KR101273264B1 - Epitope in fish nodavirus protein and a recombinant vaccine using the same - Google Patents

Abstract

The present invention provides a polypeptide comprising an epitope of a protein of a fish nodavirus protein, a vaccine, an antibody, a method of immunizing a fish using the same, and a method of ameliorating a fish nodavirus disease.

Description

Epitope of Fish Nodavirus Protein and Recombinant Vaccine Using the Same {Epitope in Fish Nodavirus Protein and A Recombinant Vaccine USing The Same}

The present invention relates to a polypeptide comprising an epitope of a protein that provides antigenicity among fish nodavirus proteins and a vaccine, an antibody, a method of immunizing a fish using the same, and a method of diagnosing a fish nodavirus disease.

Fish nodaviruses have a positive-sense single-stranded RNA as a gene and have a 25-30 nm sized icosahedron shape without an outer membrane. The virus that causes disease in fish is classified as the genus betanodavirus.

Fish nodavirus is a pathogen causing viral encephalopathy and retinopathy (VER) and viral nervous necrosis (VNN). It is known to cause.

The larvae and larvae infected with these fish nodaviruses have a 100% mortality rate. It frequently occurs in the flounder, a representative fish in Korea, and has a high mortality rate when there are many organic matters or other diseases and complex infections in the farming environment.

Thus, the development of a vaccine is required to prevent damage caused by fish nodavirus. In particular, chemicals used to treat diseases occurring in fish are being used, and there is a risk of harming the human body when humans use the fish ingested with chemicals as food. Therefore, there is a growing need for the development and use of vaccines using the fish's own immune system.

Accordingly, the present inventors came to discover the epitope of the protein of fish nodavirus and to complete the present invention while studying the economic and effective method for preventing fish nodavirus.

An object of the present invention is a polypeptide comprising an epitope of a fish nodavirus protein, a recombinant expression vector comprising a polynucleotide encoding the polypeptide, a host cell transformed with the vector, a recombinant protein produced by the host cell To provide.

It is another object of the present invention to provide a vaccine composition comprising an epitope of a fish nodavirus protein, an antibody against said epitope.

Another object of the present invention is to provide a method for immunizing fish using the vaccine composition.

Still another object of the present invention is to provide a method and diagnostic kit for diagnosing fish nodavirus disease using the antibody.

Still another object of the present invention is to provide a feed composition and a feed additive comprising the vaccine.

In one aspect of the invention, the invention provides a polypeptide comprising an epitope of a fish nodavirus protein, consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

The fish nodavirus can be isolated from the flounder.

The polypeptide may be selected from capsid protein, B1 and B2 protein of fish nodavirus.

In one embodiment of the present invention, the present invention provides a recombinant expression vector comprising a polynucleotide encoding the polypeptide, a host cell transformed with the recombinant expression vector, and a recombinant protein produced by the host cell.

The recombinant expression vector, host cell and recombinant protein can be made by methods well known in the art.

In another aspect, the present invention provides a vaccine composition comprising an epitope of a fish nodavirus protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

The vaccine may be a live vaccine, an attenuated vaccine or a dead vaccine, and the vaccine may also be used as a feed or feed additive.

The vaccine according to the present invention may further contain an adjuvant. The adjuvant can be used without limitation any known in the art. For example, sugars and amino acids as stabilizers, mineral oils, vegetable oils, alum, aluminum phosphate, bentonite, silica, muralmyl dipeptide derivatives, thymosin, interleukin, and the like can be used as stabilizers.

The vaccine may be administered intraperitoneally of fish and may be inoculated once or repeatedly during spawning. The inoculation amount is the same as a conventional fish virus vaccine dose.

In another aspect, the present invention provides a method of immunizing a fish by administering the vaccine composition to the fish. The vaccine composition may be mixed with a bacterial inactivation vaccine and administered to fish, and the bacterial inactivation vaccine may be a streptococcal inactivation vaccine. The vaccine composition and the bacterial inactivating vaccine may be mixed in a mixing ratio generally used in the art, and preferably 1: 1. In the case of a bacterial virus combination vaccine, various diseases can be prevented at once, which is advantageous in terms of economic and time advantages over a single vaccine.

There is no restriction on the fish that is the target of the immunity, and may include a flounder with high fear of Nodavirus infection.

In another aspect, the present invention provides an antibody that specifically recognizes an epitope of a fish nodavirus protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

The antibody is prepared by using a polypeptide comprising an epitope of a fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 1 as an antigen, and may be an antibody that recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

The antibody may be an antibody that is prepared by using a polypeptide containing an epitope of a fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 2 as an antigen and recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

The antibody may be an antibody that is prepared by using a polypeptide containing an epitope of a fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 3 as an antigen and recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.

The antibody may be a monoclonal or polyclonal antibody, and may be used for fish nodaviruses of fish such as halibut.

In another aspect of the invention, the invention comprises reacting the antibody or fragment of the antibody to a sample comprising a fish nodavirus, and determining that the sample exhibiting a positive response to the antibody is infected with a fish nodavirus. Provides a method for diagnosing fish nodavirus disease.

Confirmation of the positive reaction may be performed by a method of detecting a conventional antibody-antigen reaction. For example, a label selected from the group consisting of radioisotopes, fluorescent materials, luminescent materials, enzymes, chromogens, and dyes is labeled on the antibody or fragment of the antibody, and the reaction is performed by the label. The results can be detected and performed. ELISA etc. are mentioned as a specific method.

The present invention also provides a kit for diagnosing fish nodavirus diseases comprising an antibody or a fragment of the antibody that recognizes the epitope. The kit is the same as a conventional nucleic acid kit except that an oligonucleotide comprising the nucleotide sequence of SEQ ID NOS: 1 to 3 according to the present invention is used as a probe.

Using the epitope of fish nodavirus according to the present invention, it is possible to develop vaccines and antibodies effective against fish nodavirus, and to quickly diagnose fish nodavirus diseases.

Figure 1 shows a gene schematic of fish nodavirus.
Figure 2 is a result obtained by purely separating the recombinant protein expressed in E. coli using a nickel column SDS-PAGE electrophoresis.
Figure 3 shows the anti-JFNVV-WD neutralizing activity and the reactivity of the viral protein in five serum samples obtained from JFNVV-infected flounder.
Figure 4 shows the results of Western blot analysis performed with antiserum obtained from flounder injected with nodavirus.
Figure 5 shows the results of Western blot performed for the antibody (anti-recombinant protein) obtained by injecting the recombinant protein capsid, B1 and B2 protein in the flounder according to an embodiment of the present invention.
6 is a graph showing antibody titers against fish nodaviruses by ELISA and neutralization test.
7 is a graph showing the relative survival rate of the olive flounder after treatment with recombinant protein vaccine and Streptococcus inactivating vaccine according to an embodiment of the present invention.
Figure 8 shows the ELISA antibody titers of the olive flounder for a recombinant protein vaccine according to an embodiment of the present invention.
Fig. 9 shows ELISA antibody titers of halibut serum injected with recombinant protein against purely isolated nodavirus (antiserum value of infected halibut against recombinant protein).
Figure 10 shows the antibody titers of the sea bream (A) and flounder (B) injected with the vaccine according to an embodiment of the present invention.
11 to 12 show the tissue changes of the flounder two weeks after the treatment of the flounder with the vaccine according to one embodiment of the present invention.

Hereinafter, the structure of the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to the following examples.

Example  1: materials and methods

1-1: Cells and Viruses

GF-1 cells (purchased from ATCC) were subjected to 28 ° C. using L-15 medium (GibcoBRL, Carlsbad, USA) with 5% FBS (GibcoBRL, Carlsbad, USA) and 100 μg penicillin-streptomycin / ml. Was maintained. Betanodavirus JFNNV-WD isolated from cultured flounder in Korea was used (Acession No. FJ748760). (Cha et al., Phylogenic analysis of betanovirus isolated from cultured fish in Korea, Dis Aquat . Organ, 77: 181-189, 2007).

1-2: fish

Cultured olive flounder ( Paralichthys) with an average weight of 20 g olivaceus ) was used to prepare immune sera. The flounder was incubated in a 1 m ³ tank at about 25 ° C. under a flow through system. Prior to full-scale experiments, RT-PCR was performed by collecting brains and retinas from 10 randomly selected flounder fish stocks, and confirmed that the nonodavirus was negative.

1-3: virus (orus inoculum)

Virus was prepared from infected flounder (Cha et al., Dis Aquat. Organ, 77: 181-189, 2007). Infected brain and eyes are homogenized using the same volume of a minimum essential medium (MEM) Leibovitz L-15 Medium (Gibco BRL, USA) -SSN-1 cell medium (SSN-1 cells purchased from the European Cell Bank). And centrifuged at 1500 g for 15 minutes. The supernatant was obtained by passing through a 0.45 μm membrane filter and then stored at -80 ° C. The virus stock was named "JFNNV-WD" and the titer was confirmed to be 10 ⁷ TCID ₅₀ (median tissue culture infective dose) / ml.

1-4: cDNA Cloning

cDNAs were synthesized from total RNA of GF-1 cells infected with JFNNV-WD. PCR primers were prepared using the GenBank / EMBL database (GenBank accession no. FJ748760 and DQ864760) to amplify the full-length cDNAs corresponding to the capsids, B1 and B2 proteins of JFNNV-WD. Primer sequences are shown in Table 1 below.

primer order SEQ ID NO: Noda-capsid-f 5'- AAGCTT GCATGGTACGCAAGGGTGAGAA-3 ' 4 Noda-capsid-r 5'- CTCGAG GTTTTCCGAGTCAACCCTGGT-3 ' 5 Noda-B1-F 5'- GGATCC ATGTCTGTCGCATTGACGGG-3 ' 6 Noda-B1-R 5'- GTCGAC CACTTGAGTGCGACGTCGCCG-3 ' 7 Noda-B2-F 5'- GGATCC ATGGAACAAATCCAACAAGCG-3 ' 8 Noda-B2-R 5'- GTCGAC GTCCGTCTCCATCGGTTCCTC-3 ' 9

* F is forward primer, R is reverse primer, and italic letters indicate restriction enzyme positions.

PCR was performed with its cDNA using virus infected GF-1 cells as a template. Gene amplification conditions were as follows: 1 cycle at 94 ° C. for 5 minutes; 30 cycles at 92 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 1 minute; And 1 cycle at 72 ° C. for 5 minutes. The amplified PCR product was digested with restriction enzymes (Sal I (Hind III) and Xho I in the case of capsid; BamH I, Hind III (Sal I)) and then pET28a vector (Novagen, Darmstadt). , Germany) to produce plasmids. This plasmid was named "pET-B1-JFNNV", "pET-B2-JFNVV" and "pET-capsid-JFNVV". The sequence of the DNA insert was measured using an automated DNA sequencing device (Applied Biosystems, Inc., USA) at the Immunomodulation Research Center (IRC), Ulsan, Korea.

1-5: Expression and Purification of Recombinant Proteins

To express the recombinant protein of JFNNV-WD, E. coli BL21 (DE3) cells were transformed with pET-B1-JFNNV, pET-B2-JFNVV and pET-capsid-JFNVV. His-tagged proteins were used to express recombinant protein in E. coli and purified in Ni-NTA His-binding resin. This was done according to the manufacturer's manual.

1-6: Immunization and Antisera

100 ng of the purified recombinant protein per gram of flounder weight was administered twice in the flounder (20 g) abdominal cavity. Mineral oil was used as a supplement. Four weeks after the last immunization, bleeding occurred in the artery of the flounder, and serum was obtained through a centrifuge and stored at -20 ° C.

1-7: Serum Collection from Survival Fishes

20 flounders (20 g / fish) were administered intraperitoneally with a virus filtrate at a rate of 10 ⁵⁵ TCID ₅₀ / fish doses. Blood samples were collected from these fishes after 15 days of dosing. After overnight storage at 4 ° C., centrifugation at 1500 g for 5 minutes yielded serum and storage at 80 ° C. Serum was collected from five flounder untreated.

1-8: ELISA

Antisera from immunized flounder were carried out and antibody assays for capsid, B1 and B2 proteins were performed using ELISA. Microtiter plates (Nune, Roskilde, Denmark) were coated with 2 μg recombinant protein / ml for 16 hours at 4 ° C. in coating buffer (0.5 M carbonate-bicarbonate, pH 9.6). Antisera serially diluted by two-step dilution in PBS containing 0.05% Tween 20 was added and washed three times with PBS-Tween 20 (0.05%). Four wells without fish serum were used as negative controls. Rabbit anti-flounder immunoglobulin was prepared according to the methods described in the literature (Bang JD et al . , Dis Aquat. Organ ., 26: 197-203, 1996) and was used as secondary antibody. Alkaline-phosphate-conjugated goth anti-rabbit IgG (Sigma, St. Louis, USA) was added to the enzyme bound antibody and p-nitrophenyl phosphate (1 mg / ml), an alkaline phosphate substrate in 0.1 M NaHCO ₃ and 1.0 mM MgCl ₂ , pH 9.8) were detected. Absorbance was read at 405 nm. Antibody titers were expressed as the inverse of the serum dilution endpoints with OD ₄₀₅ twice as large as the negative control.

1-9: SDS-PAGE and Western Blotting

Recombinant protein was electrophoresed on 10% separatory gel under reducing conditions. The molecular weight was measured in comparison to the standard molecular weight (GIBCO-BRL). The proteins on acrylamide were transferred to a Hybond-P membrane (Amersham Bioscience, Inc., Buckinghamshire, UK) and the antisera obtained from flounder administered B1 and B2, capsid of JFNNV-WD, an infectious virion. I found it. Rabbit anti-flounder immunoglobulin (Ig) and horseradish peroxidase conjugated goth anti-rabbit IgG (Sigma) were added and the immune response was detected with an ECL detection system (Amersham Bioscience, Inc.).

1-10: Virus Neutralization Test

Antisera were serially diluted in two-step dilution with EMEM in 96-multiwell plates. 100 TCID _50/50 ㎕ of the new virus was added to each well. Plates were incubated with shaking continuously for 1 hour at room temperature. To each well, 100 μl of 1 × 10 ⁵ cells / ml of GF-1 cells were added, and each plate was further incubated at 28 ° C. for 7 days. The neutralizing antibody titer of the antiserum was expressed as the reciprocal of the highest dilution of the antiserum protecting 50% of the inoculated cells.

Example  2: vaccine manufacturing

2-1: Screening for Vaccine Genes

In order to develop a nodavirus vaccine, it is necessary to identify an immunogenic protein among the nodavirus proteins and to determine which of these proteins has a neutralizing epitope. To this end, nodaviruses were isolated from the flounder and injected into the flounder to obtain an antiserum to examine the immune response.

1 shows a schematic diagram of the nodavirus gene. As described in Example 1, full-length cDNAs corresponding to the capsids, B1 and B2 of JFNNV-WD were amplified using PCR. As shown in Figure 1, full-length B1 can be seen that corresponds to the C-terminal of protein A (aa 872-982).

Genes of the capsid, B1 and B2 proteins of Nodavirus isolated from the flounder were subcloned into pET28a expression vector and then expressed in E. coli BL21, as described in Example 1 above. Recombinant proteins expressed in E. coli were isolated using a nickel column. The recombinant protein was purified using His-tagged protein and analyzed using SDS-PAGE. The results are shown in Fig. The molecular weights of the capsid, B1 and B2 proteins predicted based on the amino acid sequence analyzer were, in turn, 37.0 KDa, 11.9 KDa and 8.5 KDa, respectively. The actual results shown in FIG. 2 showed that, due to the His-tag, each molecular weight was slightly higher than this.

To assess the immune response of flounder to fish nodavirus infection, antiserial JFNVV-WD neutralizing activity and viral protein reactivity were examined on five serum samples from JFNVV-infected flounder. As a result, as shown in Figure 3, JFNVV-infected flounder showed a high neutralizing activity when compared with the control.

In addition, Western blot analysis was performed to determine which protein of the nodavirus the antibody present in the serum. As a result of the Western blot analysis, it can be confirmed through the band shown in FIG. 4 that the antibodies of capsid, B1 and B2 are present in the serum of the flounder injected with nodavirus.

Amino acid sequence analysis of the capsid, B1 and B2 proteins revealed that the sequences of the capsid, B1 and B2 proteins were SEQ ID NOs: 1, 2 and 3, respectively.

2-2: Preparation of Vaccine

PCR products obtained using the respective primers prepared to express the capsid of Nodavirus, B1 and B2 recombinant proteins were eluted, cloned into pGEM-T easy vector, and each recombinant gene was present through colony PCR. Confirmed.

To express the recombinant protein of JFNNV-WD, E. coli BL21 (DE3) cells were transformed with pET-B1-JFNNV, pET-B2-JFNVV and pET-capsid-JFNVV. Thus, E. coli strains were synthesized to synthesize recombinant proteins.

The E. coli pET-B1-JFNNV, pET-B2-JFNVV and pET-capsid-JFNVV strains thus prepared were incubated at 37 ° C. in LB medium containing Kanamycin for 4 hours, followed by IPTG (Isopropyl-β-D -thio-galactoside) was added at 0.1 mg / mL to incubate for 6 hours to allow expression of the recombinant protein.

In order to confirm the recombinant protein thus expressed, bacterial lysate was confirmed by SDS-PAGE electrophoresis. The protein concentration was diluted to 200 ug / kg (body weight) /0.1 mL and used as a vaccine. Mass expressed proteins were separated using a nickel affinity column.

2-3: Antiserum Production and Neutralization Experiment of Recombinant Protein

Next, in order to confirm which neutralizing epitope is present in any of the three types of recombinant proteins, one antibody to each of the three types was made by extracting antisera by injecting each of the three types of recombinant proteins into a flounder. Western blot analysis was performed to confirm that these antibodies actually recognized each protein. As a result, as shown in Figure 5, it was confirmed that the antibodies to the capsid, B1 and B2 proteins were made.

In order to confirm whether the prepared antibody neutralizes infection of nodavirus, antibody titers and neutralization titers of capsids, B1 and B2 proteins, etc. of nodaviruses were determined through an Enzyme-linked immunosorbent assay (ELISA). As a result, as shown in Fig. 6, antibody titers for three types of proteins range from 2 to ⁷ It can be seen that the value of 2 ⁸ .

Next, it was analyzed through neutralization experiments whether these antibodies can neutralize nodavirus. As a result of neutralization experiment using nodavirus isolated from the flounder, the neutralization titers were 2 ^8.4 and 2 ^4.9 for the antibodies against the B1 and B2 proteins, respectively. 2 ^{15.3, which} was high. From these results, it was confirmed that the capsid protein of the three proteins isolated from the nodavirus has a relatively strong neutralizing epitope. This suggests that the capsid proteins are more suitable as antigens of vaccines of nodaviruses.

From the above results, it can be seen that the capsid, B1, and B2 proteins of nodaviruses are suitable for nodavirus vaccines as proteins having neutralizing epitopes.

Example  3: Vaccine efficacy test

3-1: Efficacy test by mixing with immunogenic recombinant protein vaccine and bacterial vaccine

The recombinant protein vaccine prepared in Example 2 (including capsid-Rep 1 and B1-Rep 2) alone or two of three recombinant proteins (capsid and B1 in a ratio of 1: 1) were mixed (Rep 1 + Rep 2), or a mixed vaccine (Streptococcus + Rep 1,2) containing a 1: 1 mixture of recombinant protein (capsid and B1 in a ratio of 1 to 1) and streptococcal inactivation vaccine The flounder was tested in which streptococcus iniae (JSL0208) and nodaviruses (JFNNV-WD (FJ748760)) were mixed and attacked.

As shown in FIG. 7, when the vaccine containing the recombinant protein and the streptococcus inactivated vaccine were mixed, the survival rate of the flounder was higher than that of each vaccine. In addition, non-specific immunity was observed in the two control groups, the recombinant protein vaccine-free group and the PBS-treated group. In addition, it was confirmed that the anti-virus protection effect of about 2 to 3 times higher in the test group using a mixture of two or more recombinant protein vaccines than when using the recombinant protein alone.

3-2: Analysis of efficacy of recombinant protein vaccine

Two weeks after injection of 0.1 ml of recombinant protein vaccine into the halibut (weight 20 ㅁ 1g) intraperitoneal and muscle, fish nodavirus cultured in GF cells at a concentration of 10 ⁵ TCID ₅₀ The survival, antibody titer and safety of the flounder against the virus were examined by infection.

ELISA antibody titers for recombinant capsid protein, B1 protein and B2 protein were measured and the results are shown in FIG. 8 and Table 2. FIG. It can be seen that the capsid proteins, proteins B1 and B2 antibodies are 5 ⁷ , 5 ⁸ and 5 ⁷ , respectively. Was investigated and the antibody against the virus Noda purified using a flatfish that serum immunized with the recombinant protein vaccine, the antibody is shown as ^57.

protein Antibody titer Capsid proteins 5 ⁷  B1 protein 5 ⁸  B2 protein 5 ⁷ Nodavirus ORF (cDNA) 5 ⁷

As a result of testing the neutralizing titer of nodavirus, as shown in Fig. 9, the neutralizing titers were 0.48 × 10 ⁵ and for the flounder sera of the nodavirus capsid, B1 and B2 protein, respectively. 0.35 × 10 ³ , 0.3 × 10 ² . In addition, the antibody titers were 5 ⁷ in all three proteins, as shown in Table 3 below.

protein Antibody titer Capsid proteins 5 ⁷ B1 protein 5 ⁷  B2 protein 5 ⁷

3-3: Identify target fish

It was confirmed that the antibody titers to the recombinant protein vaccines differ depending on fish species, which are shown in FIG. 10. Through this, it can be seen that the fish nodavirus has a high antibody value in the flounder. In addition, the antibody titer for iridovirus in rock dome is high, but the antibody titer for nodavirus is not high.

Example  4: safety investigation by vaccine administration

Histopathological examination was performed to investigate the safety of the vaccine treatment according to the present invention. Two weeks after the treatment of the recombinant protein vaccine, PBS, E. coli according to the present invention on the olive flounder, each tissue of the fish was fixed in 10% neutral formalin and subjected to H & E staining according to the conventional method. As shown in Figures 11 and 12, there is no specific degeneration change in the liver, spleen, kidney, heart, brain tissue, only due to prolonged fasting, seoid deposition in the spleen, kidney was noticeable. That is, it was confirmed that there is no toxicity of the vaccine according to the present invention.

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Claims (16)

delete delete delete delete A vaccine composition comprising an epitope of a fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 1 and an epitope of a fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 2.
The vaccine composition according to claim 5, wherein the vaccine is a live vaccine, an attenuated vaccine or a dead vaccine.
A method of immunizing fish, comprising administering the vaccine composition of claim 5 or 6 to the fish.
The method of claim 7, wherein the vaccine composition is mixed with a bacterial inactivating vaccine and administered to the fish.
The method of claim 8, wherein the bacterial inactivating vaccine is a streptococcal inactivating vaccine.
An antibody that specifically recognizes an epitope of a fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 1 and an fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 2.
The polypeptide of claim 10, wherein the antibody comprises a polypeptide comprising an epitope of a fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 1 and an epitope of a fish nodavirus protein consisting of the amino acid sequence of SEQ ID NO: 2 An antibody that is prepared by using an antigen and recognizes a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 or a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2.
delete delete The antibody of claim 10, wherein the antibody is a monoclonal or polyclonal antibody.
15. A method comprising reacting an antibody according to any one of claims 10, 11 and 14 with a sample comprising a fish nodavirus, and determining that the sample showing a positive response to the antibody is infected with a fish nodavirus. Diagnosis method of fish nodavirus disease to do.
15. A kit for diagnosing a fish nodavirus disease, comprising the antibody according to any one of claims 10, 11 and 14.

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