NO20141117A1 - Use of Specific Genes to Determine Phenotypes Susceptible to, or Resistant to, Infectious Salmon Anemia in Atlantic Salmon - Google Patents

Description

[0001] The invention relates to the use of Grb2 (for its acronym in English, "Growth factor receptor-bound protein 2"), immunoglobulin M (IgM), MAVS (for its acronym in English, Mitochondrial signaling antiviral protein) gene or mitochondrial antiviral signaling protein and Regul to determine susceptible and resistant phenotypes for infectious salmon anemia (ILA) in Atlantic salmon (Salmo salar).

Known technique

[0002] The Atlantic salmon (Salmo salar) is a highly cultivated and highly commercial species in aquaculture worldwide. However, this species is susceptible to various pathogens, resulting in losses for the salmon industry. One of the most important pathogens is the virus ILA, which can reach an accumulated mortality of between 15% and 100%. It has nevertheless been observed in the field that there are families of fish that have a reduced mortality rate against the virus (resistant families), which may prove to be a key in the development of effective treatments.

[0003] As shown by scientific evidence, today there is a clear consensus that conventional fisheries are in crisis, since amounts of catch have decreased continuously during the last 5 years ( The State of world fisheries and aquaculture, FAO ( Food and Agricultural Organization), 2012; http:// www.fao.org/ docrep/ 016/ i2727e/ i2727e. pdfj The result of vital importance since population growth has exceeded 1.7% annually over the past five decades, requiring production of larger quantities of food It should be noted that in 2009 fish accounted for 16.6% of the intake of animal protein among the world's population and some regions are almost entirely dependent on fisheries.

[0004] With this intermediate scenario, aquaculture, which involves the cultivation of various freshwater or saltwater species under controlled conditions, emerges as an alternative to the growing needs that conventional fisheries cannot meet. This industry has also presented a growth of about 7% annually (The State of world fisheries and aquaculture, FAO, 2012; http:// www. fao. org/ docrep/ 016/ i2727e/ i2727e. pdf)

[0005] Among the branches of aquaculture, salmon farming is of great economic importance for Chile and reports an annual growth rate of 20.5% between 1990 and 2008 (Informe para el Consejo Nacional de Innovacion para la Competitividad, http://biblioteca.cnic. cl/ media/ user$/ 3/ 181868/ file$/ 18813/ G_ Parada_ ACUIJinal. pdf) and places salmon in 2008 as the fourth national export product and the aquaculture industry as the third largest exporter after mining and forestry. Salmon production has grown exponentially since 1990, where it was 24 thousand tons worth 116 million USD during 2008, the production reached 445 thousand tons involving an income of 2,392 million USD for our country. The consolidation of the national salmon industry over the past two decades has led Chile to position itself as a leading salmon producer worldwide, with 31% of production, along with Norway (41%), Great Britain (10%) and Canada ( 7 %). Salmon fish with higher production in our country are Atlantic salmon (Salmo salar) with 46% of all produced salmon, Coho salmon (Oncorhynchus kisutch) with 26% and rainbow trout (Oncorhynchus mykiss) with 25% (Informe Sernapesca 2010, SalmonChile, ProChile).

[0006] The intensive nature of farming has favored the introduction of a number of diseases which have caused mortality, problems in production and economic impact on the industry. These pathogens in farmed systems are normally present in wild fish stocks; In natural environments, however, mortality is rare due to the absence of stressful conditions, such as handling, transport, high population density and changes in water quality, which are present in farming facilities ( Toranzo A. e., Magarihos B. y Romalde J. l. 2005 .A Review Of The Main Bacterial Fish Diseases In Mariculture System. Aquaculture 246:37- 61). Within the pathogens causing significant impact on this industry are bacterial: Salmon Rickettsial Septicemia (SRS) and Bacterial Kidney Disease (BKD), and viral: Infectious Pancreatic Necrosis (IPN) and Infectious Salmon Anemia ( ILA)). In recent years and especially in 2007, the national salmon industry has been seriously affected by the ILA virus, a highly contagious disease with horizontal transmission that causes a reduction in production and export levels (17% in the period 2008-2009), excretion of more than 20 thousand of the 50 thousand workers in the sector and a corresponding loss in income to USD 883 million from exports ( Carreho A. ( 2010 Impacto del virus ISA en Chile. Publicaciones Fundacion Terram 2010).

[0007] Infectious salmon anemia (ILA) is a multisystem disease with high mortality caused only by viruses of the genus Isavirus, family Orthomyxoviridae, to which the influenza virus also belongs (Dannevig BH, Falk K, Namork E. Isolation of the causa! virus of infectious salmon anemia ( ISA) in a long-term cell line from Atlantic salmon head kidney. in Gen Virol. 1995 Jun;76 ( Pt 6) :1353- 1359; Kawaoka, Y., Cox, N. J., Kaverin, N., Klenk, H. - D., Lamb, R. A., McCauley, i., Nakamura, K., Palese, P., Webster, R. G., 2005. The family Orthomyxoviridae. Disseminated around 2005, based on surface glyco protein gene sequences. Virol. J. 6, 88. 3245- 3259). The first reported outbreak of this virus dates back to 1984 on the southwest coast of Norway (Thorud K, Djupvik HO (1988) Infectious anemia in Atlantic salmon (Salmo salar L). Bull EurAssoc Fish Pathol 8:109-11). Subsequently, the disease has been reported in Canada in 1996 ( Mullins JE, Groman D, Wadowska D ( 1998) Infectious salmon anemia in salt water Atlantic salmon ( Salmo salar L.) in New Brunswick, Canada. Bull Eur Assoc Fish Pathol 18( 4) : 110- 114), in Scotland during 1998 (Rodger HD., Turnbull T, Muir F., Millar S., Richards RH. ( 1998). Infectious salmon anemia (ILA) in the United Kingdom. Bull Eur Assoc Fish Pathol 18( 4) :115- 116), in the Faroe Islands in 1999 ( Lyngøy C ( 2003) Infectious salmon anemia in Norway and the Faeroe Islands: An industrial approach. In: Miller O, Cipriniano RC ( ed.), International response to infectious salmon anemia: Prevention, control and eradication, pp 97-10), in Chile in 1999 (Kibenge F. S. B., Gårate O. N., Johnson G. Arriagada R., Kibenge MJ. T. & Wadowska D. ( 2001). Isolation and identification of infectious salmon anemia virus (ILAV) from Cohol salmon in Chile. Dis. Aquat. Org., 45, 9-18) and in Maine, USA, in the year 2000 (Bouchard D. A., Brockway K. , Giray C, Keleher W. & Merrill P. L. ( 2001 ). First report of infectious salmon anemia (ILA) in the United States. Bull. Eur. Assoc. Fish Pathol., 21, 86 - 88).

[0008] This virus has a genome consisting of eight single-stranded RNA segments with negative polarity and hemagglutination activity, receptor destructive and with adsorption capacity (Dannevig B.H., Falk K. & Namork E. (1995) ; Isolation of the casual virus of infectious salmonanemia ( ISA) in a long-term cell line from Atlantic salmon head kidney. K. Gen. Virol., 76, 1353- 1359; Eliassen T. M., Froystad M. K., Dannevig B. H., Jankowska M., Brech A., Falk K, Aspehaugv. , Vlasak R. & Endresen C. (2004) Identification and characterization of vira! structural proteins of infectious salmon anemia virus. J. Virol., 78, 3063- 3071; fakk K, Dale OB. ( 2007) : lack of receptor- destroying enzymatic activity of ISA- virus is an important virulence factor for development of ISA in Atlantic salmon. At the conference Frisk Fisk, Tromsø: J23- 25 January 1007; Norway; Mjaaland, S., E. Rimstad, K. Falck and B. H. Dannevig (1997). Genome characterization of the virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L): an orthomyxo-like virus in a teleost. J. Virol. 71:7681-7686). The nucleotide sequence of the 8 segments has been described ( Cloutier, SC, Rector, T, Brown, NE, Anderson, DA, 2002, Genomic organization of infectious salmon anemia virus. J. Gen. Virol. 83 ( Pt. 2), 421 - 428) and we have identified four basic structural proteins, including a nucleoprotein (NP) of 68 kDa, a matrix protein (MP) of 22 kDa, a hemagglutinin esterase (HE) with receptor-destroying activity of 42 kDa and a surface glycoprotein of 50 kDa with potential adsorption capacity (F), which is encoded in the genome segments; respectively 3, 8, 6 and 5 (Falk K., Aspehaugv., VlasakR. & Endresen C. (2004) Identification and characterization of vira! structural proteins of infectious salmon anemia virus. J. Virol., 78, 3063- 3071) . A total of 10 polypeptides exist, the remaining 6 present orthology to genes of influenza ( Cottet, L, Cortez-San Martin, M., Tello, M., Olivares, E., Rivas-Aravena, A., Vallejos, E. , Sandino, AM, Spencer, E., ( 2010). Bioinformatic analysis of the genome of infectious salmon anemia viruses Associated with Outbreaks of high mortality in Chile. J. Virol 84 ( 22) : 11916- 11928). A schematic representation of the ILA virus can be observed in the schematic presentation of the ILA virus, given (Bioinformatic analysis of the genome of infectious salmon anemia viruses Associated with Outbreaks of high mortality in Chile. J. Virol 84 ( 22) : 11916- 1192 ).

[0009] Sequence analysis of the genome segments revealed differences between isolated and within defined geographic areas (Blake, S., Bouchard, D., Keleher, W., Opitz, M., Nicholson, B. L, (1999). Genomic relationships of the North American isolate of infectious salmon anemia virus (ISAV) to the Norwegian strain oflSAV. Dis. Aquat. Organ. 35 (2), 139- 144; Dannevig BH., Falk K and Namork E. Isolation of the causal virus of infectious salmon anemia (ISA) in a long-term cell line from Atlantic salmon head kidney. J. Gen Virol. 1995. 76:1353- 1359; Kibenge F. S. B., Gårate O. N., Johnson G. Arriagada R., Kibenge M. J. T. & Wadowska D. ( 2001). Isolation and identification of infectious salmon anemia virus ( ISAV) from Coho salmon in Chile. Dis. Aquat. Org ., 45, 9- 18). Accordingly, there are 2 main genotypes of ILA virus: European genotype (I) and American genotype (II), which differ between 15 and 19% in their amino acid sequence fragments 5 (F) and 6 (HE). Another way of classifying ILA virus isolates lies in the hemagglutinin esterase gene, particularly in a region near the transmembrane domain of a maximum of 35 amino acids called HPR (Highly Polymorphic Region) which exhibits great variability in pathogenic isolates given by insertions or deletions ( Nylund, A., Devold, M., Plarre, H., Isdal, E., Aarseth, M., ( 2003). Emergence and maintenance of infectious salmon anemia virus ( ISAV) in Europe: a new hypothesis. Dis. Aquat. Organ. 56 ( 1), 11- 24; Mjaaland, S., Hungnes, O., Teig, A., Dannevig, B. H., Thorud, K., Rimstad, E., 2002. Polymorphism in the infectious salmon anemia virus hemagglutinin gene: importance and possible implications for evolution and ecology of infectious salmon anemia disease. Virology 20 ( 304), 379- 391). To date, we have identified 35 HPRS, of which HPR1 and HPR2 are the most prominent, while the others are variants of these, where amino acids are added at the carboxyl region of the hemagglutinin of HPR1 or the amino region of HPR2. In Chile, several types of HPRS have been found, with HPR7b being the most prominent in the years 2007-2008 and responsible for the high mortality recorded in these years in our country ( Kibenge, F. S., Godoy, M. G., Wang, Y., Kibenge, M. J. , Gherardelli, V., Mansilla, S., Lisperger, A., Jarpa, M., Larroquete, G. , Avendano, F., Låra, M., Gallardo, A. Infectious salmon anemia virus ( ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences. Virol J. 2009. 6:8- 16). A characteristic of this hypervariable region is the existence of a variant that contains all the motifs described for the other HPRS, the longest of which is called HPRO and is characterized by low pathogenicity and is not cultivated in standard cell lines such as SHK-1 (Salmon Head Kidney), (McBeath, A. J. , Bain, N., Snow, M., 2009. Surveillance for infectious salmon anemia virusHPRO in marine Atlantic salmon farms across Scotland. Dis. Aquat. Organ. 87, 161 - 169; Markussen, T., Jonassen, C. M., Numanovic, S., Braaen, S., Hjortaas, M., Nilsen, H., Mjaaland, S., 2008. Evolutionary mechanisms involved in the virulence of infectious salmon anemia virus ( ISAV), a piscine orthomyxovirus. Virology 374, 515- 527 ).

[0010] Endothelial cells are one of the main targets of the virus (Hovland T, Nylund A, Watanabe K, Endresen C.

(1995). Observation of Infectious salmon anemia virus in Atlantic salmon, Salmosalar L. J Fish Dis 17:291-6) allows the virus to replicate in various organs. In addition, we have detected the presence of the virus in polymorphonuclear cells and lymphocytes (Sommer AJ. & Mennen S. (1996). Propagation of infectious salmon anemia virus in Atlantic salmon, Salmo salar L, head kidney macrophages. J. Fish Dis., 19 , 179- 183; Kawaoka, Y., Cox, N. J., Kaverin, N., Klenk, H.-D., Lamb, R. A., McCauley, J., Nakamura, K., Palese, P., Webster, R. G., 2005. Family Orthomyxoviridae. Disseminated around 2005, based on surface glycoprotein gene sequences. Virol. J. 6, 88. 3245- 3259). In erythrocytes, the ILA virus covers them during the hemagglutination reaction by cooperating with sialic acid receptors and viral HE protein, a process similar to the hemagglutination of influenza virus in which 3 independent phenomena occur: a) adsorption of the virus in the erythrocyte membrane, b) elution of the virus, which is not always complete, and c) virus absorption via endosome of erythrocytes (Kawaoka, Y., Cox, N. J., Kaverin, N., Klenk, H.- D., Lamb, R. A., McCauley, J., Nakamura, K, Palese, P. , Webster, R. G., 2005. Family Orthomyxoviridae. Disseminated around 2005, based on surface glycoprotein gene sequences. Virol. J. 6, 88. 3245- 3259; Workenhe, S. T., Wadowska, D. W., Wright, G. M., Kibenge, M. J., Kibenge , F. S., ( 2007). Demonstration of infectious salmon anemia virus ( ISAV) endocytosis in erythrocytes of Atlantic salmon. Virol. J. 4, 13). ILA virus elution from erythrocytes has been reported in various fish species except Atlantic salmon, which only shows elution in mild types. This phenomenon would be associated with the receptor-destroying activity of the HE protein and has no effect on Atlantic salmon that would lead to serious clinical effects that these species suffer from (Falk K, Dale OB. (2007) : Lack of receptor-destroying enzymatic activity oflSA-virus is an important virulence factor for development of ISA in Atlantic salmon. Conference Fresh Fish, Tromsø: 23-25 January 2007; Norway). A schematic presentation of the ILA virus infection cycle can be found in the schematic presentation of infection of a cell by ILA virus (Cottet, L, Cortez-San Martin, M., Tello, M., Olivares, E., Rivas-Aravena, A. , Vallejos, E., Sandino, A. M., Spencer, E., ( 2010). Bioinformatic analysis of the genome of infectious salmon anemia viruses associated with outbreaks of high mortality in Chile. J. Virol 84( 22) : 11916-1192) .

[0011] The disease in Atlantic salmon can occur with a systemic and fatal nature, characterized by severe anemia and bleeding in various organs. Hematocrit is usually below 20% and in the most severe cases less than 10% (normal values between 35% to 64%). The virus mainly affects five organs: liver, kidney, heart, viscera and gills. Hepatic manifestation is characterized by the dark color of the liver caused by haemorrhagic necrosis. In the kidneys, inflammation occurs with moderate interstitial bleeding and tubular necrosis. The viscera appear dark red due to bleeding in the intestinal wall but not in the lumen. In some cases, blood accumulates in the gills in addition to pallor, particularly in the central venous sinus filaments in these. The most striking observable symptoms in cases in Chile reported in 2007 are pale gills, eye bleeding, exophthalmia, ascites in the abdominal cavity, bleeding petechiae in the skin, abdominal hysteria, protruding eyes, blocked liver and spleen, and bleeding in the basal fins ( Godoy, M. G., Aedo , A., Kibenge, M.J., Groman, D.B., Yason, C.V., Grothusen, H., Lisperguer, A., Calbucura, M., Avendano, F., Imilan, M., Jarpa, M., Kibenge, F.S., (2008). First detection, isolation and molecular characterization of infectious salmon anemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile. BMC Vet. Res. 4, 28).

[0012] The main cause of spread is via direct contact with infected specimens and cohabitation due to natural secretion and excretion of fish, implying that the transmission is horizontal. For that reason, the main route for injection is through the gills, but oral administration cannot be ruled out (Mikalsen, A. B., Teig, A., Helleman, A. L., Mjaaland, S., Rimstad, E., ( 2001). Detection of infectious salmon anemia virus (ISAV) by RT-PCR after cohabitant exposure in Atlantic salmon Salmo salar. Dis. Aquat. Organ. 47 (3), 175-181). However, it has also been reported that vertical transmission also exists due to sequence similarity with strains in Norway and Chile in 2007, suggesting the outbreak in Chile originated from Norway. Note that Chile imports large quantities of eggs from Norway, and these have been infected with the virus and brought it to the country. There is still a controversy as to whether vertical transmission actually occurs or whether it was caused by poor disinfection of the eggs (Nylund A, Plarre H, Karlsen M, Fridelt F, Ottem KF, Bratland A, Saether PA, (2007) . Transmission of infectious salmon anemia virus ( ISAV) in farmed populations of Atlantic salmon ( Salmo salar). Arch Virol 2007, 152:151- 179; Snow M., ( 2011). The contribution of molecular epidemiology to the understanding and control of vi ra I diseases of salmonid aquaculture. Veterinary Research 2011, 42:56; Vike S., Nylund S., Nylund A., ( 2009) ISA virus in Chile: evidence of vertical transmission. Arch Virol ( 2009) 154:1- 8).

[0013] In experiments with viral transmission, other species such as brown trout (Salmo trutta), rainbow trout and Coho salmon show viral replication but no clinical symptoms of the disease, which may be reservoirs of viruses or vectors for their spread (Nylund A , Jakobsen P, ( 1995). Sea trout as a carrier of infectious salmon anemia virus. J Fish Biol 47:174 - 1 76; Kibenge F. S. B., Gårate O. N., Johnson G. Arriagada R., Kibenge M. J. T. & Wadowska D. ( 2001 ). Isolation and identification of infectious salmon anemia virus ( ISAV) from Coho salmon in Chile. Dis. Aquat. Org ., 45, 9- 18; MacWilliams C, Johnson G, Groman D, Kibenge FS. Morphologic description of infectious salmon anemia virus ( ISAV)- induced lesions in rainbow trout ( Oncorhynchus mykiss) compared to Atlantic salmon ( Salmo salar). Dis Aquat Organ. 2007;78( 1) :1- 12). We also detected the virus in species different from salmon, such as cod (Gadus morhua) and pollock (Pollachius virens) (Grove S, Hjortaas MJ, Reitan U, Dannevig BH. ( 2007). Infectious salmon anemia virus ( ISAV) in experimentally challenged Atlantic cod ( Gadus morhua). Arch Virol. ;152( 10) :1829- 37; McClure CA, Hammell KL, Dohoo IR, Gagne N.( 2004) Lack of evidence of infectious salmon anemia virus in pollock ( Pollachius virens) cohabitating with infected farmed Atlantic salmon ( Salmo salar). Dis Aquat Organ. ; 61( 1- 2) :149- 52).

[0014] A parasite that must be taken into account in the development of the disease and in the salmon industry is sea lice or Caligus ( Caligus rogercresseyui). This jumping crab forms skin lesions and stress that leads to a weakening of the immune system resulting in greater susceptibility to pathogens, such as the ILA virus. Due to their direct contact with the salmon, it is believed to serve as a vector and may contribute to the spread of the virus ( Bravo S. ( 2009), " Estrategias de Manejo Integrado para el control de Caligus en la industria del salmon en Chile" ).

[0015] Finally, due to the high fish density handled by salmon farmers (up to 30 kg/m<3>) and the proximity of facilities in southern Chile, optimal conditions exist for episodes of massive outbreaks and high mortality. According to the public account Sernapesca from November 2009, there have been outbreaks in 141 salmon farms, and included in these, a total of 228 centers have detected the virus, with the month of January 2009 being the largest occurrence of the disease, that is, the highest rate of affected centers in relation to those populated with susceptible species (Atlantic salmon), the occurrence of positive centers in this month reached 45% (Sernapesca, 2009).

[0016] Teleost fish, such as salmon, exhibit an immune system that is similar but not identical to higher vertebrates, consisting of a natural and an adaptive immune system (Press CM, Evensen 0. (1999). The morphology of the immune system in teleost fishes. Fish Shellfish Immunol 309- 318; Fernåndez AB, Ruiz I, de Blas I. ( 2002) El sisterna immune de los teleosteos (II) : Respuesta immune inespecifica. Revista Aquatic disponible en: http:// www. revistaaquatic. com/ aquatic/ html/ artl707/ immune2. htm).

[0017] The fish skin is the first line of defense against pathogens, as well as the mucous membranes that enclose the gills and digestive tract. The mucous membranes that cover the skin are an important defense mechanism, since they contain a number of reactive antimicrobial compounds, such as lysozyme, proteases, complementary factors, C-reactive proteins, lectins, interferons, eicosanoids, transferrins, piscidins and various carbohydrates (Buchmann K, Lindenstrom T , Bresciani J. (2001) Defense mechanisms against parasites in fish and the prospect for vaccines. ActaParasitol ;46:71- 81; Magnadottir B. ( 2006) Innate immunity of fish (overview). Fish Shellfish Immunol 2006; 20: 137- 151; Manning MJ. (1998) Immune defense systems. In: Black KD, Pickering AD editors. Biology of farmed fish. Sheffield: Sheffield Academic Press; 1998:180- 221; Woo PTK.

( 2007) Protective immunity in fish against protozoan diseases. Parasitologia 49:185-191). As a supplement, we can mention that it is composed of a number of serum proteins with three central defense functions: a) coat pathogens and foreign particles close to the body to promote recognition and destruction by phagocytic cells (opsonization), b) initiate inflammatory responses by stimulating vasodilation and chemoattraction of the lymphocytes, and c) lysis of pathogens by perforation of their membranes (Boshra H, Lii, SunyerJO. (2006) Recent advances on the complement system of teleost fish. Fish Shellfish Immunol 20:239- 262; Janeway CA, Travers P, Walport M, Schlomchik MJ. (2005) Immunobiology; the immune system in health and disease. 6th edition, New York: Garland Science Publishing). Eicosanoids include the prostaglandins, thromboxanes and leukotrienes, and are potent pro-inflammatory mediators. They are involved in several physiological processes, such as hemostasis, immune regulation, increased phagocytosis and chemoattractants for neutrophils ( Secombes a. ( 1996) The nonspecific immune system: cellular defenses. In: Iwama G, Nakanishi T editors. The fish immune system: organism , pathogen and

environment. London: Academic Press Ltd. ; 63-103; Manning MJ. ( 1998) Immune defense systems. In: Black KD, Pickering AD ( eds.). Biology of farmed fish. Sheffield: Sheffield Academic Press; 1998:180-221). The interferons (IFNs) form a family of heterologous proteins to provide protection against viral infections (Secombes CJ. (1996) The nonspecific immune system: cellular defenses. In: Iwama G, Nakanishi T (eds.). The fish immune system: organism, pathogen and environment. London: Academic Press Ltd. ; 63- 103). There is Type I, which includes IFN and IFN-3, which is produced by cells infected with the virus and can be produced by any cell type, and type 2, which includes IFN which is a cytokine produced by T-lymphocytes ( Magnadottir B.

( 2006) Innate immunity of fish (overview). Fish Shellfish Immunol 2006;20:137-151). The response begins when the pathogen and host interact, leading to the activation of pathogen-recognition receptors (PRRs), among which the most prominent are the toll-like (TLR) family members capable of recognizing pathogen-associated molecular patterns (PAMPs) , such as lipopolysaccharide (LPS) in Gram-negative bacteria, peptidoglycan from Gram-positive bacteria, double- and single-stranded RNA viruses and CpG DNA unmethylated (lliev, D., Roach, J., MacKenzie, S., Planas, J. and Goetz, FW. (2005). Endotoxin Recognition: In fish or not in fish. FEBS Letters 579 (29):6519- 6528). To date, the presence of at least 4 TLRs has been reported, two of which have been associated with viral infections in salmon, one of which is TLR-8 in mammals whose function is to recognize single-stranded RNA. In salmon expression of TLR-8, messenger level is induced in cells treated with IFN-E and IFN-E [ Skjaeveland., D. B. lliev, & J. B. Jorgensen (2009). Identification and characterization of TLR8 and MyD88 homologs in Atlantic salmon (Salmo salar.) Developmental & Comparative Immunology. 33: 1011-1017). The other is TLR-9 which recognizes unmethylated CpG DNA as in mammals ( Skjaeveland, D. B. lliev, J. Zou T. Jorgensen & J. B. Jorgensen ( 2008). A TLR9 homolog that is up-regulated by IFN- in Atlantic salmon ( Salmon salar ).Developmental and Comparative Immunology, 32, 603-607).

[0018] Another alternative route of entry of viral RNA is through the helicase RIG-I, which acts through a molecule known as IPS-1 (also called MAVS, VISA and CARDIF) which activates a signaling cascade that ultimately leads to the activation of IFN type I ( Onomoto K, Yoneyama M, Fujita T. (2007). Regulation of antiviral innate immune responses by RIG-I family of RNA helicases. Curr Top Microbiol Immunol. 316:193- 205). In salmon, RIG-I is known to exist and it has been reported that cells transfected with cloned IPS-1 are able to induce an antiviral state (Biacchesi, S., Leberre, M., Lamoureux, A., Louise, Y., Lauret, E., Boudinot, P., and Brémont, M. (2009) MAVS Plays a Major Role in Induction of the Fish Innate Immune Response against RNA and DNA Viruses. J. Virol. 83( 16) :7815 - 7827 (Journal)).

[0019] Salmon, like most animals, unlike mammals, have no lymph nodes or bone marrow, but the thymus, kidney and spleen play this role. In kidneys, hematopoietic activity has been observed, showing stem cells that exhibit this function (Hanington PC, Tam J, Katzenback BA, Hitchen SJ, Barreda DR, Belosevic M. (2009). Development of macrophages of cyprinid fish. Dev Comp Immunol 33: 411- 429), also together with the spleen have extensive networks to capture particles transported with the blood and presented together with macrophage and lymphocyte populations with the ability to initiate immune responses [ Press CM, Evensen 0.

(1999). The morphology of the immune system in teleost fishes. Fish Shellfish Immunol 309- 318). It has also been reported that the kidneys display lymphatic activity and are the main organ for B cells and antigen production (Whyte, S. K. ( 2007). The innate immune response offish- A review of current knowledge. Fish & Shellfish

Immunology, 2007. pp. 1127-1151). The adaptive immune response can be divided into humoral and cellular immunity, largely dependent on B and T lymphocytes respectively ( Fernåndez AB, Ruiz I, de Blas I. ( 2002) El sisterna inmune de los teleosteos ( II) : Respuesta inmune inespecifica. Revista Aquatic Disponible en: http: // www . revistaaauatic . com / aauatic / html / artl707 / inmune2 . htm ).

[0020] Similar to how it occurs in mammals, humoral immunity involves the recognition and binding of antigens by B-lymphocytes, which differentiate into plasma and storage cells to produce antigen-specific antibodies or immunoglobulins, respectively, and generate rapid responses and evident in subsequent infections ( Hansen JD, Landis ED, Phillips RB. (2005). Discovery of a unique lg heavy-chain isotype (Igl) in rainbow trout: Im pl cations for a distinctive B cell developmental pathway in teleost fish. Proe NatlAcad Sei. USA. 102 :6919- 6924). In viral infections, immunoglobulins can bind to the surface of viruses to neutralize viruses, with IgM being the most predominant, which can associate with the B-lymphocyte membrane and serve as a receptor for antigen recognition, or in secreted form ( Janeway CA, Travers P, Walport M, Schlomchik MJ.

( 2005) Immunobiology; the immune system in health and disease. 6th ed., New York: Garland Science Publishing). In addition to IgM, other immunoglobulins have been described in teleost fish, such as IgD, IgT and IgZ, which have been cloned in Atlantic salmon and have sequence similarity (Randelli, E., Buonocore, F., Scapigliati, G. ( 2008). Cell markers and determinants in fish immunology. Fish Shellfish Immunol. 25, 326- 340).

[0021] On the other hand, the cell-mediated acquired immunity mainly involves the activation of T-lymphocytes, which are activated by the interaction of the major histocompatibility complex ("Major Histocompatibility Complex" (MHC)) loaded with an antigen and the T-cell receptor (TCR) ( Finelli, A., K. M. Kerksiek, S. E. Allen, N. Marshall, R. Mercado, I. Pilip, D. H. Busch, and E. G. Pamer. 1999. MHC dass I restricted T cell responses to Listeria monocytogenes, an intracellular bacterial pathogen. Immunol Res. 19:211-223.). There are two types of MHC class I and II. MHC class I is the main part of the cells and acts primarily against intracellular pathogens by activating T-lymphocyte (CD8+) cytotoxics, while MHC class II is only found on cells with antigen, such as macrophages, dendritic cells and B-lymphocytes, and they lead to the activation of T-helper lymphocytes (CD4+) (Nakanishi T, Ototake M. (1999). The graft-versus-host reaction (GVHR) in ginbuna crucian carp, Carassius auratus langsdorfii. Dev Comp Immunol 23:15- 26). The interactions between cells are not only mediated by cell-cell contact, but also by soluble factors, such as cytokines, which ensure the different types of immune responses via differential expression depending on the pathogen to show that the individual is presented to. Against intracellular pathogens, CD4+ T lymphocytes are differentiated into Th1 lymphocytes by the activity of IL-12 and INF, which are secreted by precursors expressing the gene T-bet, and this gene is the key regulator in the production of Th1. Th1 lymphocytes finally secrete molecules, such as IL-2, INF and TNF-3 which mainly promote cellular response, generating the activation of macrophages, increased antigen presentation and differentiation of CD8 + T lymphocytes. Against extracellular pathogens, CD4+ T cells differentiate into Th2 cells upon activity of IL-4, which is secreted by precursors expressing the gene GATA3. These lymphocytes secrete IL-4, IL-5, IL-6, IL,9, IL-10 and IL-13 which activate a B cell mediated response for antibodies ( Janeway CA, Travers P, Walport M, Schlomchik MJ. ( 2005 ) Immunobiology; the immune system in health and disease. 6th edition New York: Garland Science Publishing).

[0022] From the known technique, one can particularly mention the following patent documents:

[0023] WO 2004024956 describing the identification of resistant Atlantic salmon and trout susceptible to viral diseases (ILA, IPN), and bacterial, to determine the genotype of salmon with a set, type of chip, type of molecular markers containing gene sequences belonging to the major histocompatibility complex (MHC, which is the English abbreviation for "Histocompatibility Main Complex") class I and MHC class II alpha/beta. The use of the technology is within the field of reproduction.

[0024] WO0226784 refers to the use of nucleotide sequences encoding the structural proteins M1 and M2 of ILA virus for use in vaccines, generation of antibodies against proteins or diagnostic kits or detection of ILA virus.

[0025] WO2012075597 describes a method for determining the virulence of a viral infection produced by infectious pancreatic necrosis (IPN), serotype Sp from a salmon tissue sample. First, RNA is extracted from the sample, the gene region corresponding to the VP2 protein is amplified by PCR, sequencing the purified PCR product, translating the nucleotide sequence into an amino acid sequence, and finally identifying the presence of the amino acids threonine, proline, and alanine at a specific position, and in this way determine whether the IPN virus has high or medium virulence.

[0026] Furthermore, the following products are available on the market which are related to the area of the present invention.

[0027] The product Sb + Health from Salmobreed, which comprises eggs from Salmo salar (Atlantic salmon) which show resistance to viruses ILA and IPN and furunculosis. These eggs are genetically selected to deliver better growth rates and survival throughout the salmon's life cycle. This product is a selection carried out at a parental level for the salmon, on the basis of various parameters, including resistance to ILA.

[0028] The product AquaGen Atlantic QTL-innOva IPN/PD corresponds to eggs from Salmo so/or species originating from spawning salmon that are highly resistant to infectious pancreatic necrosis virus (IPN, an English abbreviation for "Infectious Pancreatic Necrosis") and pancreatic disease (PD, an English abbreviation for "Pancreas Disease"), where, in the use of the technique of marker-assisted selection, direct selection is made of a parent used in the production of eggs that have specific genetic markers or areas of quantitative character, QTL ( QTL, English abbreviation for "Quantitative Trait Loci") that is associated with resistance to a virus. The selection is carried out both at family level and at individual level, which ensures high resistance to IPN and/or PD virus in the offspring or eggs.

[0029] The vaccine subunit against infectious salmon anemia (injectable) from Controvet is a vaccine against ILA virus for 5. salar made from the recombinant proteins of this virus produced in yeast cultures. Help to prevent infectious salmon anemia in pre-smolt salmon starting from a weight of 30 grams.

[0030] Transcriptome sequencing service, LC Science, refers to an RNA sequencing service (RNA-seq), which uses high-throughput sequencing technology to determine the structures of llluminagen splicing patterns, post-transcriptional modifications, detection of novel or rare transcripts and quantification of changes in the level of transcript expression in different organisms, and corresponds to a generic service and not only focused on salmon.

[0031] The services of technical assistance, genetic programs and reproductive programs, are generally a service in the field of aquaculture-breeding of species of interest, including Atlantic salmon. Their focus has primarily been on improving the percentage of weight gain for salmonids, between 5 to 8%, including Atlantic salmon.

[0032] In addition, a number of investigations have been or are being developed within the scope of the invention, and as examples the following projects are listed below:

[0033] Project development and validation of a chip of SNP to determine the molecular basis and genetic improvement of disease resistance in Salmo salar Aqualnnovo S.A. 2012-2015. In this project, a system for genetic detection/screening is validated for the identification of Atlantic salmon resistant to a disease produced by the viruses ILA and IPN, using the technique of single nucleotide polymorphism, SNPs (an English abbreviation for "Single Nucleotide Polymorphism") in chip format.

[0034] Project development and validation of genetic markers for immune response to ILA by Fisheries and Oceans, Canada, 2007-2009, identified from a genomic point of view using DNA microarrays, immunological genetic markers involved in response to ILA. The in vivo validation of these markers provides information for the study of disease, the resistance of the lask and the response of the same to vaccines.

[0035] The present invention refers to the use of the genes Gata3, Grb2, IgM, MAVS and Regul with respect to infectious salmon anemia (ILA) in Atlantic salmon (Salmo salar).

[0036] In the present invention, the level of expression of specifically selected genes related to the response to ILA virus, in fish with susceptible and resistant phenotypes, is quantified by linking a genetic expression profile to such phenotypes.

[0037] They were selected according to ontological criteria and functional genome, genes from the group consisting of Gata3, Grb2, IgM, MAVS and Regul, and others.

[0038] To quantify the expression levels of each gene from salmon with resistant and susceptible phenotypes, real-time PCR was used from tissues obtained from salmon with resistant and susceptible phenotypes.

[0039] In general, as a result, a tendency was obtained with the lower expression of the selected genes in susceptible fish, and then Grb2, Gata3, IgM, MAVS and Regul were found to be able to be used as markers of phenotype to differentiate "resistant" and " receptive".

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Graph with accumulated mortality of fish families corresponding to resistant and susceptible phenotypes. Each challenge was performed in duplicate for greater span. Figure 2. Ribosomal bands with 18S and 28S RNA corresponding to susceptible groups (A) and resistant controls

(B), shown on agarose gel at 1%.

Figure 3. Dissociation curve for analyzed genes: annexin (A), cathepsin (B), Gata 3 (C), Grb2 (D), HSP90 (E),

IgM (F), IgZ (G), Mavs (H), Tbet (I), Regul (J). The presence of a single PCR product was observed.

Figure 4. Normalized relative expression of candidate immune genes IgM (A), Grb2 (B), HSP 90 (C), Annexin (D) and Regul (E) are shown. Bars with squares represent susceptible individuals and fine lines resistant individuals. Data are the average of 9 individuals per phenotype, normalized against 18S rRNA gene expression and adjusted for relative efficiency. (<*>) Denotes significant difference from control (<*>p <0.05,<**>p <0.01,<***>p <0.001).

Figure 5. Normalized relative expression of immune candidate genes, genes with similar expression patterns Cathepsin (A), IgZ (B), Mavs (C), Gata3 (D) and Tbet (E) are shown. Bars with squares represent susceptible individuals and fine lines resistant individuals. Data are the mean of 9 individuals per phenotype, normalized against 18S rRNA gene expression and adjusted for relative efficiency. (<*>) Denotes significant difference from control (<*>p <0.05,<**>p <0.01,<***>p <0.001).

DETAILED DESCRIPTION OF THE INVENTION

[0040] In general, one of the most important strategies with candidate genes is functional genome research, which aims to relate the sequence of a gene with the function it has. To achieve this goal, platforms such as microarray are used, which are based on the hybridization of fluorescently labeled complementary labeled DNA from two different conditions, with thousands of genes present on the microarray chip ( Belbin TJ, Gaspar J, Haigentz M, Perez .. - Soler R, Keller SM, Prystowsky MB, Chllds G, Soccl ND (2004) Indlrect measurements of differential gene expression with cDNA microarrays Biotechniques 2004, 36: 310- 314). This results in differences in the expression of the genes studied under both conditions. However, microarray data are subject to tests (genes) that each platform may possess. A new "RNA-sequencing" technology allows access to the differentially expressed RNA sequence in a broad and comprehensive manner, making it an excellent tool in the search for candidate genes ( Wang Z, Gerstein M, Snyder M. ( 2009) RNA- Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 10(l):57-63).

[0041] Differential expression patterns of certain genes, representative of a specific phenotype, can be analyzed using the Polymerase Chain Reaction (PCR). A variant of this reaction is real-time PCR, which uses fluorescent markers to monitor DNA amplification in each cycle. This is characterized by its sensitivity and specificity, which makes it suitable for detailed analysis of genes of interest (Pfaffl M.W. and Hageleit, M. (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotechnol. Lett, 23, 275-282).

[0042] In the present invention, the technique is "RNA sequencing" technology that uses Aillumina-Solea sequencing, and develops an experiment that includes families of Atlantic salmon that are resistant and susceptible to the ILA virus. The genetic status of the families was determined according to the level of heritability; which corresponds to a proportion of phenotypic variation in a population attributable to genotypic variation among individuals. The value of heritability can vary (for each trait) in different populations or even within the same population at different points in its history. For the sake of the present description, value greater than or equal to 0.35 will be classified as resistant family and less than or equal to 0.34 as susceptible family. Alongside this, the total mortalities within each family must be consistent with expectations (higher in susceptible).

[0043] The experimental results indicated that different candidates for RNA sequencing expressed genes differentially between the two phenotypes. Within the associated central ontological categories, note: a) signal transduction, b) regulation/proliferation of B cells, c) a stress response, d) T cell receptors, d) differentiation of T cells, e) immune response, f) regulation of transcription , g) regulation of endothelial cell migration, h) viral response. For each of these categories, representative candidate genes were selected. The genes, their function and their associated biological processes are detailed in Table 1.

[0044] Annexin A1 inhibits phospholipase protein 2, which releases arachidonic acid from the cell, which eventually transforms into prostaglandins, leukotrienes and thromboxanes (eicosanoids), which are associated with inflammatory processes. Note that prolonged inflammatory processes cause great damage to tissues and organs, so it is important to analyze genes related to the biosynthesis of eicosanoids (Glaser KB. ( 1995) : Regulation of phospholipase A2 enzymes: selective inhibitors and their pharmacological potential. Adv Pharmacol 1995, 32:31-66).

[0045] Cathepsin S is a cysteine protease expressed in the antigen presenting cells, which has the function of generating antigens for loading on MHCII. Deficiencies in this gene have been reported to produce damage to mitochondria and increased levels of reactive oxygen species (ROS), as well as problems with signaling with MHCII.

( Costantino CM, Hang HC, Kent SC, Hafler DA, Ploegh HL. ( 2008) Lysosomal cysteine and aspartic proteases are heterogeneously expressed and act redundantly to initiate human invariant chain degradation. J. Immunol 180:2876- 2885. [ PubMed: 18292509 ]). It is analyzed for its relationship to the loading of MHCII antigens.

[0046] Grb2 is an adapter protein linked to the activation of the MAP kinase pathway and signal transduction in lymphocytes. The signaling is positively regulated by the TCR (T-cell receptor), which activates the development of T-lymphocytes and negative signaling by the BCR (B-cell receptor), thereby inducing an immune response that is linked more cellularly than humorally. (Jang IK., Zhang i., Gu H. (2009) Grb2, a simple adapter with complex roles in lymphocyte development, function, and signaling. Inmunol Rev 232; 150- 159).

[0047] HSP90 is a protein involved in the correct folding of polypeptides, renaturation of proteins damaged by heat and as a chaperone. It is mainly expressed either by osmotic stress, by pollutants, or immune response (Pearl LH, Prodromou C (2001) Structure, function, and mechanism of the Hsp90 molecular chaperone. 2001. Adv Protein Chem 59:157- 186.). It is mainly used in this thesis as a stress marker. IgM and IgZ are immunoglobulins, the first of which is present in larger quantities in salmon and as a primary response against pathogens ( Frazer, J. K. and J. D. Capra. Immunoglobulins: structure and function. Fundamental Immunology, 1999, 4th edition W. E. Paul, publishers. Lippincott-Raven, Philadelphia. 37- 74.). With regard to IgZ, its function has not yet been described, but it is believed to be a more specific immunoglobulin (Gomez-Lucia Duato E The major histocompatibility complex. in "Manual of Veterinary Immunology" 2007. Gomez-Lucia E, Blanco MM y Doménech A (ed.). Pearson-Prentice Hall. Madrid, pp. 117-136.). Tbet and Gata 3 are antagonist genes involved in the differentiation of T cells into Thl and Th2, respectively. Both genes are key regulators in the fate of the lymphocytes T cells CD4+ and analyzed as markers of cellular and humoral immunity. Regul is a negative regulator of protein G signaling and accelerates the conversion of GTP to GDP, reducing signaling downstream of the G protein. This repressor function reduces the effect of chemokines and the migration of immune cells ( Tiffany Tran, Pedro Paz, Sharlene Velichko, Jill Cifrese, Praveen Belur, Ken D. Yamaguchi, Karin Ku, Parham Mirshahpanah, Anthony T. Reder and Croze. E. InterferonQ- lb Induces the Expression of RGS1 a Negative Regulator of G-Protein Signaling. Internationa/ Journal of Cell Biology. Jan 2010, Vol. 2010:1- 12).

[0048] The alternatives to control infection caused by the ILA virus in Atlantic salmon can be mainly grouped into three: i) vaccines, which corresponds to the alternative in use mainly due to the order from national authorities (in Chile, Sernapesca) to vaccinate all fish by smolt stage (General Law on Fisheries and Aquaculture No. 20,632 in Chile). There are currently in Chile over 10 commercial vaccines registered for ILA, which can be mono- or polyvalent molecules of inactivated viruses and mainly injectable or of the oral type. Unfortunately, there is currently no published information regarding the actual coverage of vaccines against ILA, only limited internal reports from the manufacturing companies. However, it is generally known that the coverage of vaccines in the area does not correspond to 100% ( Mikalsen, A. B., Sindre, H., Torgersen, J. and Rimstad, E. Protective effects of a DNA vaccine expressing the infectious salmon anemia virus hemagglutinin-esterase in Atlantic salmon. 2005. Vaccine 23:4895- 4905; Lauscher a., Krossøy B., Frost P., Grove S., Konig m., Bohlin J., Falk K., Austbø L, Rimstad E. (2011). Immune responses in Atlantic salmon ( Salmo salar) following protective vaccination against Infectious salmon anemia ( ISA) and subsequent ISA virus infection. Vaccine, Vol. 29, chapter 37, 6392- 6401), that is, not all vaccinated individuals fail to developing immune protection, ii) Genetics, which corresponds to an alternative classical selection within the context of genetic animal model, which is mainly based on the detection and use of individuals resistant to a given disease and subsequent design of controlled crosses. Until now, this option has been very successful but only in certain companies that have the advantage of the heterogeneous resistant, and iii) fish that vary from immune stimulation, antiviral and synthetic peptides.

[0049] Through the improvement of families due to controlled crossing, fish families resistant to ILA virus were obtained and ultimately resulted in lower accumulated mortality (less than 40% for this description and heritability (H) greater than or equal to 0.35) than what was achieved with other establishments. These families were termed resistant families. At the same time, there are also families that have high mortality (50% or more for this description and H less than or equal to 0.34) against the virus, which are classified as susceptible. Taking these two phenotypes as a basis (resistant and susceptible), the expression patterns of candidate genes and their relationship to resistance or susceptibility to the pathogen are compared.

[0050] For this, the following activities were carried out: • Quantify and evaluate associated expression profiles for candidate genes in resistant and susceptible fish with ILA virus as the initial target phenotypes. • Develop and standardize the analysis by real-time PCR quantification of the expression of candidate genes as the next target.

• Quantified as relative candidate genes in susceptible and resistant phenotypes point below.

• Analyze the suitability of some of the selected genes to serve as a marker for resistance to the ILA virus, as the ultimate target.

[0051] In the initial phase, a challenge test was performed with 2 families of S. salar (SNIR and Fanad, respectively resistant and susceptible) to determine the degree of resistance to ILA virus. For this, a Chilean viral isolate was used that was sequenced in the hypervariable region (HPR) resulting in HPR type 7b that was inoculated intraperitoneally injected in the ventral midline at 0.2 ml of inoculum per Trojan fish containing a degree of 7 ,2 x IO<4>TCID50. Only 40% of the fish tank, which was waiting for the remaining 60%, was infected by cohabitation, an infection that reflects what happens in the field.

[0052] Before the test was initiated, the health status of the fish in the source fishery was confirmed. A visit to the field was carried out to check the general condition of the fish in the selected group. At the same time, samples of 30 fish were taken for analysis in the laboratory. The analyzes cover 15 autopsies, gram analysis, Flavo-BKC, RT-PCR IPNV and RT-PCR ILA. If the selected group with every fish pathogen analyzed was positive, it was discarded and a new group of fish was searched.

[0053] In what follows, an experimental design scheme associated with the challenge of cohabitation is illustrated:

[0054] The experiment studied the reception of 528 Atlantic salmon (Salmo salar) with an average weight of 115 g, corresponding to the experimental design scheme in front of challenge by cohabitation. These fish were all individually marked by fin tagging for identification. An evaluation was also made of 363 Atlantic salmon corresponding to the "Trojan" fish, which were injected once after acclimatization with the ILA virus inoculum. All fish were transported to an experimental center and received in a 720 liter one vessel for the Trojans in 3 vessels for the cohabitants. The fish were received in brackish water with a salinity of 15 which was later increased gradually over the next 7 days to finally end up at 32. The fish remained in the 4 tanks for a period of 14 days as an acclimatization period at a temperature of 12 °C, the same temperature as the fish's origin. During this period, the fish were manually fed a commercial diet, with three rations three times daily up to 2% BW (body weight/day). Subsequently, a total of 480 individuals, which were cohabited with 330 infected individuals, were challenged with ILAv by intraperitoneal injection (40% of a total of 810 fish). It was proposed to use a total of 4 vessels of 720 litres, 3 permanent vessels and 1 temporary. In each of the 3 permanent tanks, 40 fish from each group (resistant or susceptible) were added for challenge by cohabitation, see diagram, which shows that in each tank there were 160 cohabiting fish that were marked individually by cutting the tail fin, which had an average weight of 155 g. In the temporary tank were placed 330 "Trojan" fish of the same weight, which were inoculated intraperitoneally with inoculum of ILA virus once at the end of the acclimation period. This tank is temporary and housed the Trojan fish during the period of acclimatization (14 days). Upon completion of the phase of acclimatization, the inoculation was realized by intraperitoneal injection of the virus, whose fish were anesthetized with benzocaine. The fish were fasted 24 hours before inoculation. The posterior dorsal fin ("adipose fin") was cut from these fish for easy identification and 110 fish were placed in each of the permanent tanks containing the 160 cohabiting fish. In this way, there were 3 vessels with 40% of the infected fish (Trojans) and 60% of cohabiting fish with fin markings to identify the different fish groups at all times. The phase of cohabitation lasted 35 days. Fish mortality was recorded daily. Samples of kidney, spleen and heart were extracted from dying fish.

[0055] During the entire experiment, the fish had a light period regime of 24 hours with light to ensure complete smoltification of the fish.

[0056] The fish got at 12 °C since the water temperature in the fish culture is at that temperature. This temperature was maintained during the days of acclimatization. During the entire period of cohabitation challenge, the temperature was maintained at 14 °C. The temperature was recorded automatically every minute with a "logger". Oxygen was maintained between 85% and 100% during the entire period of acclimatization and cohabitation challenge.

[0057] For the period of acclimatization and cohabitation challenge, a commercial diet, Golden Optima 4, from the company Biomar with caliber 4.0 mm was used. The feeding was carried out manually with three rations daily until the daily ration was reached.

[0058] 9 specimens for each phenotype were selected for study, the previously extracted kidney at day 15 post-infection, which was stored in "RNA later", and then stored at -80 °C for further analysis.

[0059] Extraction of RNA was performed with the previous kidney samples using the RNeasy mini kit kit (Quiagen). Each sample was disintegrated using a tissue homogenizer (TissueRuptor, Qiagen) in 1 ml of Trizol LS (Invitrogen) according to what was established by the manufacturer. The homogenized sample was incubated for 2 min at ambient temperature, then 200 µl chloroform was added, vortexed and centrifuged at 10,000 G for 15 min at 4 °C to separate the phases. The aqueous phase was transferred to a 700 µl tube with 70% ethanol and then the total volume was transferred to a mini-column (RNeasy Mini Kit, Quiagen) and was centrifuged at 9,000 G for 30 seconds. 600 µl of RW1 buffer was added and centrifuged at 9,000 G for 30 seconds. A DNase treatment step was also added with the addition of 80 µl of the digestion solution (10 µl of the enzyme DNase (Quiagen) and 70 µl of the buffer RDD according to the manufacturer's instructions) to each column. Then it was washed 2 times with 500 µl of buffer RPE, the first wash at 9,000 G for 30 seconds and the second at 9,000 G for 2 minutes to dry. Finally, the column was eluted with 50 µl of nuclease-free water, and aliquots of 10 µl of extracted RNA were prepared. RNA integrity was verified by gel electrophoresis with 1% agarose gel, stained with ethidium bromide. The presence of the bands 28s and 18s (ribosomal RNA) and the level of degradation serve as an indicator of the integrity of the samples. At the same time, RNA concentration was measured with a biophotometer (Eppendorf), which was carried out by diluting 2 ?L of the extracted RNA plus 58 ?L of nuclease-free water, and the absorbance was determined at 230, 260, 280 and 320 nm for to verify the condition and possible contamination.

[0060] MMLV (Promega) enzyme and the method from Random Primers (Promega) were used for reverse transcription. For this, 2.5 pg RNA in water was incubated (12.875 µl of the final volume from the RT reaction) at 60 °C for 10 min and then quickly transferred to ice. The RT mix consisted of 5X RT buffer, dNTPs 3.125 µl with 4 pM, 100 U ribonuclease inhibitor RNasin (Promega), 100 U with MMLV and 0.5 pg Random Primers, in a final volume of 12.125 µl of RT mix for reaction. Then the RT mixture was added to 12,875 µl RNA to achieve a final reaction volume of 25 µl pg reverse transcription was carried out in a thermocycler with the following program: 60 min at 42 °C, 5 min at 99 °C to denature the enzyme.

[0061] The primers of the selected genes were generated using the program Primer3 using preset parameters based on the database Immune Genome developed by our laboratory (Cepeda et. al 2011) and multiple alignments were performed with the program BioEdit 5.09 to finally determine the approximate exon-exon junctions. The primers were synthesized with IDT (Integrated DNA Technologies). Conditions for PCR primers were optimized for each set with varying concentrations of primers (0.5-2 pM) and cDNA directly loaded into the reaction (1 to 3 µl) to achieve the cycle ct between 20 and 30. Real-time PCR was performed using the Green qPCR kit TMSYBR Master Mix (Fermentas) in 96-well plates in the 7300 Real-Time PCR equipment System (Applied Biosystems). Dissociation curves were also performed to determine dissociation temperatures and analyze the specificity of the amplicons. Sizes were also confirmed with agarose gels. The conditions used in real-time PCR were 10 min at 95 °C with preincubation, followed by 40 cycles at 95 °C for 30 seconds, 53-62 °C for 1 min and 72 °C for 30 seconds. Finally, dissociation curves were performed from 60 °C to 95 °C. The primers for each gene are listed in Table 2.

[0062] Calibration curves were made for each gene from RNA extract of known concentration; as the point of maximum concentration, 2.5 pg of total RNA was used, which was subjected to a series of dilutions. Each standard curve consists of at least 5 points in triplicate by plotting the mean Ct from each point versus the log of the concentration of RNA. From these curves, PCR efficiency for each gene was determined to be acceptable in the range between 90% and 110%.

[0063] The linear method described by Pffafl was used for relative quantification (Pfaffl, MW (2001) A new mathematical model for relative quantifkation in Real-time PT-PCR Nuclek Acids Res 29: ... E45). This calculates the relative expression rate of a gene based on the efficiency and Ct differences in the sample versus the control. The Ct value for the gene of interest is normalized to a reference gene, which in this case is the gene 18S. The formula used for the relative quantification with Pfaffl is shown below:

[0064] This equation takes into account the PCR efficiency obtained from calibration curves for both the target gene (Emå!) and the reference gene 185 (Eref), as the difference in cT in the candidate genes, (ACTmå| <kontro"-Preve>) between control fish (none ILA challenge) and the sample (ILA exposed to challenge, belonging to one of four phenotypes challenged). The same applies to the reference gene 18S (ACTref<<tontrol>|<->»*>'<e>>).

[0065] The results obtained with the Pffafl method will be analyzed and plotted with the program Graphpad Prism 4 (Graphpad Software, Inc.). From the results of the analysis of RNA sequencing with the lllumina methodology, the best housekeeping gene was determined to be 18SA (which has less variation), which will be used as a reference gene. This is consistent with what is reported by Pfaffl, 2004 (Pfaffl, M. W.

(2004)). Quantification strategies in real time PCR. Chapter 3, pages 87-112).

[0066] The mean normalized expressions corrected for efficiency and the relative expressions will be determined by processing data for Ct obtained for each condition and gene, in the program Q-genes (Simon P.

(2003). Q-Gene: processing quantitative real-time RT-PCR data. Bioinformatics. 2003 Jul 22;19(ll):1439-40).

[0067] An analysis of variance (ANOVA) with one-way and two-way normalized expression data was performed to find the differences with this control in the case of one pathway, and to analyze the effect of the factor phenotype, and their contribution to the variance of the data in the case of two pathways , this in each of the selected genes. Tukey and Bonferroni a posteriori tests were also performed to find differences between each of the groups (phenotypes) in the study.

[0068] To confirm whether the genes in the study could be used as markers and classifiers for phenotype, an analysis of the ROC curve was carried out, and thus determine genes with greater predictive power. After selecting the most predictive genes, a discriminant analysis was performed with these in order to determine whether it is possible to classify the phenotypes in the study, an individual subjected to an analysis with real-time PCR using the predictor genes. In order to simplify the visualization and understanding of the data, an analysis of the most important components was also carried out.

[0069] The results from the challenge test are shown in Figure 1. There you can see that Trojan fish have the highest accumulated mortality (100%) compared to the other groups in the study. The most extreme values for each duplicate were selected for further analysis by real-time PCR, corresponding to a 70% cumulative mortality for the susceptible phenotype and 40% for the resistant one.

[0070] Total RNA extracted from resistant and susceptible individuals and uninfected controls was quantified spectrophotometrically. The concentration range for the samples varied between 0.8-3 g/l. The purity of the sample in light of protein and organic solvents was determined by the ratio A26onm/A280nm, which varied between 1.8 and 2.0 for protein and the ground A26onm/A230nm which varied between 1.95 and 2.5 for organic solvents. Both reasons are within the acceptable range. RNA integrity was evaluated by 1% agarose gel electrophoresis, in Figure 2 the rRNA bands 18S and 28S can be seen for samples from resistant, susceptible and control subjects.

[0071] To determine the experimental efficiency of each set of splitters, calibration curves were realized from total RNA of known concentration. The curves were made by plotting Ct against the log of the concentration of RNA at each point in the curve (dilution). Each curve consists of at least 5 points. It was determined by the correlation coefficient that the points behave linearly and that the efficiency and slopes are within the acceptable ranges (90 and 110% with respect to efficiency; -3.6 and -3.1 with respect to slope). The results are detailed in Table 3.

[0072] The method of real-time PCR with SYBR Green allowed the performance of an analysis of dissociation of each of the amplicons, which in turn allowed the determination of the melting temperature of each amplicon and confirmed the existence of a single product from the PCR. In Figure 3 are the melting curves for PCR product for each of the candidate genes, where the presence of a single PCR product is confirmed whose size was confirmed by agarose gel.

[0073] With the aim of confirming the expression levels of the candidate genes and their connection with the immune response developed in fish resistant and susceptible to ILA virus, kidney samples from moribund fish and on day 21 post-infection were analyzed. This trial period has been chosen on the basis of the modulation of an immune response and the incubation period described for the experimental induction of ILA (Joseph, T., Kibenge, M. T. and Kibenge, F. 5. B. ( 2003). Antibody-mediated growth of infectious salmon anemia virus in macrophage-like fish cell lines. J Gen Virol 2003. 84:1701- 1710; Totland GK, Hjeltnes BK and Flood PR (1996) Transmission of infectious salmon anemia (ISA) through natura! secretions and excretions from infected smelts of Atlantic salmon (Salmo salar) during their presymptomatic phase. 1996. Dis Aquat Org 26:25- 31). The genes will be presented according to their expression profile. IgM and Grb2 show a similar expression profile, where in the exposed phenotypes it shows a lower expression compared to the control and is statistically significant (p < 0.001). For the gene Grb2, the susceptible phenotype shows an expression 49 times less than the control (p < 0.001).

[0074] For the gene Annexin, individuals from the susceptible and resistant phenotypes showed a significant difference from the control, with a reduction of expression of 32 and 27 times, respectively.

[0075] For the case of Regul, a significant overexpression of the gene was observed in individuals, with a 57-fold increase in the susceptible phenotype and 54-fold in the resistant phenotype.

[0076] For the genes Cathepsin, IgZ, MAVS, Gata3 and Tbet, they have a similar expression profile similarly consistent with a low expression of the susceptible phenotype compared to the control of 36.5, 23, 33.43 and 39 times respectively.

[0077] In order to explain the variance in the data and the expression profile of each gene, a two-way ANOVA was performed, taking into account phenotype and vaccine. This analysis shows the percentage contribution to the variability of the data. The results are shown in Table 4, which shows high percentages with low interaction within the factors. For the phenotype factor, there are 2 genes that mainly contribute to the variability; Grb2 with 88.62% of IgM and 83.02%. The genes mentioned above also represent a low level of interaction (less than 10%), so that each factor separately presents greater explanatory power.

[0078] With the aim of confirming the genes best able to serve as predictors of phenotype, an ROC curve analysis was performed with the raw data for normalized relative expression, in susceptible and resistant phenotype. The results are shown in Table 5. 5 genes are observed that show 100% sensitivity and specificity, resulting in the absence of false negatives or positives. These genes are Gata3, Grb2, IgM, MAVS and Regul. These genes are confirmed as the best candidates for use as predictors of phenotype and were used to conduct a discriminant analysis to test its predictive power.

[0079] In order to check the predictive power of the genes given by the analysis of the ROC curve, a discriminant analysis was performed (Gata3, Grb2, IgM, MAVS and Regul). This was first performed by considering two phenotypes, which could discriminate 100% resistant from susceptible individuals. You can see a summary of the results in table 6. Table 6. Discriminant analysis used genes Gata3, Grb2, IgM, MAVS and Regul as predictors. It is shown that the 9 individuals are correctly classified with a rate equal to 1, which is equivalent to 100% correct classification.

Classification table:

[0080] This presentation was able to establish that there are differences in the expression of genes involved in innate and adaptive immune responses between resistant and susceptible phenotypes for ILA virus, by relative transcript quantification by real-time PCR.

[0081] With regard to the technique used (real-time PCR), it can be mentioned that there are various important factors to take into account in order to carry out a reliable quantification. One of these factors to consider is the integrity and quality of RNA, which exhibits an unstable chemical nature and is susceptible to hydrolysis by basic catalysis or RNase from its own tissue ( Houseley JM, Tollervey D. The many pathways of RNA degradation. 2009 Cell 136 763- 776; DeRose, V. J. Two decades of RNA catalysis. 2002. Chem. Biol. 9: 961- 969.). The effect of the degradation of RNA on real-time PCR results in a lower reproducibility and later Ct values (Fleige, 5. and Pfaffl, M.W. RNA integrity and the effect on the real-time. qRT-PCR performance, 2006. Mol. Aspects Med 27, 126-139). As can be seen in Figure 2, the RNA does not have significant degradation and can therefore be used in further analysis without causing errors in the relative quantification of the candidate genes.

[0082] The presence of a single PCR product was verified through the analysis of the dissociation curves. Fluorine for SYBR Green is 25 to 100 times more sensitive than ethidium bromide ( Schneeberger C, Speiser P, Kury F, Zeillinger R. Quantitative detection of reverse transcriptase- PCR products by means of a novel and sensitive DNA sta in. 1995. PCR Methods Appl 4 : 234- 238.), and will therefore be more likely to detect non-specific products in the analysis of the dissociation curves, than with electrophoresis in agarose gel stained with ethidium bromide. As can be seen in Figure 3, the dissociation curves show a single PCR product, although the products were also analyzed by agarose gel electrophoresis to verify the appearance of the expected band size.

[0083] An aspect to take into account when evaluating the levels of gene expression in biological samples from the same species and treatment is the variation between individuals, which in many cases prevents the finding of statically significant differences between experimental groups. One way to overcome the tendency introduced by heterogeneity is to increase the sample size and in this way deliver increased statistical weight to the results. In the case of this thesis, inter-individual undesirable variations may occur due to difference in efficiency of the infection by the pathogen, or genetic differences in the immune response in each individual. However, a substantial sample size (n = 9) was used to reduce bias.

[0084] As for the relative quantification of genes, expression profiles for some genes were observed for the phenotypes in the study. Two genes that show a similar expression pattern are IgM and Grb2.1, a reduction is observed in the susceptible phenotypes compared to the resistant or the control. IgM is the most dominant immunoglobulin in salmon and a primary line of defense ( Bengten E., Wilson M., Miller N., Clem L. W., Pilstrom L. and Warr GW. Immunoglobulin isotypes: structure, function, and genetics. Curr Top Microbiol Immunol. 2000. 248:189- 219), however, this immunoglobulin appears to be unspecific [ Hordvik I., Lindstrom C. D., Voie A. M., Jacob A. L. J. and Endresen C. Structure and organization of the immunoglobulin M heavy chain genes in Atlantic salmon, Salmo salar. 1997. Molecular Immunology. 34. 631- 639) and with few neutralizing antibodies, but despite this its importance lies in recognition and binding to virus to absorb it, and also complement activation, which increases the number of neutralizing antibodies, as well as B-lymphocytes and promotes activation of adaptive immunity and therefore the effectiveness of the system to fight the virus ( Olesen NJ., Jorgensen PEV. Ouantification of serum immunoglobulin in rainbow trout Salmo gairdneri under various environmental conditions. 1986. Dis Aquat Org 1. 183- 189; Boes, M. Role of natura ! and immune IgM antibodies in immune responses. 2000. Molecular Immunolology 37, 1141- 1149.). In this way, the susceptible individuals will show a lower level in the expression of IgM and slow down the activity of the immune system and show a lower activity and reaction.

[0085] Furthermore, Grb2 is an adapter molecule related to the signaling of the receptors activated by antigen, both T-lymphocytes (TCR) and B-lymphocytes (BCR). This signaling regulates both the development and activation of both lymphocytes. For antigen stimulation to occur, the receptors are activated by tyrosine kinase in the Src family, which provokes phosphorylation of several substrates and the multiple signalosome assemblies. The coordination of the signaling components in the signalosome starts the corresponding signaling cascades that control the transcription and with it the cellular target, activation or other biological processes ( Samelson, L. E. Signal transduction mediated by the Tcell antigen receptor: the role of adapter proteins. 2000. Annu. Rev. Immunol 20:371-394). To combat antigen stimulation, the activated receptor for antigen should recruit negative regulators such as negative tyrosine kinases and phosphatases in the signalosome ( Shaw A and Thomas ML Coordinate interactions of protein tyrosine kinases and protein tyrosine phosphatases in T- cell receptor- mediated signaling. 1991. Curr Opin Cell Biol; 3:862-868.). The balance of positive and negative signals is essential for satisfactory development, activation and induction of lymphocytes. It has been described that Grb2 is fundamental in the activation of Ras-MAPK and the regulation of MAPK, p38 and JNK which are involved in the selection of both positive and negative T-lymphocytes (Houtman JC, Yamaguchi H., Barda Saad M., Braiman A. , Bowden B., Appella E., Schuck P and Samelson LE. Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1. 2006. Nat Struct Mol Biol; 13:798- 805.). This regulation occurs at the start of the cascade triggered by the TCR. It is also described to be involved in the regulation of the signaling for positive selection of the T cells CD4+ and CD8+ (Jang IK., Zhang J., Gu H. ( 2009) Grb2, a simple adapter with complex roles in lymphocyte development, function, and signaling.Immunol Rev 232;150-159). B-lymphocytes have been reported to both positively and negatively regulate the signaling of the BCR as well as the regulation of the signaling of Ca<2+> (Stork B, et al. Grb2 and the non-T cell activation linker NTAL constitute a Ca( 2+)- regulating signal circuit in B lymphocytes. 2004. Immunity 2004; 21:681- 691). The low expression of the gene Grb2 in individuals with a susceptible phenotype will realize a reduction of the activation of the immune response mediated by antigen receptors, generating with this less regulation of a process of selection and development of the T and B lymphocytes, while the opposite occurs in individuals with a resistant phenotype. The differential expression of this gene in susceptible and resistant phenotypes suggests that the induction of lymphocytes T and B will be related to the resistance to the ILA virus.

[0086] While Annexin shows significant differences with control individuals for both phenotypes (susceptible and resistant), it shows a lower expression of the gene. There could be a possibility that the virus could have an inhibitory effect against this gene as described by some viruses having an immunosuppressive effect on some genes (Rønneseth A., Wergeland Hl. and Pettersen EF. Neutrophils and B<ells in Atlantic cod ( Gadus morhua L). 2007. Fish Shellfish Immunol. 3:493-503).

[0087] Regul has a high expression of this in individuals. Regul is a negative regulator of the G protein, since it accelerates the hydrolysis of GTP to the activated subunits of the G protein. This inhibitory effect induced by chemokines acts on signals, which have an important role in moving immune cells to sites of inflammation. It has been described that this gene is widely expressed in lymphocytes, which suggests a role in regulating the immune cells. A strong relationship between the expression of Regul and the migration of the B cells has also been described (C. Moratz, J. R. Hayman, H. Gu and J. H. Kehrl, "Abnormal B-cell responses to chemokines, dist ur bed plasma cell localization, and distorted immune tissue architecture in RGS1-/- mice," Molecular and Cellular Biology, vol. 24, no. 13, pages 5767- 5775. 2004). The inhibition of Regul results in a reduction of the effect of the chemokines and a reduction of the migration of the immune cells, ultimately generating a weakening of the immune response.

[0088] The following 5 genes show a similar expression pattern where the individuals with resistant phenotypes show a similar expression as control without significant differences in individuals, while susceptible individuals showed a low expression of the gene compared to the control.

[0089] Cathepsin S is a cysteine protease that has a critical role in the processing and presentation of antigens. It has been described that Cathepsin S is essential in the presentation of antigens by MHC-II and that its inhibition leads to a reduced response of T-lymphocytes CD4+ (Gupta S., Dennis i., Thurman RE., Kingston R., Stamatoyannopoulos JA. Predicting Human Nucleosome Occupancy from Primary Sequence. 2008. PLoS Comput Biol 4:1-9). Its weakness is also linked to a low mobility of antigen-presenting cells as well as B cells (Faure- André G., Vargas P., Yuseff ML, Heuzé M., Diaz i., Lankar D., Steri V., Manry i ., Hugues S., Vascotto F., Boulanger i., Raposo G., Bono MR., Rosemblatt M., Piel M., Lennon-Duménil AM.Regulation of dendritic cell migration by CD74, the MHC dass ll- associated invariant chain. 2008. Science. 322:1705-10). The susceptible phenotype exhibits a low expression of the gene, which could present problems with the presentation of antigens and mobility of immune cells, thereby primarily affecting the acquired immunity. This effect may contribute to the individuals' susceptibility to this phenotype.

[0090] MAVS is an adapter molecule that acts after the sense helicase RIG-I viral RNA by activating a signaling cascade that ultimately activates the expression of IFN type I that generates other signaling cascades that lead to the transcription of activated interferon genes (ISGs) that are those that code for proteins with antiviral function. In the case of salmon, one can mention Mx-1 which is capable of protecting cells by preventing the replication of IPNV ( Larsen, R., Rokenes, T, Robertsen, B. ( 2004). Inhibition of infectious pancreatic necrosis virus replication by atlantic salmon MX1 protein. J virol 78, 7938-7944.). In this way, individuals with low expression of MAVS exhibit less antiviral activity by inhibited signaling cascade with IFN, which may contribute to the susceptible phenotype.

[0091] IgZ is an immunoglobulin recently discovered in zebrafish about which there is not much information regarding its biological function. It has recently been described that it may be associated with mucosal immunity and neutralization of the antigens in blood (Ryo S., Wijdeven RH., Tyagi A., Hermsen T, Kono T, Karunasagar I., Rombout JH., Sakai M., Verburg-van Kemenade BM. and Savan R. Common carp have two subclasses of bonyfish specific antibody IgZ showing differential expression in response to infection. 2010. Dev Comp Immunol. 34:1183- 1190). This immunoglobulin is expressed in smaller quantities than IgM, but could contribute to the neutralization of the virus and the signaling for activation of the immune system. Due to the fact that individuals with susceptible phenotypes show a low expression of the gene, one could conclude that there is less neutralization of the virus and therefore further progression of the disease.

[0092] T-bet and Gata3 are antagonistic transcription factors involved in T-lymphocyte compromise and targeting, ultimately resulting in the development of Th1 and Th2 lymphocytes. T-bet promotes the differentiation towards lymphocytes of the Thl type, which protect against intracellular parasites and viruses, and produce factors such as IFN, IL-2 and TNF that induce activation of the macrophages and cells NK, T cells producing cytotoxicity (CTL) and complement activation, generates a response that is mainly of a cellular type (Jenner RG., Townsend MJ., Jackson I., Sun K., Bouwman RD., Young RA., Glimcher LH. and Lord GM., The transcription factors T- bet and GATA - 3 control alternative pathways of T-cell differentiation through a shared set of target genes. 2009. Proe Nati Acad Sei USA. 106:17876- 17881). Among other things, Gata3 promotes the differentiation towards lymphocytes of the Th2 type, which with the help of the action of interleukins such as IL-4, IL-5, IL-6 and IL-13 produce effects such as activation of immunity mediated by antibodies, differentiation of eosinophils, inflammatory processes and suppression of the macrophages, and mainly generates a humoral response. The effects from both pathways have an inhibitory effect on the other, this to polarize the target of the lymphocytes towards Thl and Th2. IFN-y inhibits the polarization towards Th2, while IL-4 does so with Thl (Constant SL. and Bottomly K. Induction of Thl and Th2 CD4+ T cell responses the alternative approaches. 1997. Annu Rev Immunol, 15:297- 322). In this way and as can be seen in the graphs Figure 5, it could be observed in susceptible individuals that both pathways are reduced or inhibited, which realizes that processes such as differentiation and polarization of T-lymphocytes do not take place or to a lesser extent compared to resistant individuals or controls . The susceptibility of individuals is explained for the same reason.

[0093] The analysis of the ROC curve was carried out using the data for expression relatively normalized gross resistant and susceptible individuals with the aim of finding genes that can differentiate both phenotypes by means of prediction and that these show a good level of sensitivity and specificity, which translates into an area under the curve (AUC) equal to or close to 1. When performing the analysis, there are 5 genes that present an area under the curve equal to 1, and these are Gata3, Grb2, IgM, Mavs and Regul, which show 100% success. For the same reason, these genes were chosen for a discriminant analysis, which could also distinguish both phenotypes with perfection and check the predictive effect of genes given by the ROC curve. When comparing the displayed genes for the analysis of the ROC curve with the results of the two-way ANOVA, we noticed that the genes Grb2, IgM and Regul have significant percentages in the variation of the data, whether this is associated with the effect of the phenotype in the case of IgM and Grb2, because the variations only affect the susceptible phenotype so that its impact is reduced statistically, resulting in a smaller percentage.

Claims (1)

1. Application of genes selected from the group consisting of Grb2, GATA3, IgM and MAVS and Regul, characterized in that they serve as predictors for determining susceptible and resistant phenotypes for infectious salmon anemia (ILA) in Atlantic salmon (Salmo salar), by quantification of the expression profile of these genes using real-time PCR analysis and where a sample with an expression pattern of challenge for cohabitation with ILA, which has no significant difference compared to the expression pattern of these genes in a control sample without challenge from cohabitation with ILA, is indicative of phenotypic resistance against ILA, namely having as a characteristic a mortality rate which is less than 40% for the ILA virus and a heritability of this character greater than or equal to 0.35; and a sample with a cohabitation challenge expression pattern for ILA that is found to be low compared to a control sample without challenge or cohabitation for ILA is indicative of a phenotype susceptible to ILA, namely presented as a characteristic a mortality rate exceeding 50% for The ILA virus and a heritability of less than or equal to 0.34.