JP2011016845A - Vaccine and diagnostic agent for iridovirus infection of fish, and method for producing those - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve problem that, since 1990, iridovirus infectious diseases of fishes such as Pagrus major, Lateolabrax japonicus, Seriola quinqueradiata have occurred in great quantities, and a death rate of Pagrus major has reached at most 90% and known to be wide range of damages from young to adult fishes, therefore, the infectious diseases sound alarm bells to management of culture business and correlative industry thereof, and it is an urgent need to establish preventive methods therefor.SOLUTION: Pathogen virus of the infectious diseases and subcultured strains thereof, effective vaccine for preventing the infectious diseases, a diagnostic agent useful for simple established diagnosis and early stage diagnosis, and a method for large scale production and purification of such a virus antigen are provided. Technically, the iridovirus antigen or iridovirus-originated antigen is produced in a large scale using a habituated strain prepared by subculturing the virus freshly isolated from fishes suffering from the iridovirus infectious diseases in a sensitive host, and after purifying the same, the vaccine and diagnostic agent using such an antigen as an effective ingredient are prepared.

Description

  The present invention relates to infections with fishes, for example, fishes belonging to the order of sea bass, blowfish, flounder, etc. The present invention relates to pathogenic viruses, vaccines, diagnostic agents, production methods and purification methods of the virus antigens, basic research, and clinical or applied research reagents.

  According to the 6th report of the International Virus Classification Committee, viruses belonging to the family Iridoviridae are regular icosahedrons with a diameter of 130-170 nm, have linear double-stranded DNA inside, and have no envelope. The genus varies depending on the genus. Currently, the following five genera are used, and the hosts are Lymphocystivirus and Goldfish which infects fish such as Ranavirus, flounder, yellowtail, red sea bream, sea bass, etc., whose insects are Iridovirus and Chloriridovirus, frog virus 1-like viruses "(Archives of Virology, Supplement 10, p. 95-99, 1995).

  On the other hand, the present inventors discovered and isolated a new virus from a diseased fish with symptoms different from those of a known iridovirus infection, and categorically tentatively identified iridovirus as to this infection and its pathogen virus. Various basic researches were carried out for the first time in the world in the category of Virology. The following is a summary of the history of these studies: From August to September 1990, a large number of moribund deaths of unknown origin occurred in several farms in Shikoku. We conducted external observation, autopsy, biopsy, morphological observation by electron microscope, virus isolation, etc., and reported that the pathogen of this infectious disease was an unreported new iridovirus ( Fish Disease Research, 27 (2), p.19-27, 1992). Furthermore, this iridovirus was cultured and virological analysis on biological and physicochemical properties was carried out (Fish Disease Research, Vol. 29 (1), p. 29-33, 1994). In addition, the inventors have succeeded in producing a monoclonal antibody against the virus, and have pioneered an immunological diagnostic method using the antibody (Fish Disease Research, Vol. 30 (1), p. 47-51, 1995). The iridovirus referred to in the present invention means not the known iridovirus based on the 6th report of the aforementioned International Virus Classification Committee, but the above-mentioned novel iridovirus discovered and isolated by the present inventors. . The iridovirus infection referred to in the present invention means a fish disease caused by infection with the virus. The characteristic of such infections is the extremely high mortality rate, and the appearance characteristics of the diseased fish are blackening and discoloration of the body color, bleeding threads on the body surface and eyelids, partial standing scales, slight protrusion of the eyeball and bleeding Etc. Necropsy and biopsy findings, pathological observations, virological properties and immunological characteristics are detailed in the above three reports (fish disease research).

  Techniques relating to the iridovirus of the present invention include monoclonal antibodies and hybridomas for producing the same (Japanese Patent Laid-Open No. 6-1531979), oligonucleotide primers for PCR (Polymerase Chain Reaction) (Japanese Patent Laid-Open No. 6-165777) and the like. Are known.

  However, passage and mass culture of such iridoviruses are not easy, and mass production and purification methods for the antigens are still unestablished, and the development and provision of vaccines and diagnostic agents using the virus antigens are not known. It has not been long-awaited.

  Since the first outbreak in the Shikoku region in 1990, the iridovirus infectious disease according to the present invention has been observed on a large scale, especially in red sea bream farms in western Japan, and it has reached not only larvae but also reared fish and parent fish. Has occurred. Furthermore, the mortality rate is as high as 90% in red sea bream, and it is known that the damage ranges from fry to adults and is extensive and enormous. In addition, infection and onset occur not only in red sea bream, but also in sea fishes such as sea bream, sea bream, sea bream, sea bass, yellowtail, amberjack, flatfish, striped horse mackerel, tiger puffer, pheasant groupfish, flounder, etc. Widely confirmed. Thus, iridovirus infections are now alarming the supply, demand and management of aquaculture and related industries.

  The present invention relates to a pathogen virus of an iridovirus infection and its passage strain, a vaccine effective for the prevention of such infection, a diagnostic agent useful for simple early diagnosis and definitive diagnosis, and a method for producing and purifying an iridovirus antigen. Furthermore, the above-mentioned problems are solved by providing useful reagents in basic research and clinical or applied research.

  The effects of the present invention are as follows: not only prevention of fish iridovirus infection by vaccine, but also simple definitive diagnosis and early diagnosis by a diagnostic agent using the virus antigen are possible. Furthermore, the iridovirus and its antigen subcultured to a specific host provided by the present invention, and an antibody produced using such an antigen are useful reagents in basic research and clinical or applied research of fish iridovirus infection. It is extremely useful as a material or contributes greatly to the progress of such research.

  (1) Material for virus isolation: Pathogen viruses include fish that have developed or died due to infection with an iridovirus, such as fish belonging to the order of perch, puffer fish, flounder, such as red sea bream, sea bream, sea bream, sea bream, sea bass, It can be isolated from organs excised or removed from yellowtail, amberjack, flounder, striped mackerel, tiger puffer, pheasant grouper, flounder. As such organs, for example, spleen, heart, kidney, sputum, liver and the like can be used. These virus isolation materials are, for example, chopped up with an ophthalmic scissor, and added to a common salt solution or culture medium such as a phosphate buffer solution (PBS) or MEM medium described later, and then ground with a homogenizer. After preparation of about 5-30% (W / W) tissue emulsion, it is centrifuged at low speed, and the supernatant or the resuspended solution of the sediment tissue is inoculated into the following host as a virus separation material. Such materials can be sterilized by membrane filtration, and antibiotics can be added.

  (2) Host for virus amplification / separation / passaging / acclimation / mass production: A virus-sensitive host capable of propagating a pathogen virus can be selected and used. When the fish body is used as a host, fish belonging to the order Perch, Puffer, Spotted, etc., for example, red sea bream, sea bream, sea bream, sea bream, perch, yellowtail, amberjack, flounder, striped mackerel, tiger puffer, pheasant group, flatfish, etc. It can be illustrated. Virus isolation material is inoculated intraperitoneally, subcutaneously or intramuscularly in such hosts. Moreover, when using various cell cultures as a host, well-known cell lines, such as BF-2, FHM, CHSE-214, JSKG, KRE-3, RTG-2, YTF, GF, can be mentioned, for example. . However, the present invention is not limited to these cell cultures, and various primary or subcultured cell cultures, established cell lines, etc. can be adopted as long as the host is susceptible to the virus. In addition, the susceptible hosts that can be used in virus amplification, passage, and habituation are not limited to the same host, and the host can be changed during virus amplification, passage, and habituation based on the propriety of virus propagation. That is, one or more types of hosts can be used in combination.

  (3) Medium and salt solution for virus culture and cell culture: known and commercially available mediums such as 199 medium, MEM (Eagle's Minimum Essential Medium) medium, BME (Eagle's Basal Medium) medium , Hanks' solution, Dulbecco's phosphate buffer (PBS), and the like. These compositions and preparation methods are detailed in common texts on virus experiments, cell culture, tissue culture, etc., such as “Tissue Culture Technology” (edited by the Japanese Society for Tissue Culture, published by Asakura Shoten 1982) and commercial product catalogs. It is stated. Such media and salt solutions can be modified by adding additives. Additives include conventional substances such as commercially available human serum, fetal calf serum FCS (Fetal Calf Serum), antibiotics, sodium bicarbonate solution, sodium chloride, non-essential amino acids [manufactured by Dainippon Pharmaceutical Co., Ltd.] Appropriate amount can be used. For example, these final concentrations added and mixed per liter of medium are about 3-25% (V / V) for serum, about 1-6 g for sodium bicarbonate, and about 0.1-0.25 for sodium chloride. Molar and non-essential amino acids should have the standard amounts described in the product catalog.

  (4) Temperature and time of virus culture: The culture temperature is water temperature or about 10-37 ° C, preferably about 20-30 ° C, and the culture days are about 2-21 days, preferably about 4-16 days. is there.

  (5) Pathogen virus isolation and fresh isolate: A fresh isolate can be obtained by amplifying the virus using the isolation material and host of (2) above. In order to increase the probability of isolation, intraperitoneal inoculation using the infected spleen spleen as the isolation material and red sea bream as the host is desirable. The number of passages for amplifying the virus during virus isolation is usually about 1-10 times, preferably about 1-5 times. In order to determine the isolated pathogen virus, it is necessary to identify the pathogen virus in parallel with the separation operation. Such identification can be done by assaying the isolated material organ, for example, by nucleic acid staining of organ sections or stamp specimens by nucleic acid staining or Giemsa-stained tissue cells under alcohol fixation, by electron microscopy of organ sections and fresh isolate viruses. It is possible to employ a combination of morphological observations and experiments on infection of fresh isolate virus to larvae of host fish such as red sea bream.

  (6) Acclimatization by subculture of fresh isolate and establishment of virus strain for vaccine production: The fresh isolate is selected from the host group consisting of the virus-sensitive fish body and cell culture of (2) above. A virus strain for vaccine production can be established by passage for about 1 generation or more, preferably about 5 generations or more, and acclimatization to such a host. The total number of passages for acclimation to the host for the purpose of mass production of virus antigens and stabilization of virus yield and antigenicity is about 1-100 generations, preferably about 5-30 generations. The host for antigen production is preferably a fish-derived cell from an immunological viewpoint. For example, the iridovirus Ehime-1 strain that has been passaged 3 times to red sea bream and further passaged 10 times to fish-derived GF cells is suitable as a virus strain for producing virus antigens, that is, a seed for production. In addition, in order to establish such production virus strains, in order to increase the mass production yield and quality of virus antigens, to stabilize them and to reduce production costs, the selection of virus culture hosts and virus passage methods It is necessary to set various conditions for virus culture. For example, acclimation of virus to GF cells (ATCC CCL58) and BF-2 cells (ATCC CCL 91) as production hosts, passage of inoculating and culturing virus or its infected cells on a cell culture monosheet, uninfected Passage inoculated with virus in cell suspension and co-culture, passage by mixed culture of infected and uninfected cells, passage of persistently infected cells, cell culture of lysate of infected cells by ultrasound and freeze-thaw treatment Passage to inoculate and culture monosheet, use of virus or its infected cells as seed, estimation of virus culture days based on multiplicity of infection (MOI), virus infected cells according to the present invention take round The degree of spread of the cytopathic effect (CPE) in the cell monosheet, collection of virus and its infected cells with about 50-90% as an index Can.

  (7) Preparation of antigen for vaccine: The virus is cultured using a host acclimatized with a virus strain for vaccine production, and the antigen or active ingredient for vaccine is mass-produced. When a living fish is used as a host, an about 5-30% (W / W) tissue emulsion of a virus-growing organ, for example, the spleen of an infected juvenile red sea bream is prepared by the method (1), and the tissue emulsion is prepared. As such, the supernatant obtained by low-speed centrifugation, the suspension of the sediment tissue, etc. can be used as the vaccine stock solution. When cell culture is used as the host, for example, the supernatant obtained by low-speed centrifugation of the virus culture solution, the disrupted cell suspension obtained by collecting and sonicating the infected cell fraction, the virus frozen and thawed with dry ice and an organic solvent And a suspension of the infected cells, a supernatant obtained by low-speed centrifugation of the suspension, a resuspended solution of the sediment cells, and the like can be used as a vaccine stock solution. Such vaccine stock solutions can be sterilized by membrane filtration, and antibiotics can also be added. In addition, viral antigens in these vaccine stock solutions can be concentrated and purified by ultracentrifugation using a sucrose cushion. For example, using a sucrose solution layer with a sucrose concentration of 40% (W / V) as a cushion, overlaying the vaccine stock solution, applying ultracentrifugation to sediment the virus antigen on the bottom of the centrifuge tube, and then resuspending the sediment The purified viral antigen can be prepared by immersing. The sucrose concentration that can be employed in the present invention is in the range of about 15-55% (W / V), the centrifugation conditions are about 50,000-100,000 G, and about 30-150 minutes. Furthermore, virions that are complete virus particles, incomplete virus particles, virion constituents and their post-translational modifications, virion non-structural proteins and their post-translational modifications, infection protective antigens, epitopes for neutralization reactions Etc. can be used. Such a vaccine antigen can be stabilized with a conventional inactivating agent to stabilize the steric structure. In particular, virions are desirably used as inactivated antigens by inactivating their infectivity with an inactivating agent. As the inactivating agent, for example, formalin, glutardialdehyde, β-propiolactone and the like can be added before or after preparation of the vaccine stock solution. In the case of using formalin, the amount added is about 0.0004-0.7% (V / V), the inactivation temperature is about 2-30 ° C., and the inactivation time is about 5-180 days. However, when antigenicity is impaired by inactivation, ingenuity to relax the inactivation conditions is required. Such relaxation can be achieved, for example, by reducing the amount of the inactivating agent, adding a neutral amino acid or a basic amino acid, or lowering the inactivation temperature. The free formaldehyde remaining in the inactivation step can be neutralized by adding an equal amount of sodium bisulfite, if necessary, or removed by dialysis.

(8) Preparation of vaccine: For example, the amount of antigen for vaccine is about 4-9 in terms of logarithm log (TCID ₅₀ / ml) of infectious virus amount TCID ₅₀ (Media Tissue Culture Infective Dose) The vaccine stock solution is diluted with a solution, a medium, or the like, for example, a medium BME. That is, by such dilution, the amount of antigen in the vaccine is adjusted to the amount necessary for antibody production. Further, at that time, a stabilizer for enhancing the heat resistance of the vaccine and an adjuvant as an adjuvant for enhancing the immunogenicity can be added and mixed. For example, sugars and amino acids can be used as stabilizers, and mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, and the like can be used as adjuvants. Then, it is dispensed into vials of an appropriate volume, for example, about 10-500 ml, and sealed and sealed before use as a vaccine. Such a vaccine is not only liquid, but can be used as a dry preparation by lyophilization after dispensing. In addition, the prepared vaccine preparation must be tested for safety and efficacy to ensure its quality before use. Such an assay is based on an existing preparation similar to the vaccine according to the present invention, for example, “Japanese encephalitis inactivated vaccine” defined in “Animal Biologics Standard” based on the Pharmaceutical Affairs Law (Act No. 145 of 1960), “ It can be performed in accordance with regulations such as “rabies tissue culture inactivated vaccine”.

  (9) Vaccine usage: Can be used for fish of any age at risk of infection. However, it is desirable to use it for juvenile or fry from the viewpoint of fish farming conservation. As a method of use, for example, intraperitoneal, subcutaneous, or intramuscular inoculation, an immersion method, oral administration and the like are possible. In the case of inoculation, it is desirable to use about 0.05-1.0 ml of the antigen amount of (7) above. For immunization by immersion, the vaccine is diluted about 10-100 times with breeding water or hypotonic breeding water. Can be used. The vaccine is stored in a cold, non-freezing environment, for example, in a cool dark place at about 2-8 ° C.

  (10) Preparation of diagnostic agent: As described in (6) above, a virus suspension, purified antigen, etc. are prepared, and these are used as diagnostic antigens such as sedimentation reaction, agglutination reaction, neutralization reaction, fluorescent antibody method, It can be used as an antigen for enzyme immunoassay, radioimmunoassay and the like. Furthermore, antibodies can be produced from the sera of these animals that have been inoculated intraperitoneally, subcutaneously or intramuscularly in animals such as rabbits, guinea pigs, mice, etc. with diagnostic antigens or vaccine stock solutions. Such antibodies can be used, for example, for antigen detection in the various diagnostic methods described above. The diagnostic antigens and antibodies according to the present invention are used after being diluted and adjusted so that the content of these diagnostic agents is an amount necessary for causing an antigen-antibody reaction.

  The aforementioned vaccine is extremely effective for immunity or prevention of fish iridovirus infection. In addition, the virus antigens and antibodies produced using such antigens are useful as diagnostic agents for definitive diagnosis as well as reacting with antigens and antibodies specifically and in various immunological diagnoses.

  In the following, embodiments, configurations and effects of the present invention will be specifically described with reference to experimental examples and examples. However, the present invention is not limited to these.

[Experimental Example 1]
Isolation material of pathogen virus: After removing the spleen of a dead fish infected with red sea bream virus and chopping it with an ophthalmic scissor, 9 volumes of BME medium (final concentration 300 units / ml penicillin and 300 μg / ml streptomycin) Add 10) and mix with a sterilized glass homogenizer under ice-cooling to prepare a 10% (W / W) spleen emulsion. Subsequently, this was centrifuged at a low speed (3,000 rpm, 20 minutes), and the supernatant was collected, sterilized and filtered with a membrane filter having a filter pore of 450 nm, and the filtrate was used as a pathogen virus separation material for later use. Provide.

[Experiment 2]
Isolation and passaging of pathogen virus in vivo: After inoculating 0.1 ml of each fish into the abdominal cavity of 10 red sea bream (average body length 4.0 cm, average body weight 1.5 g) with the filtrate prepared in Experimental Example 1 , Reared in an isolated breeding tank with a water temperature of 25 ± 1 ° C (40L of seawater is put in a tank with an internal volume of 60L, fresh seawater flows in at 2L / min, an equal amount of old water is discharged, and the breeding seawater is continuously exchanged) , Observe the presence or absence of onset. The affected fish begins to appear as early as the third day after the start of breeding. Each diseased fish is collected each time immediately before death, and a spleen emulsion supernatant is prepared in the same manner as described in Experimental Example 1 and used as a fresh isolate virus suspension for subsequent passages after sputum. The passaging of fresh isolate virus in red sea bream is repeated.

[Experiment 3]
Isolation and passaging of pathogen virus by cell culture: 0.5 ml of the filtrate prepared in Experimental Example 1 is inoculated into each bottle of GF cells cultured in five 25 cm ² plastic bottles and adsorbed at 25 ° C. for 30 minutes. Medium BME (final concentration 15% (V / V) fetal bovine serum, standard amount of non-essential amino acids, 1.2 mg / ml sodium bicarbonate, 100 units / ml penicillin, and 100 μg / ml streptomycin) Add 10 ml of each bottle, and incubate at 25 ° C. Replace with fresh medium every 3 days from the date of inoculation. A microscopic examination is performed daily to determine the state of cell culture and to detect the presence or absence of cytopathic effects as an indicator of pathogen virus infection. By the 14th day after the start of the culture, the cytopathic effect, that is, the cell rounding spreads to an area of about 70% of the cell monosheet. At this point, the culture medium is collected from each culture bottle and used as a fresh isolate virus suspension for subsequent passage. At the same time, the infected cell monosheet was prepared using a conventional EDTA / trypsin solution (Dulbecco's PBS excluding both Ca and Mg ions, final concentration 0.08% (W / V) EDTA 3Na and 0.125% (W / V). V) Trypsin was added to each culture bottle, and after centrifugation, the precipitate was collected by low speed centrifugation, resuspended in 2 ml of the above-mentioned medium BME, and passed as a fresh isolate virus infected cell after passage. Provide. Passage of fresh isolate virus by cell culture is carried out using both virus suspension and its infected cells as follows: inoculate both in a GF cell monosheet and culture; virus in uninfected GF cell suspension Inoculate the suspension and co-culture; inoculate 200,000 cells / ml of uninfected GF cell suspension with 1/10 volume of the infected cells and mix and culture; and prepare the infected cell suspension This is carried out by continuous infection cell culture in which the medium is diluted 5-fold with BME.

[Experimental Example 4]
Identification of fresh isolate virus: Each spleen section and each stamp specimen of the dead fish used in Experimental Example 1 and the onset fish obtained in Experimental Example 2 were nucleic acid-stained by the foilgen reaction, and the presence of atypical hypertrophic cells and the cytoplasmic part The reddish purple staining was confirmed by microscopic examination with an optical microscope. In addition, it was confirmed by electron microscopy that the virus form between the spleen of the dead fish used in Experimental Example 1 and the onset fish obtained in Experimental Example 2 was the same as that of the fresh isolate virus. . Further, an infection experiment is performed by the method described in Experimental Example 2. That is, inoculate 0.1 ml of each fish into the peritoneal cavity of 10 fresh sea bream fish and observe the symptoms of the affected fish, and perform autopsy and histopathological examination. We confirmed that the development of juvenile red sea bream by inoculation with fresh isolate virus was an iridovirus infection.

[Experimental Example 5]
Measurement of virus infectivity titer: Using the medium BME used in Experimental Example 1, the GF cell monosheet cultured in a 1 L roux bottle was detached with EDTA / trypsin solution, and then the cells were collected by low-speed centrifugation. A cell suspension of 200,000 cells / ml is prepared, and 1.0 ml is dropped into each well of a 24-well microplate. Next, 0.1 ml of virus solution diluted 10-fold in the same medium is inoculated into each hole. In addition, the virus solution of the same dilution is inoculated into 6 holes in 1 row, and after culturing in a carbon dioxide incubator at 25 ° C. for 14 days, the virus infectivity titer, TICD ₅₀ / ml was determined by a conventional method using CPE as an indicator for determination. Is measured and converted to a logarithmic value, log (TICD ₅₀ / ml). Infected cells are subjected to an ultrasonic generator (KUBOTA 200M) for 2 minutes in 20 ml of the infected cell suspension in an ice bath, the cells are crushed and suspended, and the supernatant is centrifuged at a low speed (3,000 rpm, 20 minutes). The virus infectivity titer is measured as a liquid.

[Example 1]
Establishment of vaccine antigen production strain: After the virus isolated from the isolation material obtained in Experimental Example 1 was named the iridovirus Ehime-1 strain, and after three passages to red sea bream fry using the method described in Experimental Example 2 Furthermore, the subculture is repeated and acclimatized with GF cells by the method described in Experimental Example 3. The Ehime-1 strain acclimated to GF cells by such passage is used as an antigen-producing strain in the examples described later. In addition, among the passage by various culture methods performed in Experimental Example 3, as a representative example of these, Table 1 shows the results of combined passage of red sea bream and persistently infected cell culture. Viral antigens were amplified and mass-produced by passing the virus isolation material on a virus-sensitive host.

[Example 2]
Mass production of antigen for vaccine: A vaccine is prepared using the infected cells of Ehime-1 strain obtained from Example 1 (3rd generation red sea bream and 10th generation GF cells) as seeds. First, using the medium BME used in Experimental Example 1, the infected GF cell monosheet of the Ehime-1 strain cultured in one 1 L roux bottle was detached with EDTA / trypsin solution, and then centrifuged at a low speed (3,000 rpm). , 20 minutes), the infected cells are collected and resuspended in 500 ml of the same medium BME to prepare an infected cell suspension. Next, this is dispensed into five 1 L roux bottles in 100 ml portions, followed by stationary culture at 25 ° C. for 14 days. Exchange with fresh medium every 3 days from the start of culture. However, the medium is not changed during the culture day 10-14. On the 14th day when CPE reached about 80% of the cell monosheet, the culture solution was collected and centrifuged at a low speed (3,000 rpm, 20 minutes), and the supernatant was collected to prepare 500 ml of a virus suspension. On the other hand, the infected cell monosheet is peeled off with EDTA / trypsin solution, collected by low speed centrifugation (3,000 rpm, 20 minutes), and the pooled cells from five roux bottles are resuspended in 200 ml of the above-mentioned medium to give infected cells. A suspension was obtained.

Measurement of the antigen amount for vaccine: About the virus suspension and the infected cell suspension, the infectivity titer and log (TICD ₅₀ / ml) of both are measured by the method described in Experimental Example 4. As a result, the virus suspension was 6.6, and the infected cell suspension was 6.2.

  Virus inactivation and preparation of inactivated vaccine stock solution: Formalin was added to each solution of 300 ml of virus suspension and 180 ml of infected cell suspension to a final concentration of 0.03% (V / V), and mixed at 30 at 4 ° C. Inactivated for days. After completion of the inactivation, the inactivated virus vaccine stock solution and the inactivated infected cell vaccine stock solution were respectively stored in a cold dark room at 4 ° C.

  Test of inactivated vaccine stock solution: 100 ml of each inactivated vaccine stock solution prepared from virus suspension and infected cell suspension was dispensed, and the “Biological product for animals” based on the Pharmaceutical Affairs Law (Act No. 145 of 1960) The inactivation test, staining test, abnormal toxicity denial test, protein nitrogen quantitative test, and sterility test are conducted in accordance with the regulations of “rabies tissue culture inactivated vaccine” defined in “Criteria”. As a result, it was confirmed that both inactivated vaccine stock solutions were eligible as tissue culture inactivated vaccine stock solutions, together with the results of Example 3 regarding safety and efficacy described later.

  Preparation of inactivated vaccine: 50 ml of inactivated virus vaccine stock solution and 50 ml of inactivated infected cell vaccine stock solution were each diluted 10-fold with medium BME, then dispensed 10 ml each into a 20 ml vial, sealed and sealed, Used as inactivated virus vaccine and inactivated infected cell vaccine.

[Example 3]
Safety and efficacy of inactivated vaccine: In order to confirm the safety and efficacy of each inactivated vaccine prepared in Example 2, clinical trials are conducted using red sea bream fry. In addition, the cell culture medium supernatant not inoculated with the virus and the non-infected cell suspension are treated in parallel with the vaccine production to prepare an inactivated non-infected supernatant and an inactivated non-infected cell suspension. These are used as comparative samples. A total of 150 healthy red sea bream larvae (length 4.0-8.0 cm, body weight 1.5-11.5 g) were divided into 5 groups consisting of 30 fish per group, and one vaccine was used for each sample. Each control sample is inoculated 0.1 ml / tail into the abdominal cavity of the fry. The remaining group is not vaccinated. These juveniles are reared and observed in the rearing tank described in Experimental Example 2 using one rearing tank for each group of 30 fish. On the 10th day from the day of inoculation, 0.1 ml of Echime-1 strain virulent virus suspension is inoculated into the abdominal cavity of each juvenile and directly attacked. The attack virus amount log (TCID ₅₀ / ml) is 5.0. After the attack, the fishes in the aquarium and the presence or absence of life or death are reared and observed for 14 days. As seen in Table 2 showing the results, the vaccine according to the present invention was safe and effective.

[Example 4]
Trial production of inactivated vaccine and confirmation of its safety and efficacy: Infected cells of Ehime-1 strain (3rd generation red sea bream and 13th generation GF cells) obtained in Example 1 were used as seeds. An inactivated vaccine is prepared as described. That is, after culturing the virus in GF cells, the culture solution is centrifuged at low speed, and the culture supernatant is collected to produce a 5 L virus suspension (the virus infectivity is 7.5 log TCID ₅₀ / ml). Next, after adding formalin to the suspension to a final concentration of 0.1% (V / V) and mixing, it is left standing at 4 ° C. for 30 days to inactivate to obtain a vaccine stock solution. This stock solution is subjected to various tests related to the assay, and after confirming its eligibility as a tissue culture inactivated vaccine, it is diluted 10-fold with medium BME, dispensed 100 ml each into a 200 ml vial, sealed and sealed. 400 subdivided products were produced. About 20 small-sized products that were randomly selected from the above, the regulations for “rabies tissue culture inactivated vaccine” defined in the “Animal Biologics Standards” based on the Pharmaceutical Affairs Law (Act No. 145 of 1960) Compliant with various tests related to subdivided products, ie, characteristic test, hydrogen ion concentration test, sterility test, staining test, protein nitrogen quantitative test, abnormal toxicity negative test, and qualified as a tissue culture inactivated vaccine subdivided product After confirming the safety, the remaining subdivided products were subjected to the following safety and efficacy verification tests.

  Both safety and efficacy confirmation tests are conducted as described in Example 3. In other words, healthy red sea bream fry (7.5-8.0 cm long, body weight 9.3-10.0 g) was divided into 6 groups consisting of 25 fish per group, and 4 randomly-divided products (in each vial) 1 group for each vial (signed A, B, C and D), a total of 4 groups, and 1 group for comparison (inactivated culture supernatant of uninfected GF cells) Inoculate 0.1 ml / tail intraperitoneally. The remaining group is not vaccinated. Each group 25 animals were reared and observed using one aquarium, and on the 10th day of vaccination, all groups except the vial B and D vaccinated groups were directly attacked as described in Example 3, and For 14 days, the fish in the aquarium and the presence or absence of life or death will be reared and observed. The results of the confirmation test are shown in Table 3. The vaccine according to the present invention was qualified as an inactivated vaccine and was safe and effective.

[Example 5]
Preparation of antigen for diagnosis: In the same manner as described in Example 2, after preparing a virus suspension, it is inactivated with formalin to obtain an inactivated virus suspension. Next, 9.0 ml of 40% (W / V) sucrose solution was added to each bottom of 6 ultracentrifuge tubes (manufactured by Hitachi, Ltd .: model SCP70H for swing rotor SRP28SA) to form a sucrose cushion layer, Gently layer 25 ml of the inactivated virus suspension on the sucrose layer and ultracentrifuge at 4 ° C. (90,000 g, 90 minutes). Virus particles settled on the bottom of each centrifuge tube were suspended in 5 ml of PBS, and these were pooled to obtain 30 ml of purified virus antigen solution.
Preparation of a reagent for passive agglutination reaction: 0.4 ml of washed sheep erythrocytes was suspended in 10 ml of PBS, 1.0 ml of the above antigen solution was added thereto, and the mixture was lightly stirred and then diluted with PBS to a concentration of 2.5%. (W / V) 3.0 ml of glutaraldehyde is added dropwise and mixed thoroughly. This is further stirred for 60 minutes at room temperature to adsorb the antigen to the red blood cells. Next, after washing 5 times with PBS, it was suspended in 1.0 ml of 1M glycine buffer (pH 7.2), left overnight at 8 ° C., washed again 5 times with PBS, and then a final concentration of 1% (V / V 1) PBS containing normal sheep serum was added to prepare a 1% (V / V) antigen-adsorbed erythrocyte suspension, which was used as a reagent for passive agglutination reaction.
Measurement of iridovirus antibody by passive agglutination reaction: Using the above-mentioned reagent for passive agglutination reaction, each sample serum collected from 5 iridovirus-infected fish by the microtiter method of 2-fold serial dilution, and 2 healthy fish The antibody titer of each serum is measured. The results are shown in Table 3. The diagnostic antigen of the present invention exhibited a very specific agglutination reaction, enabling a simple definitive diagnosis by measuring antibody titer.

Claims (6)

  A virus strain isolated from an iridovirus-infected diseased fish is subcultured into one or more hosts selected from a host group consisting of the fish body or cell culture having the virus sensitivity. An iridovirus infection vaccine comprising an antigen produced by growing in a fish or cell culture having an amount that provides antibody production or protection against infection.   A virus strain isolated from an iridovirus-infected diseased fish is subcultured into one or more hosts selected from a host group consisting of the fish body or cell culture having the virus sensitivity. Iridovirus infection containing an inactivated antigen prepared by inactivating an antigen produced by propagation in a fish body or cell culture with an inactivating agent in an amount capable of producing antibodies or protecting against infection Disease vaccine.   A virus strain isolated from an iridovirus-infected diseased fish is subcultured into one or more hosts selected from a host group consisting of the fish body or cell culture having the virus sensitivity. An agent for diagnosing an iridovirus infection comprising an antigen produced by growing in a fish or cell culture having an amount of an antigen-antibody reaction.   The diagnostic agent for iridovirus infection according to claim 3, wherein the iridovirus is Ehime-1 strain.   A virus strain isolated from an iridovirus-infected diseased fish is subcultured into one or more hosts selected from a host group consisting of the fish body or cell culture having the virus sensitivity. Diagnosis of Iridovirus Infectious Diseases Containing an Inactive Antigen Prepared by Inactivating an Antigen Produced by Proliferating in a Fish Body or in Cell Culture with an Inactivating Agent Agent.   The diagnostic agent for iridovirus infection according to claim 5, wherein the iridovirus is Ehime-1 strain.