgataattc tctcaagctc accaatcttg taggacattg 720

tgtctattac tatacggtat gcaatgcaaa gagagt 756

<210> 14

<211> 207

<212> PRT

<213>Tilapia mossambica lake virus

<400> 14

Ser Leu Cys Ile Ala Tyr Arg Ile Val Ile Asp Thr Met Ser Tyr Lys

1 5 10 15

Ile Gly Glu Leu Glu Arg Ile Ile Thr Arg Lys Ser Thr Leu Pro Lys

20 25 30

Asp Ser Gly Ser Gln Thr Gly Leu Phe His Arg Leu Leu Leu Glu His

35 40 45

Tyr Ser Gly Ala Ser Asn Val Trp Phe Phe Cys Ala Thr Gly Phe Thr

50 55 60

Pro Asn Thr Asn Gly Thr Thr Trp Ile Val Leu Thr Ser His Pro Thr

65 70 75 80

Asp Gly Gly Glu Lys Val Pro Leu Lys Trp Lys Tyr Glu Val Ser Pro

85 90 95

Gly Leu Pro Val Arg Arg Val Leu Ala Gln Glu Gly Thr Ala Val Arg

100 105 110

Gly Pro Lys Gly Ala Tyr Leu Val Lys Gly Asp Met His Leu Cys Ser

115 120 125

Thr Thr Phe Tyr Thr Arg Arg Glu Ala Lys Tyr Trp Leu Cys Ala Pro

130 135 140

Ser Pro Lys Phe Pro His Trp Thr Lys Arg Ser Ala Leu Val Thr Ser

145 150 155 160

Thr Arg Pro Leu Thr Glu Leu Ser Arg Val Ala Thr Tyr Leu Glu Ala

165 170 175

Ile Ser Lys Gly Ala Thr Asp Val Asn Glu Ser Trp Cys Ser Tyr His

180 185 190

Arg Val Gly Leu Val Pro Ile Pro Lys Gly Ile Thr Phe Glu Leu

195 200 205

<210> 15

<211> 635

<212> DNA

<213>Tilapia mossambica lake virus

<400> 15

catctttaca caaatggagt agcttacctc cctggggaaa atgacactga agattcactt 60

tttatttaag catttcacgg aaatgattga tagcagcaga agtgtctgcc ttaagggcca 120

tcctgtcatc ttgtgaacct tcagtgtcct tcaaagcagt tgccaggact tcctcgagaa 180

aagcgagcgc agtgggtatg tcgacacagt taattccagg gagcttaagc ttcatagctt 240

gtttggcaat atccaggtac tgttttcgat tgaattcaaa tctttgaccc ttgcggtttg 300

ttattataat tgcgtcgact gttacactga ctcgcctcga gattcgtccc agcctttcct 360

ctatccgctt tcgggtcatg ccggcgtgct taacaagcag gtgtttaaac tttgtttctt 420

ctgagaggcc ttcgccaatt gttagggaaa gcaaatccgg acttgcattg acaagaaact 480

ccagagcaac tacagtgttg actgtgtctc tagtcactgt catgccattg agcgtgaaat 540

cgtagagctt cccttggccc tctcttagcg ttggaatttg agccatactt attcctggat 600

atgctgtcca attggaaatg ttagtgtaga tgagg 635

<210> 16

<211> 190

<212> PRT

<213>Tilapia mossambica lake virus

<400> 16

Leu Ile Tyr Thr Asn Ile Ser Asn Trp Thr Ala Tyr Pro Gly Ile Ser

1 5 10 15

Met Ala Gln Ile Pro Thr Leu Arg Glu Gly Gln Gly Lys Leu Tyr Asp

20 25 30

Phe Thr Leu Asn Gly Met Thr Val Thr Arg Asp Thr Val Asn Thr Val

35 40 45

Val Ala Leu Glu Phe Leu Val Asn Ala Ser Pro Asp Leu Leu Ser Leu

50 55 60

Thr Ile Gly Glu Gly Leu Ser Glu Glu Thr Lys Phe Lys His Leu Leu

65 70 75 80

Val Lys His Ala Gly Met Thr Arg Lys Arg Ile Glu Glu Arg Leu Gly

85 90 95

Arg Ile Ser Arg Arg Val Ser Val Thr Val Asp Ala Ile Ile Ile Thr

100 105 110

Asn Arg Lys Gly Gln Arg Phe Glu Phe Asn Arg Lys Gln Tyr Leu Asp

115 120 125

Ile Ala Lys Gln Ala Met Lys Leu Lys Leu Pro Gly Ile Asn Cys Val

130 135 140

Asp Ile Pro Thr Ala Leu Ala Phe Leu Glu Glu Val Leu Ala Thr Ala

145 150 155 160

Leu Lys Asp Thr Glu Gly Ser Gln Asp Asp Arg Met Ala Leu Lys Ala

165 170 175

Asp Thr Ser Ala Ala Ile Asn His Phe Arg Glu Met Leu Lys

180 185 190

<210> 17

<211> 560

<212> DNA

<213>Tilapia mossambica lake virus

<400> 17

ccccttaatc cttaatagac cgttaacttt cttttgaaat ggactcgcgg tttgcacagc 60

taactggggt tttctgtgac gatttcactt atagcgaagg gagtcgaagg ttcctaagtt 120

cttacagtac agtagagaga cgtccaggag tccccgtaga gggtgactgt tatgactgtt 180

tgaagaataa gtggattgcc tttgagctgg aaggccagcc gcggaaattt ccaaaggcga 240

cagttcgttg cattttgaac aatgatgcca catacgtttg ctctgagcaa gagtaccagc 300

agatttgtaa ggtacaattc aaggattatt tggagatcga cggggttgtt aaagttgggc 360

acaaggcatc ctacgatgct gagctaaggg aacggctatt ggaactacca catccaagga 420

gtggcccgaa gcctcgtatc gagtgggtgg caccacccag acttgcggac atatccaagg 480

agacagctga actaaagagg caatatggat tcttcgagtg ctcaaagttc ctcgcctgcg 540

gtgaggaatg tggtcttgac 560

<210> 18

<211> 174

<212> PRT

<213>Tilapia mossambica lake virus

<400> 18

Met Asp Ser Arg Phe Ala Gln Leu Thr Gly Val Phe Cys Asp Asp Phe

1 5 10 15

Thr Tyr Ser Glu Gly Ser Arg Arg Phe Leu Ser Ser Tyr Ser Thr Val

20 25 30

Glu Arg Arg Pro Gly Val Pro Val Glu Gly Asp Cys Tyr Asp Cys Leu

35 40 45

Lys Asn Lys Trp Ile Ala Phe Glu Leu Glu Gly Gln Pro Arg Lys Phe

50 55 60

Pro Lys Ala Thr Val Arg Cys Ile Leu Asn Asn Asp Ala Thr Tyr Val

65 70 75 80

Cys Ser Glu Gln Glu Tyr Gln Gln Ile Cys Lys Val Gln Phe Lys Asp

85 90 95

Tyr Leu Glu Ile Asp Gly Val Val Lys Val Gly His Lys Ala Ser Tyr

100 105 110

Asp Ala Glu Leu Arg Glu Arg Leu Leu Glu Leu Pro His Pro Arg Ser

115 120 125

Gly Pro Lys Pro Arg Ile Glu Trp Val Ala Pro Pro Arg Leu Ala Asp

130 135 140

Ile Ser Lys Glu Thr Ala Glu Leu Lys Arg Gln Tyr Gly Phe Phe Glu

145 150 155 160

Cys Ser Lys Phe Leu Ala Cys Gly Glu Glu Cys Gly Leu Asp

165 170

<210> 19

<211> 521

<212> DNA

<213>Tilapia mossambica lake virus

<400> 19

gtccgattac tttttccgct tggtgatgtc acgatggata gaaaatacag attctgtgtc 60

agtaatcttg acagagatga gtcggtcgta cgtcactttg tgccattacc ccccttggag 120

cttgtgctgc ggcggcaaga catcacaacc tggtcaagtc tggatcctgg atcgaaaaca 180

ttgtctagga tgttcagaga tctcagagtt aatgacactg agtcagccaa cttggcagga 240

gagtgcaatg gtgataggga gctgggtcca agtagtaacg gagcacggaa ttttgcacac 300

ttcaacatcg gaaaggcagg cgccaagaag ggtcatgtgg aggatatctg acatggctgg 360

cgataggact ttatgagggc ggtccagggg caattgaagc tccgcgaacc tactgactcc 420

tcagctagaa cattatagtg aaccatgtga cattgataat ttagctttag taaaaaaatg 480

gataagcttc agctctggca aagtatgact ttaaggacgt g 521

<210> 20

<211> 116

<212> PRT

<213>Tilapia mossambica lake virus

<400> 20

Val Arg Leu Leu Phe Pro Leu Gly Asp Val Thr Met Asp Arg Lys Tyr

1 5 10 15

Arg Phe Cys Val Ser Asn Leu Asp Arg Asp Glu Ser Val Val Arg His

20 25 30

Phe Val Pro Leu Pro Pro Leu Glu Leu Val Leu Arg Arg Gln Asp Ile

35 40 45

Thr Thr Trp Ser Ser Leu Asp Pro Gly Ser Lys Thr Leu Ser Arg Met

50 55 60

Phe Arg Asp Leu Arg Val Asn Asp Thr Glu Ser Ala Asn Leu Ala Gly

65 70 75 80

Glu Cys Asn Gly Asp Arg Glu Leu Gly Pro Ser Ser Asn Gly Ala Arg

85 90 95

Asn Phe Ala His Phe Asn Ile Gly Lys Ala Gly Ala Lys Lys Gly His

100 105 110

Val Glu Asp Ile

115

<210> 21

<211> 124

<212> PRT

<213>Tilapia mossambica lake virus

<400> 21

Ser Asp Tyr Phe Phe Arg Leu Val Met Ser Arg Trp Ile Glu Asn Thr

1 5 10 15

Asp Ser Val Ser Val Ile Leu Thr Glu Met Ser Arg Ser Tyr Val Thr

20 25 30

Leu Cys His Tyr Pro Pro Trp Ser Leu Cys Cys Gly Gly Lys Thr Ser

35 40 45

Gln Pro Gly Gln Val Trp Ile Leu Asp Arg Lys His Cys Leu Gly Cys

50 55 60

Ser Glu Ile Ser Glu Leu Met Thr Leu Ser Gln Pro Thr Trp Gln Glu

65 70 75 80

Ser Ala Met Val Ile Gly Ser Trp Val Gln Val Val Thr Glu His Gly

85 90 95

Ile Leu His Thr Ser Thr Ser Glu Arg Gln Ala Pro Arg Arg Val Met

100 105 110

Trp Arg Ile Ser Asp Met Ala Gly Asp Arg Thr Leu

115 120

<210> 22

<211> 509

<212> DNA

<213>Tilapia mossambica lake virus

<400> 22

ctcttttctc agtttaccac tttatgacct accaggaata gaagcttaag gatcaagata 60

atggaagcag agggacttcg tcatcctcag gcgggctttt cttcttaagc cgcttcttaa 120

tatagaaaaa ggtcttgaag cacacaactc ttagcctccg gaatactaaa actttgatac 180

tcccatagaa gggcacctgc ttcaacagag aaacaagttt actgagcagc gagagaacca 240

tcagcgaaag caccaggtaa tagacaaact tatatttctc tagcatccca aacactattg 300

caaggatagc ttccccaaag gctgcaaatt gctccttgaa tttgcctatg ttatcagatt 360

tacagacgag ggatttaatc gagcaaaact ctcctagcgt cataccggtg acttcccgtg 420

tcaaagcttc taaatactcc caaatctcag aaacatctgc ttgagctgca tctgctaggc 480

cgatgacccc agcatagttg taaacaccc 509

<210> 23

<211> 151

<212> PRT

<213>Tilapia mossambica lake virus

<400> 23

Gly Val Tyr Asn Tyr Ala Gly Val Ile Gly Leu Ala Asp Ala Ala Gln

1 5 10 15

Ala Asp Val Ser Glu Ile Trp Glu Tyr Leu Glu Ala Leu Thr Arg Glu

20 25 30

Val Thr Gly Met Thr Leu Gly Glu Phe Cys Ser Ile Lys Ser Leu Val

35 40 45

Cys Lys Ser Asp Asn Ile Gly Lys Phe Lys Glu Gln Phe Ala Ala Phe

50 55 60

Gly Glu Ala Ile Leu Ala Ile Val Phe Gly Met Leu Glu Lys Tyr Lys

65 70 75 80

Phe Val Tyr Tyr Leu Val Leu Ser Leu Met Val Leu Ser Leu Leu Ser

85 90 95

Lys Leu Val Ser Leu Leu Lys Gln Val Pro Phe Tyr Gly Ser Ile Lys

100 105 110

Val Leu Val Phe Arg Arg Leu Arg Val Val Cys Phe Lys Thr Phe Phe

115 120 125

Tyr Ile Lys Lys Arg Leu Lys Lys Lys Ser Pro Pro Glu Asp Asp Glu

130 135 140

Val Pro Leu Leu Pro Leu Ser

145 150

<210> 24

<211> 442

<212> DNA

<213>Tilapia mossambica lake virus

<400> 24

ctctgacacc ctgtatagtt agcgttggcc tgtggatacg aacgaaatca gaaccgatat 60

taaggtgcta agactgcacg tcaagagact tctttccgaa atcttcggaa aatcgagata 120

ggtcactctg cccatcatcc tctctgtccc ttctgttttt gggattgaag tcgaccctag 180

cccactctgg gattgcagaa tcacagtcgt ccatctcgag gtcgacttcg tcaccccact 240

ctatattgtc ttcaccgctc tcgtcagcac catacctttc attcttccaa cttcgcttct 300

ttgaagcagc tttcttgccc ttcttgacct tccgacttct tagtactaaa cagcctgagc 360

tctcagcccc cgagtcactg tcacttgaca aataatctgc cacactcatc ctggcttata 420

gctatatttg gtgttagtag gg 442

<210> 25

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 25

cagggagaaa gcccactgag 20

<210> 26

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 26

ctccaattga ccccatgcct 20

<210> 27

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 27

gagtccaggg ttgctgtgaa 20

<210> 28

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 28

aaagaacctg gcccacaaca 20

<210> 29

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 29

ctcaatcctg gagtcggcaa 20

<210> 30

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 30

tcgcattgag gtctggaagg 20

<210> 31

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 31

tggcacttgt atggggagtg 20

<210> 32

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 32

gtatgctaat gtcgcgtggc 20

<210> 33

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 33

gggtgtactg tcatccgcaa 20

<210> 34

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 34

aaacctggct gtgaacacga 20

<210> 35

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 35

cctagcgtca taccggtgac 20

<210> 36

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 36

ggtcgttctg cactgggtta 20

<210> 37

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 37

cctccccaac aagacaacga 20

<210> 38

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 38

cagaagggta cttgcccagg 20

<210> 39

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 39

cctctatccg ctttcgggtc 20

<210> 40

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 40

caacgctaag agagggccaa 20

<210> 41

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 41

cgaagggagt cgaaggttcc 20

<210> 42

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 42

caacgaactg tcgcctttgg 20

<210> 43

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 43

gtcgtacgtc actttgtgcc 20

<210> 44

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 44

gcaaaattcc gtgctccgtt 20

<210> 45

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 45

ggaagcagag ggacttcgtc 20

<210> 46

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 46

gtcaccggta tgacgctagg 20

<210> 47

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 47

ctcgagatgg acgactgtga 20

<210> 48

<211> 20

<212> DNA

<213>Tilapia mossambica lake virus

<400> 48

gttggcctgt ggatacgaac 20

<210> 49

<211> 595

<212> DNA

<213>Tilapia mossambica lake virus p20 clone 4

<220>

<221> misc_feature

<222> (339)..(339)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (496)..(496)

<223> n is a, c, g, or t

<400> 49

gcaaatcttt ctccaattac cgtctaagaa tttaccagtt tttgatgact tgttgaagaa 60

tatataggaa acttgggctc agaggggccc ttggcagcct gtgcagcttt ccgcgcatgg 120

gtggcctatt tcgttgccta tcttccaaca gctcctgctg tctctttgag gcagtgatac 180

agactcttcc cacttctctt acattttatg acaccgtcct gtttctgaat ggcaatgcac 240

tcttccagca gatttgtatt ccctgtcccc tcaatcctgg agtcggcaat tgctgagagc 300

ccgtggctag ccaccaccag gaagcagctt cctgtagcnt gcagaatgca gcagcatcag 360

atatcacact gttgtcctcc ctgttaaagg aacttccaaa cacatgacca caagcttcat 420

tgaaaacctc aaggtttctt gggagttcag cttgtgcctg cgaagtactg ggtgaacttt 480

catccattgc tacctnctgg tgcaacagtg ctggcagctg ccatatctag tgtttgtagt 540

tctcaccatt tcagttttta agttagctat tctgggtaat aggagtaaac tttgg 595

<210> 50

<211> 411

<212> DNA

<213>Tilapia mossambica lake virus p20 xlone 4

<220>

<221> misc_feature

<222> (36)..(36)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (136)..(136)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (202)..(202)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (252)..(252)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (270)..(270)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (294)..(294)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (369)..(369)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (380)..(380)

<223> n is a, c, g, or t

<400> 50

ggaagcagag ggacttggtc atcctcaggc gggctnttct tcttaagccg cttcttaata 60

tagaaaaagg tcttgaagca cacaactctt agcctccgga atactaaaac tttgatactc 120

ccatagaagg gcaccngctt caacagagaa acaagtttac tgagcagcga gagaaccatc 180

agcgaaagca ccaggtaata gncaaactta tatttctcta gcatcccaaa cactattgca 240

aggatagctt cnccaaaggc tgcaaattgn tccttgaagt tgcctatgtt atcntcaatc 300

ctaacttcag tagctttcca atcacctctt ccccctgctt gggaagtttg tacccgaggg 360

cgtgaagcnt ggagctgttn ggtactgcat gtaacccagt gcacaacgac c 411

Claims

1. a kind of attenuation Rofe fish virus (TLV) of the separation generated by following methods, the method includes：

The TLV is passed at least 10 times by Rofe fry cell,

The TLV is irradiated with sub- lethal radiation dose；With

Detach the attenuated clones of infection.

2. the attenuation TLV of separation according to claim 1, wherein the TLV is passaged to 20 times less by Rofe fry cell.

3. the attenuation TLV of the separation according to claim 1 or claim 2, wherein the Asia lethal radiation dose be 3.2-6.8mJ/cm²Between UV radiation dose.

4. the attenuation TLV of separation according to claim 3, wherein the Asia lethal radiation dose is 6.8mJ/cm²。

5. the attenuation TLV of separation according to claim 1 comprising with registration number CNCM I-5075 be deposited in CNCM (bar Si De research institutes) attenuation TLV.

6. a kind of immunogenic composition comprising the attenuation TLV of separation according to any one of claims 1-5, and Adjuvant.

7. one kind is for Rofe fish virus (TLV) infection to the method for subject's vaccine inoculation comprising：

With the attenuation TLV infected subjects of separation according to any one of claims 1-5 or will be according to claim 6 institute The immunogenic composition stated is applied to subject, to subject's vaccine inoculation.

8. a kind of just glutinous sample virus of separation, have by with SEQ ID NOs.1,3,5,7,9,11,13,15,17,19,22 and The genome of each of 24 at least 90% same nucleic acid sequence compositions.

9. a kind of nucleic acid of separation comprising at least 90% same nucleic acid of nucleic acid selected from SEQ ID NOs 25-48.

10. the nucleic acid of separation according to claim 9 further comprises detectable label.

11. the nucleic acid of separation according to claim 10, wherein the detectable label is fluorescent marker, radioactivity mark Note or chemical labeling.

12. method of the one kind for detecting Rofe fish virus (TLV) infection comprising：

The subject that TLV has been infected from suspection detaches sample；

CDNA templates are generated from the sample；With

Using at least one Oligonucleolide primers from the cDNA template amplifications nucleic acid, at least one Oligonucleolide primers packet It includes and is selected from SEQ ID Nos：At least 90% same nucleic acid sequence of at least one sequence of 25-48, more control sequences institute The amplification for stating primer indicates that there are TLV in the sample.

13. described at least one according to the method for claim 12, wherein the amplification is carried out at least one primer pair Primer pair have with selected from least 90% same sequence of following primer pair：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；35 Hes of SEQ ID NOs 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；With SEQ ID NOs 47 and 48.

14. one kind for detect Rofe fish virus (TLV) infection kit comprising it is according to claim 9 at least A kind of nucleic acid of separation.

15. the kit according to claim 14 for detecting Rofe fish virus (TLV) infection, wherein described at least one Kind separation nucleic acid be have and at least one Oligonucleolide primers selected from least 90% same sequence of following primer pair It is right：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；With SEQ ID NOs 47 and 48.

16. according to the kit described in claim 14 or claim 15, further comprise detectable label.

17. a kind of inactivation Rofe fish virus (TLV) of the separation generated by following methods, the method includes：

TLV is incubated in formaldehyde；With

The TLV is passed on by Rofe fry cell, until the TLV no longer infectious.

18. a kind of immunogenic composition comprising the TLV and adjuvant of the inactivation of separation according to claim 17.

19. one kind is for Rofe fish virus (TLV) infection to the method for subject's vaccine inoculation comprising：

By the composition of the TLV of the inactivation including separation according to claim 17 or according to claim 18 exempt from Epidemic disease Immunogenic Compositions are applied to subject, to subject's vaccine inoculation.