RU2842174C2 - Compositions and methods for preventing fish myocarditis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is protein to generate immune response against fish myocarditis virus (PMCV), containing SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 46, or a sequence that is at least 90% or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least by 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 46. Disclosed are: a fusion protein for generating an immune response against fish myocarditis virus (PMCV); a nucleic acid molecule that encodes said protein or fusion protein; a vector for expression of said protein or fusion protein; host cell; a vaccine for preventing fish myocarditis virus (PMCV) infection in salmonids; method of preventing fish myocarditis virus (PMCV) infection in salmonids.

EFFECT: invention enables preparing the vaccine for preventing infection by fish myocarditis virus.

29 cl, 5 dwg, 31 tbl, 7 ex

Description

AREA OF TECHNOLOGY

[001] This invention generally relates to the field of vaccines for aquaculture.

LEVEL OF TECHNOLOGY

[002] Aquaculture has seen tremendous productivity growth of >527% over the period 1990–2018. In 2018, aquaculture accounted for about 46% of global total aquatic production (179 million tonnes) and 52% of seafood for human consumption (fish, crustaceans, molluscs, and other aquatic animals excluding aquatic mammals, reptiles, seaweeds, and other aquatic plants).

[003] Commercial aquaculture is exposed to infectious diseases caused primarily by bacteria, viruses, parasites and, to a lesser extent, fungi. Bacterial diseases can cause significant biological and therefore economic losses. Although they can usually be controlled with antibiotics, the indiscriminate use of these pharmaceuticals ultimately poses a threat to human health due to the development and transfer of resistance mechanisms among bacterial species, some of which are also human pathogens.

[004] Cardiomyopathy syndrome (CMS) is an inflammatory disease of the heart that primarily affects farmed Atlantic salmon Salmo salar L. The disease was first identified in Norway in 1985 and has since been diagnosed in both farmed and wild Atlantic salmon.

[005] CMS is most often diagnosed at a late stage of exposure to seawater in farmed salmon and is a disease that causes significant losses to the salmon industry. The clinical features of CMS range from acute death without prior clinical signs to increased mortality with non-specific signs such as impaired or abnormal swimming behavior. Diagnosis of CMS is based on the histopathological finding of characteristic inflammation and degeneration of the spongy myocardium in the atria and ventricles.

[006] The causative agent of CMS has been identified as fish myocarditis virus (PMCV), and the isolation and characterization of the virus are described in WO 2011/131600. Further understanding of the agent causing CMS was provided by the isolation and cultivation of the CMS virus disclosed in NO 2008 2869. The host cells for culturing the CMS virus disclosed in NO 2008 2869 showed low yields of CMS virus.

[007] There is still a need for further knowledge to be able to develop effective means to combat this disease and to develop effective vaccines such as recombinant vaccines.

ESSENCE OF THE INVENTION

[008] In a first aspect, the present invention relates to a protein comprising from the N-terminus to the C-terminus:

a. a sequence that is at least 90% identical, or at least 91% identical, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or 100% identical to SEQ ID NO: 34 or SEQ ID NO: 35 and

b. an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 25-33,

at the same time,

i) said fish myocarditis virus (PMCV) ORF-1 antigen does not have SEQ ID NO: 15 or a sequence at least 90% identical to SEQ ID NO: 15; and/or

ii) compared to SEQ ID NO: 1, said protein comprises an internal deletion of at least four consecutive amino acids in length.

[009] In some embodiments, the fish myocarditis virus (PMCV) ORF-1 antigen is at least 90% identical to SEQ ID NO: 11, SEQ ID NO: 12, or 46.

[0010] In some embodiments, the protein is at least 95% identical to SEQ ID NO: 25 and lacks SEQ ID NO: 14 or a sequence that is at least 90% identical to SEQ ID NO: 14. Preferably, the protein lacks one or more of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 15 or a sequence that is at least 90% identical thereto.

[0011] In some other embodiments, a protein of the invention comprises a sequence that is at least 95% identical to SEQ ID NO: 26 and lacks SEQ ID NO: 14, or a sequence that is at least 90% identical to SEQ ID NO: 14. The protein can comprise a sequence that is at least 95% identical to SEQ ID NO: 26 and lacks SEQ ID NO: 14, or a sequence that is at least 90% identical to SEQ ID NO: 14 and is further lacking one or more of SEQ ID NOs: 5, 15, or 24, or a sequence that is at least 90% identical thereto.

[0012] In other embodiments, the protein comprises a sequence that is at least 95% identical to SEQ ID NO: 27 and does not have SEQ ID NO: 14, or a sequence that is at least 90% identical to SEQ ID NO: 14. The protein can comprise a sequence that is at least 95% identical to SEQ ID NO: 27 and does not have SEQ ID NO: 14, or a sequence that is at least 90% identical to SEQ ID NO: 14 and is further lacking one or more of SEQ ID NOs: 3, 15, or 24, or a sequence that is at least 90% identical thereto.

[0013] In other embodiments, the protein comprises a sequence that is at least 90% identical to SEQ ID NO: 28. Optionally, the protein lacks one or more of SEQ ID NO: 3, 4, or 24, or a sequence that is at least 90% identical thereto.

[0014] In other embodiments, the protein comprises SEQ ID NO: 29 or SEQ ID NO: 30, or a sequence at least 95%, or 96%, or 97%, or 98%, or 99% identical to any of these sequences. Preferably, the protein lacks at least one of SEQ ID NO: 3 or SEQ ID NO: 24, or an amino acid sequence at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 24.

[0015] In other embodiments, the protein comprises SEQ ID NO: 32 or a sequence at least 95%, or 96%, or 97%, or 98%, or 99% identical thereto, and optionally does not comprise at least one of SEQ ID NO: 14 or SEQ ID NO: 15, or an amino acid sequence at least 90% identical to SEQ ID NO: 14 or SEQ ID NO: 15.

[0016] In other embodiments, the protein comprises SEQ ID NO: 33 or a sequence at least 95%, or 96%, or 97%, or 98%, or 99% identical thereto and optionally not comprising SEQ ID NO: 24, an amino acid sequence that is at least 90% identical to SEQ ID NO: 24.

[0017] In any of the embodiments described above, the protein may further lack at least one of SEQ ID NOs 2 and 13, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or 13.

[0018] In some embodiments, the protein comprises SEQ ID NO: 11 or 12, or a sequence that is at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 12.

[0019] In a second aspect, the present invention provides a fusion protein comprising a protein according to any one of the embodiments of the first aspect of the present invention and further comprising:

a) an N-terminal secretion signal sequence of a secreted or first membrane-bound protein located upstream of a protein according to any of the embodiments of the first aspect of the present invention;

b) a transmembrane domain of a second membrane-associated protein located downstream of the protein according to any of the embodiments of the first aspect of the present invention.

[0020] In some embodiments, the first membrane-bound protein is identical to the second membrane-bound protein. In some embodiments, said membrane-bound protein is human septicemia virus G protein (VHSV-G). Preferably, said N-terminal secretion signal sequence is at least 90% identical to SEQ ID NO: 7. In some embodiments, said N-terminal secretion signal sequence is at least 90% identical to SEQ ID NO: 7 and at least half of the different amino acids in said N-terminal secretion signal are conservative substitutions.

[0021] In some embodiments, said transmembrane domain is at least 90% identical to SEQ ID NO: 8. Preferably, said transmembrane domain is at least 90% identical to SEQ ID NO: 8 and at least half of the different amino acids in said N-terminal secretion signal are conservative substitutions.

[0022] In a third aspect of the invention, nucleic acid sequences encoding a protein according to any embodiment of the first aspect or a fusion protein according to the second aspect of the invention are disclosed. In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 16.

[0023] In a fourth aspect, the invention provides a cassette comprising a nucleic acid sequence according to any embodiment of the third aspect of the invention. In some embodiments, the nucleic acid sequence is under the operative control of a promoter. In a certain aspect, the promoter is a CMV promoter.

[0024] In a fifth aspect of the present invention, there is provided a vector comprising a cassette according to any embodiment of the fourth aspect of the present invention or a nucleic acid sequence according to any embodiment of the third aspect of the present invention.

[0025] In some embodiments, the vector also encodes a nucleic acid sequence that encodes an immunomodulator. In some embodiments, the immunomodulator is an interferon, such as a Salmo salar IFNb type interferon that comprises SEQ ID NO: 6 or an IFNb1 type interferon that comprises SEQ ID NO: 50, wherein said nucleic acid sequence that encodes the interferon is at least 65% identical to SEQ ID NO 18 or 19, such as at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical, and wherein the codon frequency is maintained in said nucleic acid sequence. In some embodiments, said nucleic acid sequence is 65-90% identical to SEQ ID NO: 18 or 19, and wherein the codon frequency is maintained in said nucleic acid sequence. In some embodiments, a nucleic acid sequence encoding an interferon comprises SEQ ID NO: 17 or SEQ ID NO: 20, or a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 17 or SEQ ID NO: 20, and wherein said nucleic acid sequence that encodes an interferon encodes SEQ ID NO: 6 or SEQ ID NO: 50, or an amino acid sequence that is 95% identical to SEQ ID NO: 6 or SEQ ID NO: 50.

[0026] In some embodiments, the vector may be a plasmid vector or a viral vector.

[0027] In a sixth aspect, a host cell comprising a vector according to any embodiment of the fifth aspect is provided.

[0028] In a seventh aspect, the invention provides a vaccine comprising a protein according to any embodiment of the first aspect, a fusion protein according to any embodiment of the second aspect, or a vector according to the fifth aspect of the invention. In some embodiments, the vaccine further comprises an adjuvant. In some embodiments, the vaccine also provides a carrier. In one embodiment, the carrier is a lipid or liposomal carrier.

[0029] In some embodiments, the vaccine is a multivalent vaccine and comprises one or more additional antigens, preferably selected from the group consisting of SAV (salmon alphavirus, including SAV-1, SAV-2, SAV-3, SAV-4, SAV-5 and SAV-6), ISAV (infectious salmon anemia virus), IPNV (infectious pancreatic necrosis virus), ASPV (Atlantic salmon poxvirus), IHNV (infectious hematopoietic necrosis virus), VHSV (viral hemorrhagic septicemia virus), PRV (priscin orthoreovirus), etc. Aeromonas salmonicida Salmonicida proteins, Vibrio (Listonella) anguillarum serotype O1, Vibrio (Listonella) anguillarum serotype O2a, Vibrio salmonicida, Moritella viscosa and sea lice (including Lepeophtheirus salmonis and/or Caligus rogercresseyi ).

[0030] In an eighth aspect, the invention provides a method for protecting salmonids from PMCV infection, the method comprising administering to the salmonids in need thereof a vaccine according to any embodiment of the seventh aspect of the invention. In some embodiments, the salmon weighs from 15 to 200 grams. In some embodiments, the salmon is an Atlantic salmon ( Salmo salar ), a rainbow trout ( Oncorhynchus mykiss ), a coho salmon ( Oncorhynchus kisutch ), or a chinook salmon ( Oncorhynchus tshawytscha ).

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0031] Figure 1A shows the overall structure of the Totiviridae virion. Figure 1B shows an illustration of the organization of the PMCV genome. The genome includes three large ORFs.

[0032] Fig. 2A illustrates a strategy for routing a vaccine antigen for expression on the cell surface. Fig. 2B illustrates a strategy for facilitating antigen secretion.

[0033] Fig. 3A is a photograph demonstrating detection of PMCV ORF1 in non-permeabilized cells. Fig. 3B shows the results obtained using different expression cassettes used to route full-length PMCV ORF-1 to the cell surface.

[0034] Figure 4A shows the evaluation of the fluorescence intensity of the truncated capsid variants by IF staining of transfected CHH-1 cells. Figure 4B shows an illustration of protein expression by Western blotting from a membrane fraction by immunoprecipitation (IP-Western). The two blots represent two different experiments.

[0035] Figure 5 shows the evaluation of protein expression in five different plasmid backbones by immunoprecipitation from total cell lysate of transfected CHH-1 cells and Western blotting.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

[0036] To better explain the present invention, the following definitions are provided.

[0037] The term "about" when applied to a reference number refers to the reference number plus or minus 10 percent of the stated value.

[0038] The term "the frequency of codons is conserved in the said nucleic acid sequence" describes a nucleic acid sequence in which the frequencies of at least 50% of the codons in the nucleic acid sequence of the present invention do not deviate by more than 40% from the frequencies of the same codons in the host genome. In the case where, for example, the frequency of a given codon in the host genome is, for example, 20%, then this codon is included in at least 50% of the codons whose frequency does not deviate by more than 40% from the frequency of the same codon in the host genome, if the frequency of the same codon in the nucleic acid sequence according to the present invention is between 12% (40% less than 20%) and 28% (40% more than 20%).

[0039] For example, codon frequencies in the Atlantic salmon ( Salmo salar ) genome are shown in Table 1.

TABLE 1. CODON FREQUENCY IN THE ATLANTIC SALMON GENOME

AMINO ACID CODONE For 1000 FREQUENCY GLY GGG 15.87 21.91 GLY GGA 20,24 27.94 GLY GGT 14.03 19.37 GLY GGC 22.3 30.78 ASP GAT 16.76 32.69 ASP GAC 34.51 67.31 GLU GAG 45.67 76.69 GLU GAA 13.88 23.31 VAL GTG 29.06 45.71 VAL GTA 6.19 9.74 VAL GTT 9.77 15.37 VAL GTC 18.56 29.19 ALA GCG 7.2 11.08 ALA GCA 13.42 20.65 ALA GCT 17.5 26.92 ALA GCC 26.88 41.35 ARG AGG 13.4 26.85 ARG AGA 10,12 20.28 ARG CGG 6.61 13.25 ARG CGA 4.01 8,036 ARG CGT 5.74 11.50 ARG CGC 10.02 20.08 SER AGT 11.2 13.54 SER AGC 21.01 25.40 SER TCG 5.31 6.42 SER TCA 10.15 12.27 SER TCT 13.86 16.76 SER TCC 21.18 25.61 LYS AAG 39.68 69.87 LYS AAA 17.11 30.13 ASN AAT 11.42 28.36 ASN AAC 28.85 71.64 MET ATG 27,27 100 ILE ATA 6.37 13.59 ILE ATT 11.34 24.19 ILE ATC 29.17 62.22 TRP TGG 11.53 100 CYS TGT 11.05 46.80 CYS TGC 12.56 53.20 TYR TAT 8.12 27.21 TYR TAC 21.72 72.79 LEU TTG 10.16 10.97 LEU TTA 3.44 3.71 LEU CTG 45.99 49.65 LEU CTA 7.18 7.75 LEU CTT 7.87 8.50 LEU CTC 17.98 19.41 PHE TTT 12.55 32.54 PHE TTC 26.02 67.46 G.L.N. CAG 35.96 81.21 G.L.N. CAA 8.32 18.79 HIS CAT 8.63 32,43 HIS CAC 17.98 67.57 PRO CCG 5.93 11.09 PRO CCA 13.3 24.86 PRO CCT 13.89 25.97 PRO CCC 20.37 38.08 THR ACG 6.91 11.85 THR ACA 14.69 25.18 THR ACT 12.76 21.88 THR ACC 23.97 41.09 AMINO ACID CODONE For 1000 FREQUENCY

[0040] The CGC codon codes for arginine with a frequency of 20.08% - i.e. 20.08% of the arginine residues in the Salmo salar genome are encoded by CGC. If between 12.048% and 28.112% of the arginine residues in a nucleic acid sequence are encoded by CGC, then the CGC codon is included in at least 50% of the codons whose frequency does not deviate by more than 40% from the frequency of the same codon in the Salmo salar genome.

[0041] "A therapeutically effective amount" refers to an amount of an antigen or vaccine that would elicit an immune response in a subject receiving the antigen or vaccine that is sufficient to prevent or reduce the signs or symptoms of a disease, including adverse health effects or complications thereof, caused by infection with a pathogen, such as a virus, ectoparasite, or bacterium. Humoral immunity or cell-mediated immunity or both humoral and cell-mediated immunity may be induced. The immunogenic response of an animal to a vaccine can be assessed, for example, indirectly by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms following infection with a wild-type strain. Protective immunity provided by a vaccine can be assessed by measuring, for example, a reduction in clinical signs such as mortality, morbidity, temperature, general physical condition, and the general health and performance of the subject. Protective immunity can also be assessed by assessing histopathological lesions in tissues and quantifying viral load, such as by qPCR. The amount of vaccine that is therapeutically effective may vary depending on the specific adjuvant used, the specific antigen used, or the condition of the subject, and can be determined by one skilled in the art.

[0042] "Treatment" refers to the prevention of the disorder, condition, or disease to which the term applies, or to the prevention or reduction of one or more symptoms of such disorder, condition, or disease.

[0043] The inventors of the present invention have found that the N-terminal portion of SEQ ID NO: 1 (the ORF-1 protein of fish myocarditis virus (PMCV)), i.e. amino acids 1 to about 399 of SEQ ID NO: 1, or more preferably amino acids 1 to 425, or more preferably amino acids 1 to 450, or more preferably amino acids 1 to 475, or most preferably amino acids 1-499 of SEQ ID NO: 1, are necessary and sufficient for targeting an antigen to the cell surface.

[0044] At the same time, the inventors of the present invention unexpectedly found that the antigen according to the present invention elicits a better immune response, measured by viral load and/or histological score, if

a) SEQ ID NO: 25 is deleted while SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 31 is retained; or

b) SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 31 is deleted while SEQ ID NO: 25 is retained; or

c) SEQ ID NO: 14 is deleted while SEQ ID NO: 32 is retained; or

d) SEQ ID NO: 2 is deleted while one or more of SEQ ID NO: 13, 27, 28, 29, 31 are retained.

[0045] It was also found that the presence of SEQ ID NO: 15 was not sufficient to ensure an adequate immune response if SEQ ID NO: 32 was removed.

[0046] Accordingly, in a first aspect, the present invention provides a protein that comprises from the N-terminus to the C-terminus:

a) a sequence that is at least 90% identical, or at least 91% identical, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or 100% identical to SEQ ID NO: 34 or SEQ ID NO: 35 and

b) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 25-33,

moreover:

i) said protein lacks SEQ ID NO: 15 or has a sequence that is at least 90% identical to SEQ ID NO: 15; and/or

ii) compared to SEQ ID NO: 1, said protein comprises an internal deletion of at least four consecutive amino acids in length, and, optionally, said protein comprises a linker in place of said internal deletion.

[0047] In all embodiments, the differences from the reference sequence (SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) may be deletions, insertions, or substitutions. Preferably, at least some of the substitutions are conservative substitutions. In some embodiments, at least 50% (i.e., at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or 100%) of the substituted amino acids are conservative substitutions.

[0048] In embodiments where the fish myocarditis virus (PMCV) ORF-1 antigen does not have SEQ ID NO: 15, it is understood that a larger deletion, such as a C-terminal truncation, may also be performed in the antigen. In some embodiments, such a larger C-terminal truncation comprises or consists of SEQ ID NO: 13.

[0049] In some embodiments, the protein described above comprises SEQ ID NO: 25 or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 25, wherein said PMCV ORF-1 antigen does not have SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 14 or SEQ ID NO: 15 or a sequence that is at least 90% identical to SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 14 or SEQ ID NO: 15.

[0050] In other embodiments, the protein described above comprises a sequence that is at least 95% identical to SEQ ID NO: 29, or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 29, wherein said PMCV ORF-1 antigen does not have SEQ ID NO: 5, or SEQ ID NO: 14, or SEQ ID NO: 15, or a sequence that is at least 90% identical to SEQ ID NO: 5, or SEQ ID NO: 14, or SEQ ID NO: 15.

[0051] In other embodiments, the protein described above comprises a sequence that is at least 95% identical to SEQ ID NO: 32, or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 32, wherein said PMCV ORF-1 antigen does not have SEQ ID NO: 14 or SEQ ID NO: 15, or a sequence that is at least 90% identical to SEQ ID NO: 14 or SEQ ID NO: 15.

[0052] In other embodiments, the protein comprises SEQ ID NO: 26 or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 26 and does not have SEQ ID NO: 5 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 24 or a sequence that is at least 90% identical to SEQ ID NO: 5 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 24.

[0053] In other embodiments, the protein comprises SEQ ID NO: 30 or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 30 and does not have SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 24 or a sequence that is at least 90% identical to SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 24.

[0054] In other embodiments, the protein comprises SEQ ID NO: 33 or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 33 and does not have SEQ ID NO: 24, or a sequence that is at least 90% identical to SEQ ID NO: 24.

[0055] In other embodiments, the protein comprises SEQ ID NO: 27 or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 27 and does not have SEQ ID NO: 3 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 24 or a sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 24.

[0056] In other embodiments, the protein comprises SEQ ID NO: 31 or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 31 and does not have SEQ ID NO: 3 or SEQ ID NO: 24, or a sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 24.

[0057] In other embodiments, the protein comprises SEQ ID NO: 28 or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 28 and does not have SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 14 or a sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 14.

[0058] In other embodiments, the protein comprises SEQ ID NO: 15 or a sequence at least 90% identical thereto, and any of SEQ ID NOs: 4, 5, 25, 26, 27, 29, 30, 32 (or an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, 5, 25, 26, 27, 29, 30, or 32), wherein, compared to SEQ ID NO: 1, said protein comprises an internal deletion of at least four consecutive amino acids in length, and, optionally, said protein comprises a linker in place of said internal deletion.

[0059] In other embodiments, the protein lacks SEQ ID NO: 13 or a sequence that is at least 90% identical to SEQ ID NO: 13 and comprises SEQ ID NO: 25, or a sequence that is at least 95%, or 96%, or 97%, or 98%, or 99% identical to SEQ ID NO: 25.

[0060] Compared to SEQ ID NO: 1, said fish myocarditis virus (PMCV) ORF-1 antigen may comprise an internal deletion of at least four consecutive amino acids in length. Typically, the internal deletion may be up to 250 amino acids in length. Preferably, the internal deletion is about 10 amino acids long, or 10 to 250 amino acids long, or 25 to 250 amino acids long, or 50 to 250 amino acids long, or 100 to 250 amino acids long, or 150 to 250 amino acids long, or 10 to 200 amino acids long, or 25 to 200 amino acids long, or 50 to 200 amino acids long, or 100 to 200 amino acids long, or 150 to 200 amino acids long, or about 200 amino acids long, or about 50 amino acids long, or 50 to 150 amino acids long, or 50 to 100 amino acids long, or about 100 amino acids long. In some embodiments, the internal deletion comprises or consists of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 14.

[0061] In some embodiments, a linker may be present in place of said internal deletion. The length of the internal deletion and the linker need not be the same. For example, the linker may be 3-50 amino acids long, or 3-40 amino acids long, or 3-30 amino acids long, or 3-20 amino acids long, or 3-10 amino acids long, or about 5 amino acids long, or about 10 amino acids long, or about 20 amino acids long. The exact sequence of the linker is not very important. In general, the preferred amino acids are polar uncharged or charged residues that make up about 50% of the naturally encoded amino acids. More specifically, threonine, serine, and glycine can provide good flexibility due to their small sizes, and also help maintain the stability of the linker structure in an aqueous solvent by forming hydrogen bonds with water. In some embodiments, the linker is rich in glycine and/or serine. In some embodiments, the linker is 5 amino acids in length and may comprise SEQ ID NO: 36. SEQ ID NO: 36 may be encoded by SEQ ID NO: 37.

[0062] A transmembrane domain of a second membrane-bound protein located downstream of a protein according to any one of the embodiments of the first aspect of the invention. Thus, such engineered antigens will be exposed on the cell surface where they can be recognized by the host immune system and thus a protective immune response will be generated. Accordingly, in a second aspect, the invention provides a fusion protein comprising from the N-terminus to the C-terminus:

a) the N-terminal secretion signal sequence of the secreted or first membrane-bound protein;

b) a protein according to any of the embodiments of the first aspect, as described above;

c) transmembrane domain of the second membrane-associated protein.

[0063] In some embodiments, the first protein may be selected from the group consisting of interferon a, interferon b, interferon c, interferon d, interferon gamma, interleukin-2, interleukin-4, interleukin-12. The first and second proteins may be identical to or different from each other. In some embodiments, the first and/or second protein may be an Atlantic salmon paramyxovirus fusion protein whose signal and transmembrane domain sequences are SEQ ID NOs: 38 and 39, respectively. In some embodiments, the first and second protein may be an infectious salmon anemia virus (ISAV) hemagglutinin (HE) protein whose signal and transmembrane secretion domain sequences are SEQ ID NOs: 40 and 41, respectively.

[0064] In some embodiments, the first membrane protein is identical to the second membrane protein and is the human septicemia virus G protein (VHSV-G), whose secretion signal peptide sequence and transmembrane domain sequences are SEQ ID NOs: 7 and 8, respectively.

[0065] Thus, in some embodiments, said N-terminal secretion signal sequence is at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 7.

[0066] As described above, it is preferred that mutations resulting in differences from SEQ ID NO:7 are substitutions. In some preferred embodiments, at least half (or at least 60%, or at least 70%, or at least 80%, or at least 90%) of the differing amino acids in said N-terminal secretion signal are conservative substitutions.

[0067] In embodiments where the VHSV-G protein is the source of both the N-terminal secretion signal and the transmembrane domain, said transmembrane domain is at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 8.

[0068] As described above, it is preferred that mutations resulting in differences from SEQ ID NOs 7 and 8 are substitutions. In some preferred embodiments, at least half (or at least 60%, or at least 70%, or at least 80%, or at least 90%) of the differing amino acids in said transmembrane domain are conservative substitutions.

[0069] Additional fragments of the first and/or second protein may also be included. Thus, in some embodiments, SEQ ID NO: 7 or a protein that is at least 90% identical to SEQ ID NO: 7 may be included in SEQ ID NO: 48, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 48, or a fragment thereof. It should be understood that said fragment will include SEQ ID NO: 7 or a sequence that is at least 90% identical to SEQ ID NO: 7.

[0070] Regardless, SEQ ID NO: 8 or a protein that is at least 90% identical to SEQ ID NO: 8 may be included in SEQ ID NO: 49, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 49, or a fragment thereof. It should be understood that said fragment will include SEQ ID NO: 8 or a sequence that is at least 90% identical to SEQ ID NO: 8.

[0071] Like SEQ ID NOs: 7 and 8, mutations resulting in differences from SEQ ID NOs: 48 and 49 are substitutions. In some preferred embodiments, at least half (or at least 60%, or at least 70%, or at least 80%, or at least 90%) of the differing amino acids in said N-terminal secretion signal sequence and/or in the transmembrane domain are conservative substitutions.

[0072] In other embodiments, the protein may comprise SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 46, or an amino acid sequence that is at least 90% identical, or at least 91% identical, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 46. Preferably, the differences are substitutions, and more preferably at least 50% identical (or at least 60% identical, or at least 70% identical, or at least 80% identical, or at least 90% identical, or at least 95%, or at least 99%) of the replaced amino acids are conservative substitutions.

[0073] In particular embodiments, the fusion protein sequence comprises or consists of SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 47, or an amino acid sequence that is at least 90% identical, or at least 91% identical, or at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or 100% identical to SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 47. Preferably, the differences are substitutions, and more preferably at least 50% (or at least 60% identical, or at least 70% identical, or at least 80% identical, or at least 90% identical, or at least 95%, or at least 99%) of the replaced amino acids are conservative substitutions.

[0074] In a third aspect, the invention provides a nucleic acid sequence that encodes a protein according to any of the embodiments of the first aspect or a fusion protein according to any of the embodiments of the second aspect of the invention. The nucleic acid sequence according to the third aspect of the invention may, in some embodiments, comprise SEQ ID NO: 16 or be at least 90% (at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical thereto.

[0075] In a fourth aspect, the invention provides an expression cassette comprising a nucleic acid sequence according to any embodiment of the third aspect of the invention. Preferably, the nucleic acid sequence according to any embodiment of the third aspect of the invention is under the operative control of a first promoter. The first promoter can be selected from exemplary promoters such as the simian virus 40 (SV40) early promoter, the cytomegalovirus (CMV) immediate early promoter, the human ubiquitin C (UBC) promoter, the human elongation factor 1α (EF1A) promoter, the mouse phosphoglycerate kinase 1 (PGK) promoter, and the chicken β-actin promoter in combination with the CMV early enhancer (CAGG). Typically, the expression cassette of the invention also comprises a polyadenylation signal that terminates the transcription of the nucleic acid sequence encoding the antigen.

[0076] Optionally, the cassette may also comprise a nucleic acid sequence encoding a molecular immunomodulator under the operative control of a second promoter. Typically, the second promoter should be capable of initiating transcription in the host organism. In embodiments where the host is salmon, such as Salmo salar , suitable promoters include, but are not limited to, the simian virus 40 (SV40) early promoter, the cytomegalovirus (CMV) immediate early promoter, the human ubiquitin C (UBC) promoter, the human elongation factor 1α (EF1A) promoter, the mouse phosphoglycerate kinase 1 (PGK) promoter, and the chicken β-actin promoter combined with the CMV early enhancer (CAGG). Typically, the expression cassette of the present invention also comprises a polyadenylation signal that terminates transcription of the nucleic acid sequence of the present invention.

[0077] In a fifth aspect, the present application discloses a vector comprising a cassette according to any of the embodiments of the fourth aspect of the present invention. Various vectors suitable for the present invention are known in the art, including both plasmid vectors and viral vectors. Suitable plasmids include, but are not limited to, pUC-based vectors, pVAX vectors, pcDNA vectors, NTC vectors. In a number of preferred embodiments, the vector is NTC9385R (Nature Technology Corporation) or a variant thereof, as described in the examples.

[0078] In other embodiments, the relatively new dog bone plasmid or ^dbDNATM can be used as a vector. ^dbDNATM plasmids, as well as the process for producing these plasmids, are described in at least WO2018033730, WO2016034849, WO2019193361, WO2012017210, and WO2021161051. An advantage of this approach is that the vector can be synthesized in a cell-free manner, which increases production efficiency. The cell-free process preferably involves amplifying the template by strand displacement replication. This synthesis releases single-stranded DNA, which in turn can be copied into double-stranded DNA using a polymerase. Alternatively, strand displacement can be achieved by feeding a DNA polymerase and a separate helicase. Replicative helicases can open the DNA duplex and promote leading-strand polymerase. The resulting double-stranded DNA concatemer is enzymatically cut and ligated, thus forming a dog-bone-shaped DNA construct.

[0079] Suitable viral vectors include, but are not limited to, alphaviruses such as SAV, rhabdoviruses such as VHSV and IHNV, paramyxoviruses such as ASPV, adenoviruses, poxviruses such as salmon poxvirus, and the like. These viruses can be genetically modified to remove portions of the viral genome responsible for replication. The resulting viruses will thus be infectious to fish cells and suitable for antigen production, but will not be pathogenic.

[0080] In embodiments where the cassette does not contain a nucleic acid sequence encoding a molecular immunomodulator, such nucleic acid sequence may still be present in the vector.

[0081] In some embodiments suitable for both the vector of this aspect and the cassette of the fourth aspect, the molecular immunomodulator is an interferon, preferably IFNb or the like, and is encoded by SEQ ID NO: 6 (IFN-b protein) or SEQ ID NO: 50 (IFNb1 protein), or a sequence that is at least 95% (such as at least 96% or at least 97% or at least 98% or at least 99%) identical to SEQ ID NO: 6 or 50, wherein said nucleic acid sequence is 65-90% identical to SEQ ID NO: 18 or 65-90% identical to SEQ ID NO: 19, and wherein the codon frequency is maintained in said nucleic acid sequence.

[0082] The nucleic acid sequences encoding the interferon are identical to SEQ ID NO: 18 or SEQ ID NO: 19, provided that the codon frequency is maintained in said nucleic acid sequences encoding the interferon. In some embodiments, the nucleic acid sequences encoding the interferon are 70-79% or 73-77% identical to SEQ ID NO: 18 or SEQ ID NO: 19, provided that the codon frequency is maintained in said nucleic acid sequences encoding the interferon.

[0083] As disclosed above, the frequencies of at least 50% of the codons in the nucleic acid sequence encoding the interferon do not deviate by more than 40% from the frequencies of the same codons in the host genome. In some embodiments, the frequencies of at least 40% of the codons in the nucleic acid sequence encoding the interferon do not deviate by more than 30% from the frequencies of the same codons in the host genome and/or the frequencies of at least 30% of the codons in the nucleic acid sequence encoding the interferon do not deviate by more than 25% from the frequencies of the same codons in the host genome and/or the frequencies of at least 25% of the codons in the nucleic acid sequence encoding the interferon do not deviate by more than 20% from the frequencies of the same codons in the host genome. If the host is Salmo salar , the Salmo salar codon frequencies are presented in Table 1.

[0084] In some embodiments, the nucleic acid sequences comprise SEQ ID NO: 17 or SEQ ID NO: 20, or are at least 90% identical (i.e., more than 91% identical, more than 92% identical, more than 93% identical, more than 94% identical, more than 95% identical, more than 96% identical, more than 97% identical, more than 98% identical, or more than 99% identical) to one of SEQ ID NO: 17 or SEQ ID NO: 20, and the nucleic acid sequences encode SEQ ID NO: 6 or an amino acid sequence that is at least 95% identical thereto, as described above.

[0085] In some embodiments, the vector may comprise an expression cassette as described above and an additional antigen, such as a salmon alphavirus antigen. It is also possible that the vector will comprise an additional nucleic acid sequence encoding another molecular immunomodulator.

[0086] The vector of any embodiment of this fifth aspect of the invention and/or the cassette of the fourth aspect may optionally include amino acid sequences encoding additional antigen(s). In general, these additional antigens may be of viral, bacterial, protozoan or parasitic origin. Suitable viruses include PMCV, SAV, ISA, IPNV, ASPV (Atlantic salmon poxvirus), IHNV (infectious hematopoietic necrosis virus), VHSV (viral hemorrhagic septicemia virus) and PRV. Antigens from enveloped and non-enveloped viruses may be included. In some embodiments, the antigen is a viral structural protein, a capsid protein or an outer surface protein. Also suitable are fragments of these proteins capable of eliciting a protective immune response.

[0087] Suitable bacteria include, but are not limited to, Aeromonas salmonicida sub. Salmonicida, Vibrio (Listonella) anguillarum, Vibrio salmonicida, Moritella viscosa , and Yersinia ruckeri . Again, proteins present on the outer surface of the bacteria are preferred antigens, and these proteins are capable of eliciting a protective immune response.

[0088] Suitable parasites include sea lice (family Caligidae, preferably genera Lepeophtheirus or Caligus). Proteins derived from sea lice and capable of eliciting a protective immune response have been described and include intestinal peptides or fragments thereof, including, but not limited to, SEQ ID NO: 21, or a sequence at least 90% identical thereto.

[0089] It will be understood by one skilled in the art that differences between the described amino acid sequences and the reference amino acid sequences (whether in the context of a protein according to any embodiment of the first aspect or a fusion protein according to any embodiment of the second aspect, or a molecular immunomodulator suitable in some embodiments of the fourth or fifth aspect of the invention) may be in the form of insertions, deletions or substitutions. Preferably, the mutations are substitutions, and more preferably, at least some of these substitutions are conservative substitutions.

[0090] One skilled in the art will further recognize that changes in a nucleic acid sequence that result in modifications to the amino acid sequence of the protein it encodes may have little, if any, effect on the final three-dimensional structure of the protein. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be replaced by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes that result in the substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, may also be expected to result in a protein with substantially the same functional activity.

[0091] The following six groups contain amino acids that are typical conservative substitutions for each other: [1] alanine (A), serine (S), threonine (T); [2] aspartic acid (D), glutamic acid (E); [3] asparagine (N), glutamine (Q); [4] arginine (R), lysine (K), histidine (H); [5] isoleucine (I), leucine (L), methionine (M), valine (V); and [6] phenylalanine (F), tyrosine (Y), tryptophan (W) (see, e.g., U.S. Patent Publication 20100291549).

[0092] In some embodiments, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or all 100% of the amino acids that differ from the reference sequence are conservative substitutions.

[0093] The identity of protein and/or nucleic acid sequences according to any of the embodiments described above can be assessed using any of a variety of sequence comparison algorithms and programs known in the art. For sequence comparison, typically one sequence acts as a reference sequence (e.g., a sequence disclosed herein) to which test sequences are compared. The sequence comparison algorithm then calculates the percent identity of the test sequences compared to the reference sequence based on program parameters.

[0094] The percent identity of two amino acids or two nucleic acid sequences can be determined, for example, by comparing the sequence information using a GAP computer program, i.e., the Genetics Computer Group (GCG; Madison, WI) program, version 10.0, GAP program (Devereux et al. (1984), Nucleic Acids Res. 12: 387-95). When calculating the percent identity, the sequences being compared are typically aligned in a manner that provides the greatest match between the sequences. The preferred default parameters for the GAP program include: (1) the GCG implementation of the unary comparison matrix (containing the value 1 for identity and 0 for non-identity) for nucleotides and the weighted amino acid comparison matrix of Gribskov and Burgess ((1986) Nucleic Acids Res. 14: 6745), as described in Atlas of Polypeptide Sequence and Structure , Schwartz and Dayhoff, eds., National Biomedical Research Foundation, pp. 353–358 (1979), or other comparable comparison matrices; (2) a penalty of 8 for each gap and an additional penalty of 2 for each character in each gap for amino acid sequences, or a penalty of 50 for each gap and an additional penalty of 3 for each character in each gap for nucleotide sequences; (3) no penalties for end gaps; and (4) no maximum penalty for long gaps.

[0095] Sequence identity and/or similarity can also be determined using the local sequence identification algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, and computerized implementations of these algorithms (BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, WI).

[0096] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignment. It can also construct a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method proposed by Feng & Doolittle, 1987, J. Mol. Evol. 35:351–360; the method is similar to the method described by Higgins & Sharp, 1989, CABIOS 5:151–153. Useful parameters of PILEUP include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0097] Another example of a useful algorithm is the BLAST algorithm, described in: Altschul et al. , 1990, J. Mol. Biol. 215:403-410; Altschul et al. , 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al. , 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, derived from Altschul et al. , 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which have default values. The adjustable parameters are set with the following values: overlap range = 1, overlap fraction = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values and are set by the program itself depending on the composition of the specific sequence and the composition of the specific database in which the sequence of interest is searched; however, the values can be adjusted to improve sensitivity.

[0098] An additional useful algorithm is gapped BLAST, as reported by Altschul et al. , 1993, Nucl. Acids Res. 25:3389–3402. Gapped BLAST uses BLOSUM-62 substitution scores; the threshold parameter T is set to 9; the two-hit method for running non-gapped extensions charges a cost of 10+k for gaps of length k; X _u is set to 16 and X _g is set to 40 for the database search step and 67 for the inference step of the algorithms. Gapped alignments are initiated with a score corresponding to about 22 bits.

[0099] It will be understood by one skilled in the art that there are many ways to create the amino acid sequences according to the first or second aspect of the invention, the nucleic acids according to the third aspect of the invention, the expression cassettes of the fourth aspect of the invention and the vectors of the fifth aspect of the invention. For example, nucleic acid sequences can be created using software tools, such as CLC Main Workbench, and synthesized artificially or created using site-directed mutagenesis. These sequences can be subcloned into expression cassettes and vectors using genetic engineering techniques widely available to those skilled in the art. See, for example, Molecular cloning: a laboratory manual (Sambrook & Russell: 2000, Cold Spring Harbor Laboratory Press; ISBN: 0879695773), and: Current protocols in molecular biology (Ausubel et al., 1988+ updates, Greene Publishing Assoc., New York; ISBN: 0471625949).

[00100] In a sixth aspect, a host cell is provided comprising a vector according to any embodiment of the fifth aspect.

[00101] In a seventh aspect, this application discloses a vaccine. The vaccine may comprise a vector according to any of the embodiments of the fifth aspect of the invention. The vaccine may be monovalent or multivalent. Typically, one dose of a monovalent vaccine may comprise at least 1 μg of a vector according to any of the embodiments of the fifth aspect. In some embodiments, one dose of the vaccine may comprise from 1 μg to about 25 μg of vector. In other embodiments, one dose of the vaccine may comprise from 1 to about 20 μg of vector, or from about 5 to about 20 μg of vector, or from about 5 to about 15 μg of vector, or from about 10 to about 20 μg of vector, or from about 5 to about 10 μg of vector.

[00102] Multiple antigens suitable for the vaccine of the invention have been described above. These antigens can be administered in the form of DNA vaccines, including the vectors and expression cassettes described above in the fourth and fifth aspects of the invention. In other embodiments, these antigens can be administered in the form of subunits (i.e., purified or partially purified proteins). In other embodiments, the antigens can comprise inactivated or attenuated organisms.

[00103] In addition to antigens, the vaccine of the present invention may further comprise adjuvants, i.e. substances that enhance or modulate the immune response, in addition to a molecular immunomodulator comprising SEQ ID NO: 6 or an amino acid sequence that is 95% identical to SEQ ID NO.

[00104] Suitable adjuvants are well known in the art. Suitable, non-limiting adjuvants include saponins (e.g., Quil A), alum, CpG oligonucleotides, poly I:C, oligoribonucleotides, cytokines, glycolipids such as ^BAY® 1005, quaternary amines such as dimethyldioctadecylammonium bromide (hereinafter "DDA"). Complexes comprising a saponin, a sterol (e.g., cholesterol), and optionally a phospholipid are described in the art. Combinations of CpG oligonucleotides and saponin, CpG and cholesterol, and CpG and alum have been reported to produce synergistic effects.

[00105] In addition to antigens and optional additional adjuvants, the vaccine may also contain a suitable pharmaceutical carrier. A suitable carrier will be obvious to those skilled in the art and will depend largely on the route of administration. Additional components that may be present in the present invention are adjuvants, preservatives, surfactants, chemical stabilizers, suspending or dispersing agents. Typically, stabilizers, adjuvants and preservatives are optimized to determine the best formulation for efficacy in the target subject.

[00106] In some embodiments, the vaccine may include a liposomal adjuvant and/or carrier to facilitate transport of the vector across the cell membrane and thereby result in increased expression of the antigen and/or molecular immunomodulator. A suitable non-limiting example of such a liposomal adjuvant/carrier system is described, for example, in U.S. Patent 10,456,459.

[00107] In an eighth aspect, the application discloses a method for protecting salmonids from infection, the method comprising administering to salmonids in need thereof a vaccine according to any of the embodiments of the seventh aspect of the invention. Thus, the invention also provides a vaccine according to any of the embodiments of the seventh aspect for use in protecting salmonids from infection.

[00108] It should be clear to one skilled in the art that the infection that the antigen(s) present in the vaccine determines which invention the vaccine will protect against. Thus, for example and without limitation, if the vaccine contains a nucleic acid sequence encoding a PMCV antigen, the vaccine can be used against an infection caused by PMCV.

[00109] The vaccine of the present invention can be administered to salmon by various routes, including, but not limited to, intramuscularly. Preferably, the vaccine is administered in a microdose, such that the volume of a single dose is less than 500 μl, or less than 400 μl, or less than 300 μl, or less than 200 μl, or about 100 μl, or less than 100 μl, or about 50 μl, or about 25 μl.

[00110] The vaccine disclosed herein can be used to protect multiple salmon species from infection. Suitable salmon include, but are not limited to, Atlantic salmon ( Salmo salar ), coho salmon ( Oncorhynchus kisutch ), rainbow trout ( Oncorhynchus mykiss ), sockeye salmon ( Oncorhynchus nerka ), chinook salmon ( Oncorhynchus tshawytscha ), and other species.

[00111] Salmon fish of various ages (or weights) can be vaccinated according to the present invention. In some embodiments, the salmon weigh from about 15 to about 200 grams at the time of vaccination. Thus, the weight of the salmon at the time of vaccination can be from about 25 to about 150 grams, or from about 40 to about 110 grams, or from about 50 to about 100 grams.

EXAMPLES

EXAMPLE 1 Production of a surface-targeted expression system

[00112] All successful DNA vaccines for Atlantic salmon have been derived from enveloped viruses and are based on membrane-bound antigens. Thus, exposure of the antigen to the cell surface may be important for efficacy.

[00113] PMCV is a naked virus containing a 6.7 kb dsRNA genome with three predicted open reading frames (Figure 1). The capsid protein (CP) is translated from ORF1, and ORF2 encodes RNA-dependent RNA polymerase (RdRp). The function of ORF3 is still unknown, but it encodes a protein with a cytotoxic effect when overexpressed in cell culture and is not required for viral particle assembly.

[00114] DNA vaccines expressing PMCV ORF1 (a capsid protein) and PMCV ORF3 (a protein of unknown function) have already been tested and did not provide protection (data not shown). Since PMCV is a naked virus without any of the transmembrane regions predicted for the PMCV ORF1 protein, and it has been demonstrated that the PMCV ORF1 protein will only be expressed intracellularly (data not shown), it was hypothesized that a response might be elicited if the expressed ORF1 capsid protein was targeted to the cell membrane.

[00115] The aim of this experiment was to generate a DNA vaccine expressing the PMCV capsid protein (or portions thereof) as a cell surface expressed antigen. The strategy consisted of fusing the N-terminal secretion signal peptide of the human septicemia virus G protein (VHSV-G) according to SEQ ID NO: 7 upstream of the N-terminus of the PMCV ORF1 antigen. In addition, the C-terminal transmembrane domain of VHSV-G according to SEQ ID NO: 8 was fused after the C-terminus of the antigen to incorporate the antigen onto the cell surface (von Gersdorff Jørgensen et al., 2012), as illustrated in Fig. 2A. A secreted version of the vaccine antigen was also generated using only the N-terminal secretion signal peptide and omitting the use of the C-terminal transmembrane domain (Fig. 2B).

[00116] Additional constructs were prepared similarly to the construct shown in Fig. 2A, except that the signal peptide sequence and transmembrane domain sequence of the Atlantic salmon paramyxovirus fusion protein (SEQ ID NOs: 38 and 39, respectively) or the hemagglutinin (HE) protein of infectious salmon anemia virus (ISAV) (SEQ ID NOs: 40 and 41, respectively) were used instead.

[00117] The antigen expression pathway was studied by immunofluorescence (IF) staining 6 days after transfection of CHH-1 cells with the different DNA vaccine constructs. Transfection was performed using the ^{LIPOFECTAMIN®} 3000 kit (Thermo Fisher) in 12-well plates. In this experiment, the pVAX1 expression vector (Invitrogen) was used for construction of DNA vaccines and 1 μg of plasmid DNA was used for transfection. Cells were either fixed with 3.7% formaldehyde for surface expression of antigen only or fixed with 3.7% formaldehyde followed by cell permeabilization with 0.1% Triton X-100 to stain intracellularly expressed proteins. Vaccine antigen was detected using both monoclonal and polyclonal antibodies against PMCV ORF1. Detection was performed using appropriate fluorescently labeled secondary antibodies (rabbit anti-mouse FITC (DAKO F0261) or polyclonal goat anti-rabbit Alexa Fluor Plus 488 (Invitrogen A32731). Staining of permeabilized cells detecting intracellularly expressed antigen was routinely included as a positive control for the protocol.

[00118] As shown in Fig. 3, the use of the VHSV G protein can be used to direct the expression of the PMCV ORF-1 antigen to the cell surface. The use of the signal protein and the transmembrane domain of the hemagglutinin (HE) protein of infectious salmon anemia virus also resulted in membrane staining of the PMCV ORF1 protein, whereas the use of regions of the fusion protein of Atlantic salmon paramyxovirus was unsuccessful.

[00119] In the next experiment, in vivo testing was performed in Atlantic salmon ( Salmo salar ). Two antigen variants were tested: the full-length capsid protein (G-ORF1) and a truncated capsid protein (G-ORF1-DeltaD) expressing the first half of the capsid protein (PMCV ORF1 position 1-1293 SEQ ID NO: 16). Both antigens were expressed in both a standard expression vector (pcDNA3.1) and as a salmonid alphavirus 3 (SAV-3)-based alphavirus replicon vaccine. PBS and pcDNA-eGFP were included as negative vaccine controls.

[00120] For all vaccines in this study, the genes encoding the antigen were inserted downstream of the CMV promoter in the eukaryotic expression vector pcDNA3.1(+) (Invitrogen). All plasmids were diluted in sterile phosphate-buffered saline (PBS) to 10 μg per 50 μl injection volume. All fish were given a single intramuscular injection on each side of the fish for a total dose of 20 μg. Two unimmunized control groups were included: one group received 20 μg of control vaccine (eGFP in pcDNA3.1) and the other group received 2 x 50 μl PBS.

Table 2.

Vaccine group Description Detailed description of the plasmid encoding the antigen Plasmid backbone Final concentration of the construct (µg/ml) Dose mcg
(2 x 0.05 ml) 1 G-ORF1-Delta D G-PMCV_ORF1_1-1293 (Delta D pcDNA3.1(+) 200 20 2 G-ORF1 G-PMCV_ORF1_full_1-2583 pcDNA3.1(+) 200 20 3 repSAV-G-ORF1-Delta D G-PMCV_ORF1_1-1293 (Delta D) in SAV replicon pcDNA3.1(+) 200 20 4 repSAV-G-ORF1 G-PMCV_ORF1_complete_1-2583_in the SAV replicon pcDNA3.1(+) 200 20 5 eGFP enhanced green fluorescent protein (control) pcDNA3.1(+) 200 20 6 PBS       NP

[00121] Fish (n=45 per group) were maintained in freshwater and had an average weight of 48 grams. Prior to vaccination, fish were anesthetized with tricaine (PHARMAQ), labeled by shortening of the adipose fin and/or jaw bones, allocated to groups, and injected intramuscularly twice during the same anesthetic period (one injection on each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 49 days at 12°C (12:12 light:dark), after which fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized spleen (isolate ID AL V1223 Spleen) from a CMS field outbreak in Norway. Fish were then collected at three time points; 10, 20 and 50 days post-infection (n=15 per group, per sampling point). Cardiac ventricles and kidneys were collected with RNALATER ^® from all sampled fish for subsequent RNA extraction. Hearts (ventricle and atrium) were also fixed in formalin from all fish at the last sampling point (day 50) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Bergen, Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale of 0–3 based on severity, where 0 indicates no pathology and 3 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Bergen, Norway).

[00122] Vaccines expressing the full-length PMCV ORF1 targeted to the cell surface demonstrated significant protection against challenge compared to controls (one-way ANOVA, with Dunnett's post-hoc test for atrial histology, p<0.0001). This was reflected in both increased Ct values in kidney and heart early post-challenge (results not shown) and decreased histopathology score at 50 days post-challenge (Table 3). An antigen based on the first half of the capsid protein (G-ORF1-Delta D; PMCV ORF1 position 1-1293) failed to provide protection, and no additional enhancement of protection was provided by using the salmon alphavirus replicon compared to the standard expression vector.

Table 3

Vaccine group Description Average Histoscore, Atrium Standard deviation, histoscore N p-value (compared to PBS) 6 PBS 2.29 0.78 15 NP 5 eGFP 2.21 0.67 15 0.99 4 repSAV-G-ORF1 1.49 0.88 15 0.028 3 repSAV-G-ORF1-Delta D 2.31 0.69 15 0.99 2 G-ORF1 1.11 0.83 15 0,0005 1 G-ORF1-Delta D 2.35 0.83 15 0.99

[00123] A DNA vaccine candidate targeting a full-length antigen to the cell surface resulted in a reduction in histopathological score of approximately 50% and resulted in a reduction in viral load (qPCR) in the kidney and heart.

[00124] A subsequent in vivo study compared the efficacy of DNA vaccines expressing the full-length PMCV ORF1 protein as a cell surface antigen, a secreted antigen, or an intracellularly expressed antigen. In addition, a codon-optimized version of the full-length capsid protein encoded by SEQ ID NO: 45 was included. The study included two parallel immunization tanks with five groups of Atlantic salmon each (n=30 per group, per tank).

[00125] For all vaccines in this study, the genes encoding the antigen were inserted downstream of the CMV promoter in the eukaryotic expression vector NTC9385R (Nature Technology Corporation). All plasmids were diluted in sterile phosphate-buffered saline (PBS) to 10 μg per 50 μl injection volume. All fish were given a single intramuscular injection on each side of the fish for a total dose of 20 μg. A non-immunized control group was included and received 2 x 50 μl PBS as summarized in Table 4.

Table 4

Vaccine group Description Description of the plasmid encoding the antigen Plasmid backbone Antigen expressed in final concentration of the construct (µg/ml) Dose mcg
(2 x 0.05 ml) 1 G-ORF1 G-PMCV_ORF1_full_1-2583 NTC9385R Cell surface 200 20 2 ORF1 PMCV_ORF1_full_1-2583 NTC9385R Intracellular 200 20 3 Codon-optimized TG-ORF1 Codon-optimized G-PMCV_ORF1_full_1-2583 NTC9385R Cell surface 200 20 4 G-ORF1 without TM G-PMCV_ORF1_full_1-2583 without TM domain NTC9385R Secreted 200 20 5 PBS   NP NP    

Histopathological evaluation of cardiac atrium 50 days after PMCV infection. The reported p value was calculated by comparing individual groups with the PBS control group using one-way ANOVA and Dunnett's post-test. NA = Not applicable.

[00126] The fish were maintained in fresh water and averaged 26 grams at the time of vaccination. During administration of the test vaccines, the fish were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection on each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 39 days at 12 °C (12:12 light:dark), after which the fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized heart (isolate ID AL V1273 Heart) taken from an infected animal during the CMS outbreak in Norway. Fish (n=15 per group at each time point) were then sampled at 21 days and 56 days post-challenge. Cardiac ventricles and kidneys were collected using RNALATER ^® from all sampled fish for subsequent RNA extraction. Hearts (ventricle and atrium) were also fixed in formalin from all fish at the last sampling point (day 56) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale of 0–3 based on severity, where 0 indicates no pathology and 3 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Norway).

[00127] Results showed that significant protection against PMCV infection was achieved with vaccines expressing full-length PMCV ORF1 targeted to the cell surface (codon optimized G-ORF1 and G-ORF1). Expression of PMCV ORF1 in its native form (intracellular protein) did not provide significant protection. Expression by secretion provided some protection (no differences were detected in kidneys at day 20; see Tables 5, 6).

Table 5. Reservoir 1. Viral RNA in kidneys at 3 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Ct value, PMCV Standard deviation, Ct n p-value (compared to PBS) 5 PBS 20.95 0.96 16 NP 1 G-ORF1 23.02 2.17 15 0.0042 3 G-ORF1 codon optimized 23.09 1.67 15 0.0031 4 G-ORF1 without TM 20.74 2.02 15 0.99 2 ORF1 19.92 1.41 15 0.30

Table 6. Reservoir 2. Viral RNA in kidneys at 3 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Ct value, PMCV Standard deviation, Ct n p-value (compared to PBS) 5 PBS 20.05 0.73 15 NP 1 G-ORF1 22.12 1.95 15 0,0005 3 G-ORF1 codon optimized 23.06 1.93 15 <0.0001 4 G-ORF1 without TM 20.06 1.16 15 >0.99 2 ORF1 19.90 0.67 15 >0.99

EXAMPLE 2: Obtaining and determining the antigenic activity of CMS antigen

[00128] Expression of the full-length PMCV ORF1 protein on the cell membrane provided partial protection in a challenge model. Despite this finding, in vitro antigen expression levels were observed to be markedly lower than expression levels of the control GFP construct. The project investigated whether expression of truncated versions of the capsid protein by removing regions could enhance expression levels and immunogenicity of the vaccine antigen.

[00129] The expression system described in Example 1 (using the VHSV-G signal peptide and transmembrane domain sequences) was prepared using various constructs based on the PMCV ORF-1 antigen.

[00130] Initial studies of the expression of membrane-bound PMCV capsid antigen from the VHSV-G expression cassette showed that inclusion of the first part of the PMCV ORF1 protein sequence was critical for success. When the capsid protein was divided into thirds, membrane-bound protein was successfully obtained only for constructs containing the first third (data not shown).

[00131] After generating PMCV ORF1 capsid deletion variants, they were tested by IF staining and Western blotting of transfected cells. For all deletion constructs, the deleted PMCV ORF1 sequence was replaced with a GGGGS linker (SEQ ID NO: 36). The strategy for generating the different deletion variants was as follows:

(1) In the first round, relatively large deletions were tested (IntDel 1-7; 200 amino acids in length, 100 amino acids overlapping except for IntDel7, which was 162 amino acids in length). Since inclusion of some N-terminal sequence was found to be necessary for successful targeting of the expressed protein to cell membranes, the first deletion (IntDel 1) was made at nucleotide positions 298-897 in PMCV ORF1 (SEQ ID NO: 16).

(2) In the second round, smaller deletion variants were created centered on the region covered by IntDel 4-7 (IntDel 11-18, contiguous, non-overlapping 50 amino acids in length, starting at IntDel 4). The first of these deletions (IntDel 11) was constructed at nucleotide positions 1384-1533 in PMCV ORF1 (SEQ ID NO: 16).

(3) In the third round, antigen variants with very small adjacent overlapping deletions were generated (IntDel 21-42; 10 amino acids in length, 2 amino acids overlap). The first of these deletions (IntDel 21) was constructed at nucleotide positions 1936-1965 in PMCV ORF1 (SEQ ID NO: 16).

[00132] Expression of PMCV ORF1 capsid deletion variants in CHH-1 cells by IF staining and Western blotting. The procedure for immunofluorescence staining of cells was as follows: CHH-1 cells were trypsinized and seeded in 12-well plates (150,000 cells per well and 1 ml of cell medium). Cells were incubated at 15 °C ON in a CO2 incubator (5% CO ₂ ). Cells were transfected the day after seeding using 1 μg of plasmid per well and LIPOFECTAMIN ^® 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were incubated at 15 °C for 5 days in a CO2 incubator (5% CO ₂ ) prior to immunostaining of cells. Cell medium was carefully removed from all wells and all wells were washed thoroughly with PBS. Cells were fixed by incubation with 3.7% formaldehyde (diluted in PBS) for 15 min at room temperature. Cells were then blocked with 5% nonfat dry milk in PBS for 1 h. Both mouse anti-HIS (monoclonal antibody 6x-His Tag (4A12E4) Fisher Scientific; Cat. No. 37-2900 at 1:1500 dilution) and mouse anti-PMCV ORF1 (mAb#10; dilution 1:200) monoclonal antibody were used as primary antibodies and incubated at room temperature for 1 hour. FITC-conjugated polyclonal rabbit anti-mouse immunoglobulin (DAKO F0261) was used as secondary antibody at 1:1000 dilution and incubated for another 1 hour. Cell staining was assessed by fluorescence microscopy.

[00133] Western blot assessment of expression levels of PMCV ORF1 deletion constructs was performed as follows: CHH-1 cells were trypsinized and seeded in 6-well plates (1,000,000 cells per well and 3 ml of cell medium). Cells were incubated at 15 °C ON in a CO ₂ incubator (5% CO ₂ ). Cells were transfected the day after seeding using 2.5 μg of plasmid per well and LIPOFECTAMIN ^® 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were placed back in sealed bags and incubated at 15 °C in a CO2 incubator (5% CO ₂ ) for six days before further analysis. Plates were then placed on ice and medium was removed. Cells were first washed twice with ice-cold PBS. To prepare membrane lysates, cells were first harvested by adding 200 µl 20 mM Tris-HCl, pH 7.5 with protease inhibitors, cells were gently scraped and cell suspensions were transferred to pre-chilled tubes. The well was then washed with another 100 µl for a total of 300 µl cell suspension per transfection. Since this is a detergent-free buffer, cells were lysed by passing the cells through the tip of a syringe (25G, ~5x) and centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatants were discarded and membrane pellets were dissolved in 300 µl RIPA lysis buffer (Abcam, #156034) with 1x protease inhibitor mix to release membrane-bound proteins. Samples were incubated for 30 min at 4 °C, periodically resuspending the pellet by shaking the tube. The tubes were centrifuged at 12,000 rpm for 10 min at 4 °C, the membrane fraction lysates were transferred to fresh tubes and stored on ice until immunoprecipitation. On ice, 1 μg of mouse IgG1anti 6x-His monoclonal antibody 4E3D10H2/E3 (Fisher Scientific #15442890, 1 mg/ml) was added to each sample and incubated at 4 °C overnight on a rotator. Protein G-coupled beads were prepared (25 μl/sample) by washing immobilized beads 3-5 times with ~1 ml cold PBST and resuspending in 25 μl PBST with protease inhibitors per aliquot. On ice, 25 μl of pre-equilibrated beads were added to each sample and the sample-bead mixture was incubated for 1 h at room temperature with rotation. Beads were washed with 500 μl ice-cold PBST with protease inhibitors 3-5 times to remove non-specific binding with gentle mixing of beads between each wash. After the final wash, as much buffer as possible was removed from the beads and 40 µl of 1x Laemmli Sample Buffer (BioRad #161-0737) (prepared by mixing 20 µl of 2x Laemmli Sample Buffer with 2 µl of β-mercaptoethanol and 18 µl of dH2O) were added. Samples were incubated at 70°C for 10 min, briefly centrifuged and magnetized, and the eluent was transferred to a new vial. All samples were denatured at 98°C for 5 min before loading onto a 4–20% Criterion™ TGX Stain-Free™ gel. The gel was run for approximately 45 min at 200 V and blotted using the TRANS ^- BLOT® TURBO™ Transfer System (BioRad) and TRANS- ^BLOT® TURBO™ Midi PVDF Transfer Bags #170-4157. A 7 min turbo program was used. The membrane was immediately placed in blocking buffer (5% skim milk in PBST). Blocking was performed for 1 h at room temperature. The blot was incubated with rabbit anti-PMCV delta ORF1 primary antibody diluted 1:500 in 1% skim milk/PBST overnight at 4 °C on a rotator. The following day, blots were washed 2 x 15 min with PBST and incubated with secondary polyclonal porcine anti-rabbit immunoglobulin HRP (DAKO #P0217) diluted 1:1000 in 2% skim milk and PRECISION PROTEIN™ StrepTactin-HRP conjugate 1:10,000 (to visualize the ladder) for 1 h at room temperature. The blot was washed 2 x 15 min with PBST before signal detection using Clarity western ECL substrate (BioRad #170-5060).

[00134] The results can be summarized as follows: after the first round (200 amino acid long deletions), cell membrane expression was achieved only by the antigen from the IntDel 4-7 constructs. Accordingly, the N-terminal portion is necessary for targeting the antigen to the cell membranes. The constructs generated in the second round (50 amino acid long deletions) were therefore concentrated on the region covered by IntDel 4-7. All of these constructs successfully expressed the antigen on the cell membrane, except that cell surface expression was lower for IntDel 18 (the construct lacking the amino acid sequence shown in SEQ ID NO: 15). The deletion constructs (IntDel 15-17) resulted in increased membrane expression levels. IntDel 6 (the construct lacking amino acids 500-699 of SEQ ID NO: 1) from the first round was also found to have higher expression levels. With the exception of IntDel 42 (the amino acid-deficient construct shown in SEQ ID NO: 14), which performed slightly better than the others, no additional candidates were identified in the third round. The results are shown in Fig. 4.

[00135] Based on the combined results of immunofluorescence staining and Western blot analysis, several deletion candidates were selected from the results of several vaccine trials within the project. For all vaccines in the first study, the antigen encoding genes were inserted downstream of the CMV promoter in the eukaryotic expression vector NTC9385R (Nature Technology Corporation). All plasmids were diluted in sterile phosphate-buffered saline (PBS) to 10 μg per 50 μl injection volume. All fish were given a single intramuscular injection on each side of the fish for a total dose of 20 μg. A non-immunized control group was included and received 2 x 50 μl PBS as shown in Table 7.

Table 7

Vaccine group Description Description of the plasmid encoding the antigen
(positions refer to the nucleotide position of PMCV ORF1) Plasmid backbone Final concentration of the construct (µg/ml) Dose mcg
(2 x 0.05 ml) 1 G-ORF1 G-PMCV_ORF1_full_1-2583 NTC9385R 200 20 2 G-IntDel-6 G-PMCV ORF1_IntDel6 (items 1798-2397) NTC9385R 200 20 3 G-IntDel-15 G-PMCV ORF1_IntDel15 (item 1984-2133) NTC9385R 200 20 4 G-IntDel-16 G-PMCV ORF1_IntDel16 (items 2134-2283) NTC9385R 200 20 5 G-IntDel-17 G-PMCV ORF1_IntDel17 (pos. 2284-2433) NTC9385R 200 20 6 PBS   NP    

[00136] Atlantic salmon ( Salmo salar ) (n=30 per group), maintained in freshwater and weighing an average of 20 grams, were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection into each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 48 days at 12C (12:12 light:dark), after which the fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized heart (isolate ID AL V1273 Heart) taken from an infected animal during the CMS outbreak in Norway. Fish (n=15 per group, per sampling point) were then collected at two time points; 19 days and 49 days after the trial. Heart ventricles and kidneys were collected using RNALATER ^® from all sampled fish for subsequent RNA extraction. Hearts (ventricle and atrium) were also fixed in formalin from all fish at the last sampling point (day 49) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale of 0–3 based on severity, where 0 indicates no pathology and 3 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Norway).

[00137] The viral load results at 3 weeks post-infection and histoscore at 7 weeks post-infection are shown in Tables 8 and 9, respectively. The pooled data from all results showed that IntDel variants 15, 16, and 17 (where the antigen lacked the amino acid sequences shown in SEQ ID NO 3, 4, or 5, respectively) performed better than the full-length capsid protein (G-ORF1).

Table 8. Viral RNA in kidneys 3 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Ct value, PMCV Standard deviation, Ct n p-value (compared to PBS) 6 PBS 20.61 1.49 15 NP 1 G-ORF1 23.77 2.57 15 0.0002 2 G-IntDel-6 21,23 1.41 15 0.86 3 G-IntDel-15 23.13 2.81 15 0.0033 4 G-IntDel-16 22.92 1.20 15 0.0082 5 G-IntDel-17 23.13 1.71 15 0.0032

Table 9. Histopathological lesions (histoscore) in atria 7 weeks post-challenge. The reported p value was calculated by comparing individual groups with the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Histoscore, Atrium Standard deviation, histoscore N p-value (compared to PBS) 6 PBS 1.75 0.84 15 NP 1 G-ORF1 1.60 0.83 15 0.99 2 G-IntDel-6 1.54 0.99 15 0.95 3 G-IntDel-15 0.75 0.75 15 0,012 4 G-IntDel-16 1.10 0.63 15 0.16 5 G-IntDel-17 1.32 0.87 15 0.53

[00138] An additional clinical study was conducted to evaluate three new antigen ablation candidates. For all vaccines in the first study, the genes encoding the antigen were inserted downstream of the CMV promoter in the eukaryotic expression vector NTC9385R (Nature Technology Corporation). All plasmids were diluted in sterile phosphate-buffered saline (PBS) to 10 μg per 50 μl injection volume. All fish were given one intramuscular injection on each side of the fish for a total dose of 20 μg. A non-immunized control group was included and received 2 x 50 μl PBS as shown in Table 10.

Table 10

Vaccine group Description Description of the plasmid encoding the antigen
(positions refer to the nucleotide position of PMCV ORF1) Plasmid backbone Final concentration of the construct (µg/ml) Dose mcg
(2 x 0.05 ml) 1 G-ORF1 G-PMCV_ORF1_full_1-2583 NTC9385R 200 20 2 G-IntDel-5 G-PMCV ORF1_IntDel5 (items 1498-2097) NTC9385R 200 20 3 G-IntDel-7 G-PMCV ORF1_IntDel7 (items 2098-2583) NTC9385R 200 20 4 G-IntDel-42 G-PMCV ORF1_IntDel42 (pos. 2440-2469) NTC9385R 200 20 5 PBS   NP    

[00139] Atlantic salmon ( Salmo salar ) (n=30 per group), housed in a single tank and averaging 28 g in weight, were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection into each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 58 days at 12°C (12:12 light:dark) after which the fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized heart (isolate ID AL V1289) taken from an infected animal during the CMS outbreak in Norway. Fish (n=15 per group) were then collected at two time points; 3 weeks and 7 weeks post-infection. Cardiac ventricles and kidneys were collected using RNALATER ^® from all sampled fish for subsequent RNA extraction. Hearts (ventricle and atrium) were also fixed in formalin from all fish at the last sampling point (7 weeks) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale from 0 to 4 based on severity, where 0 indicates no pathology and 4 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Norway).

[00140] The results for week 3 and week 7 are summarized in Tables 11 and 12, respectively.

Table 11. Viral RNA in kidneys 3 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Ct value, PMCV Standard deviation, Ct N p-value (compared to PBS) 5 PBS 19.44 1.13 15 NP 1 G-ORF1 20.48 1.91 15 0.38 2 G-Del5 22.58 2.69 15 <0.0001 3 G-Del7 21.12 1.99 15 0.06 4 G-Del42 21.12 1.40 15 0.06

Table 12. Histopathological lesions (histoscore) in atria 7 weeks post-challenge. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Histoscore, Atrium Standard deviation, histoscore N p-value (compared to PBS) 5 PBS 2.14 0.36 14 NP 1 G-ORF1 2.07 0.70 15 0.99 2 G-Del5 1.64 1.15 14 0.28 3 G-Del7 1.80 0.56 15 0.59 4 G-Del42 1.20 0.94 15 0.0075

EXAMPLE 3: Selection of a vaccine backbone

[00141] Plasmid backbone properties such as vector size and functional elements are also important for optimizing vaccine efficacy. Three candidate vectors were compared for in vitro antigen expression. The candidates included pcDNA3.1, pVAX1 (INVITROGEN ^TM ), NTC9385R (Nature Technology Corporation), and two NTC9385R variants that co-express innate immune stimulatory elements: a RIG-I agonist that acts as an adjuvant that induces type I interferon (NTC9385R-eRNA41H) and a RIG-I agonist and a CpG motif that stimulates Toll-like receptor 9 (NTC9385R-eRNA41H-CpG). NTC9385R NANOPLASMID ^TM is smaller in size than other traditional vectors, allowing for improved uptake and persistence in transfected cells, and contains a modified promoter that is said to enhance antigen expression. It contains an antibiotic-free marker for RNA-based sucrose selection (RNA-OUT), which replaces the use of antibiotics in the production process, and a modified origin of replication, which ensures that the plasmid can only replicate in a specific producing strain of E. coli , which is beneficial from a safety perspective. The smaller size may impact transfection efficiency, as well as the level and duration of expression. In addition, some bacterial region protein marker genes, such as resistance marker genes, have been shown to dramatically reduce vector expression. Bacterial regions larger than 1 kb have been shown to repress transgene expression in quiescent tissues such as liver, likely due to the formation of bacterial non-transcribed region-mediated heterochromatin that extends into the eukaryotic region and inactivates the promoter (Suschak et al., 2017).

[00142] Western blot analysis of in vitro expression levels of the G-ORF1 vaccine antigen in five different plasmid backbones was performed as follows: CHH-1 cells were trypsinized and seeded in 6-well plates (1,000,000 cells per well and 3 ml of cell medium). Cells were incubated at 15 °C ON in a CO ₂ incubator (5% CO ₂ ). Cells were transfected the day after seeding using 2.5 μg of plasmid per well and LIPOFECTAMIN ^® 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were placed back in sealed pouches and incubated at 15 °C in a CO ₂ incubator (5% CO ₂ ) for five days before further analysis. Plates were then placed on ice and medium was removed. Cells were first washed twice with ice-cold PBS. Total cell lysates were prepared by adding 150 µl NP40 lysis buffer (Invitrogen, #FNN021) with 1 protease inhibitor cocktail (Roche, #1697498) directly to the wells. Lysates were transferred to pre-chilled Eppendorf tubes, incubated for 30 min at 4°C with resuspending cells by periodic shaking of the tube, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was transferred to fresh tubes and stored on ice until immunoprecipitation. On ice, 1 μg of mouse IgG1anti 6x-His monoclonal antibody 4E3D10H2/E3 (Fisher Scientific #15442890, 1 mg/ml) was added to each sample and incubated at 4°C overnight on a rotator. Protein G-coupled beads were prepared (25 μl/sample) by washing immobilized beads 3-5 x ~1 ml cold PBST and resuspending in 25 μl PBST with protease inhibitors per aliquot. On ice, 25 μl of pre-equilibrated beads were added to each sample and the sample-bead mixture was incubated for 1 h at room temperature with rotation. Beads were washed with 500 µl ice-cold PBST with proteinase inhibitors 3-5 times to remove non-specific binding, with gentle mixing of the beads between each wash. After the final wash, as much buffer as possible was removed from the beads and 40 µl of 1x Laemmli Sample Buffer (BioRad #161-0737) (prepared by mixing 20 µl of 2x Laemmli Sample Buffer with 2 µl β-mercaptoethanol and 18 µl dH2O) were added. Samples were incubated at 70°C for 10 min, briefly centrifuged and magnetized, and the eluent transferred to a new vial. All samples were denatured at 98°C for 5 min prior to loading onto a 4–20% CRITERION™ TGX STAIN-FREE™ gel. The gel was run for approximately 45 min at 200 V and blotted using the TRANS ^- BLOT® TURBO™ Transfer System (BioRad) and TRANS- ^BLOT® TURBO™ Midi PVDF Transfer Bags #170-4157. A 7 min turbo program was used. The membrane was immediately placed in blocking buffer (5% skim milk in PBST). Blocking was performed for 1 h at room temperature. The blot was incubated with rabbit anti-PMCV delta ORF1 primary antibody diluted 1:500 in 1% skim milk/PBST overnight at 4 °C on a rotator. The following day, blots were washed 2 x 15 min with PBST and incubated with secondary polyclonal porcine anti-rabbit immunoglobulin HRP (DAKO #P0217) diluted 1:1000 in 2% skim milk and PRECISION PROTEIN™ StrepTactin-HRP conjugate 1:10,000 (to visualize the ladder) for 1 h at room temperature. The blot was washed 2 x 15 min with PBST before signal detection using Clarity western ECL substrate (BioRad #170-5060).

[00143] For all vaccines in the present study, the full-length PMCV ORF1 capsid gene targeted for expression at the cell membrane (G-ORF1) was inserted downstream of the CMV promoter into various eukaryotic expression vectors as shown in the table below. All plasmids were diluted in sterile phosphate-buffered saline (PBS) to 10 μg per 50 μl injection volume. All fish were given one intramuscular injection on each side of the fish for a total dose of 20 μg. A non-immunized control group was included and received 2 x 50 μl PBS as shown in Table 13.

Table 13

Vaccine group Description Description of the plasmid encoding the antigen Plasmid backbone Final concentration of the construct (µg/ml) Dose mcg
(2 x 0.05 ml) 1 G-ORF1-pcDNA3.1 G-PMCV_ORF1_full_1-2583 pcDNA3.1(+) 200 20 2 G-ORF1-pVAX1 G-PMCV_ORF1_full_1-2583 pVAX1 200 20 3 G-ORF1-NTC9385R G-PMCV_ORF1_full_1-2583 NTC9385R 200 20 4 G-ORF1-NTC9385R-eRNA41H-CpG G-PMCV_ORF1_full_1-2583 NTC9385R-eRNA41H-CpG 200 20 5 G-ORF1-NTC9385R-eRNA41H G-PMCV_ORF1_full_1-2583 NTC9385R-eRNA41H 200 20 6 PBS   NP    

[00144] Freshwater-housed Atlantic salmon ( Salmo salar ) (n=30 per group, average weight 29 g) were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection into each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 59 days at 12 °C (12:12 light:dark) after which the fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized heart (isolate ID AL V1273 Heart) taken from an infected animal during the CMS outbreak in Norway. Fish (n=15 per group) were then sampled at two time points; 20 days and 48 days post-challenge. Heart ventricles and kidneys were collected using RNALATER ^® from all sampled fish for subsequent RNA extraction. Hearts (ventricle and atrium) were also fixed in formalin from all fish at the last sampling point (day 48) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale of 0–3 based on severity, where 0 indicates no pathology and 3 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Norway).

[00145] Western blot analysis of transfected CHH-1 cells demonstrated that all three NTC9385R plasmid variants mediated higher antigen expression compared to the pcDNA3.1 and pVAX1 plasmid backbones (Figure 5). Clinical trials demonstrated that NTC9385R-based vaccines provided at least as good protection as pcDNA3.1/pVAX1-based vaccines (see Table 14 and Table 15 for three and seven weeks, respectively). The combined data set demonstrated a trend toward superior protection due to the NTC9385R-based vaccine backbones. NTC9385R plasmids with immunostimulatory elements had no additional effect.

Table 14. Viral RNA in kidneys 3 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Ct value, PMCV Standard deviation, Ct n p-value (compared to PBS) 6 PBS 21.73 1.25 15 NP 1 G-ORF1-pcDNA3.1 22.66 1.20 15 0.24 2 G-ORF1-pVAX1 22.57 1.51 15 0.35 3 G-ORF1-NTC9385R 23.78 1.71 15 0,0008 4 G-ORF1-NTC9385R-eRNA41H-CpG 23.52 1.42 15 0.0034 5 G-ORF1-NTC9385R-eRNA41H 23.03 1.28 15 0.053

Table 15. Viral RNA in kidneys 7 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Ct value, PMCV Standard deviation, Ct n p-value (compared to PBS) 6 PBS 22.45 1.53 15 NP 1 G-ORF1-pcDNA3.1 23.75 1.73 15 0.26 2 G-ORF1-pVAX1 23.77 2.01 15 0.23 3 G-ORF1-NTC9385R 23.94 2.42 15 0.14 4 G-ORF1-NTC9385R-eRNA41H-CpG 25.63 1.77 15 0,0001 5 G-ORF1-NTC9385R-eRNA41H 24.44 2.02 15 0.031

EXAMPLE 4: Selection of adjuvant

[00146] Molecular adjuvants show great promise for both enhancing immunogenicity and prolonging the duration of the immune response, and these molecular adjuvants expressing cytokines, chemokines or costimulatory molecules can be co-administered with a DNA vaccine plasmid encoding the antigen. Cells transfected with molecular adjuvant plasmids secrete the adjuvant into the surrounding area, stimulating local antigen-presenting cells (Suschak et al., 2017). The adjuvant activity of fish type I IFNs has already been demonstrated in a model of infectious salmon anemia virus (ISAV) DNA vaccination in Atlantic salmon (Chang et al., 2015). In the paper, it was demonstrated that type I IFNs enhance the antibody response against the ISAV hemagglutinin protein and provide enhanced protection against viral challenge.

[00147] The following molecular adjuvants: IFNa, IFNb, IFNc, IFNd, IFNγ, IL-2, IL-4, IL-12, and VHSV-G were tested in vivo with the full-length PMCV ORF1 vaccine antigen (G-ORF1) for adjuvant activity using a high-throughput model for rapid selection of candidates based on potency.

[00148] This model is based on the unexpected finding that differences in histopathological lesions at 50 days post-infection can be predicted by viral RNA levels in the heart and kidneys of the same groups at 20 days post-infection. Thus, for rapid screening of many groups, only one sampling point at 3 weeks post-infection is needed.

[00149] For all groups in the in vivo adjuvant screening study, the vaccine antigen and plasmid-encoded adjuvants were administered on separate plasmids. The gene encoding the antigen (G-ORF1) and the various plasmid-encoded adjuvant genes were inserted downstream of the CMV promoter in the eukaryotic expression vector pcDNA3.1(+) (Invitrogen). Plasmids were diluted in sterile phosphate-buffered saline (PBS) to 10 μg per 50 μl injection volume according to the table below. All fish were administered one intramuscular injection on each side of the fish for a total dose of 20 μg of plasmid (20 μg of each plasmid for antigen plus adjuvant). Two non-immunized control groups were included: one group received 20 μg control vaccine (eGFP in pcDNA3.1) and the other group received 2 x 50 μl PBS, as shown in Table 16.

Table 16

Group Description (Antigen) Detailed description of design A (Antigen) Description (Adjuvant) Detailed description of design B (Adjuvant) Final concentration of construct A (µg/mL) Final concentration of construct B (µg/mL) Dose mcg
(2 x 0.05 ml) 1 G-ORF1 G-PMCV_ORF1_full_1-2583 Absent.   200   20 2 eGFP enhanced green fluorescent protein (control) Absent.   200   20 3 G-ORF1 G-PMCV_ORF1_full_1-2583 IFNa1 Adjuvant IFNa1 construct 200 200 20 each 4 G-ORF1 G-PMCV_ORF1_full_1-2583 IFNb Adjuvant IFNb design 200 200 20 each 5 G-ORF1 G-PMCV_ORF1_full_1-2583 IFNc Adjuvant IFNc construct 200 200 20 each 6 G-ORF1 G-PMCV_ORF1_full_1-2583 IFNd Adjuvant IFNd design 200 200 20 each 7 G-ORF1 G-PMCV_ORF1_full_1-2583 Interleukin 2 IL-2 adjuvant construct 200 200 20 each 8 G-ORF1 G-PMCV_ORF1_full_1-2583 Interleukin 4 IL-4 adjuvant construct 200 200 20 each 9 G-ORF1 G-PMCV_ORF1_full_1-2583 Interleukin 12 IL-12 adjuvant construct 200 200 20 each 10 G-ORF1 G-PMCV_ORF1_full_1-2583 IFNg IFN gamma adjuvant construct 200 200 20 each 11 G-ORF1 G-PMCV_ORF1_full_1-2583 VHSV-G DNA vaccine based on VHSV-g protein 200 200 20 each 12 G-ORF1 G-PMCV_ORF1_full_1-2583 rORF1 (PMCV) Rec.protein PMCV ORF1 (E.coli)* 200 NP 20 13 PBS PBS control          

[00150] Freshwater-housed Atlantic salmon ( Salmo salar ) (n=15 per group, average weight 31 g) were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection into each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 50 days at 12°C (12:12 light:dark) after which the fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized heart (isolate ID ALV1223 Heart) taken from an infected animal during the CMS outbreak in Norway. Fish (n=15 per group) were sampled 21 days post-infection. Cardiac ventricles and kidneys were collected using RNALATER ^® from all sampled fish for subsequent RNA extraction. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway).

[00151] After 7 weeks of immunization and 3 weeks of challenge, the model found that while most adjuvant strategies failed to improve efficacy, one adjuvant, IFNb, significantly increased the effect size. The results are shown in Table 17.

[00152] IFNb and IFNc were selected for subsequent studies in fish. All groups in this study were administered the vaccine antigen and plasmid-encoded adjuvants on separate plasmids. The gene encoding the antigen (G-ORF1) and the plasmid-encoded adjuvant genes (IFNb or IFNc) were inserted downstream of the CMV promoter in the eukaryotic expression vector pcDNA3.1(+) (Invitrogen). Plasmids were diluted in sterile phosphate-buffered saline (PBS) to 10 μg per 50 μl injection volume according to the table below. All fish were administered one intramuscular injection per side of the fish for a total dose of 20 μg of plasmid (20 μg of each plasmid for antigen plus adjuvant). A non-immunized control group was included and received 2 x 50 µl PBS as shown in Table 18.

Table 17. Viral RNA in kidneys 3 weeks post-infection. The reported p value was calculated by comparing individual groups to the G-ORF1 control group (no adjuvant) using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Antigen construct Adjuvant Average Ct value, PMCV Standard deviation, Ct n p-value (compared to G-ORF1 alone – group 1) 13 PBS Absent. 19.49 0.96 14 0.0002 1 G-ORF1 Absent. 23.12 2.60 31 NP 2 eGFP Absent. 22.04 1.51 16 0.73 3 G-ORF1 IFNa1 23.17 3.29 15 0.99 4 G-ORF1 IFNb 26.06 3.05 15 0.0033 5 G-ORF1 IFNc 23.53 3.27 14 0.99 6 G-ORF1 IFNd 21.82 2.50 16 0.52 10 G-ORF1 IFNg 21.09 1.69 14 0.11 7 G-ORF1 Interleukin 2 20.70 3.20 13 0.04 8 G-ORF1 Interleukin 4 21.72 2.01 15 0.45 9 G-ORF1 Interleukin 12 23.03 3.24 16 0.99 11 G-ORF1 VHSV-G 20.25 2.52 15 0.004 12 G-ORF1 rORF1 (PMCV) 19.42 0.74 10 0,001

Table 18

Group Description (Antigen) Description (Adjuvant) Detailed description of design A (Antigen) Detailed description of design B (Adjuvant) Final concentration of construct A (µg/mL) Final concentration of construct B (µg/mL) Dose mcg
(2 x 0.05 ml) 1 G-ORF1 Absent. G-PMCV_ORF1_full_1-2583   200   20 2 G-ORF1 IFNb G-PMCV_ORF1_full_1-2583 Adjuvant IFNb design 200 200 20 each 3 Absent. IFNb   Adjuvant IFNb design   200 20 4 G-ORF1 IFNc G-PMCV_ORF1_full_1-2583 Adjuvant IFNc construct 200 200 20 each 5 Absent. IFNc   Adjuvant IFNc construct   200 20 6 PBS   PBS control        

[00153] Freshwater-housed Atlantic salmon (Salmo salar) (n=30/group), average weight 29 g, were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection into each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 59 days at 12°C (12:12 light:dark), after which the fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized heart (isolate ID ALV1273 Heart) taken from an infected animal during the CMS outbreak in Norway. Fish (n=15/group) were then collected at two time points; 20 days and 48 days post-infection. Cardiac ventricles and kidneys were collected using RNALATER ^® from all sampled fish for subsequent RNA extraction. Hearts (ventricle and atrium) were also fixed in formalin from all fish at the last sampling point (day 48) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale of 0–3 based on severity, where 0 indicates no pathology and 3 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Norway).

[00154] Results showed that IFNb was superior to IFNc (Table 19). The difference between the two adjuvants was most significant at the 7-week post-challenge sampling time point (data for the earlier time point not shown). The adjuvant-only group was also partially protected, reflecting the fact that interferons are important effectors of both innate and adaptive immune responses.

Table 19. Viral RNA in the heart at 7 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Antigen Adjuvant Average Ct value, PMCV Standard deviation, Ct n p-value (compared to PBS) 6 PBS PBS 23.17 2.40 15 NP 1 G-ORF1 PBS 25.64 2.61 15 0.40 2 G-ORF1 IFNb 30.77 5.61 15 0,0001 3 Absent. IFNb 30.38 4.40 15 0.0002 4 G-ORF1 IFNc 25.16 4.79 15 0.64 5 Absent. IFNc 22.48 2.15 15 0.99

EXAMPLE 5 – Adjuvant Delivery

[00155] The purpose of this experiment was to determine the best method for delivering antigen and molecular adjuvant.

[00156] Plasmid-encoded adjuvants may be delivered either on a separate plasmid or included in the same plasmid as the plasmid encoding the antigen. If the adjuvant is expressed from the same plasmid as the antigen, this can be done, for example, in the following ways:

1. From a separate but identical promoter as the vaccine antigen.

2. From a separate promoter similar (but not identical) to the vaccine antigen.

3. As a fusion protein with an antigen separated by a P2A peptide sequence to facilitate self-cleavage.

[00157] In this example, different adjuvant delivery options were tested. All groups were given cell surface expressed PMCV ORF1 antigen (G-ORF1). The adjuvant was administered by the following routes:

1. On a separate plasmid (G-ORF1+IFNb)

2. Expressed together with the antigen, downstream of the same CMV promoter and separated from the antigen by the self-cleaving peptide 2A (G-ORF1-P2A-IFNb).

3. Expressed on the same plasmid as the antigen, but downstream of the second CMV promoter (G-ORF1-CMV-IFNb),

4. One group received no adjuvant, only antigen (G-ORF1).

5. An unvaccinated control group receiving PBS was also included.

[00158] For all vaccines in the present study, the genes were inserted downstream of the CMV promoter in the eukaryotic expression vector pVAX1 (Invitrogen). The antigen used was G-ORF1 (G-PMCV_ORF1_full_1-2583). All vaccines were diluted in sterile phosphate-buffered saline (PBS) to 10 μg per 50 μl injection volume. All fish were given a single intramuscular injection on each side of the fish for a total dose of 20 μg. For fish receiving plasmid-encoded antigen and adjuvant on separate plasmids, the dose of each plasmid was 20 μg. A non-immunized control group was included and received 2 x 50 μl PBS. In addition, one group received a plasmid-encoded version of the adjuvant with altered codons, as shown in Table 20.

Table 20.

Group Design 1 Construction 2 Introduction of adjuvant Dose mcg
(2 x 0.05 ml) 1 G-ORF1 IFNb codon changed The IFNb adjuvant codon change is presented in a separate plasmid 20 each 2 G-ORF1 IFNb The IFNb adjuvant is presented in a separate plasmid. 20 each 3 G-ORF1-P2A-IFNb   IFNb is present on the same plasmid and under the same promoter as the antigen. 20 4 G-ORF1-CMV-IFNb   IFNb is present on the same plasmid but downstream of the second CMV promoter. 20 5 G-ORF1   Control group without adjuvant 20 6 PBS      

[00159] Freshwater-housed Atlantic salmon ( Salmo salar ) (n=30/group), average weight 25 g, were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection into each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 49 days at 12°C (12:12 light:dark), after which the fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized heart (isolate ID ALV1273 Heart) taken from an infected animal during a CMS outbreak in Norway. Fish (n=15/group) were then collected at two time points; 19 days and 47 days post-infection. Cardiac ventricles and kidneys were collected using RNALATER ^® from all sampled fish for subsequent RNA extraction. Hearts (ventricle and atrium) were also fixed in formalin from all fish at the last sampling point (day 47) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale of 0–3 based on severity, where 0 indicates no pathology and 3 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Norway).

[00160] The results of the clinical study are illustrated by the cardiac histogram data 7 weeks after infection (Table 21).

[00161] Antigen expression levels and adjuvant activity of the various constructs were also assessed in vitro (results not shown). The combined results suggest that the preferred approach is to introduce the adjuvant into the same plasmid as the antigen, under an identical but separate promoter as the antigen, but delivery via other plasmids may also be effective.

Table 21. Histopathological lesions (histoscore) in atria 7 weeks post-challenge. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Histoscore, Atrium Standard deviation, histoscore N p-value (compared to PBS) 6 PBS 2.46 0.54 15 NP 5 G-ORF1 1.73 1.00 15 0.20 2 G-ORF1+IFNb 1.00 0.90 15 0.0014 4 G-ORF1-CMV-IFNb 1.13 1.03 15 0.0035 3 G-ORF1-P2A-IFNb 1.73 1.22 15 0.20 1 Codon G-ORF1-CMV-IFNb_changed 1.04 0.96 15 0.003

EXAMPLE 6 – Duration of Vaccine Effectiveness

[00162] Initial studies showed that fish receiving only the adjuvant were partially protected, reflecting the fact that interferons are important effectors of both the innate and adaptive immune responses. A long-term follow-up trial of the vaccine was conducted to examine the duration of protection obtained by incorporating the molecular adjuvant into the DNA construct.

[00163] For all vaccines in the present study, the genes were inserted downstream of the CMV promoter into eukaryotic expression vectors from Nature Technology Corporation (NTC). The backbones were either NTC9385R or NTC9385R-eRNA41H-CpG. For the group receiving both plasmid-encoded antigen and adjuvant, they were either provided in separate plasmids (20 μg each plasmid) or in the same plasmid (20 μg). All fish were given a single intramuscular injection on each side of the fish (0.05 ml). A non-immunized control group was included and received 2 x 50 μl PBS. For details of the vaccine groups, see Table 22 below.

Table 22

Group Design 1 Basic design 1 Construction 2 Basic design 2 Introduction of adjuvant Dose mcg
(2 x 0.05 ml) 1 G-ORF1 NTC9385R IFNb NTC9385R Antigen + adjuvant - in two separate plasmids 20 each 2 G-ORF1 NTC9385R     Antigen only 20 3 IFNb NTC9385R     Adjuvant only 20 4 G-ORF1-CMV-IFNb NTC9385R     Antigen + adjuvant in the same plasmid 20 5 G-ORF1-CMV-IFNb NTC9385R-eRNA41H-CpG     Antigen + adjuvant in the same plasmid 20 6 PBS          

[00164] Atlantic salmon ( Salmo salar ) (n=60 per group), maintained in freshwater and averaging 17 grams in weight, were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection into each side of the fish) with 0.05 ml of the test vaccine. The fish were then maintained in a tank where immunity developed for 78 days, 120 days, or 183 days at 12°C (12:12 light:dark). At each of these time points, groups of fish (n=15 per group at 78 days post-vaccination and 183 days post-vaccination and n=30 per group at 120 days post-vaccination) were transferred to a control tank. They were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml homogenized heart (isolate ID ALV1273 Heart) collected from an infected animal during a CMS outbreak in Norway. Fish (n=15 per group) were sampled 3 weeks post-challenge. Cardiac ventricles and kidneys were collected with ^RNALATER® from all sampled fish for subsequent RNA extraction. For the cohort challenged 120 days post-vaccination, additional sampling was done 7 weeks post-challenge. In addition to heart and kidney for ^RNALATER® , hearts (ventricle and atrium) were also formalin-fixed from all fish at this last sampling point (week 7) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale of 0–3 based on severity, where 0 indicates no pathology and 3 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Norway).

[00165] Vaccine efficacy was assessed by challenge at 26 weeks post-immunization (challenge at 12, 18, and 26 weeks post-immunization). Challenge at week 18 was followed for 50 days with two sampling points, 20 and 50 days post-challenge, where cardiac and renal tissue samples were collected for qPCR analysis and cardiac histopathology assessment as described above.

[00166] The results showed that significant protection against PMCV infection was achieved with all vaccines until the final challenge, 183 days/2196 degree days after vaccination. The combination of G-ORF1 and IFNb provided better protection than the two components alone (Tables 23, 24). Overall, the results indicate that the vaccine provides durable and reliable protection.

Table 23. Reservoir 1. Viral RNA in the heart 3 weeks post-challenge with three different pre-challenge immunization times. Mean PMCV Ct with standard deviation are shown for each group. N=15 per group, per sampling site.

Vaccine group Description 12 weeks of immunization 18 weeks of immunization 26 weeks of immunization 6 PBS 27.42±2.07 29.33±1.62 29.33±1.74 2 G-ORF1 28.70±2.19 30.50±2.38 29.62±3.59 3 IFNb 33.82±3.48 32.65±3.68 28.35±6.22 1 G-ORF1+IFNb 35.88±3.25 33.25±3.36 32.21±1.95

Table 24. Reservoir 2. Viral RNA in the heart 3 weeks post-challenge with three different pre-challenge immunization times. Mean PMCV Ct with standard deviation are shown for each group. N=15 per group, per sampling site.

Vaccine group Description 12 weeks of immunization 18 weeks of immunization 26 weeks of immunization 6 PBS 27.68±2.32 27.58±2.04 28.80±1.90 4 G-ORF1-CMV-IFNb 36.73±4.69 33.75±2.80 32.93±3.31 5 G-ORF1-CMV-IFNb 33.58±3.10 30.70±2.14 32.63±4.13

Table 25. Reservoir 1. Histopathological lesions (histoscore) in atria at 7 weeks post-challenge following an 18-week immunization period. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Histoscore, Atrium Standard deviation, histoscore N p-value (compared to PBS) 6 PBS 1.54 1.08 12 NP 2 G-ORF1 0.60 0.74 15 0.01 3 IFNb 0.43 0.78 15 0.0025 1 G-ORF1+IFNb 0.43 0.65 14 0.0028

Table 26. Reservoir 2. Histopathological lesions (histoscore) in atria at 7 weeks post-challenge following an 18-week immunization period. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Histoscore, Atrium Standard deviation, histoscore N p-value (compared to PBS) 6 PBS 1.68 0.75 11 NP 4 G-ORF1-CMV-IFNb 0.07 0.18 15 <0.0001 5 G-ORF1-CMV-IFNb 0.50 0.76 8 0.0002

EXAMPLE 7 – Optimizing Adjuvant Efficacy

[00167] In an attempt to further optimize the adjuvant potential of IFNb, we identified homologous genes in Atlantic salmon and closely related species. Because salmonids have recently undergone a whole genome evolutionary duplication, many salmon genes are found in duplicates (divergent homologs). Three new genes similar but not identical to the initially tested IFNb adjuvant were selected (Table 27). These were:

- Atlantic salmon (Salmo salar) – IFNb1 (divergent paralog gene)

- Brown trout (Salmo trutta)-IFN a3-like gene

- Sockeye salmon (Onchorhynchus nerka) – IFNa3-like gene.

Table 27

Gene Genbank access number 1 2 3 4 IFNb Salmo salar (SEQ ID NO: 6) 1 6 5 11 IFNb1 Salmo salar EU768890.1 2 96.74 9 13 IFNa3-like Salmo trutta XM_029727364.1 3 97.28 95.11 11 IFNa3-like Onchorhynchus nerka XM_029635101.1 4 94.02 92,93 94.02

Pairwise comparison of alternative IFNb adjuvants is shown as amino acid identity (%) and number of different amino acids (in italics).

[00168] Since integration of the vaccine into the salmon genome via homologous recombination may be of concern, risk reduction can be achieved by reducing the sequence similarity between the adjuvant and the salmon genome. This was done by first translating the plasmid-encoded adjuvants into protein sequences. These sequences were then reverse translated from proteins back into nucleotide sequences (reverse translation) based on codon frequency distribution (the probability of codon assignment is determined by their usage frequency in Atlantic salmon). Reverse translation was performed using CLC Main Workbench (Qiagen) with the software settings for codon usage based on frequency distribution.

[00169] The new adjuvant sequences obtained after this procedure were 74-80% identical to wild-type IFNb sequences at the nucleotide level. The activity of the four original native adjuvant candidates, including four codon-altered versions (called RTs), were then compared using an in vitro assay.

[00170] Thus, the wild-type nucleic acid sequence for IFNb (SEQ ID NO: 18) was approximately 76% identical to the RT nucleic acid sequence for IFNb (SEQ ID NO: 17), and the wild-type nucleic acid sequence for IFNb1 (SEQ ID NO: 19) was approximately 78% identical to the RT nucleic acid sequence for IFNb1 (SEQ ID NO: 20).

[00171] To facilitate the testing of various IFNb candidates and to evaluate their adjuvant potential, an in vitro screening assay was developed. The principle of this assay is to quantify the expression of Mx (an IFN-induced gene) by RT-qPCR. Mx proteins belong to the large GTPases superfamily that have antiviral activity against a wide range of RNA viruses. In vivo, the expression of Mx genes is tightly regulated by the presence of type I interferons (IFNs), and their induction has been described during several viral infections. Since CHH-1 cells do not express Mx in response to IFNb stimulation (a potential result of the lack of receptors) and TO cells are not easily transfected, the assay was designed as follows:

[00172] The functionality/activity of IFNb genes was studied by transfecting CHH-1 cells with various adjuvant constructs, including non-adjuvant constructs included as a negative control. Seven days after transfection, cell culture medium containing IFN molecules secreted by the CHH-1 cell transfectants was collected and transferred to another fish cell line (TO cells) at various dilutions. The final ratio of conditioned medium used to incubate TO cells was typically 1:6; 1:60, 1:600, and 1:6000, allowing the in vitro model to also provide quantitative measurements of adjuvant activity. After three days of incubation with conditioned medium, samples were collected from the TO cells by carefully removing the cell culture medium and extracting RNA from the cells. The IFNb-induced Mx response was analyzed by qPCR using the following primers/probes:

SEQ ID NO: 42, Mx forward: GATGCTGCACCTCAAGTCCTATTA

SEQ ID NO: 43, Mx av.: CGGATCACCATGGGAATCTGA

SEQ ID NO: 44, Mx Assay: 6-FAM-CAGGATATCCAGTCAACGTT-MGB

[00173] The results are presented in Table 28 below:

Table 28

Design Ratio of CHH-1 conditioned medium transferred to TO cells 1:6 1:60 1:600 Salmo Salar IFNb wild-type (SEQ ID NO: 18 21.61 24.29 34,44 Salmo Salar IFNb RT (SEQ ID NO: 17) 20.86 22,33 26.39 Salmo Salar IFNb1 wild type (SEQ ID NO: 19) 20.94 21.77 23.66 Salmo Salar IFNb1 RT (SEQ ID NO: 20) 20.57 21.05 21.84 Salmo trutta IFNa3 like wild type 20.66 22.17 27.04 Salmo trutta IFNa3, as RT 21.49 21.96 26.82 Onchorhynchus nerka INFa3 wild type 20.99 24.04 31.26 Onchorhynchus nerka INFa3 RT 21.28 22.87 28.13 eGFP No Ct No Ct No Ct Non-template control No Ct

Real-time Ct values for Mx in TO cells incubated with different concentrations of conditioned medium from transfected CHH-1 cells. Lower Ct values indicate higher levels of IFN induction.

[00174] These results demonstrate that the RT constructs have slightly higher immunomodulatory activity compared to the wild-type sequences. The results also clearly show that IFNb1 has higher immunomodulatory activity than IFNb.

[00175] Based on in vitro studies, four RT INFb adjuvants were selected for testing as vaccine adjuvants in fish (all groups received PMCV capsid antigen (G-ORF1) and adjuvant on two separate plasmids). In addition, one group received the G-ORF1 vaccine antigen plus a plasmid encoding eGFP as a negative adjuvant group. All fish were given a single intramuscular injection on each side of the body. All fish received the antigen and plasmid-encoded adjuvant on separate plasmids, the dose of each plasmid was 20 μg. A non-immunized control group was included and received 2 x 50 μl PBS as shown in Table 29.

Table 29

Group Description (Antigen) Detailed description Antigen Antigenic backbone Description (Adjuvant) Adjuvant skeleton Final concentration A, μg/ml Final concentration B (µg/ml) Dose mcg (2 x 0.05 ml) 1 G-ORF1 G-PMCV_ORF1
_full_1-2583 NTC9385R IFNb S.salar pVAX1 200 200 20 each 2 G-ORF1 G-PMCV_ORF1
_full_1-2583 NTC9385R IFNb1_ S.salar pVAX1 200 200 20 each 3 G-ORF1 G-PMCV_ORF1
_full_1-2583 NTC9385R IFNb_ S. trutta IFNa3-like pVAX1 200 200 20 each 4 G-ORF1 G-PMCV_ORF1
_full_1-2583 NTC9385R IFNb_ O. nerka INFa3-like pVAX1 200 200 20 each 5 G-ORF1 G-PMCV_ORF1
_full_1-2583 NTC9385R eGFP pcDNA3.1(+) 200 200 20 each 6 PBS PBS control NP          

[00176] Freshwater-housed Atlantic salmon ( Salmo salar ) (n=30/group), average weight 20 g, were anesthetized with tricaine (PHARMAQ), marked by shortening of the adipose fin and/or upper jaws, grouped, and injected intramuscularly twice during the same anesthetic period (one injection into each side of the fish) with 0.05 ml of the test vaccine. Immunity was allowed to develop for 48 days at 12°C (12:12 light:dark), after which the fish were again anesthetized with tricaine (PHARMAQ) and challenged with infectious PMCV by intraperitoneal injection of 0.1 ml of homogenized heart (isolate ID ALV1273 Heart) taken from an infected animal during a CMS outbreak in Norway. Fish (n=15/group) were then collected at two time points; 19 days and 49 days post-infection. Cardiac ventricles and kidneys were collected using RNALATER ^® from all sampled fish for subsequent RNA extraction. Hearts (ventricle and atrium) were also fixed in formalin from all fish at the last sampling point (day 49) for histopathological analysis. RNA was extracted from all samples stored in RNALATER ^® and analyzed by real-time RT-PCR for the presence of PMCV RNA using a commercially available service from PHARMAQ Analytiq (Norway). Formalin-preserved hearts were dissected and histopathology was scored on a scale of 0–3 based on severity, where 0 indicates no pathology and 3 indicates the presence of severe pathology (service provided by PHARMAQ Analytiq, Norway).

[00177] The results are shown in Tables 30 and 31 below.

Table 30. Reservoir 1. Viral RNA in kidneys at 3 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Ct value, PMCV Standard deviation, Ct n p-value (compared to PBS) 6 PBS 19.89 0.79 15 NP 1 G-ORF1 + IFNb S.salar 25.42 1.51 15 <0.0001 2 G-ORF1 + IFNb1 S.salar 27.66 1.31 15 <0.0001 3 G-ORF1 + IFNb a3 S. trutta 27.13 1.09 15 <0.0001 4 G-ORF1 + IFNb a3 O. nerka 26.74 1.25 15 <0.0001 5 G-ORF1 + EGFP 23,23 2.10 15 0.028

Table 31. Reservoir 2. Viral RNA in kidneys at 3 weeks post-infection. The reported p value was calculated by comparing individual groups to the PBS control group using one-way ANOVA and Dunnett's post-hoc test. NA = Not applicable.

Vaccine group Description Average Ct value, PMCV Standard deviation, Ct n p-value (compared to PBS) 6 PBS 20.73 1.44 15 NP 1 G-ORF1 + IFNb S. salar 26.55 0.94 15 <0.0001 2 G-ORF1 + IFNb1 S. salar 26,27 0.99 15 <0.0001 3 G-ORF1 + IFNb a3 S. trutta 26.72 1.15 15 <0.0001 4 G-ORF1 + IFNb a3 O. nerka 26.43 1.09 11 <0.0001 5 G-ORF1 + EGFP 21.63 1.76 15 0.20

[00178] The overall conclusion is that all RT adjuvants tested provided similar protection, although the protection provided by the IFNb1 RT construct was slightly higher than that provided by the IFNb RT construct. Codon-altered versions of these adjuvants are preferred to reduce the risk of homologous recombination.

[00179] The best method of delivering the adjuvant is on the same plasmid as the antigen, using a separate but identical promoter as the antigen, but delivery of antigen and adjuvant using different plasmids is also effective.

[00180] All publications cited in the specification, both patent and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All such publications are herein incorporated by reference in their entireties to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[00181] Although the invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. It should therefore be understood that numerous modifications can be made to the illustrative embodiments and that other designs can be devised without departing from the spirit and scope of the invention as defined by the following claims.

[00182]

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<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q1">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Piscine Myocarditis virus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEPNTSVIATEQQQAAMREVEAAAARDEVVEKIAFAEGAMMVQTRRLPSGK

SSVGGFLGELAQNIRAMNRSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSSRVFSNTGSNTMLSLWVPA

VPGPPAVPEHWDVAPSWFVCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGA

LRAGGFNTEHSLVSFVEPLIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWG

ANDTVARIVNVEDYMREAAGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVK

VRLLDVDDRTIGPGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPAL

NYVIGGADLGMLPLIQWSVGLGAEDMAQGSIAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGV

AVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYRERGVRPGSVANWQYV

RFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQAMGEDVINGQMGQPESMMRGVALNENQ

GLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEGGTMRKRIPAIEMRENGVEG

DLMNDDLYSIGTAAGYLAVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATM

RDNGVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVTNTNHRVKVSVSGGRAVVQ

KGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDI</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>200</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..200</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q2">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of ORF-1 protein of Piscine Myocarditis

virus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..200</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q3">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>YDNQARAAQVALCREADMAECPWGGYRERGVRPGSVANWQYVRFDPTVAVG

VAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQAMGEDVINGQMGQPESMMRGVALNENQGLAAATVRR

VVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEGGTMRKRIPAIEMRENGVEGDLMNDDLYS

IGTAAGYL</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="3">

<INSDSeq>

<INSDSeq_length>50</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q4">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of ORF-1 protein of Piscine Myocarditis

virus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q5">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>EGGTMRKRIPAIEMRENGVEGDLMNDDLYSIGTAAGYLAVEGMAGAQGGI</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>50</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q6">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of ORF-1 protein of Piscine Myocarditis

virus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q7">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>WDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATMRDNGVEGEP</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>50</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q8">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of ORF-1 protein of Piscine Myocarditis

virus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q9">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>CGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVNTTNHRVK</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="6">

<INSDSeq>

<INSDSeq_length>183</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..183</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q10">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Salmo Salar</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MAVLKWLSICLTLFCQGTAASKPCRWTQFRLGKLNDVSIGLLSDMGGLFPLM

CAEESVEQMFPEDLYKNTEGEDVYVVALEAMRYVEQLYNNSLTSVTWNKTKLNMFQNVIYRQVQNLELCV

VGGVWESSGDGWSVTLKTYFNKLNTVLKEKEHSACAWEIVRKEIRENLVQFKKFIDSRVKL</INSDSeq

_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="7">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q11">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of viral hemorrhagic septicemia virus

G-protein (VHSV-G)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q12">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEWNTFFLVILIIIIKSTTP</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="8">

<INSDSeq>

<INSDSeq_length>21</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q13">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of viral hemorrhagic septicemia virus

G-protein (VHSV-G)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q14">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>VGGAFLLLVLCCCCKASPPIP</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="9">

<INSDSeq>

<INSDSeq_length>779</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..779</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q15">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fusion peptide</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..779</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q16">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEWNTFFLVILIIIIKSTTPQITQRPPVENISTYHADWDHHHHHHGGGSGGR

MEPNTSVIATEQQQAAMREVEEAAARDEVVEKIAFAEGAMMVQTRRLPSGKSSVGGFLGELAQNIRAMN

RSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSRVFSNTGSNTMLSLWVPAVPGPPAVPEHWDVAPSWF

VCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGALRAGGFNTEHSLVSFVEP

LIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWGANDTVARIVNVEDYMREA

AGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVKVRLLDVDDRTIGPGGSLP

LNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYVIGGADLGMLPLIQWS

VGLGAEDMAQGSIAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGVAVEGGFWAQPAAGAAPFP

LGGLAGWVRGGGGSAVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATMRDN

GVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVNTTNHRVKVSVSGGRAVVQKGN

KAEMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDIGGRNLIPSDWSFHWSIWPSLSGMGVVGGAFLLLV

LCCCCKASPPIPNYGIPMQQFSRSQTV</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>929</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..929</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q17">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fusion peptide</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..929</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q18">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEWNTFFLVILIIIIKSTTPQITQRPPVENISTYHADWDHHHHHHGGGSGGR

MEPNTSVIATEQQQAAMREVEEAAARDEVVEKIAFAEGAMMVQTRRLPSGKSSVGGFLGELAQNIRAMN

RSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSRVFSNTGSNTMLSLWVPAVPGPPAVPEHWDVAPSWF

VCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGALRAGGFNTEHSLVSFVEP

LIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWGANDTVARIVNVEDYMREA

AGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVKVRLLDVDDRTIGPGGSLP

LNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYVIGGADLGMLPLIQWS

VGLGAEDMAQGSIAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGVAVEGGFWAQPAAGAAPFP

LGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYRERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVV

KVMVAPVPDRAAALADMAWGKGKVQAMGEDVINGQMGQPESMMRGVALNENQGLAAATVRRVVGLENESM

QTTHWSTTEVAMNGYYGRAGATAHHAAFPLSGGGGSWDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTP

VDNVATMRDNGVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVNTTNHRVKVSVS

GGRAVVQKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDIGGRNLIPSDWSFHWSIWPSLSGMG

VVGGAFLLLVLCCCCKASPPIPNYGIPMQQFSRSQTV</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="11">

<INSDSeq>

<INSDSeq_length>666</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..666</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q19">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Piscine myocarditis virus ORF 1 with an internal

deletion and a linker</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..666</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q20">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEPNTSVIATEQQQAAMREVEAAAARDEVVEKIAFAEGAMMVQTRRLPSGK

SSVGGFLGELAQNIRAMNRSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSSRVFSNTGSNTMLSLWVPA

VPGPPAVPEHWDVAPSWFVCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGA

LRAGGFNTEHSLVSFVEPLIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWG

ANDTVARIVNVEDYMREAAGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVK

VRLLDVDDRTIGPGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPAL

NYVIGGADLGMLPLIQWSVGLGAEDMAQGSIAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGV

AVEGGFWAQPAAGAAPFPLGGLAGWVRGGGGSAVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNTVGAMG

GWTRAVTPVDNVATMRDNGVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVTNTN

HRVKVSVSGGRAVVQKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDI</INSDSeq_sequen

ce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="12">

<INSDSeq>

<INSDSeq_length>816</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..816</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q21">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Piscine myocarditis virus ORF 1 with an internal

deletion and a linker</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..816</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q22">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEPNTSVIATEQQQAAMREVEAAAARDEVVEKIAFAEGAMMVQTRRLPSGK

SSVGGFLGELAQNIRAMNRSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSSRVFSNTGSNTMLSLWVPA

VPGPPAVPEHWDVAPSWFVCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGA

LRAGGFNTEHSLVSFVEPLIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWG

ANDTVARIVNVEDYMREAAGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVK

VRLLDVDDRTIGPGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPAL

NYVIGGADLGMLPLIQWSVGLGAEDMAQGSIAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGV

AVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYRERGVRPGSVANWQYV

RFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQAMGEDVINGQMGQPESMMRGVALNENQ

GLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSGGGGSWDVVQYQLPGPDDEAR

GVMNTVGAMGGWTRAVTPVDNVATMRDNGVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFG

GYQRSVTNTNHRVKVSVSGGRAVVQKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDI</INSD

Seq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="13">

<INSDSeq>

<INSDSeq_length>162</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..162</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q23">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Piscine myocarditis virus fragment of ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..162</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q24">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>AVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVAT

MRDNGVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVTNTNHRVKVSVSGGRAVV

QKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDI</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="14">

<INSDSeq>

<INSDSeq_length>10</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..10</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q25">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Piscine myocarditis virus fragment of ORF 1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..10</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q26">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>VSGGRAVVQK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="15">

<INSDSeq>

<INSDSeq_length>50</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q27">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Piscine myocarditis virus fragment of ORF 1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q28">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>VSVSGGRAVVQKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDI</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="16">

<INSDSeq>

<INSDSeq_length>2583</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..2583</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q29">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Piscine myocarditis virus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggaaccaaacacatctgtcattgcaacggagcagcagcaggctgccatga

gagaggtggaggccgaggcggcggccagagacgaagtggtggagaagatcgcattcgctgaaggagcgat

gatggtacagacgaggaggttaccatcaggaaagtcgtcggtaggaggttttctcggcgaactggcacag

aacatacgtgccatgaatcggtcattgcacacagataccaacatgctgaccgaaggggcgatggtggaca

gagcgagggcaaaagtacacaaaatcattagggaagggaatttggactctagggtattttcaaacacggg

gagcaacactatgttgtcactgtgggtaccagcagtaccgggaccaccggcagtaccggagcattgggac

gttgcgccgtcctggttcgtatgcagaccggggaaaaagggggggataaagatcacacaaagcgcatcaa

tggcagcattaaacccactatttagaggcgcagacgtggggccaatcgggacagcagtcagggcggatgt

aaacgcattttcaatgaatgcagttctgggagcactaagagccggggatttaacaccgaacattccctg

gtgtcattcgttgaaccactaattcggatcttgctaatgggggtacaaacacaagacaggggggaccagcc

catgggattgggttggagggatgagttcgcgaatagtcaatcccctagtattcacaacaagcgggaactt

cttcccagggggaccaaatttgaggtgtggggagccaacgatacagtggccaggatagtaaacgttgag

gactacatgcgcgaggcggccggggaggggaggttcgacgctggatggggaccggaattctggggtggga

caggggacgacgcagtggcggtggtaccgataagggcagtagaagcagggctaggagaagtaaacgcagg

gtggacattggcacacatggaatacccagtcaaggttagactacttgacgtcgacgaccgaacaattgga

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatgtacccgggc

cctatgccagggtactgtacgtcgtcgtggaccaaaacgcagacaggtgtgtgggggtgagagtgcaggg

acagggtgctgtaattgacgtggacccggcgttgaattacgtgatagggggagcggatttggggatgttg

ccgttgatacagtggagtgtagggctggggggccgaggacatggcgcagggatcgattgcacagacgcagc

gatgggtgaggatgtatggaaacgaggacgattgggaatcagcgtggcatctagtgtctagcgcgtacac

agtgtacagcccggcattcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgca

ggggcagcaccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggcgc

aggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagggagagaggggt

gagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacagtggctgtaggagtagctgct

cacttctggtcggtagtgaaggtgatggtggctcccgtcccagacagagcggctgctctggcggacatgg

cgtgggggaaggggaaggtgcaagccatgggtgaggatgtgatcaacgggcagatgggacaacctgagtc

catgatgagaggggtggcgctgaacgagaaccagggactagcggcggctacagtcaggagggtggttggg

ctggagaacgagtcgatgcaaacaacgcactggagtacaacggaggtagcaatgaacgggtactacggga

gagcaggagcaacagcacaccacgctgcatttccgttgtccgagggggggacaatgcgaaaacgaatacc

agctatagatgagggagaacggggtggagggggacctgatgaacgatgatctctattcaattggaacg

gcagcggggtacctggcggtagaggggatggcaggtgcgcaggggggtatctgggacgtggtccagtacc

agctgcctgggcctgacgatgaggcgaggggggtgatgaacacggtgggggcgatggggggatggacgag

ggcggtgacaccagtagacaatgtggccaccatgagggacaacggggttgagggggaaccttgtggaata

gtgatgtctctaccaacaagtgggaccgctgtggtggataggttagctaatttcggattaccaccagcga

gggcggaattaagagaagtaccatttggcgggtaccaaagatcagtcacaaacaccaaccacagagtcaa

ggtgagtgtgagtggggggcgagcagttgttcaaaaagggaacaaagccgagatgaatccagtctttgtc

aataggacaccaggacaaacgaccctaggccaaccaacaacagacactacagggatgacaactgcagatt

ttttagatata</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="17">

<INSDSeq>

<INSDSeq_length>552</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..552</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q30">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Nucleic acid sequence encoding IFNb with codon

changes</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..552</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q31">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggcagtactgaagtggctttccatctgcctgactctgttctgccaaggaa

ccgcggcctcaaagccttgtcggtggacccagttccggctggggaaactgaatgacgtttcaattggcct

actctccgacatggggggactcttcccgctgatgtgcgccgaggagtcggtggagcagatgtttccagag

gacctctacaagaacaccgagggcgaagatgtgtacgtggttgctctggaagccatgcgttacgtagagc

agctgtacaataactcacttacctccgtcacctggaacaaaactaagctcaacatgttccagaacgttat

ctacagacaggtgcagaaccttgagctatgcgttgtaggcggggtttgggagtctagtggtgacggatgg

tccgttacacttaagacctactttaataaactgaacactgtgctgaaggagaaagagcatagcgcttgtg

cctgggagattgtccgcaaggagattagggagaacctggttcagtttaagaagtttatcgactcaagagt

gaaactgtga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="18">

<INSDSeq>

<INSDSeq_length>552</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..552</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q32">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>wild-type nucleic acid sequence encoding IFN-b

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..552</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q33">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctgtattgaaatggttgagcatttgcctgactctgttctgccaaggca

cagcagcatcaaaaccttgcaggtggacgcagtttaggttggggaagctgaacgatgtgagcataggcct

gctctcagatatgggtggactctttccacttatgtgtgcagaagaaagcgtcgaacaaatgtttccagag

gatctttacaagaacacagaggtgaggacgtctatgtggtggcattggaggctatgcgatatgtggaac

aattatacaacaacagtctgacgtctgtcacgtggaacaaaacaaaacttaacatgttccaaaacgtcat

atatcgtcaagttcaaaacttagagttatgtgtcgtaggtggtgtttgggaatcctctggagatggatgg

tcggttactctgaaaacatacttcaacaagctgaacaccgtcttgaaagagaaggaacacagcgcatgcg

catgggagattgtgcgaaaggagatcgcgaaaacttggtgcagttcaagaaattcattgacagcagagt

caagctgtga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="19">

<INSDSeq>

<INSDSeq_length>552</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..552</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q34">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Nucleic acid sequence encoding

IFNb1</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..552</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q35">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctgtattgaaatggttgagcatttgcctgactctgttctgccaaggca

cagtagcatcaaaaccttgcaggtggacgcagtttaggttggggaagctgaacgatgtgagcataggcct

gctctcagatatgggtggactctttccacttatgtgtgcagaagaaaacgtcgaacaaatgtttccagag

gatctttacaaaaacacagagggtgaggacgtctctgtggttgcattggaggctatgcgatatgtggaac

aattatacaacaacagtctgacgtctgccacgtggagcaaaacaaaacttaacatgttccaaaacgtcat

atatcgtcaagttcaaaacttagagttatgtgtcgtaggtggtgtttgggaatcctctggagatggatgg

tcggttactctgaaaacatacttcaacaagctgaacaccgtcttgaaagagaaggaatacagcgcatgcg

catgggagattgtgcgaaaggagatcgcgaaaacttggtgcagttcaagaaattcattgacagcagagt

caagctgtga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="20">

<INSDSeq>

<INSDSeq_length>552</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..552</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q36">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Nucleic acid sequence encoding IFNb1 with codon

changes</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..552</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q37">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctgtactgaaatggctgtccatctgtctgacactgttctgccagggaa

ctgtagcttcaaagccatgcaggtggactcagttccgcttggggaaactaaatgacgtgagcatcggttt

gctgtccgacatgggaggactgtttccactgatgtgtgctgaggagaacgtggagcagatgttccccgag

gacctgtataagaacaccgagggcgaggacgtgtccgtcgtggccctggaggcgatgcggtacgtcgagc

aactttacaacaactcgctcacttccgccacatggagtaagacgaagctcaacatgttccagaacgtgat

ctacagacaggtgcagaacctagagctgtgtgtggtggggggggtatgggagtccagtggggatggctgg

tccgttaccctgaagacatactttaacaagcttaataccgtgctgaaagagaaagaatactcggcgtgtg

cctgggagattgtgcggaaggaaatcagggaaaatctggtgcagttcaagaaattcattgactcaagggt

aaagttgtga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="21">

<INSDSeq>

<INSDSeq_length>114</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..114</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q38">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of sea lice gut

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..114</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q39">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MENSRADVPNIEDKIPPKIEEDNELQGNSLTVPKSSNRESSNVRRMHTAVRL

NEVIVNKSHDAKLVILNLPSPPKIMGPDKDASYMEFLEVLTEGLERVLMVRGGGREVITIYS</INSDSe

q_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="22">

<INSDSeq>

<INSDSeq_length>50</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q40">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q41">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>CRERADMAECPWGGYRERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVK</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="23">

<INSDSeq>

<INSDSeq_length>50</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q42">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q43">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>VMVAPVPDRAAALADMAWGKGKVQAMGEDVINGQMGQPESMMRGVALNEN</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="24">

<INSDSeq>

<INSDSeq_length>50</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q44">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..50</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q45">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>QGLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLS</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="25">

<INSDSeq>

<INSDSeq_length>100</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..100</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q46">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..100</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q47">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>QGLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEG

GTMRKRIPAIEMRENGVEGDLMNDDLYSIGTAAGYLAVEGMAGAQGGI</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="26">

<INSDSeq>

<INSDSeq_length>100</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..100</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q48">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..100</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q49">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>EGGTMRKRIPAIEMRENGVEGDLMNDDLYSIGTAAGYLAVEGMAGAQGGIWD

VVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATMRDNGVEGEP</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="27">

<INSDSeq>

<INSDSeq_length>100</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..100</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q50">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..100</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q51">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>WDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATMRDNGVEGEPCG

IVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVNTNHRVK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="28">

<INSDSeq>

<INSDSeq_length>100</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..100</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q52">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..100</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q53">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>CGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVNTTNHRVKVS

VSGGRAVVQKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDI</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="29">

<INSDSeq>

<INSDSeq_length>150</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..150</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q54">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..150</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q55">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>QGLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEG

GTMRKRIPAIEMRENGVEGDLMNDDLYSIGTAAGYLAVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNTV

GAMGGWTRAVTPVDNVATMRDNGVEGEP</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="30">

<INSDSeq>

<INSDSeq_length>150</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..150</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q56">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..150</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q57">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>EGGTMRKRIPAIEMRENGVEGDLMNDDLYSIGTAAGYLAVEGMAGAQGGIWD

VVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATMRDNGVEGEPCGIVMSLPTSGTAVVDRLANFG

LPPARAELREVPFGGYQRSVNTTNHRVK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="31">

<INSDSeq>

<INSDSeq_length>150</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..150</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q58">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..150</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q59">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>WDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATMRDNGVEGEPCG

IVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVTNTNHRVKVSVSGGRAVVQKGNKAEMNPVF

VNRTPGQTTLGQPTTDTTGMTTADFLDI</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="32">

<INSDSeq>

<INSDSeq_length>200</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..200</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q60">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..200</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q61">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>QGLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEG

GTMRKRIPAIEMRENGVEGDLMNDDLYSIGTAAGYLAVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNTV

GAMGGWTRAVTPVDNVATMRDNGVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSV

TNTNHRVK</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="33">

<INSDSeq>

<INSDSeq_length>200</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..200</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q62">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..200</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q63">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>EGGTMRKRIPAIEMRENGVEGDLMNDDLYSIGTAAGYLAVEGMAGAQGGIWD

VVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATMRDNGVEGEPCGIVMSLPTSGTAVVDRLANFG

LPPARAELREVPFGGYQRSVTNTNHRVKVSVSGGRAVVQKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGM

TTADFLDI</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="34">

<INSDSeq>

<INSDSeq_length>399</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..399</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q64">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Piscine myocarditis virus ORF-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..399</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q65">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEPNTSVIATEQQQAAMREVEAAAARDEVVEKIAFAEGAMMVQTRRLPSGK

SSVGGFLGELAQNIRAMNRSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSSRVFSNTGSNTMLSLWVPA

VPGPPAVPEHWDVAPSWFVCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGA

LRAGGFNTEHSLVSFVEPLIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWG

ANDTVARIVNVEDYMREAAGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVK

VRLLDVDDRTIGPGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVD</I

NSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="35">

<INSDSeq>

<INSDSeq_length>499</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..499</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q66">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of piscine myocarditis virus Orf-1

protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..499</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q67">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEPNTSVIATEQQQAAMREVEAAAARDEVVEKIAFAEGAMMVQTRRLPSGK

SSVGGFLGELAQNIRAMNRSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSSRVFSNTGSNTMLSLWVPA

VPGPPAVPEHWDVAPSWFVCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGA

LRAGGFNTEHSLVSFVEPLIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWG

ANDTVARIVNVEDYMREAAGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVK

VRLLDVDDRTIGPGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPAL

NYVIGGADLGMLPLIQWSVGLGAEDMAQGSIAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGV

AVEGGFWAQPAAGAAPFPLGGLAGWVR</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="36">

<INSDSeq>

<INSDSeq_length>5</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..5</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q68">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Linker amino acid sequence</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..5</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q69">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>GGGGS</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="37">

<INSDSeq>

<INSDSeq_length>15</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..15</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q70">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Nucleic acid sequence encoding the

linker</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..15</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q71">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ggtggcggtggctcg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="38">

<INSDSeq>

<INSDSeq_length>30</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..30</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q72">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of fusion protein of Atlantic Salmon

Paramyxovirus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..30</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q73">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MDGPKFRFVLLILLTAPARGQVDYDKLLKV</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="39">

<INSDSeq>

<INSDSeq_length>77</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..77</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q74">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of fusion protein of Atlantic Salmon

Paramyxovirus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..77</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q75">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>LAKSEKILSGINPNIINTEMVLVAVILSLVCAMVVIGIVCWLSILTKWVRSC

RADCRRPNKGPDLGPIMSSQDNLSF</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="40">

<INSDSeq>

<INSDSeq_length>26</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q76">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Hemagglutinin (HE) protein of

Infectious salmon anemia virus (ISAV)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q77">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MARFIILFLLLAPVYSRLCLRNYPDT</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="41">

<INSDSeq>

<INSDSeq_length>61</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..61</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q78">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of Hemagglutinin (HE) protein of

Infectious salmon anemia virus (ISAV)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..61</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q79">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>TFNTNQVEQPANSVLSNIFISMGVAGFGIALFLAGWKACIWIAAFMYKSRGR

IPPSNLSVA</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="42">

<INSDSeq>

<INSDSeq_length>24</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..24</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q80">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>MX forward primer</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..24</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q81">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gatgctgcacctcaagtcctatta</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="43">

<INSDSeq>

<INSDSeq_length>21</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q82">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>MX reverse primer</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q83">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cggatcaccatgggaatctga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="44">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q84">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>MX assay probe</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q85">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>caggatatccagtcaacgtt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="45">

<INSDSeq>

<INSDSeq_length>2583</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..2583</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q86">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Codon optimized nucleic acid sequence encoding

Piscine myocarditis virus ORF 1 protein</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..2583</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q87">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggagcccaacaccagcgtcattgccacagagcagcagcaggctgcaatga

gggaggtggaggccgaggccgccgctagacgaggtggtcgagaaaatcgcatttgcagagggtgcaat

gatggtgcagactcgtaggctgcccagcggcaagtcatccgtcggtggatttctcggggagctcgcccag

aacattagggcaatgaacaggtccctccatacagacactaatatgctcaccgaggcgccatggtcgaca

gagctagagctaaagtccacaagattatcagggagggtaacctcgattcaagagtcttcagcaacacagg

atcaaacaccatgctcagtctgtgggtgcccgctgtccccggtccacccgctgtgcccgagcattgggat

gtcgctcccagctggtttgtctgccggccaggaaagaaaggtggaattaagatcactcagtcagcatcta

tggccgccctgaatcctctgtttagaggagccgatgtcggcccaattggtacagccgtgcgtgcagacgt

caacgcattttccatgaatgccgtgctgggggcactgagggccggaggtttcaatacagagcacagtctg

gtctcatttgtcgagccactgattagaatcctgctgatgggagtgcagacacaggacagaggaacatcac

cttgggactgggtgggcggaatgagttcccgtattgtgaatcccctggtctttacaactagtggaaattt

ctttcccggcggacccaacctcagagtgtggggtgctaacgataccgtcgcccgtattgtcaacgtcgag

gactacatgcgcgaggccgcaggcgagggaagatttgacgccggatggggtccagagttttggggtggaa

ctggggacgatgcagtcgccgtcgtgcctattagggccgtggaggctgggctcggagaggtcaacgccgg

ttggaccctcgcccacatggagtaccccgtcaaagtccggctcctggacgtcgacgacaggaccatcggc

cccggagggagtctgccactcaacgcaaatcgggagtacaccgctgccggagctacacacgtgccaggac

catacgccaggtgctgtacgtcgtggtcgatcagaacgccgacaggtgcgtgggtgtcagggtccaggg

gcaggggggcagtcatcgacgtcgatcccgccctgaactacgtgatcgggggagccgacctgggtatgctc

cccctgatccagtggagtgtgggcctgggagccgaggacatggctcaggtagcattgcccagacccagc

gttgggtcagaatgtatgggaacgaggacgattgggagagtgcctggcatctggtctcaagtgcctatac

tgtctatagtcctgcttttcgccggagtggcgtggctgtcgagggaggcttctgggcacagcccgctgcc

ggtgcagctccatttcccctgggcgggctcgcaggatgggtgaggtatgataaccaggccagggccgctc

aggtggccctgtgcagggagcgggctgacatggcagagtgcccctggggaggctatcggggagaggggagt

gaggcccggtagtgtcgccaactggcagtacgtgcgctttgaccccaccgtggctgtcggagtggctgcc

cacttctggagtgtcgtgaaagtcatggtcgcacccgtgccagatcgtgctgcagctctggccgatatgg

cctggggaaagggaaaagtccaggccatgggggaggacgtcatcaatggccagatgggacagccagagag

tatgatgcgcggagtcgccctgaacgagaaccaggggctggcagccgctactgtgaggcgtgtcgtcgga

ctggagaatgagagcatgcagacaactcactggtcaaccacagaggtggctatgaatggttactatggca

gggccggtgccacagcacaccatgctgccttccctctctcagagggaggtacaatgaggaagcgtatccc

tgcaatcgagatgcgtgagaacggtgtggagggggacctgatgaacgatgacctctacagcatcgggact

gccgccggatatctggctgtggagggcatggccggcgctcagggcggaatttgggacgtggtgcagtatc

agctgcctggacccgatgacgaggctcgtggtgtgatgaatactgtgggagccatgggtggatggaccag

agcagtgactcccgtggacaacgtggctactatgcgtgacaatggagtggagggcgagccttgcggtatc

gtcatgtccctgccaacttccggcaccgctgtggtcgaccggctggccaactttggcctcccaccagcaa

gggctgagctcagggaggtcccattcggaggataccagcgcagcgtgacaaacactaaccacagggtgaa

ggtctccgtctctggaggcagagcagtcgtgcagaagggaaacaaggcagagatgaatcccgtgttcgtc

aacagcaacccggccagacaacactgggacagcccaccacagacactacaggcatgaccactgccgact

tcctcgacatt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="46">

<INSDSeq>

<INSDSeq_length>856</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..856</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q88">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Piscine myocarditis virus ORF 1

fragment</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..856</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q89">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEPNTSVIATEQQQAAMREVEAAAARDEVVEKIAFAEGAMMVQTRRLPSGK

SSVGGFLGELAQNIRAMNRSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSSRVFSNTGSNTMLSLWVPA

VPGPPAVPEHWDVAPSWFVCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGA

LRAGGFNTEHSLVSFVEPLIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWG

ANDTVARIVNVEDYMREAAGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVK

VRLLDVDDRTIGPGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPAL

NYVIGGADLGMLPLIQWSVGLGAEDMAQGSIAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGV

AVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYRERGVRPGSVANWQYV

RFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQAMGEDVINGQMGQPESMMRGVALNENQ

GLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEGGTMRKRIPAIEMRENGVEG

DLMNDDLYSIGTAAGYLAVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATM

RDNGVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELREVPFGGYQRSVNTTNHRVKVSGGGGSGNKA

EMNPVFVNRTPGQTTLGQPTTDTTGMTTADFLDI</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="47">

<INSDSeq>

<INSDSeq_length>969</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..969</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q90">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fusion protein containing a fragment of ORF-1

protein of Piscine Myocarditis virus</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..969</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q91">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEWNTFFLVILIIIIKSTTPQITQRPPVENISTYHADWDHHHHHHGGGSGGR

MEPNTSVIATEQQQAAMREVEEAAARDEVVEKIAFAEGAMMVQTRRLPSGKSSVGGFLGELAQNIRAMN

RSLHTDTNMLTEGAMVDRARAKVHKIIREGNLDSRVFSNTGSNTMLSLWVPAVPGPPAVPEHWDVAPSWF

VCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNAFSMNAVLGALRAGGFNTEHSLVSFVEP

LIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLVFTTSGNFFPGGPNLRVWGANDTVARIVNVEDYMREA

AGEGRFDAGWGPEFWGGTGDDAVAVVPIRAVEAGLGEVNAGWTLAHMEYPVKVRLLDVDDRTIGPGGSLP

LNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYVIGGADLGMLPLIQWS

VGLGAEDMAQGSIAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGVAVEGGFWAQPAAGAAPFP

LGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYRERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVV

KVMVAPVPDRAAALADMAWGKGKVQAMGEDVINGQMGQPESMMRGVALNENQGLAAATVRRVVGLENESM

QTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEGGTMRKRIPAIEMRENGVEGDLMNDDLYSIGTAAGYLA

VEGMAGAQGGIWDVVQYQLPGPDDEARGVMNTVGAMGGWTRAVTPVDNVATMRDNGVEGEPCGIVMSLPT

SGTAVVDRLANFGLPPARAELREVPFGGYQRSVTNTNHRVKVSGGGGSGNKAEMNPVFVNRTPGQTTLGQ

PTTDTTGMTTADFLDIGGRNLIPSDWSFHWSIWPSLSGMGVVGGAFLLLVLCCCCKASPPIPNYGIPMQQ

FSRSQTV</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="48">

<INSDSeq>

<INSDSeq_length>39</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..39</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q92">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of viral hemorrhagic septicemia virus

G-protein (VHSV-G)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..39</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q93">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEWNTFFLVILIIIIKSTTPQITQRPPVENISTYHADWD</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="49">

<INSDSeq>

<INSDSeq_length>58</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..58</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q94">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Fragment of viral hemorrhagic septicemia virus

G-protein (VHSV-G)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..58</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q95">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>NLIPSDWSFHWSIWPSLSGMGVVGGAFLLLVLCCCCKASPPIPNYGIPMQQF

SRSQTV</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="50">

<INSDSeq>

<INSDSeq_length>183</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..183</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q96">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Salmo salar</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MAVLKWLSICLTLFCQGTVASKPCRWTQFRLGKLNDVSIGLLSDMGGLFPLM

CAEENVEQMFPEDLYKNTEGEDVSVVALEAMRYVEQLYNNSLTSATWSKTKLNMFQNVIYRQVQNLELCV

VGGVWESSGDGWSVTLKTYFNKLNTVLKEKEYSACAWEIVRKEIRENLVQFKKFIDSRVKL</INSDSeq

_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (31)

1. A protein for generating an immune response against fish myocarditis virus (PMCV), comprising SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 46, or a sequence that is at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identical to SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 46. 2. The protein of claim 1, wherein at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or 100% of the amino acids that differ from SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 46, are conservative substitutions. 3. A fusion protein for generating an immune response against fish myocarditis virus (PMCV), comprising the protein of any one of claims 1 or 2 and further comprising: a) an N-terminal secretion signal sequence of a secreted or first membrane-bound protein located upstream of the protein according to any one of claims 1, 2; b) a transmembrane domain of a second membrane-bound protein located downstream of the protein according to any one of claims 1, 2. 4. The fusion protein according to claim 3, characterized in that the first membrane-bound protein is identical to the second membrane-bound protein. 5. The fusion protein according to claim 4, characterized in that said membrane-bound protein is the G protein of the hemorrhagic septicemia virus (VHSV-G). 6. The fusion protein of claim 5, wherein said N-terminal secretion signal sequence is at least 90% identical to SEQ ID NO: 7 or SEQ ID NO: 48, or wherein said secretion signal sequence comprises a portion of SEQ ID NO: 48 or a sequence that is at least 90% identical to SEQ ID NO: 48, wherein said portion comprises SEQ ID NO: 7 or a sequence that is at least 90% identical to SEQ ID NO: 7. 7. The fusion protein of claim 6, wherein at least half of the differing amino acids in said N-terminal secretion signal are conservative substitutions. 8. The fusion protein of any one of claims 3 to 7, wherein said transmembrane domain is at least 90% identical to SEQ ID NO: 8 or SEQ ID NO: 49, or wherein said transmembrane fragment comprises a portion of SEQ ID NO: 49 or a sequence that is at least 90% identical to SEQ ID NO: 49, wherein said portion comprises SEQ ID NO: 8 or a sequence that is at least 90% identical to SEQ ID NO: 8. 9. A fusion protein according to any one of claims 3 to 8, wherein at least half of the differing amino acids in said transmembrane domain are conservative substitutions. 10. A nucleic acid molecule that encodes a protein according to any one of claims 1, 2 or a fusion protein according to any one of claims 3-9. 11. A nucleic acid molecule according to claim 10, comprising SEQ ID NO: 16. 12. A vector for expressing a protein according to any one of claims 1 or 2 or a fusion protein according to any one of claims 3 to 9, containing a nucleic acid molecule according to claim 10 or 11. 13. The vector according to claim 12, additionally containing a nucleic acid sequence encoding an immunomodulator. 14. The vector according to item 13, characterized in that the immunomodulator is interferon. 15. A vector according to any one of paragraphs 12-14, characterized in that it is a plasmid vector. 16. A host cell for expressing a protein according to any one of claims 1 or 2 or a fusion protein according to any one of claims 3 to 9, comprising a vector according to any one of claims 12 to 15. 17. A vaccine for preventing infection with piscine myocarditis virus (PMCV) in salmonids in need thereof, comprising an effective amount of a vector according to any one of claims 12-15 or a vector comprising a nucleic acid molecule encoding a protein according to SEQ ID NO: 1, or an amino acid sequence 98% identical thereto. 18. A method for preventing infection with the fish myocarditis virus (PMCV) in salmon fish in need thereof, comprising administering to said salmon an effective amount of the vaccine according to claim 17. 19. The method according to item 18, characterized in that said salmon weighs from 15 to 200 g. 20. The method according to item 19, characterized in that said salmon weighs from 40 to 110 g. 21. The method according to any one of paragraphs 18-20, characterized in that said salmon is Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), chinook salmon (Oncorhynchus tshawytscha) or coho salmon (Oncorhynchus kisutch). 22. The vaccine according to claim 17 for use in a method for protecting salmonids from infection. 23. The vaccine according to claim 22, characterized in that said vector is a vector according to any of claims 12-15. 24. The vaccine according to claim 22, characterized in that the vector contains a nucleic acid molecule encoding a protein according to SEQ ID NO: 1 or an amino acid sequence that is 98% identical to it. 25. A vaccine according to any of paragraphs 22-24, characterized in that said salmon weighs from 15 to 200 g. 26. A vaccine according to any of paragraphs 22-25, characterized in that said salmon weighs from 40 to 110 g. 27. The vaccine according to any one of paragraphs 22-26, characterized in that said salmon is Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) or coho salmon (Oncorhynchus kisutch). 28. The vaccine according to claim 27, characterized in that said salmon is Salmo salar. 29. The method according to claim 21, characterized in that said salmon is Salmo salar.