WO2011138489A1

WO2011138489A1 - Use of casein hydrolysates as antiviral agents

Info

Publication number: WO2011138489A1
Application number: PCT/ES2011/070318
Authority: WO
Inventors: Isidra RECIO SÁNCHEZ; Sylvia RODRÍGUEZ SAINT-JEAN; Sara Isabel PÉREZ PRIETO; Iván LÓPEZ EXPÓSITO; Ana Isabel DE LAS HERAS SÁNCHEZ; José Angel GÓMEZ RUIZ; Mercedes Ramos González
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2010-05-06
Filing date: 2011-05-04
Publication date: 2011-11-10

Abstract

The invention relates to the use of hydrolysates of bovine milk caseins with pepsin, and compositions containing said hydrolysates, as antiviral agents. Said hydrolysates and compositions are particularly suitable for use against salmonid virus, and, specifically, against infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV).

Description

Use of casein hydrolysates as antivirals.

The present invention is within biology and medicine, and specifically refers to the use of enzymatic hydrolysates of milk caseins as antivirals, and in particular against salmonid viruses, to the compositions containing said hydrolysates, and to their process of obtaining.

STATE OF THE PREVIOUS TECHNIQUE

In recent years, the protein fraction of food has been the subject of numerous studies aimed at demonstrating the biological activity of food proteins and peptides. Most of the research carried out has been aimed at the use of these peptides as ingredients in functional foods. It has been shown that certain milk proteins and peptides derived therefrom exert different biological activities such as antimicrobial activity against different Gram-positive and Gram-negative bacteria and yeasts (Pellegrini 2003. Curr. Pharm. Design é, 1225-1238; López-Expósito y Recio 2008, Adv. Exp. Med. Biol. 606, 271-293). The use of peptides and food proteins as antimicrobial agents in other applications has aroused considerable interest given the safety of these compounds. Recently, it has begun to explore the possible antiviral activity of different fragments derived from milk proteins against some viruses that affect mammals. For example, the lactoferrin milk protein and chemical modifications thereof aimed at modifying the surface charge of the protein, have demonstrated remarkable activity against human immunodeficiency virus type 1 (HIV), or human cytomegalovirus (HCMV) (Florís et al., Curr. Pharm. Design é, 2003, 1257-1275; Pan et al., 2006. Int. Dairy J. 16, 1252-1261). Other milk proteins have demonstrated antiviral activity only after their chemical modification to give them a markedly negative or positive charge. Thus, for example, chemically modified milk and plasma proteins with succinic anhydride or acetylated to increase the negative charge, have potent activity against the HIV virus (Groenínk et al., 1997. AIDS Res Hum Retroviruses, 13, 179-85; Swart et al., 1999. J. Pept. Sci. 5, 563-576). On the contrary, milk proteins with modifications aimed at increasing their positive charge have demonstrated activity against human cytomegalovirus (Swart et al., 1999. J. Pept. Sci. 5, 563-576; Berkhout er a /., 2004. Biometals 17, 291-294; Groot et al., 2005. J. Virol. 79, 3009-3015). Lactoferrin has antiviral activity against Herpes simplex type 1 and 2 (Hasegawa et al., 1994. Jpn. J. Med. Sci. Biol. 47, 73-85), adenovirus (Arnold et al., 2002. Antiviral Res 53 , 153-158) HIV virus (Harmesen et al. J. Infect.Dis 172, 1995, 380-388; Puddu et al., 1998. Int. J. Biochem. Cell B. 30, 1055-1063; Van der Strate et al., 2000. Viral Inmunol 12, 197-203) hepatitis C virus (Tanaka et al., 1999. Jpn J Cancer Research 90, 367-203). Most studies suggest that lactoferrin inhibits the entry of the virus into the host cell rather than acting on its replication Malazzeweska et al., 2006. Medycyne Weterynaryjna 62, 1 104-1 107).

However, there are fewer studies on the antiviral activity of peptides, that is, fragments derived from milk proteins. Most studies have been carried out on lactoferricin, a peptide derived from lactoferrin. This peptide has been shown to be active against cytomegalovirus, adenovirus, feline calcivirus and herpes simplex infections, although the antiviral potency was lower than that of the precursor protein (Andersen et al., 2001. Antivíral Res. 51, 141- 149; Di Biase et al., 2003. J. Med. Virol. 69:, 495-502; McCann et al., 2003. J. Appl. Microbiol. 95, 1026-1033). Other peptides derived from lactoferrin that correspond to the f (86-258) and f (324-329) fragments are active against rotavirus (Superti et al., 2001. Biochim. Biophys. Acta-Gen. Subj. 1528, 107- 1 15), although these fragments do not contain positive charges in their sequence. Likewise, the antiviral activity of positively charged peptides derived from κ-casein such as the f (1-10), f (98-1 1 1), f (98-1 12) and other negatively charged peptides such as the Glycomacropeptide f (106-169). These fragments have no activity against HIV (Berkhout et al., 1997. AIDS Res Hum Retroviruses 1, 1 101-1 107). Glycomacropeptide f (106-169) inhibits hemagglutination of influenza virus (Kawasaki et al., 1993. Bios Biotech & Biochem, 57, 1214-1215). So far, most studies with proteins and food peptides have been done with human viruses, so there is no data on the activity of these proteins or their chemical modifications against viruses involved in fish diseases. Viruses affecting teleost fish offer great interest as aquaculture has intensified and diversified throughout the world, and movements of live animals or their products have accelerated the accidental spread of diseases, which cause serious economic losses in this sector. . These viruses constitute an innovative model for the determination of the biological activity of peptides and food proteins, which may have antiviral or immunostimulatory characteristics and that could constitute a resource as a drug or as an adjuvant for DNA vaccines. Until now the approach of the use of possible antivirals in fish of consumption has not been developed because they are toxic molecules, or little active or too expensive.

Viruses that affect teleost fish such as Aquabirnavirus, infectious pancreatic necrosis virus (IPNV) and Novirhabbdovirus, infectious hematopoietic necrosis virus (IHNV) cause systemic infections and cause high mortality in trout and other salmonids being responsible for serious losses economic in the aquaculture sector worldwide. IHNV can cause losses greater than 90% in affected farms and is endemic in the North-East Pacific of North America extending to other areas and countries (La Patra et al., 1996. Ann. Rev. Fish Dis. 6, 15- 28 .; Enzmann et al., 1992. Bull. Eur. Ass. Fish. Pathol. 12, 185 .; Bootland and Leong, Fish Diseases and Disorders. Vol. 3, Viral, Bacterial and Fungal infections. 1999 ,. 57- 121. The IPNV was isolated for the first time in North America in 1960 and has spread to Europe and Japan.This virus has been isolated from other species of non-salmonid fish as well as crustaceans and mollusks and is the most prevalent in Spanish facilities (Wolf, Fish Viruses and Fish Diseases., 1988, 1 15-157, Rodríguez et al., 1997, J Aquatic Animal Health 9: 295-300. Rodríguez ef al., 2003. Adv Virus Res 62, 1 13- 165. Regarding the use of milk casein hydrolysates there is a document (WO / 2006/131586 "Bioactive peptides identified in enzymatic hydrolyzates of milk caseins and method oí obtaining same") where the use of certain peptides is claimed for their antimícrobial activity , angiotensin converting enzyme (ACEI) inhibitory activity, antihypertensive activity and for its activity as antioxidants. However, its antiviral activity is not described. Nor has any document been found on the possible use of casein hydrolysates or any of its peptides against infectious hematopoietic necrosis virus, IHNV and against infectious pancreatic necrosis virus, IPNV. Likewise, the antimícrobial activity against bacteria of some of the peptides described in the present invention has also been included in another document (WO 00/15655) but its possible activity as antivirals has not been considered.

The hydrolysates of caseins and peptides derived from the alpha s ₂ -casein (a _s2- casein) could serve, in addition to being part of food products or pharmaceuticals, for the treatment and prevention of some diseases; particularly they could act as fish antivirals and be used in aquaculture. The invention extends the applications of milk proteins contributing to their use and revaluation.

DESCRIPTION OF THE INVENTION

The present invention provides a series of casein hydrolysates, which contain a series of peptides with antiviral activity, and in particular, with activity against infectious hematopoietic necrosis virus, IHNV and against infectious pancreatic necrosis virus, IPNV

Therefore, a first aspect of the invention relates to the use of a casein hydrolyzate in the preparation of a medicament for the treatment of a viral disease, or alternatively, a casein hydrolyzate for use in the treatment of a viral disease. . In a preferred embodiment, the casein hydrolyzate comprises at least one of the peptides that are selected from the list comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEO ID NO: 4, SEO ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or any of its combinations

In another preferred embodiment of this aspect of the invention, the casein hydrolyzate comprises at least one of the peptides that are selected from the list comprising: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO: 10, SEQ ID NO: 1 1, or any combination thereof.

In another preferred embodiment of this aspect of the invention, the casein hydrolyzate comprises at least the sequence peptide SEQ ID NO: 6

In another preferred embodiment of this aspect of the invention, the viral disease is suffered by a fish, more preferably belonging to the order Salmoniformes, and even more preferably to the genus Salmo or the genus Oncorhynchus.

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the Birnaviridae family. In another more preferred embodiment of this aspect of the invention, the Birnaviridae family virus belongs to the genus Aquabirnavirus. In another even more preferred embodiment, the virus of the genus Aquabirnavirus is infectious pancreatic necrosis virus (infectious pancreatic necrosis virus or IPNV).

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the family Rhabdoviridae. In another more preferred embodiment of this aspect of the invention, the virus of the family Rhabdoviridae belongs to the genus Novirhabdovirus. In another even more preferred embodiment, the virus of the genus Novirhabdovirus is the infectious hematopoietic necrosis virus {infectious hematopoietic necrosis virus or IHNV).

In this report, "viral disease" means a disease that is characterized by the clinical manifestation resulting from an infection caused by a virus, and whose invasive and, if appropriate, harmful action has disrupted the biology of the affected host. Methods for the detection of the type of virus that acts as a pathogen are known in the state of the art, such as, but not limited to, through all molecular identification techniques (genetic probes, in situ hybridization, on colonies (colony blotting ) and on membranes (Southern blotting), DNA microarrays and microchips, techniques based on PCR (polymerase chain reaction) such as random PCR, nested PCR and RT-PCR, ligase chain reaction (CSF) ), Q-replicase reaction, cyclic probe technique (CPT), transcription-based amplification: TAS and self-sustained sequence replication (3SR), study of nucleic acid polymorphism, using, for example, restriction analysis ( RFLP and AFLP)., Mitochondrial DNA analysis, pulsed field electrophoresis (OFAGE, CHEF, etc.), ribotypia, DNA sequencing, protein analysis of microbial origin (electrophoretic analysis, two-dimensional ectrophoresis, ...), protein immunodetection {Western blotting. Cellular immunolocation: direct and indirect immunofluorescence. Fluorescence and confocal microscopy), and flow cytometry. They are also detected through isolation in cell cultures and identification by seroneutraization.

Birnaviridae is a family of viruses that affects animals. It includes the following genres:

Genus Aquabirnavirus; type species: Infectious pancreatic necrosis virus.

Genus Avibirnavirus; type species: Infectious bursal disease virus.

Genus Entomobirnavirus] type species: Drosophila virus X. Rhabdoviridae is a family of infectious viruses for animals and plants. Among infected animals are insects, fish and mammals, including humans. Rhabdoviridae is a family of infectious viruses for animals and plants. Among infected animals are insects, fish and mammals, including humans. The family includes the following genera:

Genus Cytorhabdovirus] type species: Lettuce yellow necrosis virus.

Genus Ephemerovirus; type species: Bovine ephemeral fever virus.

Genus Lyssavirus; type species: Rabies virus.

Genus Novirhabdovirus; type species: Infectious hematopoietic necrosis virus.

Genus Nucleorhabdovirus; type species: Potato yellow dwarf virus.

Genus Vesiculovirus; type species: Vesicular stomatitis virus.

Additionally, a large number of viruses are known that have not yet been assigned to any genus.

Another aspect of the invention relates to the use of a composition, hereinafter composition of the invention, comprising an enzymatic casein hydrolyzate, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to a composition which comprises comprising an enzymatic casein hydrolyzate for use in the treatment of a viral disease. In a preferred embodiment of this aspect of the invention, the composition is a pharmaceutical composition.

In another preferred embodiment, the casein enzyme hydrolyzate of the composition comprises at least one of the peptides that are selected from the list comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or any combination thereof.

In another preferred embodiment of this aspect of the invention, casein hydrolyzate comprises at least one of the peptides that are selected from the list comprising: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID NO: 10, SEQ ID NO 1 1, or any combination thereof.

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the Birnaviridae family. In another more preferred embodiment of this aspect of the invention, the Birnaviridae family virus belongs to the genus Aquabirnavirus. In another even more preferred embodiment, the virus of the genus Aquabirnavirus is the infectious pancreatic necrosis virus (IPNV).

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the family Rhabdoviridae. In another more preferred embodiment of this aspect of the invention, the virus of the family Rhabdoviridae belongs to the genus Novirhabdovirus. In another even more preferred embodiment, the virus of the genus Novirhabdovirus is infectious hematopoietic necrosis virus (infectious hematopoietic necrosis virus or IHNV).

In another preferred embodiment, the composition of the invention further comprises another active ingredient. In another preferred embodiment, the composition of the invention further comprises a pharmaceutically acceptable carrier.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 1, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 1 for use. in the treatment of a viral disease. In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the Birnaviridae family. In another more preferred embodiment of this aspect of the invention, the Birnaviridae family virus belongs to the genus Aquabirnavirus. In another even more preferred embodiment, the virus of the genus Aquabirnavirus is the infectious pancreatic necrosis virus (IPNV).

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 2, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 2 for use. in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 3, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 3 for use. in the treatment of a viral disease.

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the Birnaviridae family. In another more preferred embodiment of this aspect of the invention, the virus of the Birnaviridae family belongs to the genus Aquabirnavirus. In another even more preferred embodiment, the virus of the genus Aquabirnavirus is the infectious pancreatic necrosis virus (IPNV).

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 4, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 4 for use. in the treatment of a viral disease.

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the Birnaviridae family. In another more preferred embodiment of this aspect of the invention, the Birnaviridae family virus belongs to the genus Aquabirnavirus. In another even more preferred embodiment, the virus of the genus Aquabirnavirus is the infectious pancreatic necrosis virus (infectious pancreatic necrosis virus or IPNV).

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 5, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 5 for use. in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 6, in the preparation of a medicament for the treatment of a viral disease, or alternatively, the peptide of amino acid sequence SEQ ID NO: 6 for use in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 7, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 7 for use. in the treatment of a viral disease.

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the family Rhabdoviridae. In another more preferred embodiment of this aspect of the invention, the virus of the family Rhabdovirídae belongs to the genus Novirhabdovirus. In another even more preferred embodiment, the virus of the genus Novirhabdovirus is infectious hematopoietic necrosis virus (infectious hematopoietic necrosis virus or IHNV).

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 8, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 8 for use in the treatment of a viral disease.

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the family Rhabdovirídae. In another more preferred embodiment of this aspect of the invention, the virus of the family Rhabdovirídae belongs to the genus Novirhabdovirus. In another even more preferred embodiment, the virus of the genus Novirhabdovirus is infectious hematopoietic necrosis virus (infectious hematopoietic necrosis virus or IHNV).

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 9, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 9 for use in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 10, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 10 for use. in the treatment of a viral disease.

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the family Rhabdoviridae. In another more preferred embodiment of this aspect of the invention, the virus of the family Rhabdoviridae belongs to the genus Novirhabdovirus. In another even more preferred embodiment, the virus of the genus Novirhabdovirus is infectious hematopoietic necrosis virus (infectious hematopoietic necrosis virus or IHNV). Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 1 1, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 1 for its Use in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 12, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 12 for use in the treatment of a viral disease.

In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the Birnaviridae family. In another more preferred embodiment of this aspect of the invention, the Birnaviridae family virus belongs to the genus Aquabirnavirus. In another even more preferred embodiment, the virus of the genus Aquabirnavirus is the infectious pancreatic necrosis virus (IPNV). In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the family Rhabdoviridae. In another more preferred embodiment of this aspect of the invention, the virus of the family Rhabdoviridae belongs to the genus Novirhabdovirus. In another even more preferred embodiment, the virus of the genus Novirhabdovirus is infectious hematopoietic necrosis virus (infectious hematopoietic necrosis virus or IHNV).

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 13, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 13 for use. in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 14, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 14 for use in the treatment of a viral disease. In another preferred embodiment of this aspect of the invention, the viral disease is caused by a virus of the Birnaviridae family. In another more preferred embodiment of this aspect of the invention, the Birnaviridae family virus belongs to the genus Aquabirnavirus. In another even more preferred embodiment, the virus of the genus Aquabirnavirus is the infectious pancreatic necrosis virus (IPNV).

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 15, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 15 for use. in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 16, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 16 for use. in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 17, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 17 for use. in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 18, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 18 for use. in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 19, in the preparation of a medicament for the treatment of a viral disease, or alternatively, the peptide of AMQ sequence SEQ ID NO: 19 for use in the treatment of a viral disease.

Another aspect of the invention relates to the use of the amino acid sequence peptide SEQ ID NO: 20, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to the amino acid sequence peptide SEQ ID NO: 20 for use. in the treatment of a viral disease.

The peptides of the enzymatic casein hydrolyzate of the invention are peptides, and can act as active ingredients in compositions, preferably in pharmaceutical compositions.

As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different effect (metabolic activity, immunological, ...) in the diagnosis, cure, mitigation, treatment, or prevention of a disease or medical condition, or that affects the structure or function of the body of man or other animals.

Therefore, another aspect of the invention relates to the use of a composition comprising a peptide that is selected from the list comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:

9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or any combination thereof, in the preparation of a medicament for the treatment of a viral disease, or alternatively, to a composition comprising a peptide which is selected from the list comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 , SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:

10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 , SEQ ID NO: 19, and SEQ ID NO: 20, or any combination thereof, for use in the treatment of a viral disease. In a preferred embodiment, the peptide is selected from the list comprising: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10. and SEQ ID NO: 1 1, or any combination thereof.

Therefore, the peptide of the invention can be formulated in a composition, useful for the treatment of a viral disease. The use of peptides as potential therapeutic agents has the advantage of their high specificity and high activity, generally presenting low toxicity and few side effects. In addition, they do not accumulate in the body, since they have a relatively short half-life (Adresi and Soto, 2002. Current Medicinal Chemistry 9: 963-978; Líen & Lowman 2003. TRENDS in Biotechnoiogy 21: 556-562). In a preferred embodiment of this aspect of the invention, the composition is a pharmaceutical composition.

The composition of the present invention can therefore be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art. Preferably it can be formulated for administration to a fish, more preferably belonging to the order Salmoniformes, and even more preferably to the genus Salmo or the genus Oncorhynchus. So, they may be, without limitation, in sterile aqueous solution or in biological fluids, such as serum. Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, and the like, and nutrients including glucose, dextrose, vitamins and minerals. Alternatively, the compositions can be prepared for administration in solid form. The compositions may be combined with various inert vehicles or excipients, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate.

Such compositions and / or their formulations may be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral. , enteral, parenteral, intranasal or dermal. Preferably, the administration is oral.

The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as age, weight, sex, tolerance, ... of the animal, including man. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of peptides, or prodrugs, derivatives or analogs of said peptide that produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said prodrugs, derivatives or analogs and the therapeutic effect to be achieved. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known to those skilled in the art. The term "medicine" refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals, and even more preferably in fish, and even more preferably in organisms of the order Salmoniformes. In the context of the present invention, therefore, the pharmaceutical composition of the invention would also be a medicament.

In this report, "fish" is understood as aquatic vertebrate animals, generally ectothermal, mostly covered with scales and equipped with fins, which allow their movement in the aquatic environment. Taxonomically they belong to three groups: agnatos or fish without jaws, chondrites or cartilaginous and osteictiose fish or bony fish.

In this report the organisms of the order Salmoniformes are cellular organisms, of the Eukaryota Superein, Metazoa kingdom, Phylum Chordata, Gnathostomata superclass, Actinopterygii class. The Salmoniformes order includes the Galaxiidae, Retropinnidae, Osmeridae, and Salmonidae families. Within the Salmonidae family are the genera Coregonus, Prosopium, Stenodus, Brachymystax, Hucho, Oncorhynchus, Parahucho, Salmo, Salvelinus, Salvethymus and Thymallus.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1: Reduction of the infective titer against the IHNV virus with protein hydrolysates obtained after different times of hydrolysis using doses of 100 μg / ml of each hydrolyzate. (A) Casein hydrolyzate (B) β-casein hydrolyzate (C) Ace ₂ -casein hydrolyzate (D) As-casein hydrolyzate. Fig. 2: Viral performance over time after infecting with IHNV the cells in the presence and absence of the total casein and _S 2-casein hydrolysates at a dose of 100 μν.

Fig. 3: Percentage of cells infected with IHNV through the detection of antiviral antigens at 48 hours post-infection by flow cytometry, in the control of non-infected cells (A), in control of infected cells (B) and in the Infected cells treated with the hydrolysates of total casein and _S 2-casein.

Fig. 4: Infectious titer against IHNV incorporating the hydrolysates to the test 30 minutes before infection, at the same time as the infection, or 60 minutes after infection.

Fig. 5: FPLC analysis of a _s2 -CN hydrolyzate

Fig. 6: Percentage of inhibition of the IHNV virus and reduction of the infective titer using different concentrations of the C, D and E fractions obtained by cation exchange (according to nomenclature used in Table 2). (A)% IHNV virus inhibition. (B) IHNV virus infectious titer reduction.

Fig. 7: Percentage of inhibition and reduction of the infective titer against the IHNV virus with different concentrations of the peptide at _s2 -casein f (183-207), chemically synthesized, and the peptide hydrolyzate of a _s2 -casein from which said peptide comes.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the specificity and effectiveness of casein hydrolyzate and the peptides they contain on fish viruses. Materials and methods

Obtaining bovine casein

The isoelectric casein was prepared by precipitation at pH 4.6 of whole milk by adding 2 M HCI to pH, 4.6 followed by centrifugation at 4500 g for 15 min. The casein precipitate was washed three times with acidified water and subsequently lyophilized. The fractions of oc _s -casein, β-casein and -casein came from different commercial houses

Obtaining bovine Os2-CN

The o¾2-CN was prepared following the method of Vreeman and Van Riel, 1990 (Neth Milk Dairy Journal 44,43-48) with some modifications. A solution in milli-Q ^{® water} (Millipore, Billerica, USA) was prepared from the lyophilisate casein at a concentration of 8% (w / v). With the help of NaOH, the pH was increased to 7, DTT {1.2%, w / w) was added and stirred at 5 ^S C to achieve complete casein dissolution. Subsequently, it was heated to 80 ⁹ C to inactivate the plasmin and upon reaching that temperature the heating was interrupted leaving the temperature to lower itself to 60 ^e C, at which time the solution was transferred to an ice bath to reach an 20 ° C temperature. Once this temperature was reached, the pH was adjusted to 6.5 and slowly and coldly added, with continuous stirring, 1-propanol until reaching a concentration of 40% (v / v). The resulting solution was left overnight in a cold room ice bath at 2 ^e C. The next day the solution was centrifuged at 17000 g for 30 minutes at 5 ^e C. The precipitate containing the _s2 -CN oc was washed with 1-40% propanol and centrifuged under the conditions described above. Finally, the precipitate was frozen and lyophilized.

Casein hydrolysis with pepsin

To carry out the hydrolysis, a 0.5% (w / v) aqueous solution of isoelectric casein, oc _s -casein, Os2-casein, β-casein, or κ-casein was prepared, adjusting the pH. at 3 with 1 M HCI. Subsequently, porcine pepsin (EC 3.4.23.1, 570 U / mg) was added to a final concentration of 3.7% (w / w). Hydrolysis was performed at 37 C for 24 hours ^and with stirring, taking aliquots at 0.5, 1, 2, 3, 5, 7 and 24 hours. To terminate the reaction, it was heated at 80 ⁹ C for 20 minutes and the pH at 7 with 1 M NaOH. The hydrolyzate was centrifuged at 16,000 g for 15 minutes, the supernatant being collected for subsequent evaluation of the antiviral activity.

Isolation of bovine os2-CN peptide fractions by ion exchange chromatography (FPLC)

Isolation of peptide fractions was carried out using FPLC equipment, and a HiLoad ™ 26/10 SP Sepharose Fast Flow cation exchange column (Pharmacia, Uppsala, Sweden). Phases A and B consisted of 10 mM NH ₄ HC0 ₃ (adjusted to pH 7.0 with HCOOH) and 1.5 M NH ₃ , respectively. Samples were prepared in phase A at a concentration of 5 img / mL, injecting a volume of 5 mL using a 50 mL Superloop ™ (Pharmacia). A flow of 5 mL / min was used. After 20 minutes with 100% of solvent A, a gradient of 0 to 50% of solvent B was applied in 60 minutes, followed by 20 minutes with 50% of solvent B. The detection was carried out at an absorbance of 214 nm . The temperature of the column and the mobile phases was 9 ^e C. The fractions of several chromatographic analyzes were collected.

RP-HPLC analysis coupled to tandem mass spectrometry online (RP-HPLC-MS / MS)

The different fractions collected from the FPLC were analyzed by RP-HPLC-MS / MS. A Ski-LC equipment (Bruker Daltonik GMBH, Bremen, Germany) was used. The HPLC equipment (series 1 100) consisted of a quaternary pump, an automatic injector, an eluent degassing system and a variable wavelength ultraviolet detector (Agilent Technologies, Waldbronn, Germany) coupled in line to a spectrometer Esquire 3000 ion trap masses (Bruker Daltonik). The column was a Hi-Pore C18 column (250 x 4.6 mm di, 5 μιη particle size) (Bio-Rad Laboratories, Richmond, CA, USA). Solvent A was a mixture of water and trifluoroacetic acid (1000: 0.37) and solvent B a mixture of acetonitrile and trifluoroacetic acid (1000: 0.27). 50 μΙ of prepared sample was injected at a concentration of 4.5 mg / ml. A flow of 0.8 ml / min was used, with a linear gradient from 0% to 50% of solvent B in 60 minutes The eluent was monitored at 214 nm and the sample flow to the mass spectrometer nebulizer was 275 μΙ / min. ESI ionization used N ₂ as a fogging agent (8 L / min), fogging pressure of 60 psi, drying temperature of 350 ^Q C and a capillary voltage of 4 KV. The mass spectra were acquired in the range of 100-2500 m / z and a fragmentation ramp between 0.3 and 2 V was used. As a data acquisition system the Esquire ControlTM 5.0 program was used and to identify the sequence of the different peptides were used Data AnalysisTM 3.0, Sequence EditorTM 2.1 and BiotoolsTM 2.1 software all from Bruker Daltonik (Bruker DaltoniK, Germany).

Biological activity:

Cells and Virus Culture.

The BF-2 cell line was chosen because of its susceptibility to various viruses. Rodríguez Saint-Jean & Pérez Prieto, 2006 [Veterinary Immunology and Immunopathology 110: 1-10). This cell line comes from sunfish fry, Lepomis macrochirus and was purchased from the American Type Culture Collection (ATCC CRL 1681). The cells were grown in Leiboviz growth medium (L15, Gibco, Invitrogen, Barcelona) at 25 ^Q C, supplemented with 100 units / mL of penicillin, 100 µg / mL of streptomycin, 2 mM of L-glutamine and 10% serum fetal bovine (FBS, Gibco). For the maintenance of the cell monolayer, the same medium was used but decreasing the percentage of fetal serum to 2%.

For the culture and propagation of the viruses to be tested, the cells 24 hours after planting and with a subconfluent monolayer, were infected with 1000 TCID ₅₀ / ml virus and incubated at 15 or 20 ^e C (depending on the virus) until the cytopathic effects (CPE) produced by the virus affected the entire cell monolayer. At that time the supernatants were collected, centrifuged at low speed (900 g) for 5 minutes to remove the cell debris and aliquots of the virus suspension were distributed in eppendorff tubes that were stored at -80 ^S C until use. The infective title of the pass Virus was determined by calculating the 50% infective dose (TCID ₅₀ / mL) detailed below.

The viruses used come from the ATCC and were the infectious pancreatic necrosis virus (IPNV, ATCC VR1318) and the infectious hematopoietic necrosis virus (IHNV, ATCC VR714). The amount of virus used in the infection should be sufficient so that at 3 days post-infection (IPNV) and 7 days (IHNV) total lysis is observed in the cells not treated with compound (virus control).

Cytoxicity of the compounds:

To determine the toxicity of the hydrolysates and their fractions in cell cultures, a cell viability test of the treated and untreated cultures was used, according to the method of Renault et al., 1991 (Diseases of Aquatic Organisms 10, 23-29 titration Colorimetric: To confluent monolayers of BF-2 cells growing in disposable 96-well plastic plates, the growth medium was removed and replaced by different concentrations of the hydrolyzed or peptides to be tested, diluted in maintenance medium. cells was added the same volume of maintenance medium without compound. the plates were incubated at 15 ^Q C for 3-7 days and after removal of the medium, the monolayers were stained with a crystal violet solution 1% (w / v) in 20% ethanol for 10 min After washing and drying in air the absorbance was measured in a microplate reader (Bio-Rad ®) at 590 nm. The results are presented as percentages of c surviving cells, where 100% represents the absorbance of the control cells. The cytotoxic concentration of the compound that causes 50% inhibition of cell growth is called CT _50.

Inhibition of cytopathic effects of viruses and determination of inhibitory dose 50.

The antiviral activity is determined by the ability of the compounds to inhibit the cytopathic effects (lysis) of the virus and protect the cell monolayer and is calculated by the percentage of viable cells after infection. Briefly, BF-2 cells (approximately 10 ⁵ ml ^"1 ) cultured in 48-well plates were infected with 1000 TCID ₅₀ / ml virus and different concentrations of the hydrolyzate or its fractions or peptides diluted in maintenance medium. The cultures were incubated at the optimum temperatures for each virus 20 ° C for IPNV and 15 ° C for IHNV. They were observed daily under a microscope and when the cytopathic effects (CPE) on infected and untreated cells were total {4 + or 100% cell monolayer involvement) the monolayers were stained with a 1% violet crystal solution ( w / v) in 20% ethanol for 10 min. Total CPEs are usually 3 days for IPNV and 7 days for IHNV. The results are presented as percentages of surviving cells, 100% represents the absorbance of uninfected and treated control cells in the same way as infected cells (control cells) and 0 are virus-infected and untreated cells with extracts ( Virus control).

Reduction of the infective titer of the viruses treated with the compounds.

The infective titer of viruses that have multiplied in the presence of different doses of hydrolysates or their derivatives is determined. Briefly, BF-2 cells (approximately 10 ⁵ mL ^"1 ) cultured in 48-well plates were infected with 1000 TCID ₅₀ / ml virus in the presence of different concentrations of the compounds, diluted in maintenance medium. As controls cells were maintained untreated but infected (control virus) and untreated and uninfected cells (control cells) The cultures were incubated at the optimum temperatures for each virus.

After the appearance of cytopathic effects in the virus controls, all cell cultures were collected with their supernatants, both treated and virus controls and sonicated to release the intracellular virus, centrifuged at very low speed for 5 minutes and they were titrated in 96-well plates of BF2 cells. In this way, the yields of infective virus in the treated and untreated cells are determined. The estimate of the number of infectious viruses or units per unit of volume is known as the titration. The compound treated virus and the untreated virus are titrated by the ₅₀ / ml TCID method (50% infective dose in cell cultures). The monolayers are inoculated with serial dilutions (decimals) of the viral suspension {without compound and with compound). After an incubation period of 3 days for IPNV and 7 for IHNV, the monolayers were stained and the infective titer was calculated. The infective titer is calculated by diluting the end point, which is the highest (decimal) dilution in which the virus infects 50% of the inoculated wells. The titer is expressed as the reciprocal of this dilution, in infectious units (TCID ₅₀ ) {Tissue culture infective dose 50%) per ml_.

Example 1: Antiviral activity against salmonid viruses (IPNV and IHNV) of total casein hydrolysates and hydrolysates of different casein fractions.

Table 1 presents the results obtained on the evaluation of the antiviral activity against salmonid viruses (IPNV and IHNV) of total casein hydrolysates and hydrolysates of different casein fractions with hydrolysis times between 0.5 to 24 hours. These results show that hydrolysis with pepsin of the casein fraction generates peptides with activity against the IHNV virus. The highest therapeutic index with a value of 30 was reached in the total casein hydrolyzate obtained after 3 hours of hydrolysis, followed by the total casein hydrolyzate with a shorter hydrolysis time (therapeutic index of 6) (products 1 to 5 , from Table 1). The hydrolyzate obtained from the purified Os ₂ -casein reached a therapeutic index of 30 after 3 hours of hydrolysis, but not at higher or lower hydrolysis times. None of the hydrolysates obtained from other casein fractions at _s -, - or β-casein reached this therapeutic index, the range of values being 2.8 to 6. These results may indicate that the activity may be mainly due to peptides derived from the a _s2- casein although it cannot be excluded that other peptides may contribute to activity. Table 1: Cytotoxicity and antiviral activity of hydrolysates of

bovine against viruses that affect salmonids.

Compound ³ Time CT ₅₀ T Virus Tested MIC ₅ or hidr (h) g / ml) IPNV (TI) IHNV (TI)

Total Casein 0 2800 NA 500 (6)

Total casein hydrolyzed 0.5 3000 NA 500 (6)

Total Casein Hydrolyzate 1 3000 NA 500 (6)

Total casein hydrolyzate 2 3000 NA 500 (6)

Total casein hydrolyzate 3 3000 1000 (3) 100 (30)

Total Casein Hydrolyzate 5 3000 NA NA

Total Casein Hydrolyzate 7 3000 NA NA

Total casein hydrolyzate 24 3000 NA NA a _s2 -casein 0 3000 NA 1000 (3) 0 A _S 2-casein hydrolyzate 0.5 3000 NA 1000 (3) 1 a _s2 -casein hydrolyzate 1 3000 NA 1000 (3)

Hydrolyzate of a _s2 -casein 2 3000 NA 1000 (3) 3 Hydrolyzate of a _s2 -casein 3 3000 1000 (3) 100 (30)

Hydrolyzate of a _s2 -casein 5 3000 NA NA5 Hydrolyzate of a _s2 -casein 7 3200 NA NA6 Hydrolyzate of a _s2 -casein 24 3200 NA NA7 β-casein 0 3000 1000 (3) 1000 (3) 8 β-Casein hydrolyzate 0 , 5 3000 1000 (3) 1000 (3) 9 Hydrolyzate of β-casein 1 3000 1000 (3) 1000 (3) 0 Hydrolyzate of β-casein 2 2800 1000 (2.8) 1000 (2.8) 1 Hydrolyzate of β-casein 3 2800 1000 (2.8) 1000 (2.8) 2 Hydrolyzate of β-casein 5 2800 NA NA3 Hydrolyzate of β-casein 7 3000 NA NA4 Hydrolyzate of β-casein 24 3000 NA NA5 κ-casein 0> NA 1000 NA6 κ-casein hydrolyzate 3> 1000 NA NA7 to _s -casein 0 NA 3000 500 (6) 8-casein hydrolyzate 1 Ô¾ NA 3000 500 (6) 9 _s -casein hydrolysed ot 2 NA 3000 500 (6 ) 0 Cvcasein hydrolyzate 3 3000 NA 1000 (3) 1 Hydrolyzate of oc _s- casein 5 3000 NA NA

All experiments were performed in triplicate in BF-2 cells

CT _{5 ()} is the dose or concentration of the compound that has 50% toxicity in cell cultures.

MIC ₅₀ is the concentration of the compound that inhibits 50% the cytopathic effects of the virus

TI is the therapeutic index = CT ₅₀ / MIC ₅₀ .

Fig. 1 shows the reduction of the infectious titer of the IHNV virus determined with a dose of 100 g / m \ of the hydrolysates. The best results were obtained with the total casein hydrolysates and with the hydrolysates obtained from the fraction of a _S 2-casein. A reduction of 1.5-2.5 log of the infective titer is observed for casein hydrolysates obtained during the first three hours of hydrolysis. For hydrolysates obtained from oc _S 2-casein activity was observed up to 7 hours of hydrolysis, the most active hydrolyzate being obtained after 3 hours (2.5 log).

Using the two most active hydrolysates, total casein 3h and _s2- casein 3h, multicyclic experiments were performed where the infective titer was determined after infecting the cells in the presence and absence of the hydrolysates at different post-infection times: 1, 2, 3, 5 and 7 days The results are shown in Fig. 2. With both hydrolysates the infective titer is significantly reduced with respect to infected control cells at all times up to 7 days after infection. It should be noted that with the Os2-casein hydrolyzate the virus is not detected in the first 48 hours after infection. These results were also confirmed by the detection of viral antigens at 48 hours by flow cytometry. Figure 3 shows the percentage of infected cells in the control of infected cells, in the control of non-infected cells and in infected cells treated with hydrolysates. At 48 hours, the percentage of infected cells in the samples treated with o ^ -casein hydrolyzate was similar to that found in uninfected cells.

In order to assess whether hydrolysates were active in the early stages of the virus cycle or during viral replication, they were performed addition time trials, in which the hydrolyzate was incorporated into the test 30 minutes before infection, at the same time as the infection, or 60 minutes after infection. It was observed that the greatest reduction in the infectious titer against IHNV was obtained when the hydrolisate was incorporated at the same time that the infection occurs and that, therefore, the hydrolyzed ones must act during the adsorption of the viruses to the cells (Figure 4).

Example 2: Antiviral activity against IHNV salmonid virus of different fractions of the cc _S 2-casein hydrolyzate.

In order to identify the peptides responsible for antiviral activity in the peptide hydrolyzate of o ^ -casein, it was fractionated by cation exchange chromatography (Figure 5) and the fragments contained in each fraction were identified by HPLC coupled online to spectrometry of tandem masses. The results of the identification are shown in Table 2. The antiviral activity against the IHNV virus was also evaluated (Figure 6). Fractions A and B have no activity against the IHNV virus, while fractions C, D, and E, exhibited antiviral activity to varying degrees, with fractions C and D being the most active. Specifically, it is fraction C that achieved a greater reduction of the infective titer (2 logarithmic units) at a lower concentration (750 μς / ητιΙ).

The majority peptide in this fraction is fragment 183-207 of the oc _s2 -casein, a peptide with a marked cationic character and that had been previously described for its antibacterial activity against Gram-positive and Gram-negative bacteria (Recio and Visser, Biochim Biophys Acta 1428, 1999, 314-326; WO 00/15655). However, the antiviral activity of this fragment had not been described. In addition, the activity of other fragments cannot be ruled out since fraction D also exhibits remarkable antiviral activity and does not contain this fragment.

The activity of the peptide against the IHNV virus was evaluated both by determining the percentage of inhibition and reducing the infective titer. These activity values for the pure peptide obtained by chemical synthesis are shown in the graphs of Figure 7, in Comparison with the activity values obtained for the hydrolyzate of a _S 2-casein from which the peptide is derived. It is observed that peptide 183-207 is active against the IHNV virus and the determination of the infective titer can be concluded that it is one of the peptides responsible for the hydrolyzate activity although the additive or synergistic activity of other peptides present cannot be ruled out. in the hydrolyzate

Table 2. Identification of the different peptides present in the fractions obtained by cation exchange chromatography. Identification made by HPLC-MS / MS. Bold shows the majority peptide in each fraction and in brackets the charge of the fragmented ion.

Ion SEQ ID

Mass

Selected Sequence Amino Acids NO: observed calculated

for MS / MS action

1058.4 1059.0 1059.4 43-51 VVRNANEEE 1 A share

972.5 973.0 973.5 89-95 YQKFPQY

B 2

2125.8 2126.4 1063.9 147-163 FTKKTKLTEEEKNRLNF 3 action

1124.6 1125.3 1125.6 191-199 KPWIQPKTK 4

3001.5 3002.5 1001.5 (3} 183-206 VYQHQKAMKPWIQPKTKVIPYVRY 5

3115.5 3115.7 1039.5 (3) 183-207 VYQHQKA KPWIQPKTKVIPYVRYL 6 action

864.6 865.0 865.6 147-153 FTKKTKL 7

2011 2011.3 1006.5 (2} 164-179 LKKISQRYQKFALPQY 8

2353.2 2353.8 1177.6 (2) 164-182 LKKISQRYQKFALPQYLKT 9

2125 2125.5 1063.5 (2) 164-180 LKKISQRYQKFALPQYL 10

2272.2 2272.6 1137.1 (2) 162-179 NFLKKISQRYQKFALPQY 1 1 action

921.5 922.0 922.5 167-173 ISQRYQK 12 E

793.2 793.8 794.3 167-172 ISQRYQ 13

1290.7 1291.5 1291.7 164-173 LKKISQRYQK 14

864.6 864.9 865.6 147-153 FTKKTKL 15

1034.6 1035.2 1035.6 164-171 LKKISQRY 16

1437.8 1438.7 1438.8 164-174 LKKISQRYQKF 17

1068.5 1069.2 1069.5 167-174 ISQRYQKF 18

1621.8 1622.9 811.9 (2) 164-176 LKKISQRYQKFAL 19

2353.2 2353.8 1177.6 (2) 164-182 LKKISQRYQKFALPQYLKT 20

Claims

one . Use of a casein hydrolyzate in the preparation of a medicine for the treatment of a viral disease.

2. Use of a casein hydrolyzate according to the preceding claim, wherein the casein hydrolyzate comprises one of the peptides selected from the list comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO : 20, or any of its combinations.

3. Use of a casein hydrolyzate according to any of claims 1-2, wherein the casein hydrolyzate comprises one of the peptides selected from the list comprising: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 SEQ ID NO: 1 1, or any combination thereof.

4. Use of a casein hydrolyzate according to any of claims 1-3, wherein the casein hydrolyzate comprises at least the sequence peptide SEQ ID NO: 6.

5. Use of a casein hydrolyzate according to any of claims 1-4, wherein the viral disease is suffered by a fish.

6. Use of a casein hydrolyzate according to claim 5, wherein the fish belongs to the order Salmoniformes.

7. Use of a casein hydrolyzate according to claim 6, wherein the fish of the order Salmoniformes belongs to the genus Salmo or the genus Oncorhynchus.

8. Use of a casein hydrolyzate according to any one of claims 1-7, wherein the virus belongs to the Birnavirídae family.

9. Use of a casein hydrolyzate according to claim 8, wherein the Birnavirídae family virus belongs to the genus Aquabirnavirus.

10. Use of a casein hydrolyzate according to claim 9, wherein the virus of the genus Aquabirnavirus is the infectious pancreatic necrosis virus.

eleven . Use of a casein hydrolyzate according to any one of claims 1-7, wherein the virus belongs to the family Rhabdoviridae.

12. Use of a casein hydrolyzate according to claim 1, wherein the virus of the family Rhabdoviridae belongs to the genus Novirhabdovirus.

13. Use of a casein hydrolyzate according to claim 12, wherein the virus of the genus Novirhabdovirus is the infectious hematopoietic necrosis virus.

14. Use of a composition comprising an enzymatic casein hydrolyzate according to any of claims 1-7, in the preparation of a medicament for the treatment of a viral disease.

15. Use of a composition comprising an enzymatic casein hydrolyzate according to the preceding claim, wherein the composition is a pharmaceutical composition.

16. Use of a composition comprising an enzymatic casein hydrolyzate according to any of claims 14-15, wherein the composition further comprises a pharmaceutically acceptable carrier.

17. Use of a composition comprising an enzymatic casein hydrolyzate according to any of claims 14-16, wherein the composition further comprises another active ingredient.

18. Use of a composition comprising an enzymatic casein hydrolyzate according to any of claims 14-17, wherein the viral disease is suffered by a fish.

19. Use of a composition comprising an enzymatic casein hydrolyzate according to claim 18, wherein the fish belongs to the order Salmoniformes.

20. Use of a composition comprising an enzymatic casein hydrolyzate according to claim 19, wherein the fish of the order Salmoniformes belongs to the genus Salmo or to the genus Oncorhynchus.

twenty-one . Use of a composition comprising an enzymatic casein hydrolyzate according to any of claims 14-20, wherein the virus belongs to the family Birnaviridae.

22. Use of a composition comprising an enzymatic casein hydrolyzate according to claim 21, wherein the virus of the Birnaviridae family belongs to the genus Aquabirnavirus.

23. Use of a composition comprising an enzymatic casein hydrolyzate according to claim 22, wherein the virus of the genus Aquabirnavirus is the infectious pancreatic necrosis virus.

24. Use of a composition comprising an enzymatic casein hydrolyzate according to any of claims 14-20, wherein the RNA virus belongs to the family Rhabdoviridae.

25. Use of a composition comprising an enzymatic casein hydrolyzate according to claim 24, wherein the virus of the family Rhabdoviridae belongs to the genus Novirhabdovirus.

26. Use of a casein hydrolyzate according to claim 25, wherein the virus of the genus Novirhabdovirus is the infectious hematopoietic necrosis virus.