WO2023128527A1 - Antiviral composition for fish against viral hemorrhagic septicemia virus, containing chitosan nanoparticles having mirna-155 encapsulated therein as active ingredient - Google Patents

Abstract

The present application pertains to chitosan nanoparticles having miRNA-155 encapsulated therein and uses thereof and provides a method for preparing the chitosan nanoparticles having miRNA-155 encapsulated therein, an antiviral composition for fish against viral hemorrhagic septicemia virus (VHSV) comprising the chitosan nanoparticles having miRNA-155 encapsulated therein as an active ingredient, and a method for preventing or treating diseases in fish caused by VHSV infection using same.

Description

Antiviral composition for fish against viral hemorrhagic sepsis virus containing chitosan nanoparticles encapsulated with miRNA 155 as an active ingredient

The present invention relates to an antiviral composition for fish against viral hemorrhagic sepsis virus (VHSV) containing chitosan nanoparticles encapsulated with miRNA 155 as an active ingredient. Priority is claimed for the submitted Republic of Korea Patent Application No. 10-2021-0191859, the disclosure of which is incorporated herein by reference.

Viral hemorrhagic septicemia virus (VHSV) is a single-stranded RNA virus, an enveloped virus belonging to the family Rhabdoviridae and Novirhabdovirus. Visual examination of flounder infected with VHSV shows blackening of the body, hemorrhage throughout the body, abdominal distension due to ascites retention, hernia, discoloration of the gills, and red spot-like bleeding on the anatomical body surface. Outbreaks caused by VHSV occur in the winter when the water temperature is low at 10-13 ℃, and it mainly occurs during the stocking period, causing mass mortality of fry. The target organs of infection are the heart and kidneys, and damage to the spleen, brain, muscles, and gills is also caused. VHSV was first reported as an important viral disease of rainbow trout, and has recently been reported in a variety of fish, such as trout, rainbow trout, silver salmon, river trout, brown trout, and steelhead trout, as well as salmonids such as cod and herring. It also occurs widely in fish in natural waters and marine cultured fish such as halibut, mainly in Europe, North America and Asia.

Viral hemorrhagic septicemia (VHS) was first reported in domestic flatfish in 2001, and has since been reported in various wild fish. It is a fatal disease. In addition, VHS is regulated as an 'OIE notifiable disease' by the Office of International Animal Health (OIE), so it is a disease subject to quarantine when distributing aquatic products. It is also a disease factor.

Research on the structure and antigenic protein of VHSV, and research on inactivated vaccines, protein recombinant vaccines, attenuated vaccines, and DNA vaccines, etc. are being conducted based on these basic studies (Korean Patent Publication No. 10-2014-0003826) . However, attenuated virus vaccines are reported to be effective under experimental conditions, but are not approved due to the risk of pathogenic recovery of the virus and the risk of spreading to the natural world. In addition, in DNA vaccine experiments in which plasmids inserted with hemorrhagic sepsis virus G or N genes were injected intramuscularly, strong immune responses were induced and mortality was reported in rainbow trout and halibut, but the safety of genetic manipulation of DNA vaccines was reported. It is not commercialized because of problems.

On the other hand, chitosan ((1,4)-2-amino-2-deoxy-β-D-glucan), a natural biopolymer obtained by deacetylating chitin (the main component of crustacean shells), has excellent biocompatibility and low immunogenicity. , And because of its low cytotoxicity compared to other polymers, it has begun to be applied in the high-tech fields, including the fiber polymer industry and medical engineering, but its application to the field of prevention or treatment of VHSV-infected fish diseases is insufficient.

In order to solve the problems described above, the inventors of the present invention continued research to find a new substance exhibiting a preventive or therapeutic effect on VHSV infectious fish disease, miRNA (micro RNA) 155 or a mimic thereof (mimic) encapsulated in chitosan nanoparticles and administered to fish, the present invention was completed by confirming the excellent effect of protecting fish from VHSV infection without causing toxicity.

One object of the present invention is to provide an antiviral composition for fish against viral hemorrhagic septicemia virus (VHSV) comprising chitosan nanoparticles encapsulated with miRNA 155 as an active ingredient.

Another object of the present invention is (a) mixing miRNA 155 and tripolyphosphate solution; (b) dissolving chitosan in an acidic solvent; and (c) mixing and stirring the mixed solution obtained in step (a) and the solution obtained in step (b) to provide a method for preparing chitosan nanoparticles encapsulated with miRNA 155.

Another object of the present invention is a method for preventing or treating VHSV-infected fish disease comprising administering to fish an antiviral composition for fish against VHSV containing the miRNA 155-encapsulated chitosan nanoparticles or the same as an active ingredient. want to provide

Another object of the present invention is to provide an antiviral use of the miRNA 155-encapsulated chitosan nanoparticles or a composition containing the chitosan nanoparticles as an active ingredient against fish-infectious VHSV.

Another object of the present invention is to provide a use of the miRNA 155-encapsulated chitosan nanoparticles or a composition containing the chitosan nanoparticles as an active ingredient for preventing or treating VHSV-infected fish disease.

Another object of the present invention is to provide a use of the miRNA 155-encapsulated chitosan nanoparticles for preparing an antiviral composition for fish against VHSV or a medicament (or pharmaceutical composition) for preventing or treating VHSV infectious fish disease. want to do

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

One aspect provides an antiviral composition for fish against viral hemorrhagic septicemia virus (VHSV) comprising chitosan nanoparticles encapsulated with miRNA 155 as an active ingredient.

The term "miRNA (micro RNA)" is a regulatory RNA molecule that regulates gene expression in the post-transcriptional stage through base binding to the 3' untranslated region (UTR) of mRNA, and is about 20 to 25 nucleotides long. (nt) means non-translated RNA. miRNAs are known to be involved in critical biological processes related to development, differentiation, apoptosis, and proliferation, as well as diseases such as diabetes, neurodegenerative diseases, and cancer.

The miRNA 155 may include the nucleotide sequence of SEQ ID NO: 1. In addition, not only the nucleotide sequence composed of SEQ ID NO: 1, but also 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 98% or more, and most specifically 99% or more of the above sequence As a nucleotide sequence showing homology, any nucleotide sequence showing substantially the same or equivalent efficacy as the miRNA 155 is included without limitation. In addition, if it is a nucleotide sequence having such homology, it is obvious that a nucleotide sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present invention.

The term "homology" refers to the degree of similarity of a base sequence or amino acid sequence, and may be expressed as a percentage according to the degree of matching with a given amino acid sequence or base sequence. In the present specification, a homologous sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is expressed as "% homology". For example, hybridization using standard software, specifically BLAST 2.0, that calculates parameters such as score, identity and similarity, or under defined stringent conditions. It can be confirmed experimentally by comparing sequences, and appropriate hybridization conditions defined are within the skill of the art and are well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

The miRNA 155 may be derived from humans or mice, but is not limited thereto, and may be artificially synthesized or manufactured in vitro. In order to synthesize or prepare the miRNA 155, chemical or biological methods or methods known in the art that are not limited thereto may be used. Thus, the miRNA 155 may include a miRNA 155 mimic. The miRNA 155 mimic may refer to a synthetic miRNA made of a sequence identical to or partially modified from the sequence of miRNA 155 in a cell.

The term "chitosan" refers to a polysaccharide, a natural polymeric material derived by deacetylation of -CH ₃ CONH of chitin obtained from crustacean shells. That is, chitosan may have a form in which an acetyl group (-COCH ₃ ) present in a chitin monomer is substituted with an amino group (-NH ₃ ). On the other hand, chitin, obtained from the shells of crustaceans such as crabs, crayfish, and shrimps, is _a natural polymer that is the second most abundant after cellulose. can For example, the chitosan may be a compound represented by Formula 1 as follows:

[Formula 1]

(In Formula 1, n is any integer selected from 1 to 10000).

The chitosan may be low molecular weight chitosan (LMWC). Specifically, the chitosan may have a weight average molecular weight of about 50,000 to about 190,000 Da.

According to one embodiment, when the weight average molecular weight of the chitosan is less than about 50,000 Da, the miRNA 155-encapsulated chitosan nanoparticles reduce the electrostatic interaction between miRNA 155 and chitosan, such as the strength of the ionic bond, , the encapsulation strength for encapsulating the miRNA 155 inside the chitosan nanoparticles may be weakened. As a result, miRNA 155 may be exposed to the external environment and its effectiveness may decrease. In addition, when the weight average molecular weight of the chitosan is greater than about 190,000 Da, the cell penetration is reduced due to the increase in the size of the chitosan nanoparticles encapsulated with the miRNA 155, thereby reducing the efficiency of delivering miRNA 155 into fish in vivo or into cells. can do.

In addition, the chitosan may exhibit biodegradability and/or biocompatibility, and may include a salt form or a chitosan derivative. For example, the chitosan derivative may be an alkylate, an acylate, an arylide, a sulfur oxide, or a phosphate of chitosan, but is not limited thereto.

Chitosan nanoparticles (CNPs) may be prepared using the chitosan, and a specific material (eg, miRNA 155) may be encapsulated or encapsulated inside the chitosan nanoparticles. A specific substance (eg, miRNA 155) encapsulated or encapsulated inside the chitosan nanoparticles can be stably protected from the external environment, such as enzymes, and can be released under specific conditions. The enzyme may be a DNA degrading enzyme (DNase) or an RNA degrading enzyme (RNase), but is not limited thereto.

The chitosan nanoparticles may include the chitosan as one component, and may further include tripolyphosphate (TPP). In addition, the chitosan nanoparticles may be one in which the chitosan and the tripolyphosphate form a cross-linked bond. The tripolyphosphate may be sodium tripolyphosphate (Na ₅ P ₃ O ₁₀ ) or potassium tripolyphosphate (K ₅ P ₃ O ₁₀ ).

The chitosan may exhibit a positive charge, and the miRNA 155 may exhibit a negative charge. Therefore, the chitosan nanoparticles encapsulated with the miRNA 155 may be one in which the chitosan and the miRNA 155 are bonded or connected through an electrostatic interaction, for example, an ionic bond. That is, the chitosan nanoparticles encapsulated with miRNA 155 are coated with chitosan on the surface of miRNA 155 by electrostatic interaction to form chitosan nanoparticles on the outside, and spherical nanoparticles or nanoparticles with miRNA 155 inside them. It may have the shape of a sphere (nanosphere). In this case, the chitosan nanoparticles formed on the outside may be one in which the chitosan and the tripolyphosphate form a cross-linked bond.

The term “encapsulation” refers to the art of enclosing or coating one material with another.

The term "nano capsule" means a nano-sized capsule, and a specific material may be contained inside the nanocapsule. The term "nanoparticle" may be used interchangeably with the term "nanocapsule".

In other words, the chitosan nanoparticle encapsulated with the miRNA 155 is a complex of the miRNA 155 and the chitosan; Alternatively, it may be a complex of the miRNA 155, the chitosan, and the tripolyphosphate.

The weight ratio of miRNA 155 and chitosan contained in the miRNA 155-encapsulated chitosan nanoparticles may be about 1:5 to 15, 1:7 to 13, 1:9 to 11, or 1:10. When the weight ratio of miRNA 155 and chitosan contained in the chitosan nanoparticles encapsulated with miRNA 155 is out of the above value or range, it is difficult to form miRNA 155 encapsulated chitosan nanoparticles having a uniform size of about 200 to 500 nm. , encapsulation efficiency, encapsulation strength, or miRNA 155 releasing ability of chitosan nanoparticles encapsulated with miRNA 155 can be significantly reduced. As a result, in the chitosan nanoparticles encapsulated with miRNA 155, the efficiency of delivering effective miRNA 155 into the body or cells of fish and the effect of preventing or treating viral infectious diseases in fish can be significantly reduced.

The term "chitosan nanoparticles encapsulated with miRNA 155" may be used interchangeably with the term "miRNA 155-CNPs".

The miRNA 155-encapsulated chitosan nanoparticles may exhibit positive surface charges. In addition, the miRNA 155-encapsulated chitosan nanoparticles may have a zeta potential of about 30 to 60 or 35 to 45 mV.

The term "zeta potential" means the magnitude of the repulsive force and attractive force between particles in units. represents density. Particles of colloidal matter in water have the property of floating rather than settling. These materials have the property of repelling each other due to the + and - ions of the particles, and this repulsive force can be called zeta potential. Therefore, the zeta potential can be applied to evaluate the degree of dispersion or aggregation of particles suspended in a liquid phase, and generally, the higher the absolute value of the zeta potential, the more stable the dispersion of the particles in the suspension can be evaluated.

According to one embodiment, since the miRNA 155-encapsulated chitosan nanoparticles exhibit a high zeta potential of about 30 to 60 mV, they can maintain a stable dispersion state in a liquid phase. This also means that the miRNA 155-encapsulated chitosan nanoparticles can maintain a very stable encapsulation state by forming a strong static interaction between the internal negative charge and the external positive charge, for example, an ionic bond. As a result, the miRNA 155-encapsulated chitosan nanoparticles exhibit a very stable nanosphere form, strongly protect the internal miRNA 155 from the external environment, and are stably dispersed in the liquid phase, making it easy to use in various formulations suitable for direct administration to fish. can be applied Therefore, even if it is not by means of transforming fish using a gene expression vector, etc., effectiveness is maintained by a simple method of directly administering (oral or parenteral administration) the miRNA 155-encapsulated chitosan nanoparticles to fish. By effectively delivering miRNA 155 into living organisms or cells of fish, viral infectious diseases in fish can be prevented or treated.

The miRNA 155-encapsulated chitosan nanoparticles may have a diameter of about 200 to 500 or 300 to 400 nm.

According to one embodiment, when the diameter of the miRNA 155-encapsulated chitosan nanoparticles is less than about 200 nm, the amount of miRNA 155 that can be encapsulated inside the chitosan nanoparticles is reduced, so that miRNA 155 can be in vivo or in vivo in fish. The efficiency of intracellular delivery may be reduced. In addition, when the miRNA 155-encapsulated chitosan nanoparticles have a diameter greater than about 500 nm, the electrostatic interaction between miRNA 155 and chitosan, for example, the strength of ionic bond, decreases and miRNA 155 is incorporated into the chitosan nanoparticles. The encapsulation strength for encapsulation may be weakened, and miRNA 155 may be exposed to the external environment, reducing effectiveness. In addition, due to the increase in the size of the nanoparticles, the cell penetration of the chitosan nanoparticles encapsulated with the miRNA 155 may decrease, and thus the efficiency of delivering the miRNA 155 into the body or cells of the fish may decrease.

The miRNA 155-encapsulated chitosan nanoparticles may have miRNA 155 releasing ability. According to one embodiment, miRNA 155-encapsulated chitosan nanoparticles may continuously release miRNA 155 as chitosan on the outside is gradually biodegraded over time. This can be understood as the release of miRNA 155 as the strong electrostatic interaction formed between chitosan and miRNA 155 by the biodegradation of chitosan, for example, the strength of ionic bond, is reduced. As a result, after the miRNA 155-encapsulated chitosan nanoparticles are absorbed into the living body or cells of the fish, miRNA 155 is continuously released to continuously maintain the effect of miRNA 155 in the living body of the fish.

The term "effective ingredient" means an appropriately effective amount of an ingredient that affects a beneficial or desirable clinical or biochemical outcome. Specifically, it may refer to chitosan nanoparticles encapsulated with an effective amount of miRNA 155.

Such an effective amount can prevent disease, alleviate symptoms, reduce the extent of a disease, stabilize (i.e., not worsen) a disease state, delay or reduce the rate of disease progression, or a disease, without limitation, for a subject. It may be an appropriate amount for improvement or palliation and relief (partial or total) of the condition. The effective amount is the type of subject, the condition of the subject, the amount of the subject, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, factors including drugs used concurrently, and It may be determined according to factors well known in other related fields.

According to one embodiment, the chitosan nanoparticles encapsulated with miRNA 155 significantly improve the stability of miRNA 155 by blocking the influence of the external environment, and can effectively deliver miRNA 155 with maintained efficacy into fish in vivo or into cells. . In addition, since the miRNA 155-encapsulated chitosan nanoparticles are very small and exhibit a positive surface charge, the bioabsorption rate, bioavailability, and cell permeability can be remarkably increased, and after being absorbed into the living body or cells of fish, the chitosan Due to the gradual biodegradation, miRNA 155 is continuously released, so that the effect of miRNA 155 can be continuously maintained in the body of fish.

The antiviral composition for fish containing chitosan nanoparticles encapsulated with miRNA 155 as an active ingredient may exhibit an antiviral effect against viral hemorrhagic sepsis virus.

The term "viral hemorrhagic septicemia (VHS)" refers to sepsis caused by infection with a specific virus that afflicts over 70 species of freshwater and marine fish throughout Europe, the United States and Southeast Asia. It occurs mainly from late winter to spring, and shows a high mortality rate of about 50 to 70% in all age groups (from fry to adult fish) of flounder at a water temperature of about 8 to 15 ° C.

The term "viral hemorrhagic septicemia virus (VHSV)" refers to a virus that causes the above viral hemorrhagic septicemia, and belongs to the rhabdovirus family as a negative single-stranded RNA virus. VHSV causes death not only in juvenile fry but also in large adult fish at the time of shipment. Infected fish, blackening of the body, retention of clear ascitic fluid in the peritoneum and abdominal cavity, hepatic congestion, splenomegaly, nephroplegia, necrosis of the liver, kidney, spleen and skeletal muscle, and gill fronds It is known that internal symptoms such as thickening of the liver, thickening of the liver, and accumulation of skeletal muscle blood cells appear, and the virus invades the fish body through the epithelial layer of the skin or endothelial cells of the gills and moves rapidly through the bloodstream to internal organs such as the kidney and spleen hematopoietic tissue. .

According to one embodiment, when the fish administered with the miRNA 155-encapsulated chitosan nanoparticles are infected with VHSV, the proliferation or replication of VHSV in fish tissue can be reduced, and the survival rate of VHSV-infected fish can be significantly increased. can In addition, according to one embodiment, when the fish administered with the miRNA 155-encapsulated chitosan nanoparticles are infected with VHSV, the antiviral response, immune response regulation, and cell death response induced by VHSV in fish tissue Gene expression of factors related to can be regulated. That is, the miRNA 155-encapsulated chitosan nanoparticles can regulate antiviral, immune, and apoptotic responses of VHSV-infected fish. Through this, the miRNA 155-encapsulated chitosan nanoparticles can exhibit excellent preventive or therapeutic effects against VHSV infectious diseases of fish.

The term "administration" means introducing a predetermined substance into fish by any suitable method, and the administration route of the antiviral composition for fish may be administered through any general route as long as it can reach the target tissue. Specifically, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration may be administered, but is not limited thereto. The dose of the antiviral composition for fish depends on the formulation method, body weight of the subject, age of the animal, sex, morbid condition, health condition, diet, administration time, administration route, administration method, excretion rate, excretion rate, severity of the disease, And the range may vary depending on reaction sensitivity, etc., and may be divided and administered once or several times a day. Ordinarily, a skilled physician can easily determine and prescribe a dose of the antiviral composition for fish that is effective for the desired treatment or prevention. For example, the daily dosage of the antiviral composition for fish may be about 0.001 to 10000 mg/kg, but is not limited thereto.

The antiviral composition for fish may be administered to fish as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The antiviral composition for fish is used for fish, and may be a feed, a feed additive, an antiviral drug, a drug for preventing or treating a viral infectious disease, or a drug for enhancing immunity. Specifically, the antiviral composition for fish may be a drug for preventing or treating viral hemorrhagic sepsis virus infectious diseases.

The term "prevention" refers to any action that suppresses or delays the onset of viral hemorrhagic sepsis virus infectious disease by administration of the composition.

The term "treatment" means any action that improves or benefits the symptoms of a disease already caused by a viral hemorrhagic sepsis virus infectious disease by administration of the composition.

The antiviral composition for fish may further include one or more veterinarily acceptable carriers. Veterinarily acceptable carriers may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and, if necessary, an antioxidant, Other customary additives such as buffers and bacteriostats may be added. In addition, a diluent, a dispersing agent, a surfactant, a binder, and a lubricant may be additionally added and formulated using a method well known in the art.

When the antiviral composition for fish is used as a feed or feed additive, a binder, an emulsifier, a preservative, etc. added to prevent quality deterioration may be further included, and amino acids, vitamins, enzymes, and probiotics to increase efficacy , Flavors, non-protein nitrogen compounds, silicate agents, buffers, coloring agents, extractants, oligosaccharides, etc. may be further included, and in addition, feed mixtures may be further included. In addition, when the antiviral composition for fish is used as a feed or feed additive, grains, roots and fruits, food processing by-products, algae, fibers, oils and fats, starches, meal, grain by-products, proteins, inorganic It can be mixed with feed or feed additives such as logistics, oils, mineral oils, oils, single cell proteins, zooplankton, and fish meal.

The fish may be normal fish that require prevention against VHSV infection or infected fish that are already infected with VHSV and require treatment. In addition, the fish may collectively refer to all fish that can be infected with the viral hemorrhagic sepsis virus without limitation. Specifically, the fish include halibut (Paralichthys olivaceus), yellowtail (Seriola quinqueradiata), red sea bream (Pagrus major), purple tiger (Takifugurubripes), sea bass (Seriola aureovittata), trout (Oncorhynchus masou), rainbow trout (Oncorhynchus mykiss), and amberjack (Seriola) dumerili), horse mackerel (Trachurus japonicus), blackspotted horse mackerel (Pseudocaranx dentex), grouper fish (Epinephelus septemfasciatus), bluefin tuna (Thunnus thynnus), carp (Cyprinus carpio), zebrafish (Danio rerio), catfish (Silurus asotus), clary It may include Clarias gariepinus, tilapia (Oreochronis niloticus), salmon (Oncorhynchus keta), Atlantic salmon (Salmo salar), and killifish (Oryziaslatipes), preferably farmed fish, but is not limited thereto. don't

The antiviral composition for fish is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. or it may be prepared by incorporating into a multi-dose container. At this time, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, but is not limited thereto.

Another aspect is (a) mixing miRNA 155 and tripolyphosphate solution; (b) dissolving chitosan in an acidic solvent; and (c) mixing and stirring the mixed solution obtained in step (a) and the solution obtained in step (b) to provide a method for preparing chitosan nanoparticles encapsulated with miRNA 155.

Chitosan used in the method is as described above.

In step (a), the tripolyphosphate may be sodium tripolyphosphate (Na ₅ P ₃ O ₁₀ ) or potassium tripolyphosphate (K ₅ P ₃ O ₁₀ ).

In step (a), miRNA 155 and tripolyphosphate may be mixed in a weight ratio of about 1:2 to 4, 1:3 to 4, or 1:3 to 3.5. According to one embodiment, when the miRNA 155 and tripolyphosphate are mixed in the above weight ratio in step (a), the miRNA 155-encapsulated chitosan nanoparticles prepared by the above method have encapsulation efficiency, encapsulation strength, or miRNA 155 Since the release function is remarkably excellent, miRNA 155, which remains effective, can be effectively delivered into the body or cells of fish, and as a result, the effect of preventing or treating viral infectious diseases in fish can be remarkably increased.

In addition, in the step (a), the concentration of the tripolyphosphate solution and the type of solvent can be allowed without any limitation as long as the concentration and solvent can dissolve the tripolyphosphate without affecting the characteristics of the miRNA 155 and the tripolyphosphate. can

In the step (b), the acidic solvent is acetic acid, malonic acid, CMEBAC (N carboxymethyl N,N diethylbenzene ammonium chloride), lactic acid, citric acid, or sodium acetate (sodium acetate), but is not limited thereto. According to one embodiment, as the acidic solvent in step (b), an acidic solution, such as an aqueous acetic acid solution, may be used. The concentration of the aqueous acetic acid solution may be about 0.01 to 2, 0.01 to 1, 0.01 to 0.1, or 0.03 to 0.07% by weight, but is not limited thereto.

In addition, in step (b), the chitosan may be dissolved in the acidic solvent at a concentration of about 1 to 5, 1 to 4, or 1 to 3 mg/mL, but is not limited thereto.

In step (c), the mixed solution obtained in step (a) and the solution obtained in step (b) are mixed in a weight ratio of about 1:1 to 3, 1:1 to 2, or 1:1 to 1.5. It may be, but is not limited thereto.

The weight ratio of miRNA 155 and chitosan in the mixture obtained in step (c) may be about 1:5 to 15, 1:7 to 13, 1:9 to 11, or 1:10. According to one embodiment, when the weight ratio of the miRNA 155 and chitosan contained in the mixture obtained in step (c) is about 1: 5 to 15, the miRNA 155-encapsulated chitosan nanoparticles prepared by the method Since the encapsulation efficiency, encapsulation strength, or miRNA 155 release ability is remarkably excellent, miRNA 155 with maintained efficacy can be effectively delivered in vivo or into the cells of fish, thereby significantly preventing or treating fish viral infectious diseases. can increase

In the step (c), chitosan nanoparticles may be formed by forming a cross-link between the chitosan and tripolyphosphate, and an electrostatic interaction, such as an ionic bond, is formed between the chitosan and miRNA 155, thereby forming the chitosan nanoparticle. miRNA 155 may be encapsulated inside the nanoparticle to form chitosan nanoparticles encapsulated with miRNA 155.

The method may further include obtaining chitosan nanoparticles encapsulated with miRNA 155 by centrifuging the mixture obtained in step (c).

According to one embodiment, the miRNA 155-encapsulated chitosan nanoparticles prepared by the above method may exhibit a preventive or therapeutic effect against viral hemorrhagic sepsis virus infectious fish disease.

In addition, according to one embodiment, when miRNA 155-encapsulated chitosan nanoparticles are prepared by the above method, significantly superior encapsulation efficiency and significantly lower polydispersity index are exhibited, and the particle size is uniform and the miRNA encapsulated very stably. 155 encapsulated chitosan nanoparticles can be prepared. As a result, the miRNA 155 encapsulated chitosan nanoparticles prepared by the above method exhibit a very stable nanosphere shape, and thus miRNA 155 inside can be strongly protected from the external environment. In addition, since the miRNA 155-encapsulated chitosan nanoparticles prepared by the above method are nano-sized and exhibit a positive surface charge, bioabsorption, bioavailability, and cell permeability can be remarkably increased, and fish After being absorbed into a living body or cells, miRNA 155 is continuously released due to the gradual biodegradation of chitosan, so that the effect of miRNA 155 can be continuously maintained in the living body of fish.

In summary, the miRNA 155-encapsulated chitosan nanoparticles prepared by the above method significantly improve the stability of miRNA 155 by blocking the influence of the external environment, and can effectively deliver miRNA 155 with maintained efficacy into fish organisms, As a result, it is possible to exhibit a significantly increased preventive or therapeutic effect against viral hemorrhagic sepsis virus infectious fish disease.

It is to be understood that any of the terms or elements mentioned in the manufacturing method as mentioned in the description of the composition are the same as those mentioned in the description of the composition above.

Another aspect is to prevent or treat viral hemorrhagic sepsis virus infectious fish disease comprising administering to fish an antiviral composition for fish against VHSV containing the miRNA 155-encapsulated chitosan nanoparticles or the same as an active ingredient. provides a way

Among the terms or elements mentioned in the prevention or treatment method, the same as those mentioned in the description of the composition or preparation method are understood to be the same as those mentioned in the description of the composition or preparation method.

Another aspect provides an antiviral use of the miRNA 155-encapsulated chitosan nanoparticles or a composition containing the miRNA 155 as an active ingredient against fish-infectious viral hemorrhagic septicemia virus (VHSV).

Another aspect provides the use of the miRNA 155-encapsulated chitosan nanoparticles or a composition containing the chitosan nanoparticles as an active ingredient for preventing or treating VHSV-infected fish disease.

Another aspect provides a use of the miRNA 155-encapsulated chitosan nanoparticles for preparing an antiviral composition for fish against VHSV or a medicament (or pharmaceutical composition) for preventing or treating VHSV-infected fish disease.

Any of the terms or elements mentioned in this application as mentioned in the description of the composition, method of manufacture, or method of prevention or treatment are the same as those mentioned in the description of the composition, method of manufacture, or method of prevention or treatment above. It is understood that

According to an antiviral composition for fish comprising chitosan nanoparticles encapsulated with miRNA 155 as an active ingredient and a method using the same according to one aspect, when fish are infected with VHSV, the proliferation or replication of VHSV in fish tissue is reduced. In addition, the antiviral response, immune response, and cell death response are regulated, and the survival rate of fish is significantly increased, so that VHSV-infected fish diseases can be prevented or treated.

In addition, the miRNA 155-encapsulated chitosan nanoparticles significantly improve the stability of miRNA 155, can effectively deliver miRNA 155 with maintained efficacy into the fish body, and do not induce toxicity in fish tissue, resulting in VHSV infectious disease in fish. It can be applied as a safe treatment that can prevent or treat very effectively.

1 is a diagram schematically showing the structure of miRNA 155-CNPs and the release of miRNA 155 from miRNA 155-CNPs according to an embodiment.

2 is a diagram schematically showing a manufacturing process of miRNA 155-CNPs according to an embodiment.

3 is a diagram showing the chemical formula of low molecular weight chitosan (LMWC) used for the preparation of miRNA 155-CNPs according to an embodiment.

4 is a diagram showing the results of (a) scanning electron microscopy (SEM) and (b) transmission electron microscopy (TEM) imaging of miRNA 155-CNPs according to an embodiment. .

5 is a graph showing the results of analyzing the particle size of miRNA 155-CNPs according to an embodiment.

6 is a graph showing the results of analyzing the zeta potential of miRNA 155-CNPs according to an embodiment.

7 is a graph showing the results of analyzing miRNA 155 release levels over time of miRNA 155-CNPs according to an embodiment.

8 is a view showing the results of evaluating (a) the encapsulation strength of miRNA 155-CNPs and (b) the integrity of miRNA 155 stored therein according to an embodiment.

9 is a diagram schematically illustrating an experimental procedure for confirming the effect of miRNA 155-CNPs on VHSV (viral hemorrhagic septicemia virus) infection of fish according to an embodiment.

10 is (a) viral mRNA copy numbers in tissues of fish infected with VHSV after administration of miRNA 155-CNPs to confirm the effect of miRNA 155-CNPs on VHSV infection of fish according to an embodiment; (VCN) and (b) a graph showing the results of measuring the level of viral gRNA copy numbers (VCN).

11 is a graph showing the results of analyzing the survival rate of VHSV-infected fish after administration of miRNA 155-CNPs in order to confirm the effect of miRNA 155-CNPs on VHSV infection of fish according to an embodiment.

12 is a view of miRNA 155-CNPs according to an embodiment in order to confirm the effect of regulating the antiviral response of fish against VHSV infection, in tissues of fish infected with VHSV after administration of miRNA 155-CNPs (a ) ifnγ; (b) irf2bpl; and (c) a graph showing the result of measuring the mRNA expression level of irf9.

13 is a view of (a) socs1a in tissues of VHSV-infected fish after administration of miRNA 155-CNPs to confirm the effect of miRNA 155-CNPs on regulating the immune response against VHSV infection in fish according to an embodiment; ; (b) il1β; (c) tnfα; (d) il6; (e) il10; And (f) a graph showing the results of measuring the mRNA expression level of cxcl18b.

14 is a diagram in the tissues of fish infected with VHSV after administration of miRNA 155-CNPs in order to confirm the effect of miRNA 155-CNPs on regulating the apoptosis response of fish to VHSV infection according to an embodiment (a) cd8a; (b) caspase3; and (c) a graph showing the results of measuring the mRNA expression level of p53.

15 is H for tissue slices obtained by terminating gill tissue filaments collected from fish to which miRNA 155-CNPs were administered, in order to confirm whether miRNA 155-CNPs induce toxicity in fish tissue according to an embodiment. & E staining is performed to show the result of confirming the pathological state of the tissue ((a): NF H ₂ O administration normal control group; (b): CNPs administration comparison control group; (c): miRNA 155-CNPs administration experimental group). EC represents one layer of epithelial cells, PL represents primary lamellar epithelium, SL represents secondary lamella, and MC represents mucous cells of the inner wall of the SL. indicate

16 is a graph showing a standard curve and standard formula used for absolute quantification (qRT-PCR) of N gene (VHSV) to measure VCN in VHSV-infected fish injected with miRNA 155-CNPs according to an embodiment. .

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of miRNA 155 encapsulated chitosan nanoparticles (miRNA 155-CNPs)

A complex of miRNA 155 and CNPs (miRNA 155-CNPs) having a structure in which miRNA 155 is encapsulated inside chitosan nanoparticles (CNPs) was prepared.

1 is a diagram schematically showing the structure of miRNA 155-CNPs and the release of miRNA 155 from miRNA 155-CNPs according to an embodiment.

2 is a diagram schematically showing a manufacturing process of miRNA 155-CNPs according to an embodiment.

3 is a diagram showing the chemical formula of low molecular weight chitosan (LMWC) used for the preparation of miRNA 155-CNPs according to an embodiment.

Specifically, about 300 μg of miRNA 155 containing the nucleotide sequence of SEQ ID NO: 1 was added to about 1.2 mL of sodium tripolyphosphate (Na ₅ P ₃ O ₁₀ ) solution (about 0.83 mg/mL) and mixed. did The miRNA 155 was purchased from GenePharma (Shanghai, China) and used. Then, the mixed solution and a low molecular weight chitosan (LMWC) solution were mixed. Specifically, about 1.5 mL of a chitosan solution obtained by dissolving low molecular weight chitosan in an aqueous solution of about 0.05% acetic acid at a concentration of about 2 mg/mL and about 1.2 mL of a mixture of the miRNA 155 and sodium tripolyphosphate were mixed. Then, the mixture was mixed while stirring at room temperature for about 1 hour. Thereafter, the mixed solution was centrifuged for about 30 minutes at about 4° C. and about 12,000 rpm, and then the supernatant was removed and the obtained precipitate was examined under a scanning electron microscope (SEM) and transmission electron microscope (transmission electron microscope). Electron microscopy: TEM) analysis was performed.

4 is a diagram showing the results of (a) scanning electron microscopy (SEM) and (b) transmission electron microscopy (TEM) imaging of miRNA 155-CNPs according to an embodiment. .

As a result, as shown in FIG. 4, it was confirmed that spherical chitosan nanoparticles (miRNA 155-CNPs) in which miRNA 155 was encapsulated were obtained.

Example 2. Characterization of miRNA 155-CNPs

2.1 Analysis of particle size, zeta potential, polydispersity, encapsulation efficiency, and miRNA 155 release level of miRNA 155-CNPs

Particle size, zeta potential, polydispersity, encapsulation efficiency, and miRNA 155 of chitosan nanoparticles (miRNA 155-CNPs) encapsulated in miRNA 155 prepared in Example 1 Emission levels were analyzed.

Specifically, about 1 mL of miRNA 155-CNPs was suspended in pH 7.4 PBS, and the particle size distribution, polydispersity index (PDI), and zeta potential of the suspension were measured using Zetasizer ^® Nano-ZS (Malvern Instruments, Malvern, UK). measured. In addition, the encapsulation efficiency (%) is the final After centrifugation to obtain miRNA 155-CNPs, the concentration of non-captured miRNA 155 present in the recovered supernatant was measured and analyzed. The concentration of non-captured miRNA 155 present in the supernatant was determined by measuring absorbance at a wavelength of about 260 nm using a NanoDrop One UV-Vis Spectrophotometer (Thermo Fisher Scientific, USA). The absorbance of the supernatant of CNP without miRNA 155 was used as a blank. Encapsulation efficiency (%) was calculated according to:

Encapsulation efficiency (%) = (mass of encapsulated miRNA 155 / initial mass of miRNA 155 used) * 100.

In addition, in order to measure the miRNA 155 release level over time of the miRNA 155-CNPs, the miRNA 155-CNPs were added to a PBS solution (pH 7.4) and mixed while stirring at about 37 ° C. for about 20 days. Samples of the mixed solution were collected and centrifuged at intervals of days, and then the amount of miRNA 155 contained in the supernatant was measured using a NanoDrop One UV-Vis spectrophotometer.

5 is a graph showing the results of analyzing the particle size of miRNA 155-CNPs according to an embodiment.

6 is a graph showing the results of analyzing the zeta potential of miRNA 155-CNPs according to an embodiment.

7 is a graph showing the results of analyzing miRNA 155 release levels over time of miRNA 155-CNPs according to an embodiment.

As a result, as shown in Figures 5 and 6, it was confirmed that the particle size of the miRNA 155-CNPs was about 341.45 ± 10.00 nm, the surface charge was positive, and the zeta potential was about 39.30 ± 3.90 mv.

In addition, as a result of analyzing the polydispersity and encapsulation efficiency of the miRNA 155-CNPs, the polydispersity index (PDI) was remarkably low at about 0.516±0.05, and the encapsulation efficiency (EE (%)) was about 98.80%. was found to be significantly higher.

In addition, as shown in FIG. 7, it was confirmed that the miRNA 155-CNPs could continuously release miRNA 155 for a certain period of time. Specifically, it was confirmed that the miRNA 155-CNPs could release about 40% or more of the total amount of miRNA 155 contained in the miRNA 155-CNPs by continuously releasing miRNA 155 for about 20 days or more. This can be understood as the fact that miRNA 155 is released as the CNPs are gradually biodegraded, and the strong electrostatic interaction formed between the CNPs and miRNA 155, eg, the strength of the ionic bond, is reduced.

Through this embodiment, the miRNA 155-CNPs, as shown in Figure 1, the surface of miRNA 155 is coated with positively charged CNPs, so that miRNA 155 exists inside, and positively charged CNPs are outside miRNA 155 inside. It was confirmed that the nanospheres or spherical nanoparticles exhibiting a surface positive charge having a structure surrounding the .

In addition, in the case of the preparation method of Example 1, remarkably excellent encapsulation efficiency and remarkably low polydispersity index are exhibited, and the particle size is uniform and very stably encapsulated nano-sized miRNA 155-CNPs can be prepared confirmed.

In addition, the miRNA 155-CNPs exhibited a very stable nanosphere shape as described above, and while strongly protecting the internal miRNA 155 from the external environment, the external CNPs gradually biodegraded over time, continuously releasing miRNA 155. It was confirmed that it could be released.

As a result, it was found that the miRNA 155-CNPs significantly improved the stability of miRNA 155 by blocking the influence of the external environment, and could effectively deliver miRNA 155 with maintained efficacy into the fish body. In addition, since the miRNA 155-CNPs have very small particles and exhibit a positive surface charge, they can increase bioabsorption, bioavailability, and cell permeability, and after being absorbed into the body or cells of fish, miRNA It was found that the effect of miRNA 155 could be continuously maintained in the body of fish by continuously releasing 155.

2.2 Analysis of encapsulation efficiency of miRNA 155-CNPs according to the weight ratio of miRNA 155 and chitosan

Chitosan nanoparticles (miRNA 155-CNPs) in which miRNA 155 was encapsulated were prepared according to the preparation method of Example 1, and various types of miRNA 155-CNPs having different weight ratios of miRNA 155 and chitosan were prepared. Afterwards, the difference in encapsulation efficiency according to the difference in the weight ratio of miRNA 155 and chitosan was compared and analyzed. Encapsulation efficiency analysis was performed in the same manner as described in Example 2.1 above.

As a result, as shown in Table 1, when the weight ratio of miRNA 155 and chitosan contained in miRNA 155-CNPs is about 1: 10, the encapsulation efficiency is about 98% or more, so miRNA 155 can be encapsulated with the best efficiency confirmed.

number	miRNA 155 (ug)	low molecular weight chitosan (mg)	weight ratio (miRNA 155: chitosan)	encapsulation efficiency (%)
One	300	6	1:20	90.45±3.38
2	300	3	1:10	98.80±0.56
3	300	1.5	1:5	85.64±0.64
4	300	0.75	1:2.5	45.35±2.15

Example 3. Evaluation of encapsulation strength of miRNA 155-CNPs and integrity of internally stored miRNA 155

The encapsulation strength of the chitosan nanoparticles (miRNA 155-CNPs) in which miRNA 155 prepared in Example 1 was encapsulated was evaluated, that is, the binding strength between CNPs and miRNA 155, and the storage ability to retain miRNA 155 inside. In addition, whether the miRNA 155 stored inside the miRNA 155-CNPs remains intact, that is, RNA integrity was evaluated.

Specifically, in order to confirm the encapsulation strength of miRNA 155-CNPs, DNA marker (100 bp); miRNA 155 (naked miR-155) about 1.5 μg; About 10 μL of CNPs; About 10 μL of suspension containing about 1.5 μg of miRNA 155-CNPs; and centrifugation supernatant (miRNA 155-CNPs SN) separated from miRNA 155-CNPs. After adding about 2 μL of 6x loading buffer to about 10 μL of each sample, each sample was loaded on about 1% agarose gel. Electrophoretic analysis was performed. In addition, in order to confirm the integrity of the miRNA 155 stored in the miRNA 155-CNPs, a DNA marker (100 bp); miRNA 155 (naked miR-155) about 1.5 μg; About 10 μL of suspension containing about 1.5 μg of miRNA 155-CNPs; miRNA 155 and about 0.1 μL of RNase A solution (10 mg/mL) were mixed and incubated at about 37° C. for about 5 minutes; A mixture obtained by incubating about 10 μL of a suspension containing about 1.5 μg of miRNA 155-CNPs and about 1.9 μL of chitosanase at about 37° C. for about 4 hours; And about 10 μL of the suspension containing about 1.5 μg of miRNA 155-CNPs and about 0.1 μL of RNase A solution were incubated at about 37 ° C. for about 5 minutes. After adding about 2 μL of 6x loading buffer to each sample of the mixture, , Each sample was loaded on about 1% agarose gel and analyzed by electrophoresis. The RNase A was inactivated by raising the temperature to about 60° C. for about 5 minutes.

8 is a view showing the results of evaluating (a) the encapsulation strength of miRNA 155-CNPs and (b) the integrity of miRNA 155 stored therein according to an embodiment.

As a result, as shown in FIG. 8, it was confirmed that the encapsulation of the miRNA 155-CNPs was very strong, and the miRNA 155 inside was not easily exposed to the external environment. In addition, it was confirmed that RNA integrity was maintained by maintaining the original state of the released miRNA 155 when the CNPs of the miRNA 155-CNPs were degraded by chitosanase. In addition, it was confirmed that the miRNA 155-CNPs protect the internally stored miRNA 155 from external enzymes by preventing miRNA 155 from being degraded by ribonucleic acid hydrolase A (RNase A).

Through this Example, it was found that the miRNA 155-CNPs had remarkably excellent binding strength between CNPs and miRNA 155 and storage ability to retain miRNA 155 therein. In addition, the miRNA 155 stored inside the miRNA 155-CNPs is not affected by the external environment (e.g., RNase A, etc.), and maintains its original state intact, maintaining the integrity of the miRNA 155-CNPs. It was found that it was stored in and then released. Therefore, the miRNA 155-CNPs are used as a therapeutic agent that can effectively prevent or treat infectious diseases in fish by protecting miRNA 155 from the external environment and delivering miRNA 155 into the body or cells of fish while maintaining the effectiveness of miRNA 155. knew it could be.

Example 4. Confirmation of the effect of miRNA 155-CNPs on VHSV infection in fish

In order to confirm the preventive or therapeutic effect of chitosan nanoparticles (miRNA 155-CNPs) encapsulated with miRNA 155 prepared in Example 1 against viral hemorrhagic septicemia virus (VHSV) infectious diseases in fish, the miRNA in fish survival rate of fish infected with VHSV after administration of 155-CNPs; levels of viral RNA copy numbers (VCN) in fish tissue; And gene expression levels of factors related to antiviral responses, regulatory and inflammatory responses, and apoptotic responses induced by VHSV in fish tissues were analyzed.

9 is a diagram schematically illustrating an experimental procedure for confirming the effect of miRNA 155-CNPs on VHSV (viral hemorrhagic septicemia virus) infection of fish according to an embodiment.

Specifically, as shown in FIG. 9, after ip injection (intraperitoneal injection) of the miRNA 155-CNPs into fish (zebrafish), about 14 hours later, about 20 μL of VHSV culture medium (about 10 ⁵ TCID ₅₀ /mL/ fish) were infected by ip injection (n=10). Next, tissues (gills, muscles, kidneys, etc.) were collected from the VHSV-infected fish about 57 hours after VHSV infection, and the survival rate of the VHSV-infected fish was analyzed for about 120 hours after VHSV infection, and the miRNA 155 - The preventive or therapeutic effects of CNPs against VHSV infectious diseases in fish were analyzed. As a normal control, normal fish not injected with both miRNA 155-CNPs and VHSV were used. As comparative controls, fish infected with only VHSV injection without miRNA 155-CNPs and miRNA 155 inhibitor injected instead of miRNA 155-CNPs were used. Then, fish infected by injecting VHSV were used.

4.1 Analysis of the survival rate of VHSV-infected fish and the level of viral RNA copy numbers (VCN) in VHSV-infected fish tissues

As described above, the miRNA 155-CNPs were i.p. About 14 hours after injection, VHSV infection, and about 57 hours after VHSV infection, tissues (gills, muscles, kidneys, etc.) were taken and viral mRNA copy numbers (VCN) per tissue weight (g) were calculated. Viral gRNA copy numbers (VCN) levels were measured.

Specifically, VCN was calculated by absolute quantification (qRT-PCR) of the N gene transcript of VHSV from RNA isolated from each tissue. The standard curve and standard formula used are shown in FIG. 16 . The specific VCN level measurement method was performed according to a known method (Avunje et al., 2011).

In addition, the miRNA 155-CNPs i.p. About 14 hours after the injection, VHSV infection was performed, and survival of the fish infected with VHSV was observed for about 120 hours after VHSV infection, and then the survival rate was analyzed.

10 is (a) viral mRNA copy numbers in tissues of fish infected with VHSV after administration of miRNA 155-CNPs to confirm the effect of miRNA 155-CNPs on VHSV infection of fish according to an embodiment; (VCN) and (b) a graph showing the results of measuring the level of viral gRNA copy numbers (VCN).

11 is a graph showing the results of analyzing the survival rate of VHSV-infected fish after administration of miRNA 155-CNPs in order to confirm the effect of miRNA 155-CNPs on VHSV infection of fish according to an embodiment.

As a result, as shown in FIG. 10, when the fish administered with the miRNA 155-CNPs were infected with VHSV, compared to the control group not administered with the miRNA 155-CNPs, the viral RNA copy numbers (VCN ) level was confirmed to decrease.

In addition, as shown in FIG. 11, it was confirmed that the fish administered with the miRNA 155-CNPs exhibited a remarkably high survival rate of about 80% or more, similar to that of the normal control group, even if they were infected with VHSV. On the other hand, in the case of control fish not administered with the miRNA 155-CNPs, when infected with VHSV, it was confirmed that they exhibited a significantly low survival rate of about 50% or less.

Through this example, it was confirmed that the proliferation or replication of VHSV in fish tissue can be reduced and the survival rate can be remarkably increased when VHSV infection occurs in fish administered with the miRNA 155-CNPs. As a result, it was confirmed that the miRNA 155-CNPs exhibit preventive or therapeutic effects against VHSV infectious diseases in fish.

4.2 Analysis of changes in gene expression levels of factors related to antiviral response, immune response regulation, and apoptosis response induced by VHSV in fish tissue

As described above, the miRNA 155-CNPs were i.p. About 14 hours after injection, after infection with VHSV, about 57 hours after infection with VHSV, tissues (gills, muscles, kidneys, etc.) were collected and the antiviral response and immune response induced by VHSV in each tissue Changes in gene expression levels of factors related to regulation and cell death responses were analyzed. Changes in mRNA expression levels of ifnγ, irf2bpl, and irf9 were analyzed in relation to antiviral responses, and changes in mRNA expression levels of socs1a, il1β, tnfα, il6, il10, and cxcl18b were analyzed in relation to immune response regulation. , Changes in mRNA expression levels of cd8a, caspase3, and p53 in relation to the apoptosis response were analyzed.

Specifically, the mRNA expression level can be analyzed by a known method. For example, after reverse transcription of mRNA isolated from each tissue using RT-PCR to obtain cDNA, the antiviral reaction using the cDNA as a template , immune response regulation, and mRNA expression levels can be analyzed by amplifying genes with primers capable of amplifying genes of factors related to apoptosis response. Primers capable of amplifying genes of factors related to the antiviral response, immune response regulation, and cell death response can all be purchased from existing vendors.

12 is a view of miRNA 155-CNPs according to an embodiment in order to confirm the effect of regulating the antiviral response of fish against VHSV infection, in tissues of fish infected with VHSV after administration of miRNA 155-CNPs (a ) ifnγ; (b) irf2bpl; and (c) a graph showing the result of measuring the mRNA expression level of irf9.

13 is a view of (a) socs1a in tissues of VHSV-infected fish after administration of miRNA 155-CNPs to confirm the effect of miRNA 155-CNPs on regulating the immune response against VHSV infection in fish according to an embodiment; ; (b) il1β; (c) tnfα; (d) il6; (e) il10; And (f) a graph showing the results of measuring the mRNA expression level of cxcl18b.

14 is a diagram in the tissues of fish infected with VHSV after administration of miRNA 155-CNPs in order to confirm the effect of miRNA 155-CNPs on regulating the apoptosis response of fish to VHSV infection according to an embodiment (a) cd8a; (b) caspase3; and (c) a graph showing the results of measuring the mRNA expression level of p53.

As a result, as shown in FIG. 12, the fish administered with the miRNA 155-CNPs showed antiviral activity in all tissues of the gills, muscles, and kidneys when compared to VHSV-infected fish not administered with the miRNA 155-CNPs. It was confirmed that the mRNA expression of ifnγ and irf9, which are response-related factors, decreased. On the other hand, it was confirmed that the mRNA expression level of irf2bpl measured in kidney tissue was increased compared to the VHSV-infected fish not administered with miRNA 155-CNPs, unlike the level measured in gill and muscle tissue.

In addition, as shown in FIG. 13, the miRNA 155-CNPs-administered fish were VHSV-infected, compared to VHSV-infected fish not administered miRNA 155-CNPs, related to the regulation of immune responses in all tissues of the gills, muscles, and kidneys. It was confirmed that the mRNA expression of the factors il10 and cxcl18b decreased, whereas the mRNA expression of il1β and tnfα increased. In addition, it was confirmed that the mRNA expression levels of socs1a and il6 measured in kidney tissue were increased compared to VHSV-infected fish not administered with miRNA 155-CNPs, unlike those measured in gill and muscle tissue.

In addition, as shown in FIG. 14, fish administered with the miRNA 155-CNPs were infected with cd8a and caspase3, which are apoptosis response-related factors in gills and muscle tissue, compared to VHSV-infected fish not administered with miRNA 155-CNPs. , and p53 mRNA expression were all decreased, whereas in renal tissue, the mRNA expression of all of these factors was increased.

Through this example, when the fish administered with the miRNA 155-CNPs were infected with VHSV, gene expression of factors related to the antiviral response, immune response regulation, and apoptosis response induced by VHSV in fish tissues was increased. It was confirmed that it was regulated. As a result, it was confirmed that the miRNA 155-CNPs exhibit preventive or therapeutic effects against VHSV infectious diseases in fish by regulating the antiviral response, immune response, and cell death response of fish.

Example 5. miRNA 155-CNPs In vivo safety analysis

In order to analyze the safety of chitosan nanoparticles (miRNA 155-CNPs) encapsulated with miRNA 155 prepared in Example 1, after administering the miRNA 155-CNPs to fish, whether or not they induce toxicity in fish tissues was analyzed.

Specifically, about 48 hours after ip injection of the miRNA 155-CNPs solution at a concentration of about 100 μg/mL into zebrafish, the gill tissue of the fish was collected. H & E staining was performed on tissue sections obtained by terminating the collected tissue, and the safety of the miRNA 155-CNPs was analyzed by comparing the pathological state of the tissue with a control group. As a normal control group, normal fish to which NF H ₂ O was administered instead of the miRNA 155-CNPs were used, and fish to which CNPs were administered instead of the miRNA 155-CNPs were used as a comparative control group.

15 is H for tissue slices obtained by terminating gill tissue filaments collected from fish to which miRNA 155-CNPs were administered, in order to confirm whether miRNA 155-CNPs induce toxicity in fish tissue according to an embodiment. & E staining is performed to show the result of confirming the pathological state of the tissue ((a): NF H ₂ O administration normal control group; (b): CNPs administration comparison control group; (c): miRNA 155-CNPs administration experimental group). EC represents one layer of epithelial cells, PL represents primary lamellar epithelium, SL represents secondary lamella, and MC represents mucous cells of the inner wall of the SL. indicate

As a result, as shown in FIG. 15, it was confirmed that no difference was found in the pathological state between the tissue of the miRNA 155-CNPs-administered fish and the tissue of the normal fish.

Through this Example, it was confirmed that the miRNA 155-CNPs could be applied as a safe therapeutic agent capable of preventing or treating VHSV infectious diseases in fish because they did not induce toxicity in fish tissue.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (8)

1. An antiviral composition for fish against viral hemorrhagic septicemia virus (VHSV) comprising chitosan nanoparticles encapsulated with miRNA 155 as an active ingredient.
2. The composition according to claim 1, wherein the miRNA 155 comprises the nucleotide sequence of SEQ ID NO: 1.
3. The method according to claim 1, The chitosan nanoparticles will have a diameter of 200 nm to 500 nm, the composition.
4. The method according to claim 1, wherein the composition is a feed, feed additive, antiviral drug, a drug for preventing or treating a viral infectious disease, or a drug for enhancing immunity, the composition.
5. The method according to claim 1, wherein the composition is a composition for the prevention or treatment of viral hemorrhagic sepsis virus infectious disease.
6. (a) mixing miRNA 155 and tripolyphosphate solution;

   (b) dissolving chitosan in an acidic solvent; and

   (c) A method for preparing miRNA 155-encapsulated chitosan nanoparticles comprising mixing and stirring the mixed solution obtained in step (a) and the solution obtained in step (b).
7. The method according to claim 6, wherein the miRNA 155-encapsulated chitosan nanoparticles exhibit a preventive or therapeutic effect against viral hemorrhagic sepsis virus infectious fish disease.
8. A method for preventing or treating viral hemorrhagic sepsis virus infectious fish disease comprising administering the composition of any one of claims 1 to 5 to fish.

Images