Vaccine against cyprinid herpesvirus 2

Abstract

The purpose of the present invention is to provide a method for controlling CyHV-2 infectious diseases in fish. The present invention pertains to a composition for use in controlling cyprinid herpesvirus 2 (CyHV-2) infectious diseases in fish, the composition containing attenuated CyHV-2.

Description

Vaccine against Cyprinid herpesvirus-2 The present invention relates to a composition for controlling fish infectious diseases caused by herpesvirus, and a method for controlling fish infectious diseases caused by herpesvirus, which includes administering or inoculating the composition to fish. As the main diseases occurring in goldfish farms, Herpesviral hematopoietic necrosis (HVHN: goldfish herpesvirus disease) can be mentioned. This disease is an infectious disease caused by Cyprinid herpesvirus 2 belonging to the genus Cyprinivirus of the family Alloherpesviridae of the order Herpesvirales (Cyprinid herpesvirus 2 (CyHV-2), or goldfish hematopoietic necrosis virus). It was first reported in Japan in 1992, and since then, its occurrence has been reported in the United States, Taiwan, New Zealand, India, the United Kingdom, China, etc. (Non-Patent Documents 1 to 7). The hosts are goldfish (Carassius auratus), gibel carp (Carassius auratus gibelio), and European crucian carp (Carassius carassius). Under experimental conditions, infection of Carassius auratus langsdorfii has also been reported (Non-Patent Document 8), and its mortality rate exceeds 80%. The preferred water temperature for occurrence is 15°C to 25°C, and it occurs frequently from April to July in Japan. Goldfish infected with this disease swim slowly and have poor feeding, with gill discoloration and white spots on the body surface. Pathohistologically, necrosis of organ tissues, hematopoietic tissues of the kidney and spleen, and submucosal tissues of the intestine can be seen (Non-Patent Document 1). As a countermeasure against this disease, temperature elevation treatment is known. By raising the breeding water temperature of infected individuals to 34°C at which the virus cannot proliferate, virus proliferation can be stopped and the mortality rate of the infected group can be reduced. It is also known that individuals that survive after treatment acquire immunity against the virus (Non-Patent Document 9). However, there are problems such as high equipment costs for temperature elevation and the possibility that fish after treatment may become virus carriers (Non-Patent Document 10). CyHV-1 and CyHV-3 are known as herpesviruses that also infect cyprinid fish, and reports on genomic analysis have been made (Non-Patent Document 18). CyHV-3, which is genetically close to CyHV-2, uses carp as a host. Reports have been made on the preparation of vaccines and virus detection methods as countermeasures against infectious diseases caused by CyHV-2 and CyHV-3, which are known to cause significant damage worldwide (Patent Documents 1 to 3 and Non-Patent Documents 11 to 17). WO2014 / 185439A1 JP2018-078903A JP2011-024584A Jung, S.J. et al., J. Fish Dis. 18, 211-220 (1995). Goodwin, A.E. et al., J. Aquat. Anim. Health 18, 11-18 (2006). Chang, P.H. et al., Fish Pathol. 34, 209-210 (1999). Hine, P.M. et al., Surveillance 33, 3-5 (2006). Sahoo, P.K. et al., Acta Trop. 161, 8-17 (2016). Jeffery, K.R. et al., J. Fish Dis. 30, 649-656 (2007). Xu, J. et al., Vet. Microbiol. 166, 138-144 (2013). Nanjo, A. et al., J. Fish Dis. 40, 157-168 (2017). Tanaka, M., Bulletin of the Saitama Prefectural Agriculture and Forestry Research Center 5, 37-43 (2005). Saitama Prefectural Fisheries Research Institute, Hyperthermia Treatment of Goldfish Herpes Disease (https: / / www.pref.saitama.lg.jp / b0915 / kenkyuseika / kingyoherupesu-syouon.html) January 19, 2021.Zhou, Y. et al., Fish & Shellfish Immunology, 47(2), 1024-1031, (2015). Ito, T. et al., Bull. Eur. Ass. Fish Pathol., 33(5) 158-164 (2013). Yuan, X. et al., Journal of Virological Methods 280 (2020)113877. Zhang, L. et al., Fish & Shellfish Immunology 49 (2016) 344-350. Huo, X. et al., Vaccines 2020, 8, 567; doi:10.3390 / vaccines8040567. Ito, T. et al., Veterinary Microbiology 175 (2015) 139-144. Goodwin, A.E. et al., Dis. Aquat. Org. 69, 137-143, 2006. Davison, A.J. et al., Journal of Virology 87(5) 2908-2922 (2013).。 Vaccination is one of the effective methods to prevent the impact of viral infections on farmed fish. All the vaccines reported for CyHV-2 infection require inoculation by injection, which has been a problem due to the high labor cost. Therefore, the development of a vaccine that can be inoculated by a simple method has been strongly desired. Live attenuated vaccines confer immunity by inoculating live attenuated pathogens directly into the body. Live attenuated vaccines in the fishery field have a great advantage in that they can be inoculated by the immersion method of contacting fish bodies with water containing the vaccine, enabling low-cost vaccination. However, since the mechanism of virus transmission in the aquatic environment has not been clarified and there is a concern about the reversion of pathogenicity due to the return of the virulence of the pathogen, live attenuated vaccines have not been approved as fishery drugs in Japan. In addition, there is always a risk that the pathogenicity will return due to subsequent mutations of the attenuated virus, and it has been extremely difficult to produce sufficient attenuated mutations in the virus. The present invention provides a composition for controlling CyHV-2 infection in fish, and a method for controlling CyHV-2 infection including administering or inoculating the composition to fish. As used herein, the treatment method includes a control method, a treatment method, and a prevention method. The control method includes a treatment method and a prevention method. In one aspect, the present invention provides a vaccine that can be inoculated by a simple method for controlling CyHV-2 infection in fish. In one embodiment, the present invention provides an attenuated live vaccine for controlling CyHV-2 infection in fish with a low risk of pathogenic reversion. In view of the above, the inventor of the present case has found that a passage using animal cells other than the host is performed to produce an attenuated strain of CyHV-2, and this can be used as a vaccine. Based on this finding, the present invention has been completed. In one aspect of the present invention, the following inventions are provided. [1]A composition for use in controlling CyHV-2 infection in fish, containing attenuated Cyprinid herpesvirus-2 (CyHV-2). [2]The composition according to [1], wherein the attenuated CyHV-2 is CyHV-2 cultured using koi-derived cells or gibel carp-derived cells as a host. [3]The composition according to [1] or [2], wherein the attenuated CyHV-2 is CyHV-2 cultured using koi-derived KF-1 cells or gibel carp-derived CFS cells as a host. [4]The composition according to any one of [1] to [3], wherein the attenuated CyHV-2 is CyHV-2 subcultured using gibel carp-derived CFS cells as a host and then subcultured using koi-derived KF-1 cells as a host. [5]The composition according to any one of [1] to [4], wherein the attenuated CyHV-2 is CyHV-2 subcultured 1 to 20 times using gibel carp-derived CFS cells as a host and then subcultured 1 to 20 times using koi-derived KF-1 cells as a host. [6] The composition according to any one of [1] to [5], wherein the fish is goldfish (Carassius auratus), gibel carp (Carassius auratus gibelio), European carp (Carassius carassius) or langsdorf carp (Carassius auratus langsdorfii). [7] The composition according to any one of [1] to [6], which is used by administration by the immersion method. [8] The composition according to any one of [1] to [7], wherein the attenuated CyHV-2 has a mutation in ORF25D as compared with CyHV-2 ST-J1 (GenBank No. NC_019495). [9] The composition according to any one of [1] to [8], wherein the CyHV-2 infection is herpesviral hematopoietic necrosis.

[10] The composition according to any one of [1] to [9], which contains cells infected with attenuated Cyprinid herpesvirus-2 (CyHV-2), or a derivative thereof.

[11] The composition according to any one of [1] to

[10] , which is used as a vaccine.

[12] A method for producing attenuated Cyprinid herpesvirus-2 (CyHV-2), comprising: subculturing CyHV-2-infected Carassius auratus langsdorfii-derived cells as a host, and subculturing CyHV-2-infected Cyprinus carpio-derived cells obtained by the above culture as a host, the production method.

[13] A method for controlling CyHV-2 infection in fish, comprising: inoculating fish with a composition containing an effective amount of attenuated Cyprinid herpesvirus-2.

[14] The method according to

[13] , wherein the composition is the composition according to any one of [1] to

[11] . The present invention provides a vaccine that has a high control effect against CyHV-2 infection in fish, has high safety for the fish to be treated, and can be inoculated by a simple method. Figure 1 is a graph showing the survival rate of goldfish in the evaluation test of the vaccine of Example 4. 29 days after the vaccine treatment, each test group (n = 20) was immersed and inoculated with virulent CyHV-2. Figure 2 is a diagram showing the amount of virus in the fish body after virus inoculation of Example 5. After immersing and inoculating the goldfish with the vaccine candidate strains P7-P8 and the virulent strain Sat1 P4, the fins and body kidneys were collected, and the virus DNA copy number was measured by quantitative PCR. The right side shows the group inoculated with the virulent strain, and the left side shows the group inoculated with the attenuated strain. In the virulent group, all fish died 5 days after inoculation, so data 8 days later could not be obtained. Figure 3 is a graph showing the change in the amount of virus DNA when the vaccine candidate P7-P8 was passaged in the fish body. The line in the graph represents the detection limit of quantitative PCR. The first generation was inoculated with P7-P8, and the second and subsequent generations were inoculated with the virus re-isolated from the fish body. In the second generation, 3 out of 5 fish were negative for quantitative PCR, and in other test groups, 5 out of 5 fish were positive for quantitative PCR. The present invention will be specifically described below, but the present invention is not limited thereto. Unless otherwise specifically defined in this specification, scientific terms and technical terms used in connection with the present invention shall have the meanings generally understood by those skilled in the art. In one aspect of the present invention, the virus strain used for the production of attenuated CyHV-2 may be, in addition to the wild-type CyHV-2, a variant or subtype of CyHV-2 into which natural or artificial mutations have been introduced. As CyHV-2, a virus isolated from a fish body infected with CyHV-2 or a virus propagated in vitro using cultured cells may be used. In one aspect of the present invention, as the attenuated CyHV-2, CyHV-2 cultured using cells derived from common carp (Cyprinus carpio) or cells derived from crucian carp (Carassius auratus langsdorfii) as a host can be used. In one embodiment, attenuated CyHV-2 can be prepared by subculturing CyHV-2 using koi-derived KF-1 cells, specifically koi fin-derived KF-1 cells, and / or crucian carp-derived CFS cells as a host. In one aspect of the present invention, subculture using CFS cells derived from gimbuna as a host can be performed, for example, 1 or more times, 2 or more times, 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, or 10 or more times, and the upper limit can be, for example, 20 or less times, 19 or less times, 18 or less times, 17 or less times, 16 or less times, 15 or less times, 14 or less times, 13 or less times, 12 or less times, or 11 or less times. Specifically, the subculture can be performed 1 to 20 times, 2 to 20 times, 3 to 20 times, 4 to 20 times, 5 to 20 times, 6 to 20 times, 7 to 20 times, 4 to 15 times, 4 to 14 times, 4 to 13 times, 4 to 12 times, 4 to 11 times, or 4 to 10 times. In addition, subculture using KF-1 cells derived from carp as a host can be performed, for example, 1 or more times, 2 or more times, 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, or 10 or more times, and the upper limit can be, for example, 15 or less times, 14 or less times, 13 or less times, 12 or less times, 11 or less times, 10 or less times, 9 or less times, 8 or less times, 7 or less times. Specifically, the subculture can be performed 1 to 3 times, 1 to 4 times, 1 to 5 times, 1 to 6 times, 1 to 7 times, 1 to 8 times, 1 to 9 times, 1 to 10 times, or 1 to 10 times. In one aspect of the present invention, subculture using CyHV-2 obtained by subculture using CFS cells derived from gimbuna as a host can be performed using KF-1 cells derived from carp as a host. For the medium and the like used for subculture, a medium known to those skilled in the art can be used. For example, M199 medium (Medium 199), L-15 medium (Leibovitz's L-15 Medium), MEM (Eagle’s Minimum Essential Medium) medium, BME (Eagle’s Basal Medium) medium, Hanks' solution, Dulbecco's phosphate buffered saline (PBS), etc. can be used. In one aspect of the present invention, for the subculture of CyHV-2 using goldfish-derived CFS cells as the host, M199 medium (Medium 199), L-15 medium (Leibovitz's L-15 Medium), MEM (Eagle’s Minimum Essential Medium) medium can be used as the medium. For the subculture of CyHV-2 using carp-derived KF-1 cells as the host, M199 medium (Medium 199), L-15 medium (Leibovitz's L-15 Medium), MEM (Eagle’s Minimum Essential Medium) medium can be used as the medium. Additives may be added and mixed to the medium used for subculture. Examples of additives include commonly used substances such as commercially available fetal bovine serum FBS (Fetal Bovine Serum), calf serum, antibiotics, sodium bicarbonate solution, sodium chloride, non-essential amino acids, HEPES, etc. In one aspect of the present invention, components derived from fish such as goldfish and carp, for example, goldfish serum and body kidney extract, can be added to the medium used for subculture. The virus-containing liquid obtained by recovering the virus from the cultured cells used for virus propagation and passage can be used as the vaccine bulk solution. In one embodiment, the culture containing the cultured cells can be centrifuged, and the recovered supernatant can be used as the vaccine bulk solution. The centrifugation conditions are, for example, 4 to 25 °C, 500 to 3000 × g, and 3 to 10 minutes. In another embodiment, the supernatant obtained by low-speed centrifugation of the culture solution, the disrupted cell suspension obtained by collecting and sonication of the infected cell fraction, the suspension of the virus and its infected cells frozen and thawed with dry ice and an organic solvent, the supernatant obtained by low-speed centrifugation of such a suspension, the resuspended solution of the sedimented cells, etc. can also be used as the vaccine bulk solution. Note that such a vaccine bulk solution can be sterilized by membrane filtration, and antibiotics can also be added. In one aspect of the present invention, the composition contains cells infected with attenuated herpesvirus-2 (CyHV-2) of Cyprinidae fish, or derivatives thereof, and examples of the cell-derived substances include a virus concentrate obtained by treating the cells, a disrupted cell suspension, and a virus-containing solution obtained by further treating them. In one aspect, the composition of the present invention can be administered together with an adjuvant as an adjuvant for enhancing immunogenicity. For example, muramyl dipeptide derivatives, thymosin, interleukin, etc. can be used as the adjuvant. In one embodiment of the present invention, the vaccine formulation or the vaccine bulk solution may contain a stabilizer for enhancing the stability of the vaccine, and may also contain other additives, such as preservatives, pH adjusters, bactericides, diluents, surfactants, etc. Examples of the stabilizer include saccharides, amino acids, and serum. In one aspect, the composition of the present invention may be made into a solid by lyophilization. In that case, a dispersion medium for stabilizing the vaccine can be added. Examples of the dispersion medium include fetal bovine serum, calf serum, etc. In one aspect, the composition of the present invention can be used as an attenuated live vaccine. The vaccine can be administered to fish by the immersion method. For example, vaccination can be performed by accommodating the fish in breeding water containing 0.001 to 100 mL, specifically 0.01 to 10 mL of the vaccine bulk solution per liter of the breeding water. The time for accommodating fish in the breeding water is appropriately set by those skilled in the art in consideration of factors such as the content of the vaccine contained in the breeding water. In one embodiment, the accommodation time can be set with a lower limit of, for example, 1 second or more, 1 minute or more, 30 minutes or more, 1 hour or more, 2 hours or more, and an upper limit of, for example, within 24 hours, within 10 hours, within 4 hours, within 2 hours, within 1 hour. The water temperature of the breeding water containing the vaccine is not particularly limited as long as the virus can infect and proliferate, for example, in the range of 15 to 30 °C. In one embodiment of the present invention, for vaccination by the immersion method, the titer of the vaccine is, for example, 10 ２ TCID 50 / L or more, specifically 10 ３ TCID 50 / L or more as the lower limit, and 10 ７ TCID 50 / L or less as the upper limit, and the breeding water adjusted in this way can be used. In one embodiment, vaccination can be performed by immersing the fish body in high-concentration vaccine-containing water for a short time. The short-time immersion can be carried out, for example, by directly immersing the fish body scooped up with a net into the vaccine-containing water. After immersion, it can also be returned to the breeding water after holding for, for example, 10 seconds to 3 minutes, specifically 30 seconds to 2 minutes, preferably 1 minute. In one embodiment, vaccination can be performed by bringing the vaccine-containing water into contact with the fish body by spraying or the like. Vaccination by spraying or the like can be carried out, for example, by directly spraying the vaccine-containing water on the fish body scooped up with a net. After vaccination, it can also be returned to the breeding water after holding for, for example, 10 seconds to 3 minutes, specifically 30 seconds to 2 minutes, preferably 1 minute. The composition of the present invention may be liquid or solid. In one embodiment, the composition is a powder obtained by drying a liquid containing a live attenuated virus and is diluted with water before use for administration. In one aspect of the present invention, the composition can be used as a vaccine for fish that may be infected with CyHV-2 and can be used for fry, juveniles, or adult fish. In one embodiment, it can be used for fish with a body weight of, for example, 0.5 to 50 g, or 1 to 20 g, specifically 2 to 10 g, such as goldfish. In one embodiment, two or more fish can be housed in the rearing water and vaccinated by the immersion method at one time. In that case, fish with an average body weight of, for example, 1 to 15 g, specifically 2 to 10 g, such as goldfish can be targeted. In one embodiment of the present invention, vaccine-containing water is prepared in a large aquarium, a large number of fish are housed therein, and then normal rearing water is continuously added to the aquarium to dilute and remove the vaccine, whereby, for example, up to 100, 500, or 1000 fish can be vaccinated. In one embodiment of the present invention, vaccine-containing water is prepared in a large aquarium, a large number of fish are housed therein, and then normal rearing water is continuously added to the aquarium to dilute and remove the vaccine, whereby more than 1000 fish, for example, up to 2000, 3000, 4000, 5000, 10000 or more fish can be vaccinated. In one embodiment of the present invention, in addition to administration by the immersion method, the composition can also be administered, for example, by spray, intraperitoneal, subcutaneous, or intramuscular inoculation, oral administration, or mixed feed administration. Fish infected with CyHV-2 do not show characteristic symptoms in appearance, but their behavior becomes inactive and they stop moving on the water surface or at the bottom of the pond. It is also known to cause severe anemia. Infection with CyHV-2 in the fish body is observed in all organs such as fins, gills, heart, intestinal tract, spleen, brain, and kidney. In one aspect of the present invention, the administration target of the composition is not particularly limited as long as it is a fish infected with CyHV-2 and having the above-mentioned pathological conditions. However, the preferred administration targets are Cyprinidae fish, and more preferred administration targets are goldfish and crucian carp. For example, they are goldfish (Carassius auratus), gibel carp (Carassius auratus gibelio), European crucian carp (Carassius carassius) or Carassius auratus langsdorfii. The most preferred administration targets are goldfish or gibel carp. Examples of goldfish include goldfish generally referred to as high-grade varieties such as wakin, ranchu, edo nishiki, sakura nishiki, tosakin, azuma nishiki, jikin and ryukin. When the composition of the present invention is used as a vaccine, those skilled in the art can appropriately set the dosage, administration method and number of administrations of the vaccine, as well as the type, size and age of the fish to be administered. Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited thereto. Example 1: Subculture of CyHV-2 using CFS cells derived from Carassius auratus langsdorfii Virus strain used CyHV-2 isolated from goldfish fin cells (GFF cells, Li X, et al., J Shanghai Fish Univ 12:12-18 (2003)) created from diseased fish that occurred in a goldfish farm in Saitama Prefecture in 1999 was subcultured 23 times using Ryukin tail fin cells (RyuF-2 cells, Shibata et al. Dis. Aquat. Organ. 115, 223-232 (2015)) by the following method, and the obtained strain was used in this test (CyHV-2 Sat-1 strain). Disperse RyuF-2 cells in M199 medium (added with 5% FBS) and 25 cm ２Seeded into a flask and inoculated by adding the CyHV-2 virus culture solution (100 μL) to the cell culture solution, followed by culturing at 25°C. When the cells became spherical due to virus infection and 80% of the cells detached from the bottom of the culture vessel, the culture solution was collected as the virus solution. This virus solution was inoculated into the same cells in a newly prepared 25 cm ２ flask. This virus passage operation was performed a total of 7 times. After the 8th passage, it was passaged in RyuF-2 cells using a medium containing goldfish kidney extract (added according to the method of Shibata et al

[2015] ). To genetically homogenize the virus (CyHV-2 Sat-1 strain) passaged 23 times in total, the following limiting dilution method using a 96-well plate was used to perform two rounds of clone purification. Cloning of the virus using the limiting dilution method RyuF-2 cells (100 μL) dispersed in M199 medium (supplemented with 5% FBS) were seeded into each well of a 96-well plate and incubated overnight at 25°C. The virus was serially diluted two-fold with M199 medium (supplemented with 5% FBS), and this serially diluted virus solution (50 μL) was inoculated into the RyuF-2 cells in the 96-well plate. Wait for about two weeks until the cytopathic effect (CPE), which is evidence of virus growth, has appeared completely, and collect the virus solution that showed CPE at the highest dilution (C1-P0: first clone). This virus was similarly used to collect the virus solution from the well that showed CPE at the highest dilution by limiting dilution in a 96-well plate seeded with RyuF-2 cells again (C2-P0). This virus solution was inoculated into the RyuF-2 cells in a 25 cm ２ flask and the virus cultured at 25°C was stored at -80°C as the C2-P1 virus. This virus was further passaged once in RyuF-2 cells (Sat-1 C2-P2: second clone and then one passage). Subculture using CFS cells The above C2-P1 virus was passaged in CFS cells (Hasegawa S et al. In Vitro Cell Dev Biol Anim 33: 232-233 (1997)) derived from fin cells of clone ginbuna S3n provided by Professor Teruyuki Nakanishi of Nihon University. The CFS cells were dispersed in L-15 medium (supplemented with 5% FBS) and seeded into a 25 cm ２ flask. The cells were inoculated by adding the C2-P1 virus solution (100 μL) to the cell culture medium, and further adding goldfish kidney extract and culturing at 25°C. When the cells became spherical due to virus infection and 80% of the cells were detached from the bottom of the culture vessel, the culture medium was collected as the virus solution. This virus passage operation was performed 20 times in total. Example 2: CFS cell passage CyHV-2 genome sequence Virus preparation P4, P10, and P20 strains passaged 4, 10, and 20 times respectively in CFS cells of CyHV-2 (Sat1 C2P2) were used. As a comparative control, the strain before passage in CFS cells (CyHV-2 Sat1 C2P2) was used. Each virus was concentrated by high-speed centrifugation according to the following procedure. The virus was cultured in four 75 cm ２ flasks. After confirming that the CPE had spread throughout, the culture medium was transferred to a 50 mL centrifuge tube and centrifuged (4°C, 7000 rpm, 30 minutes). The supernatant was collected and transferred to a new 50 mL centrifuge tube, and then centrifuged again (4°C, 12500 rpm, 30 minutes). After confirming the white virus pellet, the supernatant was discarded. TNE buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.5; 500 μL) was added to suspend the virus, and it was incubated overnight at 4°C. Then, the virus suspension was transferred to a 1.5 mL tube and stored at -80°C. Subsequently, Proteinase K (Takara Bio, 5 mg / ml, 5 μL) was added to the virus suspension (500 μL), and the mixture was treated at 55°C for 5 hours. DNA of each virus was extracted using phenol / chloroform / isoamyl alcohol (PCI) and chloroform / isoamyl alcohol (CIA) according to the following procedure. To the Proteinase K-treated sample (500 μL), phenol / chloroform / isoamyl alcohol (PCI, 500 μL) was added and mixed by inversion, followed by centrifugation (4°C, 12,500 rpm, 10 minutes). The upper layer was collected with a pipette and transferred to a new 1.5 mL tube. Then, CIA (500 μL) was added and centrifuged (4°C, 12,500 rpm, 10 minutes). The upper layer was collected in the same manner as above and transferred to a new 1.5 mL tube. After that, 2-propanol (500 μL) and sodium acetate (5 M, 50 μL) were added, and the mixture was allowed to stand at room temperature for 5 minutes. Then, it was centrifuged at 4°C, 15,000 rpm for 15 minutes, and the supernatant was discarded with a decanter. Then, 70% ethanol (500 μL) was added, centrifuged (20°C, 15,000 rpm, 5 minutes), and the supernatant was discarded. It was air-dried for 30 - 60 minutes, TE buffer (Tris-HCl 10 mM, EDTA 0.1 mM; pH = 8.0, 50 μL) was added and suspended, and the obtained sample was stored at -30°C. Sequence The genomic sequence of the virus was performed using Illumina's Miseq. After preparing the library with the Nextera XT DNA preparation kit (Illumina), sequencing was performed using the 500-cycle MiSeq Reagent Nano Kit v2 (Illumina) as a cartridge. Subsequently, the sequencing data was analyzed with CLC Genomics Workbench Software (CLC bio). The same isolate CyHV-2 ST-J1 (GenBank No. NC_019495) was set as the reference sequence. Amino acid substitutions and base insertions and deletions in the CFS cell passage strain were detected by comparison with the virulent CyHV-2 Sat1 C2P2 strain. In ORF25D, ORF113, ORF115, and ORF132 of the CyHV-2 genome, mutations common to all three attenuated strains tested were confirmed. In ORF25D, in addition to four amino acid substitutions, a large deletion of 342 bases occurred. In ORF113 and ORF132, deletions of 5 bases and 1 base occurred, respectively. In ORF115, a single base substitution occurred (Table 1). ORF25 is conserved in all cyprinid herpesviruses and constitutes a gene family that may have evolved from a common ancestor. In CyHV-3, which is known to cause koi herpesvirus disease, it has been reported that artificially deleting ORF25 results in no death in vivo and reduced growth in vitro. Therefore, the large base deletion in ORF25D commonly observed in all attenuated CyHV-2 strains this time is highly likely to be involved in its attenuation. In addition, in ORF113 and ORF132, frameshifts due to base deletions have occurred, and they may also be involved in attenuation. The mutations in ORF10, ORF67, ORF68, and ORF127 were not observed in the P4 strain with a small number of passages in CFS cells, but were only observed in the P10 and P20 strains with a large number of passages (Table 1). Therefore, these mutations are considered to be involved in the adaptation of CyHV-2 to CFS cells. Example 3: Production of vaccine candidate virus by subculturing CyHV-2 using KF-1 cells derived from koi fins As a result of the vaccine test using the CFS cell subculture strain, the P20 strain with a large number of passages had a slightly low efficiency, and the P4 strain with a small number of passages was not acclimated to CFS cells and had a low titer. Therefore, using the P7 and P10 strains at the middle number of passages as the original strains, they were dispersed in MEM medium (supplemented with 5% FBS) the previous day and 25 cm 2 To the KF-1 cells, which are cells derived from koi fins seeded in a culture flask, each virus culture solution (100 μL) was added and inoculated. After thorough mixing, the cells were cultured at 25°C. The goldfish kidney extract prepared by the method described below was added to the medium for culture. Kidneys were collected from multiple healthy goldfish, added with 9 times the volume of M199 medium (supplemented with 5% FBS), homogenized, centrifuged (4 °C, 3000 rpm, 15 minutes), and the supernatant was filtered sequentially through filters with pore sizes of 0.45 μm, 0.22 μm, and 0.1 μm, and the filtrate was stored at -80 °C. At the third passage, it was confirmed that CPE had spread sufficiently in KF-1 cells, and from the fourth passage onwards, goldfish kidney extract was not added to the medium. KF-1 cells were passaged 4 times and 7 times respectively to generate 4 strains of P7-P4, P7-P7, P10-P4, and P10-P7. The notation of the above strains indicates that the number before the hyphen represents the number of passages in CFS cells, and the number after the hyphen represents the number of passages in KF-1 cells. Subsequently, each virus was cloned using the limiting dilution method shown below. After seeding KF-1 cells in a 96-well plate, MEM medium (containing 5% FBS, 100 μL) was added to each well and incubated overnight at 25 °C. The virus solution was diluted 3-fold for each column of the 96-well plate, and the virus solution (50 μL) was added to each well. Thirteen days after virus inoculation, KF-1 cells were newly seeded in a 6-well plate, MEM medium (containing 5% FBS, 2 mL) was added to each well, and incubated overnight. Fourteen days after virus inoculation, among the wells with CPE positive in the 96-well plate, the culture supernatant was collected with a pipette from the well with the highest dilution factor and inoculated into KF-1 cells in the 6-well plate. When CPE spread throughout, the culture solution was collected, centrifuged (4 °C, 2000 rpm, 5 minutes), the supernatant was collected and stored at -80 °C. Four strains of P7-P5, P7-P8, P10-P5, and P10-P8, which were passaged once more in KF-1 cells, were used as vaccine candidate viruses. The virus titers in each KF-1 cell were 10 3.9 、10 4.1 、10 4.1 、10 3.9 TCID 50 / mL. The viral titer was measured by the microtiter method. KF-1 cells (100 μL) were seeded in a 96-well plate, and the next day, a serially diluted virus solution (50 μL) diluted 1 / 10-fold was inoculated. The cells were cultured at 25°C for 2 weeks, and the TCID 50 was calculated from the number of wells showing CPE. Example 4: Confirmation test of the control effect of the vaccine Twenty goldfish (produced at the Yoshida Station of the Tokyo University of Marine Science and Technology, average fish weight: 5.5 g) were placed in 1 L of rearing water adjusted so that the vaccine titer was 10 1.1 TCID 50 / L, and inoculated by the immersion method for 2 hours. The water temperature was 25°C, and the fish were reared in a flowing water system with aeration for 28 days. Commercial feed was provided during the rearing period. There was no fish death during the above rearing period. Twenty-nine days after vaccination, the fish were challenged with the virulent CyHV-2 Sat1 P4. Twenty goldfish were placed in 1 L of rearing water adjusted so that the virus titer was 10 1.0 TCID 50 / mL, and inoculated by the immersion method for 2 hours. In the control group, MEM (containing 5% FBS, 1 mL) was added to the rearing water and the same operation was performed. The water temperature was 25°C, and the fish were reared with aeration in flowing water. After rearing and observing for 21 days and counting the number of deaths, the vaccine efficacy rate (RPS) was calculated by the following formula. The test results are shown in Figure 1. Vaccine efficacy rate (RPS) = {1 - (mortality rate in the vaccinated group / mortality rate in the control group)} × 100 As a result of the challenge, the final survival rates were 80% in the P7-P5 group, 90% in the P7-P8 group, 90% in the P10-P5 group, and 50% in the P10-P8 group (Figure 1). The survival rate after challenge in the non-vaccinated group was 0%, and the respective vaccine efficacy rates (RPS) are shown in Table 2. The groups inoculated with P7-P5, P7-P8, and P10-P5 showed a high vaccine efficiency of 80% or more. However, the vaccine efficiency of P10-P8 was 50%, which was lower than that of other vaccine strains. It is considered that the P10-P8 strain had a large number of passages in cultured cells, mutations accumulated, and its infectivity to goldfish decreased, resulting in a lower efficiency. From the above results, the P7-P8 strain, which showed a high vaccine efficiency in this challenge test and was relatively easy to grow in KF-1 cells, was selected as a vaccine candidate and used in subsequent experiments. Example 5: Confirmation test of the growth of CyVH-2 P7-P8 strain in fish It was confirmed how much the vaccine candidate virus CyHV-2 P7-P8 could grow in fish and compared with the virulent strain. Six goldfish (Wakin, average fish weight 5.5 g, produced at Yoshida Station, Tokyo University of Marine Science and Technology) were used per test group. The viruses used were the attenuated CyHV-2 P7-P8 and the virulent CyHV-2 Sat1 P4. The virus solution (0.5 mL) was added to the breeding water (500 mL), and inoculation was performed by the 2-hour immersion method. Four days and eight days after inoculation, three goldfish were sampled each time, the fins and body kidneys were removed and placed in 1.5 mL tubes, and the weights of each tissue were measured. Lysis buffer (0.1 M NaCl, 0.05 M Tri-HCl, 0.1 M EDTA and 1% [w / v] SDS, pH 8.0; 500 μL) was put into the tube, 5 μL of Proteinase K (Takara Bio, 5 mg / mL) was added, and the mixture was incubated overnight at 37 °C. Then, DNA was extracted with PCI / CIA and subjected to quantitative PCR by the following method to measure the virus DNA copy number in each tissue. The sequences of the primers and Taqman probe for quantitative PCR were referred to the quantitative PCR analysis method of CyHV-2 by A.E. Goodwin et al. (Non-Patent Document 17). The sequence of the probe used is shown in Table 3. The plasmid of the CyHV-2 polymerase sequence used for the calibration curve was prepared using the above primers and ECOSTM Competent E. coli DH5α (Nippon Gene) and used. PureYield TMPlasmid solutions extracted using the Plasmid Miniprep Systems (Promega) and with their concentrations measured using the NanoDrop ND-1000 (Thermo SCIENTIFIC) were serially diluted 10-fold with sterile distilled water until they reached 1.0×10 ８ to 1.0×10 ２ copies per microliter. After thawing the DNA solutions extracted from each sample, the DNA solutions were dispensed into 96-well plates to a total volume of 20 μl with Takara Bio's Premix Ex Taq TM (Probe qPCR, 10 μl), Forward primer (10 μM, 0.4 μl), Reverse primer (10 μM, 0.4 μl), Taqman probe (5 μM, 0.8 μl), DNA solution (2 μl), and sterile distilled water (6.4 μl), and quantitative PCR was performed using the LightCycler (registered trademark) 480 Instrument II (Roche). The PCR conditions were 30 seconds at 95°C, 40 cycles of 5 seconds at 95°C and 20 seconds at 60°C, and incubation at 50°C. Four days after virus inoculation, in the group inoculated with the virulent strain Sat1 P4, the amount of viral DNA per unit tissue weight showed a very high value of 10 ８ to 10 １０ copies / mg, while in the group inoculated with the vaccine candidate strain P7-P8, it showed a low value of 10 ４ to 10 ６ copies / mg (Figure 2). Eight days after inoculation, in the group inoculated with the vaccine candidate strain P7-P8, it showed a value of 10 ３ to 10 ５ copies / mg, which was decreased compared to that four days after inoculation (no data were available eight days after inoculation in the virulent strain group because all fish died five days after inoculation). A viral DNA amount exceeding 10 ６ copies / mg per unit tissue weight is assumed to be a lethal viral growth amount in goldfish. Also in this experiment, in the group inoculated with the vaccine candidate strain, the amount of viral DNA in the tissue was 10 ６plateaued around copies / mg. P7-P8 does not lead to death in goldfish, and it is thought that the virus proliferates to about 10 ６ copies / mg and the state persists for some time, conferring immunity to goldfish. Example 6: In vitro test for evaluating the risk of pathogenicity reversion It was verified whether pathogenicity reversion would be observed by culturing CyHV-2 P7-P8, which had been passaged in non-host-derived cells and attenuated, in cells derived from goldfish, the original host. The virus used was CyHV-2 P7-P8. The cells used were RyuF-2 cells derived from goldfish, and the culture medium used was M199 medium (Sigma) containing 5% FBS as the culture solution. The experimental fish used were 15 goldfish (Wakin, produced at Yoshida Station, Tokyo University of Marine Science and Technology) with an average fish weight of 5.3 g for each test group. Virus passage 25 cm ３ Using a 25 cm flask, RyuF-2 cells (Shibata et al. Dis. Aquat. Organ. 115, 223-232 (2015).) were newly seeded and the culture solution (5 mL) was added. The next day, a virus solution (50 μL) of CyHV-2 P7-P8 was added to the culture flask of RyuF-2 and cultured at 25 °C. When CPE spread throughout, the culture solution was collected, centrifuged (4 °C, 2000 rpm, 5 minutes), and the supernatant was collected. The collected virus solution (50 μL) was immediately inoculated into the 25 cm 2 RyuF-2 cells prepared the previous day. The above operation was repeated 5 generations. The collected virus solution was stored at -80 °C. Infection experiment CyHV-2 P7-P8-P1, P7-P8-P3, and P7-P8-P5, which were passaged 1, 3, and 5 times with CyHV-2 P7-P8 in RyuF-2 cells, were used in the infection experiment. The virus infection titers in RyuF-2 cells were 10 9.2 , 10 8.7 , 10 7.3It was TCID50 / mL. Although the infectious titers differed among the respective cultured viruses, in this experiment, the infectious titer was not considered. Goldfish were placed in the mixture of virus culture solution (1 mL) added to breeding water (1 L) and immersed for 2 hours for inoculation. They were bred at a water temperature of 25°C, and the number of deaths was counted over 21 days. M199 medium containing 5% FBS was used for the non-infected control group, and virulent CyHV-2 Sat1 P4 was used for the infected control group. CyHV-2 P7-P8 attenuated by subculture in non-host-derived cells was subcultured 1, 3, and 5 times in RyuF-2 cells derived from goldfish, the original host, to obtain CyHV-2 P7-P8-P1, P7-P8-P3, and P7-P8-P5. The mortality rates of the goldfish inoculated with them were all 0% (Table 4). Also, during the observation period, no abnormalities were observed in the appearance or behavior of the inoculated fish. From this, it was confirmed that the risk of pathogenicity reversion by subculture using goldfish-derived cells of the P7-P8 strain was low. Example 7: In vivo test for evaluating the risk of pathogenicity reversion The presence or absence of pathogenicity reversion when the vaccine candidate strain P7-P8 was subcultured in goldfish was verified. CyHV-2 P7-P8 was used as the virus. The experimental fish used were goldfish (Wakin with an average fish weight of 5.5 g, produced at Yoshida Station, Tokyo University of Marine Science and Technology). The experimental method was set to meet the pathogenicity reversion confirmation test method in the Animal Biological Products Testing Standards and General Test Methods publicly released by the Ministry of Agriculture, Forestry and Fisheries (Ministry of Agriculture, Forestry and Fisheries. Animal Biological Products Testing Standards and General Test Methods Pathogenicity Reversion Confirmation Test Method [https: / / www.maff.go.jp / nval / kijyun / pdf / st06230.pdf]) and was carried out as follows. For 15 goldfish, the undiluted solution of CyHV-2 P7-P8 (0.05 mL per fish, 10 4.1 TCID 50 / mL, KF-1 cells) were inoculated intraperitoneally using a 27G×1 injection needle (TERUMO) and a 1 mL tuberculin syringe (TERUMO). Five out of 15 fish were used as the sampling group, and 10 fish were used as the observation group. They were reared in a 6 L water tank with a flowing water system at 25 °C with aeration. The observation period for the observation group was 21 days. Four days after virus inoculation, all five goldfish in the sampling group were taken, and caudal fins, body kidneys, and spleens were excised from each individual. DNA was extracted from the excised tissue pieces, and the viral DNA copy number in each tissue was measured by quantitative PCR. Also, the excised body kidneys and spleens were pooled, and MEM (containing 5% FBS) at 100 times the tissue weight was added and homogenized on ice. Then, centrifugation was performed at 3000 rpm for 15 minutes at 4 °C. The supernatant was collected and filtered using a 0.45 μm filter. The obtained filtrate was temporarily stored at 4 °C and inoculated onto 15 new goldfish in the same manner as above within 24 hours after sampling. This operation was repeated a total of 6 times. As a negative control group, a test group was prepared in which the same operation was performed using MEM containing 5% FBS. CyHV-2 P7-P8 was inoculated into goldfish bodies to verify whether there was a recurrence of pathogenicity in the re-isolated virus. In this experiment, virus inoculation and re-isolation were performed over 6 generations, but no death of goldfish or abnormalities in appearance and behavior were observed in any of the test groups (Table 3). By quantitative PCR, from the organs of the first-generation goldfish inoculated with CyHV-2 P7-P8, virus DNA copy numbers of about 10 ４ ~10 ６ copies / mg were detected, similar to the test results described in Example 5. However, the copy numbers decreased in the second generation, and individuals below the detection limit of quantitative PCR were also observed. The third generation increased compared to the second generation, but from the fourth generation onwards, it was almost flat, and no signs of increased virus growth ability were observed (Figure 3). Also, no fish death was observed in any of the test groups, and no abnormalities in behavior or appearance were confirmed (Table 5). From the above results, it was confirmed that CyHV-2 P7-P8 has a low risk of pathogenicity recurrence during in vivo passage in fish.

Claims

1. A composition for use in controlling CyHV-2 infection in fish, comprising an attenuated CyHV-2, the attenuated CyHV-2 having a mutation in ORF25D compared to CyHV-2 ST-J1 (GenBank No. NC_019495), and administered by immersion.

2. The composition according to claim 1, wherein the attenuated CyHV-2 is CyHV-2 cultured in a carp-derived cell or a ginbuna-derived cell as a host.

3. 3. The composition according to claim 1, wherein the attenuated CyHV-2 is CyHV-2 cultured in a carp-derived KF-1 cell or a ginbuna-derived CFS cell as a host.

4. The composition according to any one of claims 1 to 3, wherein the attenuated CyHV-2 is CyHV-2 that has been passage-cultured in ginbuna-derived CFS cells as a host and then passage-cultured in carp-derived KF-1 cells as a host.

5. The composition according to any one of claims 1 to 4, wherein the attenuated CyHV-2 is CyHV-2 that has been subcultured 1 to 20 times using ginbuna-derived CFS cells as a host and then subcultured 1 to 20 times using carp-derived KF-1 cells as a host.

6. The composition according to any one of claims 1 to 5, wherein the fish is goldfish (Carassius auratus), Prussian carp (Carassius auratus gibelio), European crucian carp (Carassius carassius), or silver crucian carp (Carassius auratus langsdorfii).

7. The composition according to any one of claims 1 to 6, wherein the CyHV-2 infection is herpes virus hematopoietic necrosis.

8. The composition according to any one of claims 1 to 7, comprising a cell infected with attenuated Cyprinidae herpesvirus-2 (CyHV-2) or a derivative thereof.

9. A composition according to any one of claims 1 to 8 for use as a vaccine.

10. A method for producing an attenuated Cyprinid herpesvirus-2 (CyHV-2), comprising the steps of: Subculture of ginbuna-derived cells infected with CyHV-2 as a host; and the production method comprising subculturing, as a host, carp-derived cells infected with the CyHV-2 obtained by the above culture, wherein the attenuated CyHV-2 has a mutation in ORF25D compared to CyHV-2 ST-J1 (GenBank No. NC_019495) and is used by administration by the immersion method.

11. A method for preventing CyHV-2 infection in fish, comprising: inoculating fish with a composition comprising an effective amount of attenuated cyprinid herpesvirus-2; The method, wherein the composition is a composition according to any one of claims 1 to 9.