JPWO2010024284A1 - Fish antiviral vaccine, fish immunostimulant, and fish immunization method - Google Patents

Abstract

A fish antiviral vaccine containing double-stranded RNA and a pathogenic virus as active ingredients and a fish immunostimulant containing double-stranded RNA as active ingredients. According to the present invention, double-stranded RNA is administered or administered, and exposed to various types of pathogenic viruses, so-called live viruses that are not treated at all, or attenuated viruses, and incorporated into them. It is possible to induce, activate, modify or enhance the immunity of the target fish after inducing the antiviral state or simultaneously with inducing the antiviral state.

Description

  The present invention administers double-stranded RNA and administers various types of pathogenic viruses, so-called live viruses that do not treat them at all, or attenuated viruses, thereby immunizing target fish. The present invention relates to a fish antiviral vaccine capable of inducing, activating, modifying, or enhancing, fish immunostimulants that induce an antiviral state, and a method for conferring fish immunity.

  For example, in fish, infectious hematopoietic necrosis virus (IHNV), which targets salmon fish, and viral hemorrhagic septic virus (VHSV), which targets salmon and marine fish. ), Various pathogenic viruses or opportunistic pathogenic viruses such as fish Nodavirus (Fish Nodavirus) and red seabream Iridovirus (RSIV), which are marine fishes, are known and all have high pathogenicity.

  Among them, infectious hematopoietic necrosis (IHN), which is a causative agent of IHNV, is one of salmonid fish diseases and is a very serious disease that is extremely lethal. IHNV belongs to the genus Novirhabdovirus belonging to the Rhabdoviridae family, and has a negative single-stranded RNA genome consisting of about 11 k nucleotides, which are nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein, respectively. It contains 6 genes in the order of 3'-NPMG-NV-L-5 'encoding (G), virion nonstructural protein (NV), and polymerase (L). The pathogenicity of IHNV is generally targeted at juvenile fish, but in recent years, it has been revealed that it also extends to rainbow trout that has grown to a size that can be shipped to the market.

  In rainbow trout farms and the like, conventionally, specific pathogen-free (SPF) fish have been produced by disinfecting fish eggs with iodine or sterilizing the breeding seawater. In addition, enzyme immunoassay (ELISA) has been put to practical use in diagnosing IHNV infection in spawning fish that are not infected with a specific pathogen. However, it is difficult to prevent horizontal infection of IHNV in aquaculture ponds, and development of IHNV vaccines is indispensable. Conventionally, killed vaccines, attenuated vaccines, and genetically modified vaccines with IHNV G protein Several types of IHNV vaccines such as DNA vaccines having the IHNV G protein gene have been developed (Non-patent Document 1, etc.).

  On the other hand, Viral Nervous Necrosis (VNN), which is a causative agent of fish nodavirus, is extremely lethal in more than 20 of the marine teleosts that have been cultivated, and is very serious for marine teleosts. It can be said that it is one of the diseases. The fish nodavirus belongs to the genus Betanodavirus belonging to the Nodaviridae family, and is a spherical virus having a diameter of 25 to 30 nm lacking an envelope, and is composed of a single outer shell protein and two plus-strand ss-RNA molecules. Nodaviruses are four genotype viruses: striped jacked nervous necrosis virus (SJNNV), tiger puffer necrosis virus (TPNNV), Matsukawa neuronecrosis virus (BFNNV; Barfin Flounder NNV), and A nodavirus (TNNV; Turbot NNV), which has been classified as a red-spotted grouper NNV (RGNNV) and isolated from halibut, has been reported as a new genotype virus.

  In seedling production sites, etc., it is important to select SPF spawning fish in order to prevent vertical infection of fish nodavirus, but for fish grown in aquaculture ginger, horizontal infection of fish nodavirus through the breeding seawater In the past, several types of fish nodavirus vaccines such as a formalin-inactivated vaccine, a subunit vaccine containing a recombinant outer shell protein, and a virus-like particle vaccine have been developed (Non-patent Document 2). etc).

  Viral Hemorrhagic Septicemia (VHS), a pathogen caused by VHSV, was first observed in Japan in 1999 in cultured flounder in the Seto Inland Sea. It is regarded as one of the most serious diseases in cultured flounder in Asia. VHSV is a negative single-stranded virus belonging to the genus Novirhabdovirus belonging to the Rhabdoviridae family and having an envelope.

  In flounder seed and seedling production areas, vertical infection of VHSV is prevented and larvae that are not infected with VHSV are stably produced. However, with regard to growing fish that are bred in farmed ginger, Due to the problem of infection, several types of VHSV vaccines have been developed, such as killed vaccines, attenuated vaccines, genetically modified vaccines with VHSV glycoproteins, and DNA vaccines with VHSV G protein genes. (Non-Patent Document 3 etc.).

  In recent years, viruses such as IHNV, fish nodavirus, VHSV, infectious salmon anemia virus (ISAV), infectious pancreatic necrosis virus (IPNV), aquaeovirus (Aquareovirus), It has been reported that an antiviral state is induced by mixed infection with a virus such as aquabirnavirus (ABV; Aquabirnavirus) and picornavirus (Picornavirus). It has also been reported that synthetic double-stranded RNA polyinosine polycytidic acid (polyI: C), a Toll-like receptor (TRL) agonist, induces an antiviral state in various cell types. An adjuvant using double-stranded RNA and a vaccine containing double-stranded RNA have been developed (Patent Document 1 and Patent Document 2).

JP 2005-82581 A JP 2005-97267 A

Lorenzen N. et al. (2002) Dev. Comp. Immunol., 26: 173-179 Tanaka S. et al. (2001) J. Fish Dis., 24: 15-22 Lorenzen N. et al. (2002) Fish Shellfish Immunol., 12: 439-453

  However, in the inventions disclosed in Patent Document 1 and Patent Document 2, an inactivated virus or an inactivated antigen that is a subunit antigen is used, and not only a so-called live virus that has not been treated at all, but also an attenuated virus. No virus is used.

  The present invention administers double-stranded RNA and administers various types of pathogenic viruses, so-called live viruses that do not treat them at all, or attenuated viruses, thereby immunizing target fish. It is an object of the present invention to provide a fish antiviral vaccine capable of inducing, activating, modifying, or enhancing the above, a fish immunostimulating agent for inducing an antiviral state, and a method for conferring fish immunity.

  The present inventors have found that immunity induction against a pathogenic virus can be induced by inoculating fish with double-stranded RNA and a pathogenic virus, and have completed the following inventions.

  (1) A fish antiviral vaccine comprising double-stranded RNA and a pathogenic virus as active ingredients.

  (2) The fish antiviral vaccine according to (1), wherein the pathogenic virus is a non-attenuated pathogenic virus.

  (3) the non-attenuated pathogenic virus is a non-attenuated virus of infectious hematopoietic necrosis virus (IHNV), pheasant nerve necrosis virus (RGNNV) and / or viral hemorrhagic sepsis virus (VHSV), (2) The fish antiviral vaccine according to 1.

  (4) The fish antiviral vaccine according to any one of (1) to (3), wherein the double-stranded RNA is Poly (I: C).

  (5) The fish antiviral vaccine according to (4), wherein the dose of Poly (I: C) is 12.5 μg or more.

  (6) A fish immunostimulant containing double-stranded RNA as an active ingredient.

  (7) The fish immunostimulator according to (6), which contains a pathogenic virus as an active ingredient.

  (8) The fish immunostimulant according to (7), wherein the pathogenic virus is a non-attenuated pathogenic virus.

  (9) the non-attenuated pathogenic virus is a non-attenuated virus of infectious hematopoietic necrosis virus (IHNV), pheasant nerve necrosis virus (RGNNV) and / or viral hemorrhagic sepsis virus (VHSV), (8) The fish immunostimulant described in 1.

  (10) The fish immunostimulator according to any one of (6) to (9), wherein the double-stranded RNA is Poly (I: C).

  (11) The fish immunostimulator according to (10), wherein the dose of Poly (I: C) is 12.5 μg or more.

  (12) A fish immunization method wherein Poly (I: C) is administered 14 days before taking in a pathogenic virus or simultaneously.

  (13) The fish immunization method according to (12), wherein the pathogenic virus is a non-attenuated pathogenic virus.

  According to the present invention, double-stranded RNA is administered or administered, and exposed to various types of pathogenic viruses, so-called live viruses that are not treated at all, or attenuated viruses, and incorporated into them. It is possible to induce, activate, modify or enhance the immunity of the target fish after inducing the antiviral state or simultaneously with inducing the antiviral state.

In Example 1- Example 3, it is a figure which shows the survival test result which inoculates IHNV after Poly (I: C) administration. A shows the results of the survival test in Example 1, B shows the results of Example 2, and C shows the results of the survival tests in Example 3. In Example 4, it is a figure which shows the measurement result of an IHNV specific antibody. A shows the measurement results of IHNV-specific antibodies in the blood after the primary inoculation of IHNV, and B shows the results of the secondary inoculation of IHNV. In Example 6 and Example 7, it is a figure which shows the survival test result which inoculates RGNNV after Poly (I: C) administration. A shows the survival test result in Example 6, and B shows the survival test result in Example 7, respectively. In Example 8, it is a figure which shows the measurement result of a RGNNV specific antibody. A shows the measurement results of RGNNV-specific antibodies in blood after primary RGNNV inoculation, and B shows the results after secondary RGNNV inoculation. In Example 10, it is a figure which shows the result of the effective dose test of Poly (I: C) in Mahata. A shows the survival rate with respect to the elapsed days, and B shows the survival rate on the 18th day after administration with respect to the Poly (I: C) dose. In Example 11, it is a figure which shows the result of the effective primary inoculation amount test of RGNNV. In Example 12, it is a figure which shows the result of the duration test of the antiviral effect by Poly (I: C) in Mahata. A shows the survival rate with respect to the elapsed days, and B shows the survival rate with respect to the elapsed days from Poly (I: C) administration to RGNNV administration. In Example 13, it is a figure which shows the examination result of the administration site | part of Poly (I: C) in Mahata. In Example 14 and Example 15, it is a figure which shows the survival test result which inoculates VHSV after Poly (I: C) administration. A shows the survival test result in Example 14, and B shows the survival test result in Example 15, respectively. In Example 16, it is a figure which shows the measurement result of a VHSV specific antibody. A shows the measurement results of VHSV-specific antibodies in blood after primary inoculation of VHSV, and B shows the results of secondary inoculation of VHSV. In Example 17 and Example 18, A is a figure which shows the result of the effective dose test of Poly (I: C) in Mahata, and B is the persistence of the antiviral effect by Poly (I: C) in Mahata. It is a figure which shows the result of a period test.

  Hereinafter, the fish antiviral vaccine, fish immunostimulant, and fish immunization method according to the present invention will be described in detail. The fish antiviral vaccine according to the present invention comprises double-stranded RNA and a pathogenic virus as active ingredients.

  The fish antiviral vaccine according to the present invention generates immunity by incorporating a so-called live virus or attenuated virus that is not processed at all into the living body, and corresponds to a so-called live vaccine. In the case of a general fish vaccine, inactivated microorganisms and viruses are used. In the fish antiviral vaccine according to the present invention, a live virus or an attenuated virus is used.

  That is, in the present invention, “pathogenic virus” refers to a virus having infectivity (infectivity), pathogenicity, and immunogenicity, and does not include a so-called inactivated virus used for an inactivated (death) vaccine. Infectivity refers to the property that the virus can stably propagate in the host fish, and pathogenicity refers to the property that causes disease when an infectious virus infects the host fish. By immunogenicity is meant, respectively, the property that results in an antibody and / or cell-mediated immune response when taken up by fish.

  Non-attenuated viruses can be mentioned as preferred pathogenic viruses in the present invention. In the present invention, “non-attenuated viruses” refer to viruses that are not treated at all, so-called live viruses (live viruses), and particularly virulence ( Does not contain so-called attenuated viruses with reduced virulence. In addition, virulence means the strength of how easily an infectious disease is caused when the pathogen is infected, and how easily it becomes severe when the pathogen is infected.

  The type of non-attenuated virus is not particularly limited as long as it has infectivity, pathogenicity, and immunogenicity to fish.For example, fish rhabdovirus, fish iridovirus, fish nodavirus, and members of ABV genus including IPNV Virus, ISAV, aquareovirus, picornavirus, etc., and fish rhabdoviruses include IHNV, VHSV, Hirame Rhabdovirus (HIRRV), carp spring viremia virus (SVCV) Examples of fish iridovirus include RSIV and Lymphocystis Disease Virus (LCDV), and examples of fish nodavirus include SJNNV, TPNNV, BFNNV, RGNNV, and TNNV.

  The double-stranded RNA in the present invention is not particularly limited, and examples thereof include Poly (I: C), Poly (C: G), Poly (U: A), and the like. Preferred double-stranded RNA is Poly (I: C). In addition, as an effective dose of Poly (I: C) in the present invention, a dose in the case where a survival rate of 50% or more in one group can be exemplified, and a preferable dose is 12.5 μg or more. A preferable dose is 25 μg or more, and a more preferable dose is 50 μg or more.

  The fish immunostimulant in the present invention contains double-stranded RNA as an active ingredient. In the present invention, “immunostimulatory agent” refers to any substance that induces an antiviral state by being taken into the body of fish, and thereafter, or simultaneously with it, an immune response (antibody and / or cell-mediated). Includes any substance that induces, activates, modifies, enhances, etc.

  The fish immunostimulant according to the present invention may further contain a pathogenic virus as an active ingredient, and examples of such a pathogenic virus include non-attenuated pathogenic viruses.

  The aspect of the fish antiviral vaccine and the fish immunostimulant in the present invention is not particularly limited. For example, for a fish antiviral vaccine, an aspect in which a double-stranded RNA and a pathogenic virus are contained in a suspension or solution, etc. , Each of which is administered to the target fish as a suspension or solution, etc., and functions as an antiviral vaccine in the body of the target fish, or after double-stranded RNA is administered to the target fish as a suspension or solution, or In addition to the administration, the target fish can be exposed to a pathogenic virus and incorporated therein, so that it can function as an antiviral vaccine in the body of the target fish. In addition, for fish immunostimulants, an embodiment in which double-stranded RNA is contained in a suspension or solution, an embodiment in which these contain a pathogenic virus, a suspension in which each of the double-stranded RNA and the pathogenic virus is suspended or A mode in which the target fish is administered as a solution to the target fish and functions as an immunostimulator in the body of the target fish, or after the double-stranded RNA is administered to the target fish as a suspension or solution, etc. And the like can be mentioned by functioning as an immunostimulant in the body of the target fish.

  In the present invention, “immunity activation” means “immunity induction”, “immunomodulation”, “immunity stimulation”, “immunity enhancement”, “immunity enhancement”, “immunity activity”, “immunity activation”, “immunity enhancement” , Or “immune enhancement”, and “immunity stimulating agent” is “immunity inducer”, “immunomodulator”, “immunostimulatory agent”, “immunity enhancer”, “immune enhancer” ”,“ Immunoactive agent ”, or“ immune enhancing agent ”. Further, “fish antiviral vaccine” and “fish immunostimulant” are used interchangeably with “fish antiviral vaccine” and “fish immunostimulator”, respectively.

In the present invention, Reed LJ et al. Can be used for measurement of survival rate, virus infectivity titer, antibody detection, effective dose of Poly (I: C), duration of antiviral state by Poly (I: C), and the like. al. (1938) Am J Hygiene 27: 493-497, TCID ₅₀ (Median Tissue Culture Infectious Dose) method is used.

  The fish immunization method according to the present invention is a method in which Poly (I: C) is administered 14 days before or at the same time when pathogenic virus is incorporated. It is characterized by the timing of administration of pathogenic virus showing survival rate and Poly (I: C). That is, it is preferable to administer Poly (I: C) for 14 days before taking in the pathogenic virus or simultaneously, more preferably to administer Poly (I: C) from 7 days to the day before taking in the pathogenic virus. More preferably, Poly (I: C) is administered for 4 days to 1 day before taking up.

  Examples of the fish antiviral vaccine, fish immunostimulant, and fish immunization method according to the present invention will be described below. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Example 1> IHNV primary inoculation test after Poly (I: C) administration Experimental Materials The following materials were prepared as experimental materials used in Example 1.

(1) Infectious Hematopoietic Necrosis Virus (IHNV)
IHNV RtNag96 was used as IHNV. This IHNV RtNag96 was isolated from rainbow trout infected with infectious hematopoietic necrosis (IHN) by Nagano Prefecture in 1996 {Nishizawa T. et al. (2006) Dis Aquat Org 71: 267-272}. This was obtained by using Eagle MEM medium (MEM ₁₀ ; Gibco) containing fetal bovine serum (FBS; Gibco) 10% (volume / volume), penicillin G100 IU / mL, and streptomycin sulfate 100 μg / mL. (EPC; Epithelioma Papulosum Cyprini) cells were cultured at 15 ° C. Thereafter, the culture supernatant obtained by centrifugation at 12000 × g and 4 ° C. for 10 minutes was aliquoted and stored frozen at −80 ° C.

(2) Poly (I: C)
Poly (I: C) (Sigma) was prepared to 1 mg / mL with DEPC water (Nippon Gene) immediately before administration.

(3) Rainbow trout (Oncorhynchus Mykiss)
As the rainbow trout, a specific pathogen-free (SPF) rainbow trout fry having a body weight of 1.3 g ± 0.2 g, which was provided by the Nagano Prefectural Fisheries Experiment Station, was used. From 2 weeks before the start of the experiment to the end of the experiment, it was kept in a 10 L tank at 10 to 11 ° C. and a flow rate of 24 cycles / day.

  Hereinafter, the experimental material was used not only in Example 1 but also in Example 2 and Example 3. Hereinafter, these are simply referred to as “IHNV”, “Poly (I: C)”, and “rainbow trout”.

2. IHNV primary inoculation test after administration of Poly (I: C)
In order to evaluate the resistance to IHNV by prior administration of Poly (I: C), the following experiment was conducted. The “primary inoculation” means that the individual is inoculated with the virus for the first time.

(1) Group setting 240 rainbow trout, Poly (I: C) -IHNV group (140), Poly (I: C) -Mock group (60), Cont.-IHNV group (20), and Cont .-Mock group (20 animals) divided into 4 groups. Here, “Poly (I: C) -IHNV group” is a group in which IHNV is first inoculated after administering Poly (I: C), and “Poly (I: C) -Mock group” is Poly (I: C ) After administration of MEM ₁₀ ; “Cont.-IHNV group” means DEPC water and IHNV primary inoculation group; and “Cont.-Mock group” means DEPC water and MEM ₁₀ Each group is shown as an administration group. In the following, “Cont.” Means control, and “Mock” means “no virus”.

(2) Administration test First, after acclimatization for 2 weeks, Poly (I: C) -IHNV group and Poly (I: C) -Mock group were each treated with 50 μL of 1 mg / mL Poly (I: C), Cont. In the -IHNV group and the Cont.-Mock group, 50 μL each of DEPC water alone was intraperitoneally administered. Two days later, Poly (I: C) -IHNV group and Cont.-IHNV group were mixed with 50 μL of 10 ^5.0 TCID ₅₀ (TCID ₅₀ ; 50% tissue culture infectious dose) IHNV in Poly (I: C ) -Mock group and Cont.-Mock group were each primary inoculated intraperitoneally with 50 μL of MEM ₁₀ each. The day of primary inoculation of these IHNV or MEM ₁₀ was taken as day 0 and observation was made until day 21, and the survival rate of each group is shown in FIG. 1A. Moreover, the relative survival rate (RPS) was calculated based on the survival rate of each group {Amend DF. Et al. (1981) Dev. Biol. Stand. 49: 447-454}.

  As shown in FIG. 1A, the survival rate after 21 days of the Poly (I: C) -IHNV group was 95.7%, which was extremely higher than that of the Cont.-IHNV group. The RPS of the Poly (I: C) -IHNV group relative to the Cont.-IHNV group was 95.2%. From this, it was revealed that Poly (I: C) had resistance to IHNV by pre-administration. Moreover, since the survival rate of the Poly (I: C) -Mock group was 100%, it was confirmed that there was no influence of toxicity or the like due to Poly (I: C) administration. There were no deaths in the Cont.-Mock group.

(3) Sample collection
For detection of IHNV-specific antibodies by ELISA, on the 21st day after the primary inoculation of IHNV or MEM ₁₀ , tails from surviving fish in the Poly (I: C) -IHNV and Poly (I: C) -Mock groups Venous blood was collected. In addition, kidneys were collected from surviving fish in each group in order to measure the virus infectivity titer.

<Example 2> Secondary inoculation test 1 (21st day) of IHNV
Using the surviving fish of Example 1, an IHNV secondary inoculation test was further performed 21 days after the primary inoculation of IHNV, and resistance to IHNV was evaluated as follows. Here, “secondary inoculation” means inoculating the same virus twice.

(1) Group setting 40 surviving rainbow trout in the Poly (I: C) -IHNV group used in Example 1, 40 surviving rainbow trout in the Poly (I: C) -Mock group, and a new untreated Forty SPF rainbow trout were used. Poly (I: C) -IHNV-IHNV group (20 mice), Poly (I: C) -IHNV-Mock group (20 mice), Poly (I: C) -Mock-IHNV group (20 mice) Poly (I: C) -Mock-Mock group (20 mice), Naive-IHNV group (20 mice), and Naive-Mock group (20 mice), and 6 groups were set.

Here, the “Poly (I: C) -IHNV-IHNV group” means a group in which the Poly (I: C) -IHNV group in Example 1 was secondly inoculated with IHNV, “Poly (I: C) -IHNV- “Mock group” refers to the group in which MEM ₁₀ was administered to the Poly (I: C) -IHNV group of Example 1, and “Poly (I: C) -Mock-IHNV group” refers to the Poly (I: C ) -Mock group secondary inoculated with IHNV, “Poly (I: C) -Mock-Mock group” means a group in which MEM ₁₀ was administered to the Poly (I: C) -Mock group of Example 1, “ The “Naive-IHNV group” represents a group in which rainbow trout to which nothing has been administered is primarily inoculated with IHNV, and the “Naive-Mock group” refers to a group in which rainbow trout to which nothing has been administered has been administered MEM ₁₀ .

(2) Administration test On the 21st day after the primary inoculation of IHNV or MEM ₁₀ of Example 1, Poly (I: C) -IHNV-IHNV group, Poly (I: C) -Mock-IHNV group, and Naive-IHNV There are 3 groups of 10 ^5.0 TCID ₅₀ IHNV, 50 μL each, Poly (I: C) -IHNV-Mock group, Poly (I: C) -Mock-Mock group, and Naive-Mock group Were secondly inoculated intraperitoneally with 50 μL of MEM ₁₀ each. Observation was conducted until day 21 with the day of secondary inoculation as day 0, and the survival rate of each group is shown in FIG. 1B. Further, RPS was calculated in the same manner as in Example 1 based on the survival rate of each group.

  As shown in FIG. 1B, in the Poly (I: C) -IHNV-IHNV group, the survival rate after 21 days was 100%. On the other hand, in the Poly (I: C) -Mock-IHNV group and Naive-IHNV group, individuals who died from the 7th day after the second inoculation appeared, and the survival rate on the 21st day after the second inoculation was 10%. there were. Poly (I: C) -IHNV-IHNV group RPS for Poly (I: C) -Mock-IHNV group and Poly (I: C) -IHNV-IHNV group RPS for Naive-IHNV group 100%. In the Poly (I: C) -IHNV-Mock group, only one individual died on the 9th day, and the survival rate on the 21st day was 95%. In the Poly (I: C) -Mock-Mock group and Naive-Mock group, there were no deaths.

  From the results of Example 1 and Example 2, the resistance to IHNV was shown by primary inoculation with IHNV on the second day after administration of Poly (I: C), and further, the second day with IHNV on the 21st day. It became clear that even if inoculated, it can survive. On the other hand, it has been clarified that even when primary inoculation with IHNV was carried out 21 days or more after administration of Poly (I: C), it did not have resistance to IHNV and resulted in death.

(3) Sample collection
For detection of IHNV-specific antibodies by ELISA, Poly (I: C) -Mock-Mock group, Poly (I: C) -IHNV-Mock group, 21 days after secondary inoculation with IHNV or MEM ₁₀ Blood was collected from the surviving fish of the Poly (I: C) -IHNV-IHNV group. In addition, kidneys were collected from each group of surviving fish in order to determine the viral infectivity titer.

<Example 3> Secondary inoculation test 2 of IHNV (Day 49)
Using the surviving fish of Example 1, an IHNV secondary inoculation test was further performed on the 49th day after the primary inoculation of IHNV, and resistance to IHNV was evaluated as follows.

(1) Group setting 40 surviving rainbow trout in the Poly (I: C) -IHNV group used in Example 1 and 40 new SPF rainbow trout to which nothing was administered were used. These were divided into Poly (I: C) -IHNV-IHNV group (20 mice), Poly (I: C) -IHNV-Mock group (20 mice), Naive-IHNV group (20 mice), and Naive-Mock group ( Each group was divided into 20 groups, and 4 groups were set. In addition, "Poly (I: C) -IHNV-IHNV group", "Poly (I: C) -IHNV-Mock group", "Naive-IHNV group", and "Naive-Mock group" are shown in Example 2. It is the same as that.

(2) Administration test On Day 49 after the primary inoculation with IHNV or MEM ₁₀ in Example 1, 10 ^5.0 TCID was obtained for 2 groups of Poly (I: C) -IHNV-IHNV group and Naive-IHNV group. Secondary injection of 50 μL of ₅₀ IHNV into each group and 50 μL of MEM ₁₀ into two groups, Poly (I: C) -IHNV-Mock group and Naive-Mock group, respectively. The day of the second inoculation was set as day 0 again, and observation was performed until day 21. The survival rate of each group is shown in FIG. 1C. Further, RPS was calculated in the same manner as in Example 1 based on the survival rate of each group.

  As shown in FIG. 1C, there are no deaths in the three groups of Poly (I: C) -IHNV-IHNV, Poly (I: C) -IHNV-Mock and Naive-Mock groups, and the Naive-IHNV group Only individuals from the 6th day died, and on the 16th day, all individuals in the Naive-IHNV group died. Therefore, the survival rate after 21 days in the Naive-IHNV group was 0%. The RPS of the Poly (I: C) -IHNV-IHNV group relative to the Naive-IHNV group was 100%.

  From the results of Example 1, Example 2 and Example 3, resistance to IHNV was demonstrated by primary inoculation with IHNV on the second day after administration of Poly (I: C), and further 49 days It was clarified that even if IHNV was secondarily inoculated in the eye, it could survive.

<Example 4> Detection of IHNV-specific antibody Experimental Materials The following materials were prepared as experimental materials used in Example 4.
IHNV and Viral Hemorrhagic Septicemia Virus (VHSV) described in Example 1
As virus antigens for antibody detection by ELISA, IHNV described in Example 1 and VHSV Obama25 isolated from natural flounder (Paralichthys Olivanceus) captured in Obama Bay, Wakasa Bay, Japan were used {Takano R. et al. (2000) Bull. Eur. Assoc. Fish Pathol. 20: 186-192 etc.}.

2. Antibody detection by ELISA Using the blood sampled in Example 1 and Example 2, IHNV-specific antibodies were detected by ELISA.

  Specifically, the sampled blood was coagulated at 4 ° C. for 16 hours, and centrifuged at 12000 × g for 10 minutes at 4 ° C. to obtain serum. This serum was stored frozen at −20 ° C. until use. IHNV-specific antibodies were detected by the method of Kim et al. {Kim WS. Et al. (2007) Dis. Aquat. Org. 78: 55-59, Kim WS. Et al. (2008) Fish Pathol. 43: 112-116}.

That is, the target antigen IHNV and the control antigen VHSV were centrifuged at 19000 × g for 30 minutes at 4 ° C. The resulting IHNV supernatant or VHSV supernatant containing about 10 ^8.0 TCID ₅₀ / mL was diluted 10-fold with PBS, and overnight standing at 4 ° C. This was added 50 [mu] L / well to ELISA plates did. Thereafter, the plate was washed 3 times with PBS containing 0.05% Tween 20 (T-PBS), PBS containing 5% skim milk was added, and blocking was performed at 25 ° C. for 1 hour. Further, after washing three times with T-PBS, the serum (primary antibody) obtained in Example 1 and Example 2 was diluted 40-fold with PBS containing 5% skim milk, and this was added at 50 μL / well. It left still at 25 degreeC for 1 hour. Thereafter, anti-rainbow trout IgM rabbit serum (secondary antibody), followed by HRP-labeled anti-rabbit IgG porcine polyclonal antibody (tertiary antibody; DAKO) was added, and the mixture was allowed to stand at 25 ° C. for 30 minutes.

The secondary antibody and tertiary antibody used were diluted 1000 times with 5% skim milk immediately before use. Wash 3 times with T-PBS, add 50 μL of chromogenic substrate solution containing 1 mg / mL o-phenylenediamine, 0.03% hydrogen peroxide, 100 mM sodium hydrogen phosphate, and 50 mM citric acid, 25 The reaction was carried out at 30 ° C. for 30 minutes. Thereafter, 2N sulfuric acid was added to stop the color reaction, and the absorbance was measured at ₄₉₂ nm (OD ₄₉₂ ) using a microplate reader (MTP-300; corona). The absorbance calculated by subtracting the absorbance using VHSV from the absorbance using IHNV was defined as the absorbance (OD ₄₉₂ ) for an IHNV-specific antibody {Kim WS. Et al. (2008) Fish Pathol. 43: 112 -116}.

  The results are shown in FIGS. 2A and 2B. FIG. 2A shows the results using the serum derived from the sample of Example 1 (3), and FIG. 2B shows the results obtained using the serum derived from the sample of Example 2 (3). That is, FIG. 2A shows the results on the 21st day after the primary inoculation of IHNV, and FIG. 2B shows the results after 21 days from the second inoculation of IHNV on the 21st day after the primary inoculation of IHNV.

  As shown in FIG. 2A, the ELISA absorbance of serum at 21 days after the primary inoculation of IHNV was 0.02 or less in the Poly (I: C) -Mock group, whereas the Poly (I: C) -IHNV group From 0 to 0.03, the Poly (I: C) -IHNV group showed a slightly high tendency.

  As shown in FIG. 2B, the ELISA absorbance of serum at 21 days after the secondary inoculation of IHNV was 0.01 or less in the Poly (I: C) -Mock-Mock group, whereas Poly (I: C) The absorbance of the -IHNV-Mock group is 0 to 0.80, the average value is 0.15, and the absorbance of the Poly (I: C) -IHNV-IHNV group is 0.06 to 0.58. The average value was 0.24.

  From these results, the Poly (I: C) -IHNV-Mock group in FIG. 2B shows higher absorbance than the Poly (I: C) -IHNV group in FIG. 2A. It was shown that IHNV primary inoculation after administration of C) produced 21-42 days after primary inoculation. Furthermore, the Poly (I: C) -IHNV-IHNV group shows higher absorbance than the Poly (I: C) -IHNV-Mock group, so that IHNV-specific antibodies are promoted by secondary IHNV inoculation. It has been shown.

<Example 5> Measurement of virus infectivity titer Virus infectivity titer was measured using kidneys collected from living fish in Example 1 (3) and Example 2 (3). Specifically, the collected kidney was ground with 9 times volume (weight / weight) Hank's balanced salt (HBSS; Gibco), filtered through a 0.45 μm HA filter (Advantec), and filtrated using HBSS. A 10-fold dilution series was prepared. Each dilution series was inoculated into 4 wells of EPC cells that had been cultured in a 96-well microplate. After culturing at 15 ° C. for 14 days, observe the denaturation (CPE) of the cells in the holes inoculated with each dilution, calculate the 50% tissue culture infectious dose (TCID ₅₀ ) and convert it to the virus infection titer per mL of the test did.

In the results of Example 1 (3), that is, using the kidney collected on the 21st day after the primary inoculation of IHNV, there was no IHNV infection in the Poly (I: C) -IHNV group, whereas in the Cont.-IHNV group IHNV infectivity titers of 10 ^7.1 to 10 ^7.6 TCID ₅₀ / g (per tissue) were detected in the kidney. In addition, IHNV was not detected in the Poly (I: C) -Mock group and the Cont.-Mock group.

As a result of using Example 2 (3), that is, kidneys collected on the 21st day after the secondary inoculation of IHNV, all samples in the Poly (I: C) -IHNV-IHNV group had 10 ^1.5 TCID50 / The following values were obtained for g (per tissue). This was significantly lower than the value indicating IHNV infection, indicating no IHNV infection.

<Example 6> Fish nodavirus RGNNV inoculation test after Poly (I: C) administration Experimental Materials The following materials were prepared as experimental materials used in Example 6.
(1) Red-spotted Grouper Nervous Necrosis Virus (RGNNV), a fish nodavirus
RGNNV SgNag05 was used as RGNNV. This RGNNV was isolated from Mahata infected with viral neural necrosis (VNN) at the Nagasaki Prefectural Fisheries Experiment Station {Nishizawa T. et al. (2008) Dis Aquat Org, 79: 19-25, Kokawa Y. et al. (2008) Aquaculture, (in press)}. Using Leibovitz L-15 medium (L-15; Gibco) containing FBS (Gibco) 10% (volume / volume), penicillin G150 IU / mL, and streptomycin sulfate 100 μg / mL, at 25 ° C. in SSN-1 cells. And cultured. The culture supernatant obtained by centrifuging this at 12000 × g and 4 ° C. for 10 minutes was aliquoted and stored frozen at −80 ° C.

(2) Poly (I: C)
Poly (I: C) (Sigma) was prepared to 2 mg / mL with DEPC water (Nippon Gene) immediately before administration.

(3) Mahata (Epinephelus Septemfasciatus)
The Mahata used was an SPF Mahata grown at Nagasaki Prefectural Fisheries Experiment Station and having an average weight of 115 ± 25 g (60 to 140 g). From the experiment start date to the end of the experiment, each test group was bred using UV sterilized water in a 40 L tank at 25 ° C. and a flow rate of 24 cycles / day.

  Hereinafter, the experimental materials described above were used not only in Example 6 but also in Examples 7 to 9. Hereinafter, these are simply referred to as “RGNNV”, “Poly (I: C)”, and “Mahata”.

2. RGNNV primary inoculation test after administration of Poly (I: C)
In order to evaluate the resistance to RGNNV by prior administration of Poly (I: C), the following experiment was conducted.

(1) Group Setting 160 Mahatas were added to Poly (I: C) -RGNNV group (40 animals), Poly (I: C) -Mock group (40 animals), Cont.-RGNNV group (40 animals), and Cont. .-Mock group (40 animals) divided into 4 groups. Here, "Poly (I: C) -RGNNV group" is the group that was first inoculated with RGNNV after Poly (I: C) administration, and "Poly (I: C) -Mock group" is Poly (I: C) The group that received L-15 after administration, “Cont.-RGNNV group” was the group that received primary RGNNV after DEPC water administration, and “Cont.-Mock group” was administered L-15 after DEPC water administration Each group is shown.

(2) Administration Test First, 100 μL each of 2 mg / mL Poly (I: C) was added to the Poly (I: C) -RGNNV group and the Poly (I: C) -Mock group, the Cont.-RGNNV group and the Cont. -To the Mock group, 100 μL of DEPC water alone was intramuscularly administered. Two days later, Poly (I: C) -RGNNV group and Cont.-RGNNV group had 10 ^4.3 TCID ₅₀ RGNNV in 100 μL each, Poly (I: C) -Mock group and Cont.-Mock group. Each group received a primary inoculation of 100 μL of L-15 in each muscle. Observation was performed without feeding until day 18 with the day of primary inoculation of RGNNV or L-15 as day 0, and the survival rate of each group is shown in FIG. 3A. Further, RPS was calculated in the same manner as in Example 1 based on the survival rate of each group.

  As shown in FIG. 3A, there was no death in the Poly (I: C) -RGNNV group, whereas in the Cont.-RGNNV group, death occurred from the fourth day of RGNNV primary inoculation due to the onset of typical VNN. Individuals appeared and all died on the 8th day. In addition, there were no deaths due to primary RGNNV in the Poly (I: C) -Mock group and Cont.-Mock group. From this, it became clear that pre-administration of Poly (I: C) has resistance to RGNNV. In addition, since there was no death in the Poly (I: C) -Mock group, it was confirmed that there was no influence of toxicity or the like due to Poly (I: C) administration. In the Poly (I: C) -Mock group, one death occurred due to an accident.

(3) Sample collection
For detection of RGNNV-specific antibodies by ELISA, Cont.-Mock group, Poly (I: C) -Mock group, and Poly (I: C) -RGNNV 21 days after the primary inoculation of RGNNV or L-15 Tail vein blood was collected from each 10 surviving fish in the group.

<Example 7> Secondary inoculation test of RGNNV Using the surviving fish of Example 6, an RGNNV inoculation test was further conducted on the 21st day after the primary inoculation of RGNNV, and resistance to RGNNV was evaluated as follows.

(1) Group setting 40 surviving Mahatas in the Poly (I: C) -RGNNV group used in Example 5, 40 surviving Mahatas in the Poly (I: C) -Mock group, and a new untreated group Forty SPF Mahatas were used. Poly (I: C) -RGNNV-RGNNV group (20 mice), Poly (I: C) -RGNNV-Mock group (20 mice), Poly (I: C) -Mock-RGNNV group (20 mice) Poly (I: C) -Mock-Mock group (20 mice), Naive-RGNNV group (20 mice), and Naive-Mock group (20 mice), and 6 groups were set.

  Here, the “Poly (I: C) -RGNNV-RGNNV group” is a group in which the Poly (I: C) -RGNNV group of Example 5 was secondly re-inoculated with RGNNV, “Poly (I: C) -RGNNV The “-Mock group” is a group in which L-15 was administered to the Poly (I: C) -RGNNV group of Example 5, and the “Poly (I: C) -Mock-RGNNV group” is the Poly (I : C) -Mock group in which RGNNV was secondarily inoculated, “Poly (I: C) -Mock-Mock group” was administered to the Poly (I: C) -Mock group of Example 5 with L-15 Group, “Naive-RGNNV group” is a group that has received RGNNV primarily in Mahata who has not been administered anything, and “Naive-Mock group” is a group in which L-15 has been administered to Mahata who has not been administered anything Each is shown.

(2) Administration test On the 21st day after the primary inoculation of RGNNV or L-15 of Example 6, the Poly (I: C) -RGNNV-RGNNV group, the Poly (I: C) -Mock-RGNNV group, and Naive- the three groups of RGNNV group by 100 [mu] L of ^{10 4.3} TCID ₅₀ / 100μL of RGNNV, Poly (I: C) -RGNNV-Mock group, Poly (I: C) -Mock -Mock group, and Naive-Mock group In each of the three groups, 100 μL of L-15 was secondary inoculated into each muscle. Observation was conducted without feeding until day 14 with the day of secondary inoculation as day 0, and the survival rate of each group is shown in FIG. 3B.

  As shown in FIG. 3B, there were no deaths in the Poly (I: C) -RGNNV-RGNNV group. On the other hand, the Poly (I: C) -Mock-RGNNV group had a survival rate of 5% on the 14th day after the second inoculation, and the Naive-RGNNV group all died within 6 days after the second RGNNV inoculation. In addition, there were no death cases due to secondary RGNNV inoculation in the three groups of the Poly (I: C) -RGNNV-Mock group, the Poly (I: C) -Mock-Mock group, and the Naive-Mock group. From this, it was clarified that the group inoculated with RGNNV primary after administration of Poly (I: C) was resistant to RGNNV secondary inoculation. In the Poly (I: C) -RGNNV-Mock group, one death occurred due to an accident.

  From the results of Example 6 and Example 7, resistance to RGNNV was shown by primary inoculation of RGNNV on the second day after Poly (I: C) administration, and the resistance was further increased for 21 days thereafter. It became clear that it persisted even after the passage. On the other hand, it was revealed that even when RGNNV was inoculated after 21 days or more after administration of Poly (I: C), it did not have resistance to RGNNV and resulted in death.

(3) Sample collection
Poly (I: C) -Mock-Mock group and Poly (I: C) -RGNNV-Mock group on day 14 after secondary inoculation with RGNNV or L-15 for detection of RGNNV-specific antibody by ELISA And tail vein blood collection from fish with Poly (I: C) -RGNNV-RGNNV group. In addition, in order to measure the virus infectivity titer, brains were extracted from live or dead fish in each group.

<Example 8> Detection of RGNNV-specific antibody Using the blood sampled in Examples 6 and 7, RGNNV-specific antibody was detected by ELISA. Specifically, serum was obtained in the same manner as in Example 4, and RGNNV was immobilized on an ELISA plate instead of IHNV, and ELISA was performed based on the method of Kim et al.

  The results are shown in FIGS. 4A and 4B. FIG. 4A shows the results obtained using the serum derived from the sample of Example 6 (3), and FIG. 4B shows the results obtained using the serum derived from the sample of Example 7 (3). That is, FIG. 4A shows the results on the 21st day after the primary inoculation of RGNNV, and FIG. 4B shows the results after 14 days have passed since the secondary inoculation of RGNNV on the 21st day after the primary inoculation of RGNNV.

  As shown in FIG. 4A, the ELISA absorbance of serum at 21 days after the primary inoculation of RGNNV was 0.08 or less in the Cont.-Mock group and the Poly (I: C) -Mock group, whereas Poly (I : C) The absorbance in the -RGNNV group was 0.2 to 0.86, and the average value was 0.33. From these results, it became clear that antibodies against RGNNV were produced by primary inoculation with RGNNV after Poly (I: C) administration.

  Moreover, as shown in FIG. 4B, the ELISA absorbance of serum on the 14th day after the secondary inoculation of RGNNV is 0.01 to 1.08 in the Poly (I: C) -RGNNV-RGNNV group, and the average value is 0.41. On the other hand, the absorbance in the Poly (I: C) -RGNNV-Mock group is 0.11 to 0.49, and the average value is 0.28. The Poly (I: C) -Mock-Mock group The absorbance at was 0.09 or less. That is, a high tendency was shown in the Poly (I: C) -RGNNV-RGNNV group.

  From these results, since the Poly (I: C) -RGNNV-RGNNV group shows higher absorbance than the Poly (I: C) -RGNNV-Mock group in the same manner as the IHNV-specific antibody of Example 4, RGNNV It was shown that production of specific antibodies was promoted by secondary inoculation of RGNNV.

<Example 9> Measurement of virus infectivity titer Using the brain collected from living fish in Example 7 (3), the virus infectivity titer was measured by the method described in Example 5.

As a result of using Example 7 (3), that is, the brain extracted 14 days after the second inoculation of RGNNV, in the dead fish of Poly (I: C) -Mock-RGNNV group and Naive-RGNNV group, the RGNNV infectivity titer Was 10 ^8.3 to 10 ^9.1 TCID ₅₀ / g (per tissue). On the other hand, in the live fish of the Poly (I: C) -RGNNV-RGNNV group, the RGNNV infectivity titer was 10 ^1.8 TCID ₅₀ / g (per tissue) which was below the detection limit of 10 ^4.3 TCID ₅₀ / g (per tissue). ) That is, the live fish in the Poly (I: C) -RGNNV-RGNNV group showed a clearly lower infectious titer than the dead fish in the Poly (I: C) -Mock-RGNNV group and Naive-RGNNV group. In addition, the RGNNV infectivity titer was not detected in the live fish of the Poly (I: C) -Mock-Mock group.

  From these results, it was shown that the fish surviving by the primary inoculation of RGNNV after Poly (I: C) administration had acquired immunity against RGNNV.

<Example 10> Effective dose test of Poly (I: C) Seven groups of 12 SPF mahatas with an average body weight of 71 ± 10 g, reared by the same method as in Example 6 (3), were prepared. First, each group was given 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.063 mg / mL Poly (I: C) solution or DEPC water, respectively. 100 μL each was administered intramuscularly. Two days later, 100 μL each of 10 ^4.3 TCID ₅₀ of RGNNV was primarily inoculated into the muscle. Observation was performed until day 18 with the day of primary inoculation of RGNNV as day 0, and the survival rate of each group is shown in FIGS. 5A and 5B. FIG. 5A shows the survival rate with respect to the number of days elapsed, and FIG. 5B shows the survival rate on the 18th day after administration with respect to the Poly (I: C) dose.

  As shown in FIG. 5A and FIG. 5B, the survival rate on the 18th day after RGNNV inoculation is 90% or more in the group with Poly (I: C) dose of 50 μg or more, whereas in the group with 25 μg or more in the group In the group of 50% and 12.5 μg or less, all died by the 18th day after RGNNV inoculation. From this result, it was shown that Poly (I: C) is preferably 25 μg or more, more preferably 50 μg or more, as a dose for inducing an antiviral state. No side effects were observed for Mahata with a Poly (I: C) dose of 200 μg.

<Example 11> Effective primary inoculation amount test of RGNNV Six groups of 12 groups of SPF Mahata reared by the same method as in Example 6 (3) and having an average body weight of 53 g were prepared. All groups were intramuscularly administered with 100 μL of 2 mg / mL Poly (I: C) solution. After the 2 days, the RGNNV each _{^{L-15,10 0.3 TCID 50, 10}} 1.3 TCID 50, 10 2.3 TCID 50, 10 3.3 TCID 50 and ^{10 4.3} TCID _50, in each group 100 μL of each was inoculated into the muscle. On day 21 with the day of primary inoculation of RGNNV or L-15 as day 0, 100 μL of 10 ^4.3 TCID ₅₀ of RGNNV was secondarily inoculated into the muscles of all groups. Observation was performed until day 21 with the day of secondary inoculation of RGNNV as day 0, and the survival rate of each group is shown in FIG.

As shown in FIG. 6, the survival rate on the 21st day after the secondary inoculation of RGNNV was 25% in the group in which the primary inoculation amount of RGNNV was 10 ^1.3 TCID ₅₀ or less and the group to which L-15 was administered. 65% in the group of the ^{10 2.3} TCID _50, 83% in the group of the ^{10 3.3} TCID _50, was 100% in the group of the ^{10 4.3} TCID _50. This result, Poly (I: C) as RGNNV primary inoculum to induce an antiviral state by inoculating RGNNV after administration, preferably 10 ^3.3 TCID ₅₀ or more, more preferably 10 ^4.3 TCID It was shown to be over ₅₀ .

<Example 12> Antiviral effect duration test using Poly (I: C) SPF Mahata, average weight 78 ± 15 g, prepared in the same manner as in Example 6 (3), 20 per group Six groups were prepared. All groups were intramuscularly administered with 100 μL of 2 mg / mL Poly (I: C) solution. In each group, 100 μL each of 10 ^4.3 TCID ₅₀ RGNNV was injected intramuscularly at the same day, 1 day, 2 days, 4 days, 7 days, and 14 days after Poly (I: C) administration. Primary inoculation. Observation was performed until day 14 with the day of primary inoculation of RGNNV as day 0, and the survival rate of each group is shown in FIGS. 7A and 7B. FIG. 7A shows the survival rate with respect to the elapsed days, and FIG. 7B shows the survival rate with respect to the elapsed days from Poly (I: C) administration to RGNNV administration.

  As shown in FIGS. 7A and 7B, the survival rate of the group inoculated with RGNNV at the same time as Poly (I: C) administration, 1 day, 2 days and 4 days after administration was 95% or more. On the other hand, the survival rates of the group inoculated primarily with RGNNV 7 days and 14 days after Poly (I: C) administration were 75% and 20%, respectively. From these results, it was clarified that the antiviral effect of Poly (I: C) persists for at least 7 days but then decreases. That is, it means that administration of Poly (I: C) at the same time or 7 days before RGNNV inoculation effectively induces resistance to RGNNV having high viral activity.

<Example 13> Examination of Poly (I: C) administration site Four groups of 10 groups of SPF Mahata reared by the same method as in Example 6 (3) and having an average body weight of 53 g were prepared. Three of them were each administered 100 μL of a 2 mg / mL Poly (I: C) solution intramuscularly, in the nasal cavity and in the gills. The remaining 1 group was administered with 100 μL of DEPC water intramuscularly as a control. Two days later, 100 μL of 10 ^4.3 TCID ₅₀ of RGNNV was primaryly inoculated into the site to which the Poly (I: C) solution or DEPC water was administered, ie, intramuscular, nasal cavity, and gill respectively. Observation was performed until day 21 with the day of primary inoculation of RGNNV as day 0, and the survival rate of each group is shown in FIG.

  As shown in FIG. 8, the survival rate on the 21st day after the primary inoculation of RGNNV is 100% in the group of Poly (I: C) administration in the intramuscular and nasal cavity groups, whereas in the group of Ella the same site. In the 50% control group, all died by day 10 after RGNNV inoculation. From this result, it was shown that Poly (I: C) is preferably gill, more preferably nasal cavity and / or muscle as the administration site for inducing the antiviral state.

<Example 14> Fish rhabdovirus VHSV inoculation test after administration of Poly (I: C) Experimental Materials The following materials were prepared as experimental materials used in Example 14.
(1) Viral Hemorrhagic Septicemia Virus (VHSV), a fish rhabdovirus
VHSV Obama25 was used as VHSV. Fathead minnow-derived epithelial cell line using Eagle MEM medium (MEM ₁₀ ; Gibco) containing FBS (Gibco) 10% (volume / volume), penicillin G 150 IU / mL, and streptomycin sulfate 100 μg / mL (FHM) Cultured at 20 ° C. in cells. Thereafter, the culture supernatant obtained by centrifugation at 12000 × g and 4 ° C. for 10 minutes was aliquoted and stored frozen at −80 ° C.

(2) Poly (I: C)
Poly (I: C) (Sigma) was prepared to 2 mg / mL with DEPC water (Sigma) immediately before administration.

(3) Flounder (Paralichthys Olivanceus)
As the flounder, SPF flounder with a body weight of 31.7 ± 2.9 g provided by the Nagasaki Prefectural Fisheries Experiment Station was used. This flounder was grown in ultraviolet light sterilized seawater at 16 ± 1 ° C. in an isolated breeding tank. Prior to the experiment, it was confirmed that some of them were not infected with VHSV by virus culture using FHM cells. During the experiment, each test group was reared in a 40 L tank at 16 ± 1 ° C. and a flow rate of 14 cycles / day using ultraviolet sterilized seawater.

  Hereinafter, the experimental materials described above were used not only in Example 14 but also in Examples 15 to 18. Hereinafter, these are simply referred to as “VHSV”, “Poly (I: C)”, and “flounder”.

2. Primary inoculation test of VHSV after administration of Poly (I: C)
In order to evaluate the resistance to VHSV by prior administration of Poly (I: C), the following experiment was conducted.

(1) Group setting 126 flounder, 3 groups of Poly (I: C) -VHSV group (42), Poly (I: C) -Mock group (42) and Cont.-VHSV group (42) Divided into. Here, “Poly (I: C) -VHSV group” is the group that received VHSV primary after Poly (I: C) administration, and “Poly (I: C) -Mock group” is Poly (I: C) The group to which MEM ₁₀ was administered after administration and the “Cont.-VHSV group” indicate a group in which VHSV was primarily inoculated after DEPC water administration, respectively.

(2) Administration test First, 100 μL each of Poly (I: C) -VHSV group and Poly (I: C) -Mock group with 2 mg / mL Poly (I: C), Cont.-VHSV group and Cont. -To the Mock group, 100 μL of DEPC water alone was intramuscularly administered. Two days later, Poly (I: C) -VHSV group and Cont.-VHSV group had 10 ^4.3 TCID ₅₀ VHSV 100 μL each, Poly (I: C) -Mock group 100 μL MEM ₁₀ Each was inoculated primary into the muscle. The day of primary inoculation with VHSV or MEM ₁₀ was defined as day 0, and the animals were reared with sufficient feeding once per day until day 28. The survival rate of each group is shown in FIG. 9A. The RPS based on the survival rate of each group is 100%.

  As shown in FIG. 9A, the survival rate after 28 days in the Poly (I: C) -VHSV group and the Poly (I: C) -Mock group was 100%, whereas in the Cont.-VHSV group, Due to the onset of VHS, some individuals died from the 4th day of the primary VHSV inoculation, and all died on the 9th day. From this, it was shown that it had resistance to VHSV by pre-administering Poly (I: C). In addition, since there was no death in the Poly (I: C) -Mock group, it was shown that there was no influence of toxicity or the like by Poly (I: C) administration.

(3) Sample collection
For detection of VHSV specific antibodies by ELISA, tails from surviving fish in Poly (I: C) -Mock and Poly (I: C) -VHSV groups 25 days after primary inoculation of VHSV or MEM ₁₀ Venous blood was collected.

<Example 15> Secondary inoculation test of VHSV Using the surviving fish of Example 14, a VHSV secondary inoculation test was further performed on the 28th day after the primary inoculation of VHSV, and resistance to VHSV was evaluated as follows.

(1) Group setting 42 surviving flounder in the Poly (I: C) -VHSV group used in Example 14 and 21 new SPF flounder to which nothing was administered were used. These were divided into Poly (I: C) -VHSV-VHSV group (21 mice), Poly (I: C) -VHSV-Mock group (21 mice) and Naive-VHSV group (21 mice), respectively. Set.

Here, “Poly (I: C) -VHSV-VHSV group” means a group in which VHSV was secondly inoculated to the Poly (I: C) -VHSV group of Example 14, “Poly (I: C) -VHSV- The “Mock group” is a group in which MEM ₁₀ was administered to the Poly (I: C) -VHSV group of Example 14, and a “Naive-VHSV group” was a group in which VHSV was primarily inoculated to flounder that had not been administered anything. Show.

(2) Administration test On the 28th day after the primary inoculation of VHSV of Example 14, Poly (I: C) -VHSV-VHSV group and Naive-VHSV group had 10 ^4.3 TCID ₅₀ VHSV in 100 μL each. The (I: C) -VHSV-Mock group was secondarily inoculated with 100 μL of MEM ₁₀ in each muscle. Observation was performed until day 20 with the day of secondary inoculation as day 0, and the survival rate of each group is shown in FIG. 9B.

  As shown in FIG. 9B, in the Poly (I: C) -VHSV-VHSV group and the Poly (I: C) -VHSV-Mock group, the survival rate after 20 days was 100%. On the other hand, in the Naive-VHSV group, all died within 6 days after the second inoculation of VHSV. From this, it was shown that the group in which VHSV was first inoculated after administration of Poly (I: C) was resistant to VHSV secondary inoculation.

  From the results of Example 14 and Example 15, the resistance to VHSV was shown by primary inoculation with VHSV on the second day after administration of Poly (I: C), and the resistance was further increased for 28 days. It became clear that it persisted even after passing.

(3) Sample collection
For detection of VHSV-specific antibodies by ELISA, on the 20th day after the secondary inoculation with VHSV or MEM ₁₀ , Poly (I: C) -VHSV-Mock group and Poly (I: C) -VHSV-VHSV group Blood was collected from the surviving fish.

<Example 16> Detection of VHSV-specific antibody Using the blood sampled in Example 14 and Example 15, detection of VHSV-specific antibody by ELISA was performed. Specifically, serum was obtained in the same manner as in Example 4, and VHSV was immobilized on an ELISA plate instead of IHNV, and ELISA was performed based on the method of Kim et al. (2008). Marine fish do not use a control antigen because it is not necessary. Moreover, the criteria based on the method of Kim et al. Are that ELISA absorbance is 0.1 or less, negative (no specific antibody is present), and 0.1 to 0.2 border (specific antibody is present) Whether or not) and 0.2 or more are positive (specific antibodies are present).

  The results are shown in FIGS. 10A and 10B. FIG. 10A shows the results obtained using the serum derived from the sample of Example 14 (3), and FIG. 10B shows the results obtained using the serum derived from the sample of Example 15 (3). That is, FIG. 10A shows the results on the 25th day after the primary inoculation of VHSV, and FIG. 10B shows the results after the secondary inoculation of VHSV on the 28th day after the primary inoculation of VHSV and another 20 days.

  As shown in FIG. 10A, the ELISA absorbance of serum on the 25th day after the primary inoculation of VHSV was 0.1 or less in the Poly (I: C) -Mock group and its average value was 0.03, whereas Poly The absorbance in the (I: C) -VHSV group was 0.02 to 1.15, and the average value was 0.13. When judged according to the criteria based on the method of Kim et al. (2008), all individuals were negative in the Poly (I: C) -Mock group, and 40% negative in the Poly (I: C) -VHSV group. Those at the border were 36% and positive were 24%.

  From these results, it was clarified that the antibody against VHSV was produced by the primary inoculation of VHSV after Poly (I: C) administration, but the amount was small on the 25th day after the primary inoculation of VHSV. .

  As shown in FIG. 10B, the ELISA absorbance of serum on the 20th day after the secondary inoculation of VHSV is 0.17 to 2.14 in the Poly (I: C) -VHSV-VHSV group, and the average value is 0.59. In contrast, the absorbance in the Poly (I: C) -VHSV-Mock group was 0.16 to 1.86, and the average value was 0.55. That is, a high tendency was shown in the Poly (I: C) -VHSV-VHSV group. In addition, when judged according to the criteria based on the method of Kim et al. (2008), 19 out of 21 individuals (90% or more) were positive in the Poly (I: C) -VHSV-VHSV group, and the remaining 2 were borders. In the Poly (I: C) -VHSV-Mock group, 20 out of 21 individuals (95%) were positive, and the remaining 1 individual (5%) was bordered.

  From these results, as with the IHNV-specific antibody of Example 4 and the RGNNV-specific antibody of Example 8, the Poly (I: C) -VHSV-VHSV group is more than the Poly (I: C) -VHSV-Mock group. The high absorbance indicated that production of VHSV-specific antibodies was promoted by secondary VHSV inoculation. In addition, since the Poly (I: C) -VHSV-Mock group in FIG. 10B shows higher absorbance than the Poly (I: C) -VHSV group in FIG. 10A, the VHSV-specific antibody is Poly (I: C). It was shown that the VHSV primary inoculation after the administration produced a large amount on the 26th to 48th days after the primary inoculation.

<Example 17> Effective dose test of Poly (I: C) Six groups of 10 SPF flounder having an average body weight of 32 g were prepared. First, 100 μL each of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL Poly (I: C) solution or DEPC water was intramuscularly added to each group. Administered. Two days later, 100 μL of 10 ^4.3 TCID ₅₀ of VHSV was first inoculated into each muscle. Observation was performed up to day 28, with the day of primary inoculation of VHSV as day 0, and the survival rate of each group is shown in FIG. 11A. In the meantime, flounder was individually raised using UV sterilized seawater in a 2 L tank at a flow rate of 360 cycles / day based on the method of Kokawa et al. (2008).

  As shown in FIG. 11A, the survival rate on the 28th day after the VHSV inoculation was 100% in the group with a Poly (I: C) dose of 25 μg or more, whereas it was 90% in the group with 12.5 μg. there were. In the group administered DEPC water, all died by day 9. From this result, it was shown that Poly (I: C) is preferably 12.5 μg or more, more preferably 25 μg or more, as a dose for inducing an antiviral state.

<Example 18> Duration test of antiviral effect by Poly (I: C) SPF flounder having an average body weight of 32 g was prepared, and 7 groups of 10 groups were prepared. All groups were intramuscularly administered with 100 μL of 2 mg / mL Poly (I: C) solution. In each group, 100 μL each of 10 ^4.3 TCID ₅₀ VHSV at the timing of 2 days before, 1 day, 2 days, 4 days, 7 days, and 14 days after Poly (I: C) administration, The primary inoculation was intramuscular. Observation was made until day 21 with the day of primary inoculation of VHSV as day 0, and the survival rate of each group is shown in FIG. 11B.

  As shown in FIG. 11B, the survival rate of the group first inoculated with VHSV 2 days before Poly (I: C) administration was 0%. That is, it was shown that even if Poly (I: C) was administered 2 days after the primary inoculation of VHSV, no antiviral effect was obtained. The survival rate of the group inoculated with VHSV at the same time as Poly (I: C) administration or 14 days after administration was 50%, and after 1, 2, 4 and 7 days after Poly (I: C) administration The survival rate of the group inoculated with VHSV was 100%. From these results, it was shown that administration of Poly (I: C) on the day before or 14 days before VHSV inoculation effectively induces resistance to VHSV having high viral activity.

  As described above, the results of experiments on rainbow trout inoculated with IHNV after prior administration of Poly (I: C) in Examples 1 to 5, Poly (I: C) in Examples 6 to 9 in advance The experimental results of Mahata inoculated with RGNNV after administration and the experimental results of Japanese flounder with VHSV inoculation after administration of Poly (I: C) in Example 14 to Example 16 were in agreement. From this, it became clear that cross immunity can be acquired by Poly (I: C). In addition, antibodies against this virus were produced by the primary inoculation, and production was further promoted by the secondary inoculation of the virus, so it was revealed that immunity against this inoculated virus was acquired.

Claims (13)

  A fish antiviral vaccine containing double-stranded RNA and a pathogenic virus as active ingredients.   The fish antiviral vaccine according to claim 1, wherein the pathogenic virus is a non-attenuated pathogenic virus.   3. The fish antiviral vaccine according to claim 2, wherein the non-attenuating pathogenic virus is infectious hematopoietic necrosis virus (IHNV), pheasant nerve necrosis virus (RGNNV) and / or viral hemorrhagic sepsis virus (VHSV).   The fish antiviral vaccine according to any one of claims 1 to 3, wherein the double-stranded RNA is Poly (I: C).   The fish antiviral vaccine according to claim 4, wherein the dose of Poly (I: C) is 12.5 µg or more.   A fish immunostimulant containing double-stranded RNA as an active ingredient.   The fish immunostimulant of Claim 6 which contains a pathogenic virus as an active ingredient.   The fish immunostimulant according to claim 7, wherein the pathogenic virus is a non-attenuated pathogenic virus.   The fish immunostimulant according to claim 8, wherein the non-attenuated pathogenic virus is infectious hematopoietic necrosis virus (IHNV), pheasant nerve necrosis virus (RGNNV) and / or viral hemorrhagic sepsis virus (VHSV).   The fish immunostimulant according to any one of claims 6 to 9, wherein the double-stranded RNA is Poly (I: C).   The fish immunostimulant of Claim 10 whose dosage of Poly (I: C) is 12.5 micrograms or more.   A method for immunizing fishes comprising administering Poly (I: C) for 14 days before taking in a pathogenic virus or simultaneously.   The fish immunization method according to claim 12, wherein the pathogenic virus is a non-attenuated pathogenic virus.

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