JP2009108004A - Beta-nodavirus infection inhibitor for fish - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a drug effective for beta-nodavirus infection of useful fishes like farmed fishes. <P>SOLUTION: The beta-nodavirus infection inhibitor for fishes contains an endosome acidification inhibitor such as NH<SB>4</SB>Cl, chloroquine, bafilomycin A1, and monensin as the active ingredients. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a technique for inhibiting the beta nodavirus infection of fish organisms.

  The Nodaviridae family consists of the genera Alphanodavirus and Betanodavirus that mainly infect insects and fish, respectively. A virus belonging to the genus Betanodavirus is a pathogen of viral encephalopathy and retinopathy, also called viral neuronecrosis (VNN). Nodaviruses are small (25-30 nm in diameter), spherical, and non-enveloped viruses whose genome consists of two single-stranded plus-sense RNA molecules (Non-patent Document 1). RNA1 (3.1 Kb), the larger genome segment, encodes RNA-dependent RNA polymerase (RdRp) (Non-Patent Documents 2-4), and RNA2 (1.4 Kb), the smaller genome segment, constitutes the virion. It encodes the capsid protein (Non-patent Document 5). Recently, a subgenomic RNA (called RNA3) synthesized from RNA1 has been found to encode the B2 protein. This protein is an RNAi antagonist highly conserved in betanodavirus (Non-patent Documents 6-8).

  VNN has destroyed many species of marine aquaculture around the world (Non-Patent Document 9). Betanodaviruses have been isolated from over 30 species of marine fish from 14 families, and based on phylogenetic analysis of capsid protein sequences, there are four genotypes: Striped Jack Nervous Necrosis Virus (SJNNV), Barfin Flounder Nervous Necrosis Virus (BFNNV), Tiger Puffer Nervous Necrosis Virus (TPNNV), and Redspotted Grouper Nervous Necrosis Virus (RGNNV) (Non-Patent Documents 5, 10, and 11). In order to reduce the significant economic losses caused by viruses in the aquaculture industry, there is an urgent need for effective control methods for betanodavirus infection. A vaccine capable of preventing VNN has been developed, and immunization using a part of the recombinant betanodavirus capsid protein expressed in E. coli has been attempted (Non-patent Documents 12 and 13). In addition, it has been reported that immunization with betanodavirus virus-like particles (VLP) induces a protective immune response against VNN (Non-patent Document 14). However, VNN occurs mainly in juvenile fish that cannot be easily vaccinated due to their small body. Therefore, the development of drugs that inhibit betanodavirus infection is extremely important. The mechanism of betanodavirus infection is still unclear, but electron microscopy of betanodavirus-infected cells suggests that betanodavirus entry into fish cell lines depends on the endocytotic pathway ( Non-patent document 15).

In addition, endosome acidification inhibitors such as ammonium chloride (NH ₄ Cl) and chloroquine have been applied to many viruses (Non-patent Documents 16 and 17). Chloroquine first synthesized as an antimalarial drug is human immunodeficiency virus (HIV) (Non-Patent Documents 18 and 19), influenza virus (Non-Patent Document 20), and SARS coronavirus (SARS-CoV) It has been shown to have antiviral activity against a wide range of viruses including 21). For enveloped viruses, chloroquine and NH ₄ Cl can be used for glycosylation of viral membrane proteins (Non-patent Document 22), translocation of glycoproteins to the plasma membrane (Non-patent Documents 23 and 24), and viral nucleocapsid formation ( Non-patent document 16) was inhibited. It was also suggested that terminal glycosylation of angiotensin converting enzyme-2 (ACE-2), which is a functional receptor for SARS-CoV, is inhibited by chloroquine and NH ₄ Cl (Non-patent Document 22).
Mori K, Nakai T, Muroga K, Arimoto M, Mushiake K, Furusawa I (1992) Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187: 368-71 Guo YX, Chan SW, Kwang J (2004) Membrane association of greasy grouper nervous necrosis virus protein A and characterization of its mitochondrial localization targeting signal.J Virol 78: 6498-6508 Nagai T, Nishizawa T (1999) Sequence of the non-structural protein gene encoded by RNA1 of striped jack nervous necrosis virus.J Gen Virol 80: 3019-22 Tan C, Huang B, Chang SF, Ngoh GH, Munday B, Chen SC, Kwang J (2001) Determination of the complete nucleotide sequences of RNA1 and RNA2 from greasy grouper (Epinephelus tauvina) nervous necrosis virus, Singapore strain.J Gen Virol 82: 647-53 Nishizawa T, Mori K, Furuhashi M, Nakai T, Furusawa I, Muroga K (1995) Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish.J Gen Virol 76: 1563-9 Fenner BJ, Goh W, Kwang J (2006) Sequestration and protection of double-stranded RNA by the betanodavirus b2 protein.J Virol 80: 6822-33 Fenner BJ, Thiagarajan R, Chua HK, Kwang J (2006) Betanodavirus B2 is an RNA interference antagonist that facilitates intracellular viral RNA accumulation.J Virol 80: 85-94 Iwamoto T, Mise K, Takeda A, Okinaka Y, Mori K, Arimoto M, Okuno T, Nakai T (2005) Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2. J Gen Virol 86: 2807 -16 Ikenaga T, Tatecho Y, Nakai T, Uematsu K (2002) Betanodavirus as a novel transneuronal tracer for fish.Neurosci Lett 331: 55-9 Mori K,, Mangyoku T, Iwamoto T, Arimoto M, Tanaka S, Nakai T (2003) Serological relationships among genotypic variants of betanodavirus.Dis Aquat Organ 57: 19-26 Nishizawa T, Furuhashi M, Nakai T, Muroga K (1997) Genomic Classification of Fish Nodaviruses by Molecular Phylogenetic Analysis of the Coat Protein Gene.Appl Environ Microbiol 63: 1633-1636 Husgag S, Grotmol S, Hjeltnes BK, Rodseth OM, Biering E (2001) Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV Dis Aquat Organ 45: 33-44 Tanaka S, Mori K, Arimoto M, Iwamoto I, Nakai T (2001) Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis. J Fish Dis 24: 15-22 Thiery R, Cozien J, Cabon J, Lamour F, Baud M, Schneemann A (2006) Induction of a protective immune response against viral nervous necrosis in the European sea bass Dicentrarchus labrax by using betanodavirus virus-like particles.J Virol 80: 10201 -7 Liu W, Hsu CH, Hong YR, Wu SC, Wang CH, Wu YM, Chao CB, Lin CS (2005) Early endocytosis pathways in SSN-1 cells infected by dragon grouper nervous necrosis virus.J Gen Virol 86: 2553-2561 Koyama AH, Uchida T (1989) The effect of ammonium chloride on the multiplication of herpes simplex virus type 1 in Vero cells.Virus Res 13: 271-81 Savarino A, Boelaert JR, Cassone A, Majori G, Cauda R (2003) Effects of chloroquine on viral infections: an old drug against today's diseases? Lancet Infect Dis 3: 722-7 Savarino A, Gennero L, Sperber K, Boelaert JR (2001) The anti-HIV-1 activity of chloroquine. J Clin Virol 20: 131-5 Savarino A, Lucia MB, Rastrelli E, Rutella S, Golotta C, Morra E, Tamburrini E, Perno CF, Boelaert JR, Sperber K, Cauda R (2004) Anti-HIV effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors. J Acquir Immune Defic Syndr 35: 223-32 Ooi EE, Chew JS, Loh JP, Chua RC (2006) In vitro inhibition of human influenza A virus replication by chloroquine.Virol J 3: 39 De Clercq E (2006) Potential antivirals and antiviral strategies against SARS coronavirus infections. Expert Rev Anti Infect Ther 4: 291-302 Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollin PE, Ksiazek TG, Seidah NG, Nichol ST (2005) Chloroquine is a potent inhibitor of SARS coronavirus infection and spread.Virol J 2: 69 Dille BJ, Johnson TC (1982) Inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine. J Gen Virol 62: 91-103 Ferreira DF, Santo MP, Rebello MA, Rebello MC (2000) Weak bases affect late stages of Mayaro virus replication cycle in vertebrate cells.J Med Microbiol 49: 313-8

  Although selection and disinfection of spawning fish that are presumed to be free of viruses is currently being carried out to prevent the spread of betanodavirus, these procedures prevent the reoccurrence of betanodavirus infection on the same farm. Is not enough. In addition, as described above, the prevention and treatment of infection by vaccines are also being studied, but since VNN is caused by betanodavirus infection mainly in small juvenile fish, there are doubts about the effectiveness and effectiveness of vaccines. is there.

  Therefore, a new anti-beta nodavirus drug that can be administered to fry by the oral method or the bath method is desired.

  This invention is made | formed in view of the situation as mentioned above, Comprising: It aims at providing the effective chemical | medical agent with respect to beta nodavirus infection to useful fish organisms, such as a cultured fish.

  The present invention provides a betanodavirus infection inhibitor for fish organisms characterized by comprising an endosomal acidification inhibitor as an active ingredient.

  In addition, the present invention provides a method for inhibiting betanodavirus infection in fish organisms, which comprises administering an effective amount of an endosomal acidification inhibitor to fish organisms.

In these infection inhibitors and infection inhibition methods, the endosomal acidification inhibitor is one or more selected from the group consisting of ammonium chloride (NH ₄ Cl), chloroquine, bafilomycin A1 and monensin. This is a preferred embodiment.

In the following description, an endosome acidification inhibitor may be described as a “lysosome-directing substance”. In addition, ammonium chloride may be simply referred to as NH ₄ Cl.

  According to the present invention, it is possible to effectively prevent betanodavirus infection in cultured larvae and the like.

In the present invention, the beta-nodavirus infection inhibitor contains an endosomal acidification inhibitor (specifically, NH ₄ Cl, chloroquine, bafilomycin A1 and monensin) as active ingredients. It may be present, or may be mixed with other fish drug components (for example, excipients for making the drug form into pellets or pastes).

  As an administration method of the infection inhibitor of the present invention, any of the oral method, the drug bath method, or a combination of both methods may be used, and it may be appropriately selected according to the state of the target fish organism and the culture environment. . For example, in the case of hatched larvae before opening, a medicinal bath method may be selected. In the case of fry after opening, particularly immediately after opening, it is preferable to use the oral method and the medicinal bath method in combination. It is also effective to administer orally to infected or uninfected parent fish to treat and protect the parent fish, as well as prevent vertical infection of eggs.

  In addition, in the case of oral administration, the infection inhibitor of the present invention can be mixed with feed and orally inoculated into fish organisms. In this case, vitamins, minerals, antioxidants, antibiotics, antibacterial agents and the like can be further added to the feed mixed with the endosome acidification inhibitor. The feed amount of the feed containing the infection inhibitor of the present invention is preferably about 3% by mass of the fish body weight per day.

  The blending amount of the endosome acidification inhibitor may be appropriately determined according to the type of fish organisms and the like. For example, in the case of the oral method, it is preferable to blend approximately 1 mg / kg to 10 mg / kg in the feed. Moreover, in the case of a chemical bath method, it is preferable to mix about 1 mg / ton to 50 mg / ton with respect to the amount of water in the water tank.

  The fish organisms to which the infection inhibitor of the present invention can be applied are not particularly limited, and saltwater fish, particularly striped mackerel, yellowtail, thailand, coho salmon, mackerel, flounder, pheasant grouper, matsukawa, kue, mahata, flounder, blackfish, trough puffer, Amberjack etc. are mentioned.

  Hereinafter, the effects and the like of the present invention will be specifically described with reference to examples.

(1) Materials and methods (1-1) Cells, viruses, drugs
E-11 cells were maintained at 25 ° C. in Leibovitz L-15 medium (Invitrogen, Carlsbad, Calif.) Supplemented with 5% fetal calf serum (FBS). As a fish nodavirus, a strain isolated from Mahata in Nagasaki in 2001 was used. This virus has been confirmed to belong to the RGNNV genotype by RNA2 nucleotide sequence analysis. E-11 cells were inoculated with betanodavirus and the virus was harvested when almost all of these cells in the monolayer showed cytopathic effect (CPE). NH ₄ Cl, chloroquine, bafilomycin A1, and chlorpromazine were purchased from Sigma (St. Louis, Mo.), and monensin was purchased from Wako (Osaka).
(1-2) Virus infection and titer measurement
E-11 cells were inoculated with virus for 1 hour at 28 ° C. with or without the addition of drugs. It was then maintained at 28 ° C. in growth medium containing 2% FBS. Unless otherwise stated, cells were pretreated with a single substance at 28 ° C. for 1 hour and inoculated with virus at a multiplicity of infection (MOI) of 1. Viral titer was expressed as a 50% cytopathic endpoint (TCID ₅₀ ) assay using E-11 cells.
(1-3) Detection of viral RNA by RT-PCR Total RNA was prepared from RGNNV-infected cells (5 × 10 ⁵ ) using Trizol reagent (Invitrogen). (+) RNA1, (-) RNA1, and 18S rRNA were reverse transcribed from RNA samples by M-MLV reverse transcriptase (Invitrogen). At this time, RGRNA1-2490R (5'-gtcagtgtagtctgcatactg-3 ': SEQ ID NO: 1) is included in (+) RNA1, and RGRNA1-1868F (5'-tgcgtgagttcgtcgagttt-3': SEQ ID NO: 2) 18S rRNA-R (5′-gctggaattaccgcggct-3 ′: SEQ ID NO: 3) was used as the 18S rRNA. The primer pairs used for PCR amplification were RGRNA1-1868F and RGRNA1-2490R for RNA1 and 18S rRNA-F (5'-cggctaccacatccaaggaa-3 ': SEQ ID NO: 4) and 18S rRNA for 18S rRNA. -R. PCR products were run on agarose gel electrophoresis and visualized by ethidium bromide staining. Band intensity was quantified using Image J software (NIH).

(2) Results (2-1) Effects of NH ₄ Cl and chloroquine on the onset of betanodavirus-induced CPE Using the appearance of virus-induced CPE as an index, the effect of lysosome-directing substances on betanodavirus was examined. NH ₄ Cl and chloroquine diffuse into endosomes and act as proton sinks to inhibit endosomal acidification (Ohkuma S, Poole B (1978) Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci USA 75: 3327-31). E-11 cells were inoculated with RGNNV at MOI = 1 in the presence of various concentrations of these substances (FIG. 1). Uninfected E-11 cells showed a flat adherent shape (FIG. 1a) and virus-infected cells showed a typical CPE shape and separated from the dish by 6 days after inoculation (FIG. 1b). In contrast, E-11 cells infected with RGNNV in the presence of NH ₄ Cl (FIGS. 1c-e) or chloroquine (FIGS. 1f-h) suppressed the development of CPE in a dose-dependent manner. CPE was completely inhibited at concentrations of 1 mM NH ₄ Cl (FIG. 1e) and 1 uM chloroquine (FIG. 1h), respectively. The effect of NH ₄ Cl and chloroquine on viral infection may be due to its own cytotoxicity. Therefore, the cytotoxicity of these substances to E-11 cells was examined using the WST-1 assay. The degree of cell growth and morphological changes by NH ₄ Cl and chloroquine were not affected at concentrations up to 12.5 mM and 25 uM, respectively (data not shown).
(2-2) Effect of lysosome-directed substance treatment on betanodavirus adsorption to cells To examine the mechanism of the inhibitory effect of test substances on betanodavirus infection, adsorption of betanodavirus to E-11 cells The effect of the inhibitor on the effect was investigated. As shown in FIGS. 2A and B, (+) RNA1 bands were detected in a viral load-dependent manner in cells inoculated with RGNNV at 28 ° C. for 1 hour. Viral (+) RNA1 was still detected in E-11 cells infected with the virus in the presence of 1 mM NH ₄ Cl and 1 uM chloroquine (FIG. 2C). The intensity of (+) RNA1 detected in the presence of drug was almost similar to that of control cells (FIG. 2D). From these results, it was suggested that none of the drugs inhibited the adsorption of betanodavirus to cells.
(2-3) NH ₄ Cl that inhibits betanodavirus entry into cells
In order to investigate the detailed mechanism of the inhibitory effect of NH ₄ Cl and chloroquine, we investigated the accumulation of (+) RNA1 as the viral genome and (-) RNA1 as the template in infected cells. E-11 cells were inoculated with the virus with or without these substances, and the infected cells were incubated for a specified time, and then total RNA was prepared from the cells. Since (-) RNA1 is synthesized using (+) RNA1 as a template, the presence of (-) RNA1 in cells was used as an indicator of viral genome replication. As shown in FIGS. 3A and B, the detection of (+) RNA1 band in control cells gradually increased, reaching a maximum at 18 hours after inoculation. No significant (-) RNA1 bands were detected in the cells 1 hour after inoculation, but the intensity of these bands increased gradually by 9 hours after inoculation and increased significantly after 9-12 hours inoculation. These data suggest that beta-nodavirus begins synthesis of (-) RNA1 as soon as it enters the cell after attachment to the cell membrane. No significant (−) RNA1 band was detected in cells inoculated and cultured in the presence of 1 mM NH ₄ Cl or 1 uM chloroquine. Furthermore, the band intensity of (+) RNA1 was constant over the sampling period.

Next, NH ₄ Cl was added to virus-infected E-11 cells at some time after virus infection (FIG. 3C). When NH ₄ Cl was added to a final concentration of 1 mM at the same time as inoculation, (+) RNA1 accumulation was not detected as compared to the control that only measured cell adsorption, as shown in Panel B. It was. However, significant addition of (+) RNA1 was detected when NH ₄ Cl was added to the cells 1 hour after inoculation. When NH ₄ Cl was added 3 hours after inoculation, the amount of (+) RNA1 accumulated in the cells was almost the same as the amount of (+) RNA1 in uninfected cells. Considering these results, NH ₄ Cl inhibited the early entry stage of beta nodavirus into the cell and had no effect on viral replication after the beta noda virus genome entered the cell.
(2-4) Inhibitory effect on virus replication by lysosome-directing substance The amount of infectious virus released into the culture supernatant after betanodavirus infection in the presence of lysosome-directing substance was measured. As shown in FIG. 4A, infectious virus decreased as the concentration of NH ₄ Cl increased. At a concentration of 160 uM NH ₄ Cl, the number of infectious viruses was reduced to approximately 90% of the control. In cells treated with a concentration of 630 uM NH ₄ Cl, almost complete loss of virus production was observed. Chloroquine had the same effect as NH ₄ Cl on virus production (FIG. 4B). In the presence of 200 nM chloroquine, infectious virus was reduced to less than 5% of the control. A specific inhibitor of vacuolar H ⁺ -ATPase (Perez L, Carrasco L (1994) Involvement of the vacuolar H (+)-ATPase in animal virus entry. J Gen Virol 75: 2595-606) The sex substance bafilomycin A1 also showed an inhibitory effect on virus production in a dose-dependent manner (FIG. 4C). Virus-induced CPE was not observed in cells treated with 1 nM bafilomycin A1 for 6 days after inoculation (Table 1). In addition, the lysosome-directing substances monensin and chlorpromazine also inhibited the onset of virus-induced CPE at the concentrations shown in Table 1. These results strongly suggested that various lysosome-directing substances could be used as inhibitors against betanodavirus infection.

(3) Discussion In this example, a lysosome-directing substance as an effective antiviral substance against betanodavirus infection in fish cell culture was examined. The reason for using these substances was that the presence of betanodavirus in intracellular vesicles was observed by electron microscopy of Liu et al. (Non-patent Document 15). Initially, these substances were used to investigate whether NH ₄ Cl and chloroquine could inhibit the development of CPE after beta nodavirus infection. Both substances completely inhibited virus-induced CPE at non-cytotoxic concentrations (Figure 1). NH ₄ Cl and chloroquine had no effect on virus attachment to cells (FIGS. 2C and D). Furthermore, when NH ₄ Cl was added to the medium 1 hour after inoculation, (+) RNA1 accumulation was not affected, so NH ₄ Cl did not inhibit polymerase activity but was infected at a very early stage. Was shown to be inhibited (FIG. 3C). In this example, the effective amount of NH ₄ Cl (1 mM) and the effective amount of chloroquine (1 uM) are the effective amounts used in another virus (Brindley MA, Maury W (2005) Endocytosis and a low-pH step are required for productive entry of equine infectious anemia virus.J Virol 79: 14482-8; Chu VC, McElroy LJ, Chu V, Bauman BE, Whittaker GR (2006) The avian coronavirus infectious bronchitis virus undergoes direct low-pH-dependent fusion activation J Virol 80: 3180-3188; Lecot S, Belouzard S, Dubuisson J, Rouille Y (2005) Bovine viral diarrhea virus entry is dependent on clathrin-mediated endocytosis. J Virol 79: 10826-9; Stuart AD, Brown TD (2006) Entry of feline calicivirus is dependent on clathrin-mediated endocytosis and acidification in endosomes. J Virol 80: 7500-9). Betanodavirus infection was also inhibited by bafilomycin A1 and monensin (FIG. 4 and Table 1), confirming that lysosome-directing substances are useful in preventing betanodavirus infection.

NH ₄ Cl or a result of the check the CPE inhibition of beta nodavirus infected cells by chloroquine. Cells were inoculated with RGNNV in the presence of the indicated concentrations of NH ₄ Cl (ce) or chloroquine (f to h). Cell morphology was photographed 6 days after inoculation. Control cells were infected with the indicated MOI and incubated without substance (ab). Results of confirming that NH ₄ Cl and chloroquine do not affect the adsorption of betanodavirus to E-11 cells. (A) Cells (3-4 × 10 ⁵ ) were inoculated with RGNNV at the indicated MOI and the cells were washed 4 times to remove unbound virus. Total RNA was prepared, (+) RNA1 and 18S rRNA were amplified by RT-PCR, and then agarose gel electrophoresis was performed. (B) The band intensity of (+) RNA1 was normalized with respect to the 18S rRNA of the cells and expressed as the ratio of (+) RNA1 compared to the cell band intensity at MOI = 1. Data were expressed as mean and standard deviation from 3 independent experiments. (C) 1 mM NH ₄ Cl or 1 uM chloroquine cells inoculated with 1 hour virus in the presence, respectively, were washed in media containing 1 mM NH ₄ Cl or 1 uM chloroquine. Next, total RNA was prepared and RT-PCR was performed. Control cells were inoculated with MOI = 0.1 or 1 without substance. (D) The band intensity of (+) RNA1 was calculated as the ratio of (+) RNA1 compared to the band intensity of the control with the normalization shown in panel B and MOI = 1. Data were expressed as mean and standard deviation from 3 independent experiments. Results of confirming that NH ₄ Cl and chloroquine inhibit early stages of beta-nodavirus infection. (A) Cells were inoculated with RGNNV in the presence of 1 mM NH ₄ Cl (middle panel) and 1 uM chloroquine (lower panel). Control cells were inoculated and incubated without substance (upper panel). At the specified time after inoculation, total RNA was extracted, and (+) RNA1, (−) RNA1 and 18S rRNA were detected by RT-PCR. (B) RNA1 band intensities were normalized as (+) RNA1 relative values (black circles) and (-) RNA1 relative values (white circles), which were observed 1 hour after inoculation. Calculated. Data were expressed as the average from two independent experiments. (C) Cells were inoculated with virus and incubated throughout the experiment with or without 1 mM NH ₄ Cl (grey bar) or not (white bar). At the specified time after inoculation, NH ₄ Cl was added to the virus-infected cells to a final concentration of 1 mM (black bar). 12 hours after inoculation, total RNA was extracted and RT-PCR was performed. The band intensity of (+) RNA1 was normalized and displayed as the ratio of (+) RNA1 compared to the band intensity of cells infected only with virus (absorption only). Data were expressed as mean and standard deviation from 3 independent experiments. E-11 cells were inoculated with RGNNV in the presence of various concentrations of NH ₄ Cl (A), chloroquine (B), and bafilomycin A1 (C) as shown. The culture supernatant was collected 4 days after the inoculation, and the infectious titer was measured by the TCID ₅₀ measurement method. Relative virus production was calculated as the percentage of virus titer relative to the virus titer of the supernatant of infected cells in the absence of material. Data were expressed as the average from two independent experiments performed in primary and secondary.

Claims (4)

A betanodavirus infection inhibitor for fish organisms, characterized by comprising an endosomal acidification inhibitor as an active ingredient. The infection inhibitor according to claim 1, wherein the endosomal acidification inhibitor is one or more selected from the group consisting of ammonium chloride (NH ₄ Cl), chloroquine, bafilomycin A1 and monensin. A method for inhibiting fish betanodavirus infection, which comprises administering an effective amount of an endosomal acidification inhibitor to a fish organism. The infection inhibition method according to claim 3, wherein the endosomal acidification inhibitor is one or more selected from the group consisting of ammonium chloride (NH ₄ Cl), chloroquine, bafilomycin A1 and monensin.