CA2442346A1 - Nucleic acids encoding isav polypeptides - Google Patents

Abstract

Infectious Salmon Anemia Virus (ISAV) nucleic acid molecules and polypeptide s are disclosed, as well as host cells and transgenic fish transformed by expression vectors containing such nucleic acids. The nucleic acid molecules can encode antigenic epitopes capable of eliciting an immune response in a host cell or animal, such as an immune response against ISAV, and the polypeptides themselves can be antigenic epitopes and also induce such an immune response.

Description

NUCLEIC ACIDS ENCODING ISAV POLYPEPTIDES
FIELD
This invention relates to Infectious Salmon Anemia Virus (ISAV), more specifically to ISAV nucleic acid sequences and the peptides these nucleic acids encode. This invention also relates to the use of ISAV peptides in producing an immune response in fish.
BACKGROUND
Global aquaculture production is estimated at 39.4 million tons annually, is worth $52.5 billion (LJS), and contributes over 20% of the total fish harvest.
Although the United States contributes only 2% of global production, the aquaculture industry in this country is gaining momentum and importance. For example, farm-raised salmon are a prominent industry in the Pacific Northwest and Maine.
As the natural fisheries provided by the open seas decline globally, and the world's population is projected to grow to ~ billion people by 2025, cultured finfish products will be in increasing demand as an important protein source. Some of the factors that must be successfully accommodated to sustain the economic viability and increase the productivity of finfish culture include maintaining adequate culture facilities, complying with regulatory and environmental requirements and countering the many infectious pathogens and diseases that can threaten farmed populations of aquatic animals. Of these variables, the economic impact of disease on cultured finfish operations has become increasingly important. One of the primary means for raising finfish culture efficiency is through the development of reliable treatments against infectious pathogens and thus improve the overall health of farmed species.
Infectious salmon anemia (ISA), formerly called Hemorrhagic Kidney Syndrome (HKS), has caused massive economic losses in the Atlantic salmon farming industry in Norway, Atlantic Canada, and Scotland. Mortality from ISA
disease is variable, ranging from 10% to more than 50%. Clinical signs of the disease axe apparent in Atlantic salmon, but other salmonids can act as non-symptomatic reservoirs for the virus. The pathological changes associated with ISA

_2_ are characterized by severe anemia, leukopenia, ascites and hemorrhaging of internal organs with subsequent necrosis of hepatocytes and renal interstitial cells.
The infectious agent is an enveloped virus (ISAV) which replicates in endothelial cells in vivo and buds from the cell surface. The virus has a single-stranded RNA
genome consisting of 8 segments with negative polarity, and the structural, morphological, and physiochemical properties of the virus suggest that ISAV is related to members of the O~thonzyxoviridae family (see, e.g., Falk, et al., J. Virol. 71:9016-23 (1997)).
ISA originally appeared in Norway in 1984 (Thorud and Djubvik, 1988). In 1996 and 1998, the disease was diagnosed on fish farms in Atlantic Canada and Scotland, respectively. Subsequent to the appearance of clinical disease in Canada, ISAV surveillance programs were instituted in New Brunswick. A central aspect of the Canadian ISAV management approach involves the depopulation of ISAV-infected cages that are found through participation in the surveillance protocols. The Canadian government and Canadian salmon producers themselves have developed several compensation programs to offset losses from eradication measures, which has helped lower the incidence of new cases of both virus and disease at previously negative marine sites. Recent Canadian outbreaks are currently confined to the Bay of Fundy area of Maritime Canada. However, the Norwegian disease pattern has shown that the virus spreads from population to population principally by exposure to body fluids from infected fish, through untreated water coming from fish processing plants or through shared equipment that hasn't been properly disinfected at marine sites. Thus, Atlantic salmon netpens at neighboring Maine marine sites are at considerable risk of encountering ISA virus.
Historically, the elimination of ISA disease in other countries through the attempted eradication of ISA virus has proven to be futile. Given the many unknown factors involved in disease transmission, including ties between the ISA
pathogen and wild reservoirs of virus, outright elimination of ISA and the virus (ISAV) does not appear to be an achievable goal. However, as shown over time in several other international epizootics of ISA, mortality from ISA can be decreased through the development of biosecurity protocols and good management techniques.
Nonetheless, the development of effective treatments against ISAV remains a high priority for salmon producers in the U.S. and elsewhere.

Fish that survive ISA demonstrative a protective immune response indicating that prophylactic treatment against ISA is possible. Whole killed viral formulations have been shown to be effective against other viral diseases of fish, but the disadvantage of such an approach is that virulent virus may remain in the formulation if extreme care is not taken during the manufacturing process.
Additionally, the immune.response conferred is often brief and may need to be boosted. Finally, killed virus formulations are prepared by growing virus in large amounts in cell culture or in the actual animal species, and either method is expensive. Furthermore, if the titer of the amplified virus is low, then achieving the appropriate antigenic dose within the final formulation requires the addition of more virus and raises the cost of production. Thus, a need remains for an effective ISAV
vaccine.
SUMMARY
ISAV nucleic acid molecules are disclosed. In some embodiments, the nucleic acid molecule has a sequence at least 70% identical to SEQ ID NO: 1, a nucleic acid sequence at least 85% identical to SEQ ID NO: 3, or a nucleic acid sequence at least 85% identical to SEQ ID NO: 11, or a sequence consisting essentially of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 11. In particular embodiments, the nucleic acid molecule is operably linked to a heterologous nucleic acid, such as an expression control sequence. In one specific non-limiting example, the nucleic acid sequence is included in a vector.
Host cells and transgenic fish transformed by such nucleic acids also are disclosed. In some embodiments, the nucleic acid molecule encodes an antigenic epitope capable of eliciting an immune response in the cell or fish, such as an immune response against ISAV. Particular fish and fish cells include (but are not limited to) rainbow trout, coho salmon, Chinook salmon, amago salmon, chum salmon, sockeye salmon, Atlantic salmon, arctic char, brown trout, cutthroat trout, brook trout, catfish, tilapia, sea bream, seabass, flounder, or sturgeon.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the results of an efficacy trial of Atlantic salmon treated with whole killed ISAV and challenged with live ISAV.
FIG. 2 is a digital image of the results of SDS-PAGE analysis of purified ISAV proteins.
FIG. 3 is a graph illustrating the results of a humoral immune response to whole killed ISAV in Atlantic salmon.
FIG. 4 is the amino acid sequence alignment of the RNA binding domain of NP from influenza virus A and B with the putative NP RNA binding domain from ISA virus. This alignment was predicted using the Clustal W system.
FIG. 5 is a graph illustrating the titration of ISAV-specific antibodies from Atlantic salmon infected with ISAV.
FIG. 6 is a graph illustrating the ISAV-specific antibodies in sera obtained from Atlantic salmon infected with ISAV or rainbow trout injected with a nucleic acid encoding an ISAV-specific protein.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
The nucleic acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
SEQ ID NO: 1 shows a 2.4 kbp nucleic acid fragment of ISAV (segment 1) with a partial open reading frame (orf) encoding the PI protein.
SEQ ID NO: 2 shows the partial amino acid sequence of the PI protein encoded by SEQ ID NO: 1.
SEQ ID NO: 3 shows a 2.4 kbp nucleic acid fragment of ISAV (segment 2) with a 2127 by orf encoding the PBI protein.
SEQ ID NO: 4 shows the amino acid sequence of the PBI protein, measuring 709 aa, encoded by SEQ ID NO: 3.
SEQ ID NO: 5 shows a 2.2 kbp nucleic acid fragment of ISAV (segment 3) with a 1 X51 by orf encoding the NP protein.

SEQ ID NO: 6 shows the amino acid sequence of the NP protein, measuring 617 aa, encoded by SEQ ID NO: 5.
SEQ ID NO: 7 shows a 1.9 kbp nucleic acid fragment of ISAV (segment 4) with a 1737 by orf encoding the P2 protein.
SEQ ID NO: 8 shows the amino acid sequence of the P2 protein, measuring 579 aa, encoded by SEQ ID NO: 8.
SEQ ID NO: 9 shows a 1.6 kbp nucleic acid fragment of ISAV (segment 5) with a 1335 by orf encoding the P3 protein.
SEQ ID NO: 10 shows the amino acid sequence of the P3 protein, measuring 445 aa, encoded by SEQ ID NO: 9.
SEQ ID NO: 11 shows a 1.5 kbp nucleic acid fragment of ISAV (segment 6) with an 1185 by orf encoding the HA protein.
SEQ ID NO: 12 shows the amino acid sequence of the HA protein, measuring 395 aa, encoded by SEQ ID NO: 10.
SEQ ID NO: 13 shows a 1.3 kbp nucleic acid fragment of ISAV (segment 7) with a 771 by orf encoding the P~ protein and a 441 by orf encoding the PS
protein.
SEQ ID NO: 14 shows the amino acid sequence of the P4 protein, measuring 257 aa, encoded by SEQ ID NO: 13.
SEQ ID NO: 15 shows the amino acid sequence of the PS protein, measuring 147 aa, also encoded by SEQ ID NO: 13 SEQ ID NO: 16 shows a 1.0 kbp nucleic acid fragment of ISAV (segment 8) with a 705 by orf encoding the P6 protein and a 552 by orf encoding the P7 protein.
SEQ ID NO: 17 shows the amino acid sequence of the P6 protein, measuring 235 aa, encoded by SEQ ID NO: 16.
SEQ ID NO: 18 shows the amino acid sequence of the P7 protein, measuring 184 aa, also encoded by SEQ ID NO: 16.

DETIALED DESCRIPTION
Abbreviations as = amino acid by = base pair ISA = infectious salmon anemia ISAV = infectious salmon anemia virus kbp = kilo-base pair orf = open reading frame PCR = polymerase chain reaction RT = reverse transcription Tef gas The following explanations of terms are provided in order to facilitate review of the embodiments described herein. Explanations of common terms also can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; Lewin, Nucleic acids 1~II, Oxford University Press: New York, 1999; and Dictio~ra~y of Bioscience, Mcgraw-Hill: New York, 1997.
The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a nucleic acid" includes single or plural nucleic acids and is considered equivalent to the phrase "comprising at least one nucleic acid."
The term "or" refers to a single element of stated alternative elements or a combination of two or more elements. For example, the phrase "a first nucleic acid or a second nucleic acid" refers to the first nucleic acid, the second nucleic acid, or both the first and second nucleic acids.
As used herein, "comprises" means "includes." Thus, "comprising A and B" means "including A and B," without excluding additional elements.
The standard one- and three-letter nomenclature for amino acid residues is used.
Amplification of a nucleic acid. Any of several techniques that increases the number of copies of a nucleic acid molecule. An example of amplification is the _7_ polymerase chain reaction (PCR), in which a sample containing the nucleic acid is contacted with a pair of oligonucleotide primers under conditions that allow for the hybridization of the primers to nucleic acid in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.
The amplification products (called "amplicons") can be further processed, manipulated, or characterized by (without limitation) electrophoresis, restriction endonuclease digestion, hybridization, nucleic acid sequencing, ligation, or other techniques of molecular biology. Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in European Patent Appl. 320 308; gap filling ligase chain reaction amplification, as disclosed in U.S. Patent No. 5,427,930; and NASBATM RNA transcription-free amplification, as disclosed in U.S. Patent No.
6,025,134.
Conservative amino-acid substitution. Conservative amino acid substitutions in a polypeptide, such as an ISAV polypeptide, include those listed in Table 1 below.

_$_ Table 1 Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu Non-conservative substitutions are those that disrupt the secondary, tertiary, or quaternary conformation of a polypeptide. Such non-conservative substitutions can result from changes in: (a) the structure of the polypeptide backbone in the area of the substitution; (b) the charge or hydrophobicity of the polypeptide; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in polypeptide properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; or (c) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine. In particular embodiments, a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is not substituted for (or by) an electronegative residue, for example, glutamyl or aspaxtyl.
Analog or homolog. An analog is a molecule that differs in chemical structure from a parent compound. A homolog differs by an increment in the chemical structure (such as a difference in the length of a nucleic acid or amino acid chain), a molecular fragment, a structure that differs by one or more functional groups, or a change in ionization.
Antigen. A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes.
Animal. A living, mufti-cellular, vertebrate organism, including, for example, mammals, birds, reptiles, and fish. The term "aquaculture animal"
includes all species suitable for aquaculture farming, such as fish, cephalopods, and crustaceans, including the specific species described herein. Similarly, the term "subject" includes both human and veterinary subjects, such as aquaculture animals.
cDNA (complementary DNA). A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription.
cDNA can be synthesized in a laboratory by reverse transcription from messenger RNA extracted from cells.
Complementarity. A nucleic acid that performs a similar function to the sequence to which it is complementary. The complementary sequence does not have to confer replication competence in the same cell type to be complementary, but merely confer replication competence in some cell type.
Delivery of compositions. For administration to animals, purified active compositions can be administered alone or combined with an acceptable carrier.
Preparations can contain one type of therapeutic molecule, or can be composed of a combination of several types of therapeutic molecules. The nature of the carrier will depend on the particular mode of administration being utilized. For instance, parenteral formulations usually comprise injectable fluids that include physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), which can be added to an aquaculture environment, conventional non-toxic solid carriers can include, for example, mannitoh, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, compositions to be administered to fish can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
It is also contemplated that the nucleic acids could be delivered to cells subsequently expressed by the host cell, for example through the use viral vectors, plasmid vectors, or liposomes administered to fish.
Compositions of the present invention can be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of the nucleic acids, or an active fragment thereof, can be readily determined.
For use in treating viral infections, compositions are administered in an amount effective to inhibit viral infection or progression of an existing infection, or administered in an amount effective to inhibit or alleviate a corresponding disease.
In one embodiment, infection is completely prevented.
Typical amounts initially administered would be those amounts adequate to achieve tissue concentrations at the site of action which have been found to achieve the desired effect ih vitro. The compositions can be administered to a host ih vivo, for example through systemic administration, such as intravenous, intramuscular, or intraperitoneal administration. The compositions also can be administered intralesionahhy, through scarification of the skin, intrabuccal adminitstration, cutaneous particle bombardment, or by immersion in water containing a nucleic acid composition described herein (for uptake by the fish). Additionally, the nucleic acid compositions can be administered by encapsulation with a nanoparticle matrix composed of a nucleic acid in methacrylic acid polymer, and an attenuated bacteria (such as Yersinia rucked, Edwa~dsiellc~ ictaluri, Aeromonas salmonicida, or Yibrio anguillarurn) carrying the nucleic acid for delivery by immersion administration (see, e.g., U.S. Patent No. 5,877,159, herein incorporated by reference).
Effective doses for using compositions can vary depending on the severity of the condition to be treated, the age and physiological condition of the fish, mode of administration, and other relevant factors. Thus, the final determination of the appropriate treatment regimen can be made by someone at the site of the fish, such as an operator or employee of an aquaculture facility. Typically, the dose range will be from about 1 ~,g/kg body weight to about 100mg/kg body weight, such as about wg/kg body weight to about 900 ~.g/kg body weight, or from about 50 ~,g/kg body 10 weight to about 500 ~.g/kg body weight, or from about 50 ~g/kg body weight to about 150 ~,g/kg body weight, such as about 100 ~,g/kg body weight. Nanogram quantities of transforming DNA have been shown to be capable of inducing an immune response in fish (see, e.g., Corbeil, S., et al., Tlaceine 18(25):2817-(2000), herein incorporated by reference).
The dosing schedule can vary from a single dosage to multiple dosages given several times a day, once a day, once every few days, once a week, once a month, annually, biannually, biennially, or any other appropriate periodicity. The dosage schedule can depend on a number of factors, such as the species' or subject's sensitivity to the composition, the type and severity of infection, route of administration, and the volume of the container that contains the fish. In the case of a more aggressive disease, compositions can be administered by alternate routes, including intramuscularly and by environmental uptake. Continuous administration also can be appropriate in some circumstances, for example, immersing fish or other aquaculture animals in water containing the composition.
Hybridization conditions. "Stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization probe and the target sequence. "Stringent conditions" can be broken down into particular levels of stringency for more precise measurement.
Thus, as used herein, "moderate stringency" conditions are those under which DNA
molecules with more than 25% sequence variation (also termed "mismatch") will not hybridize; conditions of "medium stringency" are those under which DNA
molecules with more than 15°/~ mismatch will not hybridize, and conditions of "high stringency" are those under which DNA sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which DNA sequences with more than 6% mismatch will not hybridize.
Hybridization. Oligonucleotides hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding between complementary nucleotide units. For example, adenine and thymine are complementary nucleotides that pair through formation of hydrogen bonds.
"Complementary" refers to sequence complementarity between two nucleotide units.
For example, if a nucleotide unit at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide unit at the same position of a nucleic acid molecule, then the oligonucleotides are complementary to each other at that position. The oligonucleotide and the nucleic acid molecule are complemtary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotide units that can hydrogen bond with each other.
Nucleic acid molecules and nucleotide sequences derived from the disclosed molecules also can be defined as nucleotide sequences that hybridize under stringent conditions to the sequences disclosed, or fragments thereof.
"Specifically hybridizable" and "complementary" are terms which indicate a sufficient degree of complementarity, such that stable and specific binding occurs between an oligonucleotide and the target nucleic acid. An oligonucleotide need not be 100% complementary to the target to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target molecule interferes with the normal function of the target and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired (for example, under physiological conditions in the case of ih vivo assays) or under conditions in which the assays are performed.
Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization, method of choice, and the composition and length of the hybridizing nucleic acid used. Generally, the temperature of hybridization and the ionic strength (especially the Na+
concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Gold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ).
Epitope. A site on an antigen at which an antibody can bind, the molecular arrangement of the site determining the combining antibody. A portion of an antigen molecule that determines its capacity to combine with the specific combining site of its corresponding antibody in an antigen-antibody interaction.
Nucleotide molecules that hybridize. Nucleotide molecules and sequences which are derived from the disclosed nucleotide molecules as described above also can be defined as nucleotide sequences that hybridize under stringent conditions to the nucleotide sequences disclosed, or fragments thereof.
Genetic fragment. Any nucleic acid derived from a larger nucleic acid.
Hcterologous. Originating from a different organism or distinct tissue culture, such as from a different species or cell line.
Homologs. Two nucleotide sequences that share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species.
Tsolated. An "isolated" biological component (such as a nucleic acid, polypeptide, protein, or organelle) has been substantially separated, produced apart from, or purified away from other biological components (for example, other chromosomal and extrachromosomal DNA and RNA, and polypeptides) found in the cell of the organism in which the component naturally occurs. Nucleic acids, polypeptides, and proteins that have been "isolated" thus include nucleic acids and polypeptides purified by standard purification methods. The term also embraces nucleic acids, polypeptides, and proteins that are chemically synthesized or prepared by recombinant expression in a host cell.
Nucleic acid. A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form. Unless otherwise limited, this term encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. An "oligonucleotide" (or "oligo") is a linear nucleic acid of up to about 250 nucleotide bases in length. For example, a polynucleotide (such as DNA or RNA) which is at least 5 nucleotides long, such as at least 15, 50, 100, or even more than 200 nucleotides long.
Operably linked. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous. Where necessary to join two protein coding regions, the operably linked sequences are in the same reading frame.
Expression control sequence. A nucleic acid sequence that affects, modifies, or influences expression of a second nucleic acid sequence.
Promoters, operators, repressors, and enhancers are examples of expression control sequences.
ORF (open reading frame). A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
Ortholog. Two nucleotide sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.
Parenteral. Administered outside of the intestine and not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
Polypeptide. Any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
Polypeptide sequence homology. In certain embodiments, a polypeptide is at least about 70% homologous to a corresponding sequence (such as SEQ ID
NO:1) or a native polypeptide (such as HA), such as at least about 80% homologous, and even at least about 95% homologous. Such homology is considered to be "substantial homology."

Polypeptide homology is typically analyzed using sequence analysis software, such as the programs available from the Genetics Computer Group (Madison, WI, see the Genetics Computer Group website) Portion of a nucleic acid sequence. At least 10, 20, 30, 40, 50, 60, 70, 80, or more contiguous nucleotides of the relevant sequence.
Promoter. A promoter is one type of expression control sequence composed from an array of nucleic acid sequences that directs transcription of a nucleic acid.
A promoter includes necessary nucleic acid sequences near the start site of transcription, such as a TATA element. A promoter also can include distal enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription. A promoter can be constitutive or inducible. An inducible promoter directs transcription of a nucleic acid operably coupled to it only under certain environmental conditions, such as in the presence of metal ions or above a certain temperature.
Protein Purification. Polypeptides can be purified by any method known to one of skill in the art. Exemplary, non-limiting methods are described in:
Guide to Proteiv~ Purification: Methods Enzymologyl, ed. Deutscher, Academic Press, San Diego, 1997; and Scopes, Protein Purification: Principles and Practice, 3'd ed.,Springer Verlag, New York, 1994.
Purified. The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid is one in which the nucleic acid is more enriched than the nucleic acid is in its natural environment within a cell. In one embodiment, a preparation is purified if a component, such as a nucleic acid, represents at least 50% of the total amount of that component (e.g. the nucleic acid content) of the preparation.
Recombinant. A recombinant nucleic acid is one that has a sequence that is not naturally occurring, or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or artificial manipulation of isolated segments of nucleic acids, for example by genetic engineering techniques. Similarly, a recombinant protein is one encoded for by a recombinant nucleic acid molecule.

The term recombinant includes nucleic acids that have been altered solely by deletion of a portion of the nucleic acid.
Resistance to infection. Animals resistant to infection will demonstrate decreased symptoms of infection compared to non-resistant animals. Evidence of resistance to infection can appear as, for example, lower rates of mortality;
increased life-spans measured after exposure to the infective agent; fewer or less intense physiological symptoms, such as fewer lesions; or decreased cellular or tissue concentrations of the infective agent. In one embodiment, resistance to infection is demonstrated by a heightened immune response.
Sequence identity. The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homlogy); the higher the percentage, the more similar are the two sequences.
Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Bio.
48:443, 1970; Pearson and Lipman, Methods in Molec. Biology 24: 307-331, 1988;
Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-90, 1988; Huang et al., Compute~Applications i~c BioSciences 8:155-65,1992; and Pearson et al., Methods ih Molecular Biology 24:307-31,1994. Altschul et al. (1994) presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.
Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biological Information (NBCI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at the NCBI website.
Homologs of the nucleic acids and polypeptides described herein are typically characterized by possession of at least 70% sequence identity counted over the full length alignment with a disclosed sequence using the NCBI Blast 2.0, gapped blastp set to default parameters. Such homologous nucleic acids or peptides will possess at least 70%, at least 80%, or even at least 90% or 95% sequence identity determined by this method. When less than the entire sequence is being compared for sequence identity, homologs will possess at least 70%, such as at least 85%, or even at least 90% or 95% sequence identity over short windows of 10-20 amino acids. Methods for determining sequence identity over such short windows are described at the NCBI website. These sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs or other variants could be obtained that fall outside of the ranges provided.
In addition to the peptide homologs described above, nucleic acid molecules that encode such homologs are encompassed by alternative embodiments. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. Stringent conditions, as described above, are sequence dependent and are different under different environmental parameters.
Nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequence that all encode substantially the same polypeptide.
Nucleic acid molecules demonstrating substantial similarity may be of different types. A DNA molecule can demonstrate some degree of identity to an RNA molecule by comparing the sequences, where a T residue on the DNA
molecule is considered identical to a U residue on the RNA molecule.
Substantially similar. When optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 50%, 60%, 70%, 80% or 90 to 95% of the nucleotide bases.

Therapeutic agent. Includes treating agents, prophylactic agents, and replacement agents made from nucleic acid and/or amino acid compositions described herein.
Therapeutically effective amount or effective amount. A quantity sufficient to achieve a desired effect irc situ, in vitro, in vivo, or within a subject being treated. For instance, the effective amount can be the amount necessary to inhibit viral proliferation or to measurably alter progression of disease. In general, this amount will be sufficient to measurably inhibit virus (ISAV) replication or infectivity.
An effective amount can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount can depend on the composition applied or administered, the subject being treated, the severity and type of the affliction, and the manner of administration.
The compositions disclosed have application in various settings, such as aquaculture, environmental containment, or veterinary settings. Therefore, the general term "subject being treated" is understood to include all fish that are or may be infected with a virus or other disease-causing microorganism that is susceptible to neutralization by the compositions described herein.
Transduced, transformed, and transfected. A virus or vector "transducer"
a cell when it transfers nucleic acid into the cell. A cell is "transformed"
by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication. Transfection is the uptake by eukaryotic cells of a nucleic acid from the local environment and can be considered the eukaryotic counterpart to bacterial transformation.
As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into a cell.
Transgene. An exogenous gene supplied by a vector.
Transgenic. Of, pertaining to, or containing a gene, ORF, or other nucleic acid native to another species, microorganism, or virus. The term "transgenic"
includes transient and permanent transformation, where the nucleic acid integrates into chromosomal DNA, including the germ line, or is maintained extrachromosomally.
Variants of Amino Acid and Nucleic Acid Sequences. The production of proteins disclosed herein (for example, HA) can be accomplished in a variety of ways. DNA sequences which encode for the protein, or a fragment of the protein, can be engineered such that they allow the protein to be expressed in eukaryotic cells, bacteria, insects, and/or plants. In order to accomplish this expression, the DNA sequence can be altered and operably linked to other regulatory sequences.
The final product, which contains the regulatory sequences and the nucleic acid, is referred to as a vector. This vector can then be introduced into the eukaryotic cells, bacteria, insect, and/or plant. Once inside the cell, the vector allows the protein to be produced.
The DNA can be altered in numerous ways without affecting the biological activity of the encoded protein. For example, PCR can be used to produce variations in the DNA sequence which encodes an ISAV peptide. Such variants can be variants that are optimized for codon preference in a host cell that is to be used to express the protein, or other sequence changes that facilitate expression.
At least two types of cDNA sequence variant can be produced. In the first type, the variation in the cDNA sequence is not manifested as a change in the amino acid sequence of the encoded polypeptide. These silent variations are simply a reflection of the degeneracy of the genetic code. In the second type, the cDNA
sequence variation does result in a change in the amino acid sequence of the encoded protein. In such cases, the variant cDNA sequence produces a variant polypeptide sequence. In order to preserve the functional and immunologic identity of the encoded polypeptide, certain embodiments utilize amino acid substitutions that are conservative.
Variations in the cDNA sequence that result in amino acid changes, whether conservative or not, can be minimized in order to preserve the functional and immunologic identity of the encoded protein. Variant amino acid sequences can, for example, be 70, 80%, 90%, or even 95% identical to the native amino acid sequence.
Vector. A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector can include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A
vector can also include one or more therapeutic genes and/or selectable marker genes and other genetic elements. A vector can transduce, transform or transfect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating, or the like. Plasmids are often used as vectors to transform fish cells.
ISAV Specific Nucleic Acids and Polypeptides Polypeptides and nucleic acid molecules are disclosed herein, as are and treatments for protecting fish, shellfish, and other aquacultured organisms against ISAV. The nucleic acids include segments of the ISAV genome, such as the segments described herein and summarized in Table 5 below, or fragments thereof.
Also included are fragments of the ISAV genome that overlap the individual segments summarized in Table 5.
ISAV polypeptides are described herein, as are nucleic acids that encode the ISAV polypeptides. ISAV polypeptides include, but are not limited to, P1, PB1, (nucleotprotein) NP, P2, P3, hemaglutinin (HA), P4, P5, P6, and P7. Thus, polypeptides having a sequence as set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 18, an antigenic fragment thereof, or a conservative variant thereof, are provided herein.
Polypeptides can be divided into sections, such as an N-terminal and a C-terminal portion. Thus, in one embodiment, polypeptide fragments are provided that include the N-terminal or the C-terminal portion of an ISAV polypeptide.
Antigenic fragments of an ISAV polypeptide are provided herein. An antigenic fragment is any ISAV polypeptide that can produce an immune response in fish. The immune response can be a B cell or a T cell response, or induction of a cytokine.
Also provided herein are nucleic acids that encode and ISAV polypeptide. In one embodiment, a nucleic acid is provided that encodes a P1 polypeptide. One specific non-limiting example of a P 1 polypeptide is the sequence set forth as SEQ
ID N0:2, a fragment, or a conservative variant thereof.
In another embodiment, a nucleic acid is provided that encodes a hemaglutinin (HA) polypeptide. One specific, non-limiting example of an HA
polypeptide is the sequence as set forth as SEQ ID N0:12, a fragment, or a conservative variant thereof.
In a further embodiment, a nucleic acid is provided that encodes a PB 1 polypeptide. One specific, non-limiting example of an PB 1 polypeptide is the sequence as set forth as SEQ ID N0:4, a fragment, or a conservative variant thereof.
Nucleic acids are also disclosed herein that are substantially similar to particular segments, such as nucleic acids that are at least 70% identical to SEQ ID
N0: 1, at least 85% identical to SEQ ID NO: 3, or at least 85% identical to SEQ ID
N0: 11. Thus, in one embodiment, a nucleic acids is provided that is are at least 75%
identical to SEQ ID NO: 1, at least at least 80% identical to SEQ ID N0: 1, at least 85% identical to SEQ ID NO: 1, at least 90% identical to SEQ ID N0: 1, or at least 95% identical to SEQ ID NO: 1. In another embodiment, a nucleic acid is provided that is at least 90% identical to SEQ ID NO: 3, at least 95% identical to SEQ
ID NO:
3, or at least 99% identical to SEQ ID N0:3. In a fiu-ther embodiment, a nucleic acid is provided that is at least 90% identical to SEQ ID NO: 1 l, at least 95%
identical to SEQ ID NO: 11, or at least 99% identical to SEQ ID N0:11.
In yet another embodiment, nucleic acids are provided that consist essentially of an ISAV nucleic acid sequences, such as a nucleic acid having a sequence as set forth as SEQ ID NO: 1, SEQ ID N0: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID N0:
9, SEQ ID N0: 1 l, or SEQ ID N0: 16.
The nucleic acids disclosed herein can be operably linked to a heterologous nucleic acid, such as an expression control sequence. In one embodiment, the expression control sequence is a promoter, such as an interferon response element, beta-actin, a cytokine promoter, a cytomegalovirus promoter, or a fish viral promoter. In particular embodiments, the promoter is an inducible promoter, such as a heat shock promoter, or a promoter induced by a hormone or a metal ion.
Nucleic acid compositions can contain other elements, such as additional expression control elements, structural sequences, origins of replication, or multiple coding sequences.

In particular embodiments, an expression control sequence operably linked to a nucleic acid encoding an ISAV polypeptide is included in a vector, including, but not limited to, a plasmid, a viral vector, a phagemid, or a cosmid. Cloning vectors include, but are not limited to, those described in U.S. Patent No. 5,998,697.
Viral vectors include, but are not limited to, retroviral or adenoviral vectors.
The nucleic acid compositions described herein can be utilized irc vitro, in vivo, or in situ. For example, a nucleic acid at least 70% identical to SEQ ID
NO: 1 could be used to study an antigenic epitope of interest for in vitro production and manipulation, or to study its effect on cell physiology or activity ih vivo, or for tissue-specific expression analysis in situ. Particular uses of these nucleic acid compositions also are illustrated in the Examples below.
In some embodiments, the nucleic acid molecule encodes an antigenic sequence, such as an antigenic sequence for pathogens of aquacultural animals.
Aquacultural animals include fish (both bony and cartilaginous fish), shellfish and other arthropods, and molluscs. Particular exemplary aquicultural animals include, but are not limited, to the following: salmonids, such as rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisz~teh), Chinook salmon (D.
tshawytcha), amago salmon (O. rhodurus), chum salmon (O. keta Walbaum), sockeye salmon (O.
nerka), Atlantic salmon (Salmo salary, arctic char (Salveli~us alpinus), brown trout (Salmo t~utta), cutthroat trout (Salmo cla~kii), and brook trout (Salveliv~us fontinalis); catfish (Ictalu~us punctatus); tilapia (Oreochf~omis hiloticusand and Oreochromis mozambicus); sea bream (A~ehosaf°gus ~homboidalis), seabass (Dicentrarcha~s lab~ax); flounder (Paralichthys defztatus); sturgeon (Scaphirhy~chus albus); eels (including members of the order Anguilliformes, class Actinopterygii, such as Conger spp., Ariosonza spp., Gnathophis spp., Colocorcge~ spp., Anguilla spp., Nesso~hamphus spp., Cynoponticus spp., Aha~chias spp., Echidna spp., Enchelyco~e spp., Gym~othor ax spp., and Uropte~ygius spp.); cephalopods (octopi and squids); crustaceans (including lobsters, prawns, shrimp, crabs, and crayfish in the order Decapoda); and bivalves (clams and oysters, such as Ostrea edulis and Pisidiurn spp.). In some embodiments, the aquaculture animal is a fish, such as a salmonid. In particular embodiments, In some embodiments, the nucleic acid molecule encodes a polypeptide that is an antigenic sequence, such that upon introduction in fish, an immune response is induced against ISAV.
Eliciting a~ Immune Respo~rse in Fish Some embodiments employ nucleic acid compositions containing nucleic acid sequences encoding antigenic epitopes. In such embodiments, the nucleic acid composition includes an expression control sequence operably linked to a nucleic acid sequence encoding an antigenic epitope, thus driving expression of the nucleic acid sequence and eliciting an immune response to the antigenic epitope in the fish.
In particular embodiments, the antigen expressed is a polypeptide encoded by ISAV, which elicits an immune response in the fish against ISAV.
In any such embodiment, the fish utilized can belong to a particular species, such as rainbow trout, coho salmon, Chinook salmon, amago salmon, chum salmon, sockeye salmon, Atlantic salmon, arctic char, brown trout, cutthroat trout, brook trout, catfish, tilapia, sea bream, seabass, flounder, and sturgeon.
Exemplary, non-limiting uses of these nucleic acid compositions are described in the Examples below.
In certain embodiments, any of the nucleic acid compositions described herein is used to transform fish tissue to produce a transgenic fish. In such embodiments, a nucleated cell of the transgenic fish is transformed with a nucleic acid sequence substantially similar to the nucleic acid sequences described herein (for example, SEQ ID NOS.: 1, 3, 5, 7, 9, 1 l, 13, and 16). In particular embodiments, the nucleic acid is at least 70% identical to SEQ ID NO: 1, at least 85% identical to SEQ ID NO: 3, or at least 85% identical to SEQ ID NO: 11, operably linked to a heterologous nucleic acid sequence.
If it encodes an antigenic epitope, expression of the nucleic acid sequence can induce an immune response to the antigenic epitope within the fish or other aquaculture animal. In such embodiments, the animal exhibits an increased resistence to infection by ISAV as compared to a non-transformed animal of the same species.
In alternative embodiments, the animal subject is treated with a polypeptide composition that functions as an antigenic epitope and induces an immune response within that subject, such as the polypeptides a sequence as set forth as SEQ
ID NO:

2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 18, an antigenic fragment thereof, or a conservative variant thereof. In some embodiments, the antigenic polypeptide is a fusion protein, such as a polypeptide as set forth in SEQ
ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 18, an antigenic fragment thereof, or a conservative variant thereof, coupled to a fusion partner. Such fusion partners include, but are not limited to, antigens from other fish viruses (for example, glycoprotein from rhabdovirus, birnavirus, reovirus, nodavirus, herpes virus, or infectious pancreatic necrosis virus), ISS DNA elements, or T-cell epitopes.
The antigenic polypeptides can be obtained by recombinant methods, such as expression in eukaryotic or bacterial cell culture, or can be chemically synthesized.
In particular embodiments, the antigenic polypeptides are recombinantly expressed in a non-mammalian eukaryotic cell culture, such as a fish cell culture, for example a CHSE-214, TO, SHIM, RTG-2, or EPC cell culture. Thus, an antigenic polypeptide can be prepared by transforming fish cells with a nucleic acid vector encoding an antigenic polypeptide (including one that is'a fusion protein), as described above, culturing the host cells under conditions suitable for expressing the antigenic polypeptide, and then recovering the antigenic polypeptide from the cell culture.
Additionally, such cell cultures can be transformed with multiple nucleic acid vectors, thus expressing multiple antigenic polypeptides.
Recovered antigenic polypeptides can then be purified and readied for delivery to the subject (as described above), and the antigenic polypeptide can be combined with a pharmaceutically acceptable salt, carrier, adjuvant, or diluent, and/or other active or inactive ingredients, to form a pharmaceutical composition.
The amount or concentration of the antigenic polypeptide within the pharmaceutical composition can vary according to factors such as the effectiveness of the antigenic polypeptide in inducing an immune response within the species of the subject, the severity of the disease or condition to be treated, the route or frequency of administration, or other relevant factors. These compositions also can be tested for immunogenicity prior to delivery to a subject using an in vitro assay, such as one of the assays described in the Examples below.
Once prepared, an effective amount of the antigenic polypeptide or pharmaceutical composition is delivered to the subject via a suitable route of administration, for example, intramuscular, intraperitoneal, oral, immersion, or ultrasound administration. An effective amount is any amount that enhances the immunocompetence of the subject treated and elicits some immunity against ISAV, for example, by delaying, inhibiting, or even preventing the onset or progression of ISA. In some embodiments, the subject's immune system is stimulated by at least about 15%, such as by at least about 50%, or even at least about 90%.
EXAMPLES
The following examples are intended to illustrate the invention, but not to limit it in any mamler, either explicitly or implicitly. While these examples are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art alternatively can be used.
Example 1 Vaccine Trial to Test the Efficacy of the Whole Killed ISAV Vaccine Atlantic salmon, each weighing about 90 g, were anaesthetized and intraperitoneal injected with 0.2 ml of a solution of whole killed ISAV. Four groups of twenty fish in each group were studied; two groups were injected with whole killed ISAV and two groups were injected with an equivalent amount of saline.
Following vaccination, the salmon were acclimated to saltwater at 12°C and held for 79~ degree days prior to challenge with ISAV. Twenty-four native Atlantic salmon were anaesthetized, fin clipped for identification, and intraperitoneal injected with 1 ml ISAV infected CHSE-214 cell culture supernatant (1x10 TCIDSO/ml).
Six of these fish were added to each of the four tanks containing the groups of either the vaccinated or control fish. Saline-injected control salmon experienced a cumulative mortality of 57.5% when challenged with ISAV by cohabitation.

Vaccinated salmon had a cumulative mortality of 17.5 %. The RPS value of the ISAV whole killed vaccine was 70.0%.

Humoral Immune Response to Whole Killed ISAV
Atlantic salmon were vaccinated with the two different serials of whole killed ISAV in MV4 at two different antigen doses, and sera was collected at degree-days, 696 degree-days, and 972 degree-days after vaccination.
ISAV-specific antibodies in the sera were detected by enzyme-linked immunosorbent assay (ELISA). ISAV antigen was dried onto wells of an ELISA
plate overnight at 37°C. Wells were rinsed three times with PBS/Tween and, serial dilutions of anti-ISAV Atlantic salmon sera were added in triplicate. After one hour, wells were rinsed three times with PBS/Tween. A second antibody, mouse anti-salmon immunoglobulin, was added to each well, the plates were incubated for one hour, and then rinsed with PBS/Tween in triplicate. After incubation with a third antibody, goat anti-mouse IgG conjugated to alkaline phosphatase, the wells were washed with PBS/Tween and developer containing p-nitrophenyl phosphate was added. The absorbance was measured at 405 nm.
FIG. 3 illustrates the results of this humoral response trial. Antibody levels were reported as a percent of the value obtained with mAb 10A3 to normalize the variation between ELISA plates. A lx dose of the whole killed ISAV vaccine serial 327 elicits a humoral immune response at 972 degree-days post-vaccination.

Virus and RNA Purification Virus was prepared by inoculating CHSE-214 cell monolayers in a 6300 cm2 Cell Factory~ (Nalge Nunc International, Rochester, NY, USA) with ISA virus.
Following complete cell lysis, the cell culture supernatant was harvested from the Cell Factory~ as disclosed by the manufacturer and filtered through a sterile 0.45 micron filter to remove extraneous cell debris. After dialysis against solid polyethylene glycol to reduce the volume, the cell culture supernatant was centrifuged for two hours at 24,000 rpm using a SW28 rotor and a Beckman L8-70M ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA, USA). The pelleted virus was resuspended in TNE, layered on a 2S, 3S and 4S% sucrose gradient and centrifuged for 3 hours at 27,000 rpm using a SW28 rotor and a Beckman L8-70M
ultracentrifuge. Virus at the interface of the 3S and 4S% sucrose layers was S collected, resuspended in THE and centrifuged for two hours at 24,000 rpm using a SW28 rotor and a Beckman L8-70M ultracentrifuge. The fraction collected from the 3S-4S% interface was enriched with material that reacted with an ISAV-specific monoclonal antibody. Viral RNA was isolated from the pelleted virus using Trizol (Gibco) as described by the manufacturer and then used to construct cDNA
libraries.
Purified ISAV was resuspended in SDS-sample buffer. The solubilized proteins were separated by SDS-PAGE on a S% stacking gel and a 12% resolving gel and visualized by Coomassie blue staining. As shown in FIG. 2, after SDS-PAGE, four distinct protein bands were evident: 72 kDa, 47 kDa, 42 kDa, 2S
kDa.
Seven proteins from purified ISAV were subjected to N-terminal amino acid 1 S sequence analysis. The proteins of purified ISAV were separated by SDS-PAGE, blotted onto PVDF membrane (BioRad Laboratories, Hercules, CA) and stained with 0.1% Coomassie blue R-2S0 in 40% methanol/1% acetic acid. The stained protein bands were cut out of the membrane and subjected to N-terminal amino acid sequence analysis using an Applied Biosystems model 470A gas-phase sequencer (Applied Biosystems, Tnc., Foster City, CA) or an Applied Biosystems model 473 liquid-phase sequencer with on-line phenylthiohydantoin analysis. The results of this sequencing analysis are shown in Table 2.
Table 2: N-terminal amino acid sequence analysis of ISAV proteins Protein Sequence analysis Similarity analysis MW

(kDa) 2S KVSFDMA; SLQGPVA (internal No similarity found sequence) 3 S N-terminally blocked N/A

38 N-terminally blocked N/A

40 RLXLRNHPDTTWIGDSRSDQSRXNQ Putative segment 7 ISAV;

(N-terminal sequence) segment 4 Influenza C

42 RLXLRNHPDTTWIGDSRSDQSRXNQ HA (segment 6) (N-terminal sequence) 47 EPXIXENPTXLAI (N-terminal sequence)S:E-7 (segment S) 72 N-terminally blocked N/A

_ 28 _ Construction of cDNA libraries Strategies for Cloning the ISAT~ Gehome.
Approach 1: First strand cDNA was synthesized from ISA vRNA by reverse transcription with the ISAV-specific primer (SEQ ID NO: 19):
5'-AAGCAGTGGTAACAACGCAGAGTAGCAAAGA-3' RNA (100 ng) isolated from purified ISAV or CHSE-214 cells (control) was mixed with ISAV primer (20 pmol/~,l), incubated at 80 °C for 5 min and then combined with the following in a total of 20 ~.1: 4 ~,l Sx first strand buffer (Gibco Invitrogen Corp., Carlsbad, CA), 2 ~,1 10 mM dNTP mix (Boehringer Mannheim), 1 ~,l 0.1 M DTT (Gibco) and 1 ~,1 Superscript II reverse transcriptase (15 U/~,1; Gibco).
The mixture was incubated at 25 °C for 10 min and then at 42 °C for 1 hr.
The first strand ISAV cDNA products synthesized by reverse transcription were PCR amplified using the ISAV primer and random hexamers. To the first strand reaction, the following components were added in a total of 100 ~,1:
1.5 q1 10 mM dNTP mix (Boehringer Mannheim, 1.25 ~,l ISAV primer (20 pmol/~,1), 1 ~,1 random hexamers (25 pmol/~,1; Gibco), 10 q1 l Ox PCR buffer with Mg2+
(Boehringer Mam~heim), 1 q1 Taq (5 U/ql; Boehringer Mannheim). After 35 cycles of 94 °C for 30 sec, 59 °C for 45 sec and 72 °C for 1 min, the PCR products were extended for 10 min at 72 °C. The amplified cDNA products were separated by agarose gel electrophoresis, gel purified and then cloned into the pGEM-T
vector as described by the manufacturer (Promega).
Approach 2: First strand cDNA was synthesized from ISA vRNA by reverse transcription with random hexamer primers. RNA (100 ng) isolated from purified ISAV or CHSE-214 cells (control) was mixed with random hexamers (50 ng/~.1;
Gibco), incubated at 65 °C for 5 min, placed on ice for 2 min and then combined with the following in a total of 20 ~,1: 4 ~.1 Sx first strand buffer (Gibco), 2 ~.l 10 mM dNTP mix (Boehringer Mamlheim), 1 ~,l 0.1 M DTT (Gibco) and 1 q1 Superscript II reverse transcriptase (15 U/~1; Gibco). The mixture was incubated at 25 °C for 10 min and then at 50 °C for 50 min.

The TimeSaver cDNA synthesis kit (Pharmacia) was used for second strand cDNA synthesis. The first strand reaction was added to the second strand reaction mix, incubated at 12 °C for 30 min and then at 22 °C for 1 hr.
After spin column purification, the blunt ended, double stranded cDNAs were cloned into dephosphorylated, SmaI digested pUCl8 (Pharmacia) as outlined by the manufacturer.
For both libraries, E. coli DHSa, (Gibco) was transformed with the ligation reactions and the ampicillin-resistant colonies containing either pGEM-T or pUC 18 with cloned ISAV cDNA were selected by blue/white screening. The white colonies were transferred to 96 well plates containing 200 ~,1 LB/ampicillin (250 p,g/ml)/15°l0 glycerol per well, grown overnight at 37 °C and stored at 20 °C.
RT PCR amplification of segments 2, 6 and 8 fi°om ISATr CCBB.
First strand cDNAs for segments 2, 6 and 8 were synthesized from ISA virus RNA by reverse transcription using primers outlined in Table 3 and conditions described above. PCR amplification was used for second strand cDNA synthesis;
after 30 cycles of 95 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min, the PCR
products were extended for 10 min at 72 °C (see Table 3 for primers).
RT-PCR
products were gel purified as described by the manufacturer (Qiagen).
Table 3. RT and PCR oligonucleotide DNA primers for RNA segments 2, 6 and 8 of ISA virus isolate CCBB.
Segment Primer name Primer sequence (5'-3') 2 seg 2-5'F-mRNA GAACGCTCTTTAATAACCATG
seg 2-3'R-mRNA TCAAACATGCTTTTTCTTC
6 HA forward AGCAAAGATGGCACGATTC
HA reverse TGCACTTTTCTGTAAACGTACAAC
8 seg 8-5'F-mRNA AAGCAGTGGTAACAACGCAGAGTCTATCTACCATG
seg 8-3'R-mRNA TTATTGTACAGAGTCTTCC
Selection and Identification of ISAV Clones from the cDNA Libraries.

The contents of one 96-well plate were transferred to one Hybond N+
membrane (Amersham) then placed on top of an LB agar plate containing ampicillin (250 ~.g/ml). Clones were grown on the filters at 37 °C overnight and the filters were processed on soaking pads saturated with the following solutions: 0.5 N
NaOH (7 min); 1 M Tris-HCl pH 7.4 (2 min); 1 M Tris-HCl pH 7.4 (2 min); 0.5 M
Tris-HCl pH 7.4, 1.5 M NaCI (4 min). The filters were transferred to a bath of 2x SSC (lx SSC is 0.15 M NaCI, 0.015 M Na3 citrate), 1% sodium dodecyl sulfate (SDS) and then soaked in 2x SSC. After a brief wash in chloroform, the filters were air dried and then baked at 80 °C for 2 hrs. Prehybridization of the filters for 2 hr in 6x SSC, 0.5% SDS, Sx Denhardt's and 0.lmg/ml E. coli tRNA (Sigma) was followed by hybridization with a probe labelled with [a32P] dCTP by nick translation. Nick translation was done as outlined by the manufacturer (Amersham).
The libraries were initially screened using gel purified, RT-PCR amplified cDNA
for segments 2, 6 or 8 of ISAV isolate CCBB. The remaining segments were identified using probes consisting of gel purified, restriction enzyme fragments digested from the plasmids of randomly selected library clones. Library clones were grouped based on the probe to which they hybridized (see Table 4). Eight distinct cDNA hybridization groups were identified. Of these, two groups were found in cDNA library l and all but one segment of the ISAV genome in cDNA library 2 (see Table 4).
Plasmid DNA isolated from representative clones of each group using Qiaprep columns (Qiagen) was sequenced at the University of Maine Core Sequencing Facility. Only those sequences that matched other orthomyxovirus sequences or that did not match non-viral sequences were analyzed further.

Table 4. Summary of groups formed from screening ISA virus cDNA libraries.
Origin of Number of positive clones Probe probe cDNA librarycDNA library approach approach 2 S:E-6 approach 0 33 Segment RT-PCR 0 41 1-1#2; 5-1#1approach 212; 1144 6; 43 2: C-5; approach 0 5; 3 8 4:D-8 2 S:E-7 approach 0 14 Segment RT-PCR 0 0 2:B-10 approach 0 50 Segment RT-PCR 6 10 Library from approach 1 had a total of 1364 clones ' Library from approach 2 had a total of 768 clones Norther ~ Blot Hyb~idizatio~.
Northern blot analysis was used to correlate each representative sequence with a specific ISAV genomic segment. Total RNA was isolated from CHSE-214 cell monolayers or CHSE-214 cell monolayers infected with ISAV using Trizol (Gibco) as outlined by the manufacturer. The RNA was separated on a 2% agarose gel containing formaldehyde and transferred onto Hybond N+ membrane (Amersham) in lOx SSC by capillary action as described in Fourney et al., Focus, 10:5-7 ( 1992).
The probes used for Northern blot analysis were gel purified, restriction enzyme fragments digested from the plasmids of appropriate cDNA library clones.
The probes were labelled with [a32P]dCTP (NEN) by nick translation (Amersham) and hybridized to the blots at 42°C for 18 hr in ULTRAhybTM (Ambion).
The membranes were washed 2 x 5 min in 2X SSC-0.1% SDS at 42 °C and then 2 x 15 min in O.1X SSC- 0.1% SDS at 42 °C. The results were recorded on Kodak X-OMAT AR film.
The probes used in the Northern blot hybridization experiments were derived from four clones constructed using approach 2, one clone constructed using approach 1 and RT-PCR products of the three known segments (see Table 3). A
single RNA blot was consecutively probed with each of the eight individual probes.
One probe was hybridized to the Northern blot and the results were visualized by autoradiography. The next probe was hybridized to the same Northern blot, the results were visualized and compared with the results from the previous hybridization. By repeating this process with each of the eight probes, each individual probe and its corresponding nucleotide sequence was correlated with a specific RNA segment.
Eight RNA segments were identified; segments l and 2 were both approximately 2400 nucleotides in length. The ISAV RNA segment corresponding to each cDNA clone is summarized in Table 5. The genome segments are numbered with respect to their mobility in agarose gels, from the slowest to the fastest and comprise a genome of 14,500 nucleotides.
Table 5. RNA segments of ISAV isolate CCBB, their genes and encoded proteins Length Nascent Molecular of SegmentClone segments Length Encoded polypeptide~'~'eight of (kb) C~ (bp) protein length Predicted (aa) (kDa) 1 S:E-6 2.4 1749 P1 -2 PB 1 2.4 2127 PB 1 709 80.5 3 1-1#2/5-5#12.2 1851 NP 617 68.0 4 2:C-5/4:D-81.9 1737 P2 579 65.3 5 S:E-7 1.6 1335 P3 445 48.8 6 HA 1.5 1185 HA 395 43.1 7 2:B-10 1.3 771 P4 257 28.6 441 PS 147 16.3 8 NS 1.0 705 P6 235 26.5 552 P7 184 20.3 Based on the average length determined from Northern blot hybridization analyses with 2-5 replicates per probe.
Purified cellular RNA was separated on a 2% agarose gel and transferred to a Hybond N+ membrane. Lanes 1-7 & 10 contain cellular RNA from CHSE cells infected with ISA virus isolate CCBB; lane 8 contains cellular RNA from ISA
virus isolate ME-Ol; lane 9 contains cellular RNA from CHSE cells infected with ISA
virus isolate NB-99; and lane 11 contains cellular RNA from naive CHSE cells.
The RNA blot was consecutively hybridized with radioactively labeled DNA
probes specif c for one of the ISA virus RNA segments. The results recorded by autoradiography after the addition of each single probe to the same RNA blot are shown in lanes 1-1 I. The probes are identified by segment (according to Table above): lane 1, segment 3; lane 2, segment 4; lane 3, segment 6; lane 4, segment 1;
lane 5, segment 5; lane 6, segment 7; lane 7, segment 8; lanes 8-11, segment 2.
Molecular weight standards on the left are in kbp. The RNA segments are labeled on the right.
Construction of Full-Length Closes of Each ISAV Genome Segmev~t.
Full-length cDNA sequence for each of the ISAV RNA segments, with the exception of segment l, was generated by rapid amplification of cDNA ends (RACE) PCR using the RLM-RACE kit (Ambion). The PCR products were cloned into either pCR~2.1-TOPO~ or pGEM-T as directed by the manufacturers (Invitrogen or Promega, respectively) and then sequenced. AssemblyLIGN 1Ø9b (Oxford Molecular Group) was used to order the overlapping sequenced DNA
fragments for construction of the full-length sequence.
PCR primers were designed from the consensus sequence obtained for each ISAV RNA segment and used to amplify full-length cDNA sequence for each segment with the exception of segment 1. The PCR product for each segment was cloned into pGEM-T as directed by the manufacturer (Promega) and DNA from three representative clones was sequenced. The computer programs contained in MacVectorT"" 6.5.3 (Oxford Molecular Group) were used to identify open reading frames and regions of local similarity. The nucleotide and predicted amino acid sequence for each open reading frame were analyzed by BLAST searches through the National Center for Biotechnology Information server (Altschul et al., 1990;
Pearson & Lipman, 1988) or the Influenza database (Los Alamos National Laboratory). The most likely cleavage sites for signal peptidase in HA and 5:E-were determined using SignalP V 1.1 (Nielsen et al., 1997).
The length of each gene, the corresponding encoded polypeptide(s) and the predicted molecular weights of the translated proteins are summarized in Table 1.
Only partial sequence from segment 1 was obtained. The cDNA sequence of segments 1-6 was predicted to encode one open reading frame. Segments 7 and 8 each were predicted to encode two proteins.
Comparison of the cDNA nucleotide and predicted amino acid sequences for the ISA virus genome to those listed in the GenBank and Influenza databases showed that RNA segments 1 and 5 of ISA virus isolate CCBB were unique. RNA
segments 2, 3, 4 and 6 were found to encode the putative proteins PB1, NP, PA
and HA, respectively. The predicted sequences of the P6 and P7 proteins encoded on RNA segment 8 were similar to the sequences of the two open reading frames (orf) on segment 8 from other ISA virus isolates.
The protein sequence of the partial open reading frame encoded on segment 1 was unique. The predicted amino acid sequence of PB 1, encoded by RNA
segment 2, was 82.2 to 84.5% similar to the amino acid sequences of PB 1 proteins from Norwegian (AJ002475) and Scottish (AF262392) ISA virus isolates. The assignment of NP to the open reading frame encoded on RNA segment 3 was based on nucleotide sequence similarity to the influenza A NP RNA binding region (see FIG. 4) and to the putative NP sequence described by Snow & Cunningham (2001).
The sequence for the CCBB ISA virus NP was highly conserved, sharing 96.6%
identity to that reported for the Scottish NP (AJ276858). The predicted protein sequence of P2 from RNA segment 4 had 99% identity to the putative PA sequence (AF306548) described by Ritchie et al. (2001). The nucleotide sequences for segment 5 of the Scottish (AF429988), Norwegian (AF429987) and Maine (AF429986) isolates of ISAV were 76.4, 76.0 and 99.7% similar to the corresponding sequence of ISAV isolate CCBB.
The predicted translation of the open reading frame encoded by RNA
segment 6 shared 84.8 to 84.3 % identity to the predicted HA protein sequences for TSA virus isolates from Norway (AF302799) and Scotland (AJ276859), and 99.2%
identity to the Maine ISA virus isolate (AY059402). The nucleotide sequence for ISAV CCBB segment 7 had 99.6% identity with a reported ISAV sequence (AX083264). The P4 and PS proteins encoded on segment 7 had 99.2 to 99.3%
identity to the translations predicted for orfl and orf2 from the reported sequence (AX083264). The nucleotide sequence for segment 8 of the Norwegian (AF429990) and ME/OI (AF429989) isolates of TSAV was 88.7-99.9% identical to the corresponding sequence from ISAV isolate CCBB. Our results confirmed that segment 8 encoded two proteins as previously reported by Mjaaland et al.
(1997).
The amino acid sequence translated from the largest open reading frame was 75.6-97.9% identical to the sequence previously reported fox Norwegian (AF262382), Scottish (AJ242016) and Canadian (AJ242016) isolates of ISA virus.
FIG. 4 shows the amino acid sequence alignment of the RNA binding domain of NP from influenza virus A and B with the putative NP RNA binding S domain from ISA virus as predicted using the Clustal W system. ISAV NP, as 307 from accession number AF404345; Inf A NP, as 90-188 from accession number P1567S; Inf B NP, as 149-249 from accession number P04666. Identical amino acids and amino acid residues with similarity in physical and chemical properties axe indicated as * and ~, respectively. The NP RNA binding domain from influenza viruses A and B was taken from Kobayashi et al. (1994).

Humoral Immune Response Anti-ISA virus antibodies were generated in Atlantic salmon injected with tissue culture supernatant from ISA virus-infected CHSE cell monolayers. Anti-ISA
virus antibodies were also generated in rainbow trout vaccinated with I?NA
vaccines expressing a ISAV-specific antigens. Mouse polyclonal and monoclonal antibodies (mAbs) to ISA virus were generated by Rob Beecroft (Immuno-Precise Antibodies Ltd.). ISAV-specific immunoreactive antigens were detected by IFAT, ELISA, Western blot and serum neutralization assays.
Indirect Fluorescent Antibody Technique (IFAT) IFATs were used to screen the ISAV-specific monoclonal antibodies (mAb).
CHSE-214 cells infected with ISAV were fixed to a glass slide with 100%
acetone, 2S blocked with 3% skim milk buffer, incubated with ISAV-specific mAb 10A3, washed and reacted with TRITC-labelled goat anti-mouse antibody (Sigma). The slide was washed, air dried, and fixed to a glass slide with Cytoseal 60 (Stephens Scientific).
Viral-infected cells were stained a deep red whereas control slides of naive CHSE cells were negative by IFAT with mAb 1 OA3.

ELISA
The levels of ISAV-specific antibodies in sexum from Atlantic salmon infected with ISAV or rainbow trout vaccinated with a DNA vaccine were determined by enzyme-linked immunosorbent assay (ELISA). DNA vaccines tested were pISA-HA (NA), pISA-HA (Nor), pISA-seg7, and PISA-seg8.
ISAV antigen was dried onto wells of an ELISA plate overnight at 37°C.
Wells were rinsed three times with PBS/Tween and then serial dilutions of anti-ISAV sera were added in triplicate. After 1 hr, wells were rinsed three times with PBS/Tween. The second antibody, mouse anti-salmon/rainbow trout immunoglobulin (Rob Beecroft), was added to each well. The plates were incubated for 1 hr and then rinsed with PBS/Tween in triplicate. After incubation with the third antibody, goat anti-mouse IgG conjugated to alkaline phosphatase, the wells were washed with PBS/Tween and developer containing p-nitrophenyl phosphate was added. The absorbance was measured at 405 nm.
ISAV-specific antibodies were detected in sera from fish injected with either live ISAV (Fig. 4 and 5) or with DNA vaccines expressing ISAV-specific antigens (Fig. 5).
Antibody studies are conducted in Atlantic salmon vaccinated with an ISAV
DNA vaccine, an ISAV recombinant vaccine or a whole killed ISAV vaccine {DNA
vaccines: pISA-NP, pISA-Ac, pISA-HA (NA), pISA-HA (Nor), PISA-seg7;
recombinant vaccines: rHA-1; whole killed vaccines: lx, 2x and 4x doses of formalin killed ISAV). Sera samples are collected from 5 fish/timepoint at 4, 6, 8, 10 and 12 weeks post-vaccination.
FIG. 5 shows the titration of ISAV-specific antibodies from Atlantic salmon infected with ISAV. Fish 1 had not been exposed to ISAV and, thus, the serum was used as a negative control. Fish 45 was injected with ISAV, and the ELISA
results indicated that the corresponding serum contained ISAV-specific antibodies.
Sera from fish 1 and fish 45 were negative when tested by ELISA using plates coated with CHSE-214 cells.
FIG. 6 shows ISAV-specific antibodies in sera obtained from Atlantic salmon infected with ISAV or rainbow trout injected with a nucleic acid encoding an ISAV-specific DNA vaccine. Sera were collected at 4, 6, 8, 10 and 12 weeks post-injection with 1 ~.g DNA vaccine or post-infection with at least 1x103 TCIDSO
live ISAV/fish. Levels of ISAV-specific antibodies were expressed as a percentage of the mAb values to normalize variations between ELISA plates. ISAV-specific antibodies were detected at various times post-treatment. However, the levels of ISAV-specific antibodies were much higher in fish that had been exposed to live virus relative to those injected with the nucleic acid.
SDS-PAGE and Western Blot Analysis:
Whole cell lysates of naive and ISAV-infected CHSE cells as well as purified ISAV were screened for the presence of immunoreactive antigens with mAb 10A3 and sera from Atlantic salmon infected with ISAV. SDS-polyacrylamide gel electrophoresis (PAGE) was carried out by the method of Laemmli (1970).
Proteins were solubilized with SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and separated by SDS-PAGE on 5% stacking gel and 12% resolving gel.
Immunoreactive protein bands were visualized by Western blot analysis.
Briefly, proteins separated by SDS-PAGE were electrophoretically transferred to nitrocellulose (Bio-Rad Laboratories). The membranes were blocked with 3% skim milk buffer and then incubated with either mAb 10A3 or sera from Atlantic salmon infected with ISAV followed by an incubation with goat anti-mouse immunoglobulin G conjugated to alkaline phosphatase or mouse anti-salmon immunoglobulin (Rob Beecroft), respectively. In the latter case, a final incubation with goat anti-mouse immunoglobulin G conjugated to alkaline phosphatase was required. The immunoreactive proteins were visualized following development with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.
Immunoreactive polypeptides encoded by the RNA segments were identified by Western blot analysis performed on naive and ISAV-infected CHSE-214 cells (see Table 6 below). Sera, collected from Atlantic salmon injected with live ISA
virus, reacted with the 72 and 42 kDa proteins of ISA virus (Table 6). Similar analyses were performed with ISA virus-specific mouse polyclonal and monoclonal antibodies.

Table 6. ISAV immunoreactive proteins detected by Western blot analysis Sera Immunoreactive proteins (kDa) CHSE CHSE/ISAV Purified ISAV
mAb 10A3 - 42 42 Mouse polyclonal - 42, 36, 25, 15, 42, 25, 15 11, 9 Atlantic salmon - 72, 42 Not done conyalescent *Serum from Atlantic salmon infected with ISAV reacted with a 72 kDa and a 42 kDa protein and was neutralizing. These two proteins are potential vaccine candidates.
Of the six immunoreactive proteins present in the cellular preparation of ISA
virus and recognized by the mouse polyclonal sera, three were present in the purified ISA virus sample (42, 25 and 15 kDa; Table 5). Only the 42 kDa protein was recognized by the monoclonal antibody (Table 5). For each serum tested, no reaction was observed with the naive GHSE sample indicating that the immunoreactive proteins were derived from ISA virus.
See°urrz Neutralization Assay Ten-fold dilutions of ISAV in PBS were incubated with PBS or serum from naive or ISAV-infected Atlantic salmon for 1 hr at 15°C. Aliquots of 100 ~1 of the serum/virus mixture were transferred in quadruplicate to 96-well cell culture plates seeded with CHSE-214 cells, incubated at 15°C and monitored for CPE.
Table 7 summarizes the results.
Table 7: Summary of serum neutralization studies Sample Neutralization ISAV/PBS -ISAV/sera from naive Atlantic salmon -ISAV/sera from ISAV-infected Atlantic salmon +
*Serum from ISAV-infected Atlantic salmon is neutralizing. Studies to determine if serum from Atlantic salmon infected with ISAV isolate CCBB is neutralizing with the Scottish, Norwegian and Maine isolates of ISAV are underway.
Having illustrated and described the principals of the invention by several embodiments, it should be apparent that those embodiments can be modified in arrangement and detail without departing from the principles of the invention.
Thus, the invention includes all such embodiments and variations thereof, and their equivalents.

SEQUENCE LISTING
<l10> Clouthier, Sharon C
Anderson, Eric D
<120> NUCLEIC ACIDS ENCODING ISAV POLYPEPTIDES
<130> 3777-62574 <160> 25 <170> PatentIn version 3.1 <210> 1 <211> 1749 <212> DNA
<213> synthetic construct <400> 1 ggatccccggcgtttacttcttaaacacgaaagaaatagtgactgcagaagggaaagttg60 atgaaacaagaggacccttagaaaggacatcagctcctcttatgagagatatctccaggc120 tgatacaagaaacaatagaagaagtggaaacaggaggagacccctctttttcagtaagaa180 gtgaaggaggttctaaaatagaaggaagaatcgccttttcattgcactcagaggtgtcca240 cattgaaaatgaggatagcacttgaacagaaactggccagatatgagtacatgggagaaa300 accttctcacacttgtcaaaaacacttctatagacagaatgcagcctgattctgcaatga360 tggggaaaatggtgttagaaagtcttagaacacacacggtatcctctgagcagttgaatg420 ggagaatgattactgtccaatcgcaaggcctagaaacaatagcgatatcgagtccttttg480 ~atgtggaatacgacgatggatacgtattcacaaggatgaaaggagactttgtggcaatcg540 gaagagactacaagggagctatcttatgcttcagagaaggacaagggacattcttcagtg600 ggagaggcaactggtctgggctcatggagagatgtctagttgaaatgagactatgtccat660 gcttttacagctgcacctggcaagactaccctgacaaaaagagtctctacgaaaaagcaa720 cgttcgaagccaaacagatagtctttgctatgggagaaagtgttggagtagatgtcagag780 taaacacagatggtgaaataggagacaagggaatttccttactaacaagggaaagagagg840 acaaatacatgtcaaaagtgtcttatgagtgcagagtggtcagtgggaaactggtgatgg900 gtttggacaaaatgagcagagtcgcaaaagggaacctagaagtagtgagggaaaaaggag960 atgacacaagtcagtcagattccttttatgaaggtgtactacaggtaggtagcatgattg1020 ggaccacaatggagagtttaaaacagcagttacaaggacctgtgggaatttggagagcat1080 caggtgtttcagctatggaaaggtgcatgaagagagggcaaagcaaaactgtagtggcaa1140 gtgctagatacacattccaaaagatgatggaaaagatggcatctggtagggaagtgtcta1200 aatacagtttgataattgtcatgaggtgctgcattggtttcacgtctgaagctaataaga1260 gagctttaactaacatctctggaactggatactacattagtgttgcacaacctaccgtag1320 taaaactagcaggagaatggttgatcacacctgtgggtaggtctaagacaggggaagttc1380 agtatgtatctgccaagctgaagaaggggatgaccacagggaagctagaattgattaaga1440 aagcagatagatctgacttggacaacttcccagaaccgtcggctgatgaactgttgagag1500 aaggaacaattgtgttaatgcaaatcggaaaagacaaatggttatgtagggtaagaacag1560 gtgataggagagtgaggaccgacacagacatacagagggcagaagcaaaatctcaggttg1620 aaaaagaagatttgatggatgaatatggtgtttaaaataagtggttgtaaaaattgaatg1680 ttgtttcttttgctttttgagcctttgacgatacttttaataaataaaatgtccattttg1740 tccgatccc 1749 <210> 2 <211> 550 <212> PRT
<213> synthetic construct <400> 2 I1e Pro Gly Val Tyr Phe Leu Asn Thr Lys Glu Ile Val Thr Ala Glu Gly Lys Val Asp Glu Thr Arg G1y Pro Leu Glu Arg Thr Ser Ala Pro Leu Met Arg Asp Ile Ser Arg Leu Ile G1n Glu Thr Ile Glu Glu Val Glu Thr Gly Gly Asp Pro Ser Phe Ser Val Arg Ser Glu Gly Gly Ser Lys Ile Glu Gly Arg Ile Ala Phe Ser Leu His Ser Glu Val Ser Thr Leu Lys Met Arg Ile Ala Leu Glu Gln Lys Leu Ala Arg Tyr G1u Tyr Met Gly Glu Asn Leu Leu Thr Leu Val Lys Asn Thr Ser Ile Asp Arg Met Gln Pro Asp Ser Ala Met Met Gly Lys Met Val Leu Glu Ser Leu Arg Thr His Thr Val Ser Ser Glu Gln Leu Asn Gly Arg Met Ile Thr Va1 Gln Ser Gln Gly Leu Glu Thr Ile A1a Ile Ser Ser Pro Phe Asp Val Glu Tyr Asp Asp Gly Tyr Val Phe Thr Arg Met Lys Gly Asp Phe Val Ala Ile Gly Arg Asp Tyr Lys Gly Ala Ile Leu Cys Phe Arg Glu Gly Gln Gly Thr Phe Phe Ser Gly Arg Gly Asn Trp Ser Gly Leu Met Glu Arg Cys Leu Val Glu Met Arg Leu Cys Pro Cys Phe Tyr Ser Cys Thr Trp Gln Asp Tyr Pro Asp Lys Lys Ser Leu Tyr Glu Lys Ala Thr Phe G1u Ala Lys Gln Ile Val Phe Ala Met Gly Glu Ser Val Gly Val Asp Val Arg Val Asn Thr Asp Gly Glu Ile Gly Asp Lys Gly Ile Ser Leu Leu Thr Arg Glu Arg Glu Asp Lys Tyr Met Ser Lys Val Ser Tyr G1u Cys Arg Va1 Val Ser Gly Lys Leu Va1 Met Gly Leu Asp Lys Met Ser Arg Va1 Ala Lys Gly Asn Leu Glu Val Val Arg Glu Lys Gly Asp Asp Thr Ser Gln Ser Asp Ser Phe Tyr Glu Gly Val Leu Gln Val Gly Ser Met I1e Gly Thr Thr Met Glu Ser Leu Lys Gln G1n Leu Gln G1y Pro Val Gly Ile Trp Arg Ala Ser Gly Val Ser Ala Met Glu Arg Cys Met Lys Arg Gly G1n Ser Lys Thr Val Val Ala Ser Ala Arg Tyr Thr Phe Gln Lys Met Met Glu Lys Met Ala Ser G1y Arg Glu Val Ser Lys Tyr Ser Leu Ile Ile Val Met Arg Cys Cys Ile Gly Phe Thr Ser Glu Ala Asn Lys Arg Ala Leu Thr Asn Ile Ser G1y Thr Gly Tyr Tyr Ile Ser Val Ala Gln Pro Thr Val Val Lys Leu Ala Gly Glu Trp Leu I1e Thr Pro Va1 Gly Arg Ser Lys Thr Gly Glu Val Gln Tyr Val Ser Ala Lys Leu Lys Lys Gly Met Thr Thr Gly Lys Leu Glu Leu Ile Lys Lys Ala Asp Arg Ser Asp Leu Asp Asn Phe Pro Glu Pro Ser Ala Asp Glu Leu Leu Arg Glu G1y Thr Ile Va1 Leu Met Gln Ile Gly Lys Asp Lys Trp Leu Cys Arg Val Arg Thr Gly Asp Arg Arg Val Arg Thr Asp Thr Asp Ile Gln Arg Ala Glu Ala Lys Ser Gln Val Glu Lys Glu Asp Leu Met Asp Glu Tyr Gly Val <210>

<211>

<212>
DNA

<213>
synthetic construct <400>

ccatggccgcgggattgaacgctctttaataaccatggaaactctagtaggagggctgct60 gactggagaagattctctgatcagtatgtcaaacgatgtatcttgtctttatgtttacga120 tggaccaatgagagttttctctcagaacgcattaatgccaactctgcaaagtgtaaaaag180 aagtgaccaattttccaaagggaaaacaaagagatttatcattgacctgttcggaatgaa240 gagaatgtgggacatcggaaacaaacagttggaagacgagaacttagacgaaactgtagg300 cgtggctgacttggggctggtgaaatatctaatcaacaacaagtacgatgaagcagaaaa360 gacaagtttaaggaagtcaatggaagaagcattcgaaaaatccatgaacgaagaatttgt420 ggttttaaacaaaggaaagtctgcaaacgacatcatttcagacacaaatgcgatgtgcaa480 attctgtgtaaagaactggatagtggcaacaggtttcaggggaagaacgatgtcagattt540 aattgaacaccatttcagatgcatgcaagggaaacaggaggtgaaaggatacatttggaa600 acacaagtacaacgaaaggcttaaaagaaaacagctaagcaaagaagaagtgaaattcga660 cagagaagaatatacttcaagaagcttcagactactctctttcttgaagaacagcgagag720 gaccaaactcgagccgagagcagtgttcacagcaggagttccatggagggcattcatctt780 cgtcctagaacagacaatgctggtggtaaacaaactggacccgaattcagtgatatggat840 gggaagtgatgcaaagataaacaccacaaactccaggataaaggaaatagggatgaaaaa900 tcaaggacaaacactagtgacactcacaggagataactccaaatacaacgagagcatgtg960 cccagaggtgatgatggtgttcctaagagaactaggaataaaaggaccaatgttggaagt1020 actggactatgcgctgtggcaattttcacagaagagtgtaaaacctgtcgcacctataaa1080 gaagagaaccggcaagtctaccgtggtgataaaagcagattccgttaaggagtgtagaga1140 tgccttcaacgaaaaggaactggagctgattcaaggagttgaatggatggacgacggatt1200 tgtgagagtgaggagaggaatgttgatgggaatggcaaacaacgcttttaccacagcttc1260 tacaattgcctcctcttttagtttcacaccagaagctgtgtacacattacagagctcaga1320 cgacttcgttacaggtagctgtggaagagacgtgcaacacgcaagacaaaggctagagat1380 ggctcttaaagtgagcaaagccgcaggtctgaacgtatcacagaagaagtcattctacgt1440 tgaagggacaactttcgagttcaactctatgttcgtaagagacggtaaagtgatggcaaa1500 cggaggaaactttgagaacatgacagttcctggaggattaggaccatctacagatctctt1560 tgtcgtggggaaacaagcaagaaactccatgttgagaggcaacctatccttcagccaggc1620 gatggagatgtgcaaaataggaatcacaaatgttgagaaagtttactatggaaacagaaa1680 ataccaggagctgaaaaatgagataagagagaaatgtggagaagaaacgatgtccatacc1740 agagagcatgggaggagacaggaaaccaagaccgtgggaattacctcagagctttgatgg1800 aattgccttaaaagaagctgtgaacagaggacattggaaagctgccaagtacatcaaatc1860 ttgctgcagcatagagttcgatgaagaaggagaccaatcttgggacacttcgaaaacagc1920 acttgtggtcataaggaaaaatgaaacggacatgagaagaagaactgttaaaacgaggaa1980 cccaaaagataaaatcttcaatgatgcaatgaacaaggccaaaaggatgtacgaaacagt2040 cgtggacaga aacccattac taggtctgaa ggggaaggga ggtagactga cagtaaaaga 2100 cttgaaagca aggaagctta ttgatgaagt agaagttgtt aagaagaaaa agcatgtttg 2160 aaatcactag tgcggccgcc tgcag 2185 <210> 4 <211> 726 <212> PRT
<213> synthetic construct <400> 4 His Gly Arg Gly Ile Glu Arg Ser Leu Ile Thr Met Glu Thr Leu Val Gly Gly Leu Leu Thr Gly Glu Asp Ser Leu Ile Ser Met Ser Asn Asp Val Ser Cys Leu Tyr Val Tyr Asp Gly Pro Met Arg Val Phe Ser Gln Asn Ala Leu Met Pro Thr Leu Gln Ser Val Lys Arg Ser Asp Gln Phe Ser Lys Gly Lys Thr Lys Arg Phe Ile Ile Asp Leu Phe Gly Met Lys Arg Met Trp Asp Ile Gly Asn Lys Gln Leu Glu Asp Glu Asn Leu Asp Glu Thr Val Gly Val Ala Asp Leu Gly Leu Val Lys Tyr Leu Ile Asn Asn Lys Tyr Asp G1u Ala Glu Lys Thr Ser Leu Arg Lys Sex Met Glu Glu Ala Phe Glu Lys Ser Met Asn Glu Glu Phe Val Val Leu Asn Lys Gly Lys Ser Ala Asn Asp Ile Ile Ser Asp Thr Asn A1a Met Cys Lys Phe Cys Val Lys Asn Trp Ile Val Ala Thr Gly Phe Arg Gly Arg Thr Met Ser Asp Leu Ile Glu His His Phe Arg Cys Met Gln Gly Lys Gln Glu Val Lys Gly Tyr Ile Trp Lys His Lys Tyr Asn Glu Arg Leu Lys Arg Lys Gln Leu Ser Lys Glu Glu Val Lys Phe Asp Arg Glu Glu Tyr Thr Ser Arg Ser Phe Arg Leu Leu Ser Phe Leu Lys Asn Ser Glu Arg Thr Lys Leu Glu Pro Arg Ala Val Phe Thr Ala Gly Val Pro Trp Arg Ala Phe Ile Phe Val Leu Glu Gln Thr Met Leu Val Val Asn Lys Leu Asp Pro Asn Ser Val Ile Trp Met Gly Ser Asp Ala Lys Ile Asn Thr Thr Asn Ser Arg Ile Lys Glu Ile Gly Met Lys Asn Gln Gly Gln Thr Leu Val Thr Leu Thr Gly Asp Asn Ser Lys Tyr Asn Glu Ser Met Cys Pro Glu Val Met Met Val Phe Leu Arg G1u Leu Gly Ile Lys Gly Pro Met~Leu Glu Val Leu Asp Tyr Ala Leu Trp Gln Phe Ser Gln Lys Ser Val Lys Pro Val Ala Pro Ile Lys Lys Arg Thr Gly Lys Ser Thr Val Val Ile Lys Ala Asp Ser Val Lys Glu Cys Arg Asp Ala Phe Asn Glu Lys G1u Leu Glu Leu I1e Gln Gly Val Glu Trp Met Asp Asp Gly Phe Val Arg Val Arg Arg G1y Met Leu Met Gly Met Ala Asn Asn Ala Phe Thr Thr Ala Ser Thr Ile Ala Ser Ser Phe Ser Phe Thr Pro Glu Ala Val Tyr Thr Leu Gln Ser Ser Asp Asp Phe Val Thr Gly Ser Cys Gly Arg Asp Val Gln His Ala Arg Gln Arg Leu Glu Met Ala Leu Lys Val Ser Lys Ala A1a Gly Leu Asn Val Ser Gln Lys Lys Ser Phe Tyr Val Glu Gly Thr Thr Phe Glu Phe Asn Ser Met Phe Va1 Arg Asp Gly Lys Val Met Ala Asn Gly Gly Asn Phe Glu Asn Met Thr Val Pro Gly Gly Leu Gly Pro Ser Thr Asp Leu Phe Val Val Gly Lys G1n Ala Arg Asn Ser Met Leu Arg Gly Asn Leu Ser Phe Ser Gln Ala Met G1u Met Cys Lys Tle G1y Ile Thr Asn Val Glu Lys Val Tyr Tyr Gly Asn Arg Lys Tyr Gln Glu Leu Lys Asn Glu Ile Arg Glu Lys Cys G1y Glu Glu Thr Met Ser Ile Pro Glu Ser Met Gly Gly Asp Arg Lys Pro Arg Pro Trp Glu Leu Pro Gln Ser Phe Asp Gly 21e A1a Leu Lys Glu Ala Val Asn Arg Gly His Trp Lys Ala Ala Lys Tyr Ile Lys Ser Cys Cys Ser Ile G1u Phe Asp Glu Glu Gly Asp Gln Ser Trp Asp Thr Ser Lys Thr Ala Leu Val Val Ile Arg Lys Asn G1u Thr Asp Met Arg Arg Arg Thr Val Lys Thr Arg Asn Pro Lys Asp Lys I1e Phe Asn Asp Ala Met Asn Lys A1a Lys Arg Met Tyr Glu Thr Val Val Asp Arg Asn Pro Leu Leu G1y Leu Lys G1y Lys Gly Gly Arg Leu Thr Val Lys Asp Leu Lys Ala Arg Lys Leu Ile Asp Glu Val Glu Val Val Lys Lys Lys Lys His Va1 Asn 705 7l0 715 720 His Cys Gly Arg Leu Gln <210> 5 <211> 2046 <212> DNA
<213> synthetic construct <400>

agcaaagattgctcaaatcccaaaaataatacagaaaacgtataagagatggccgataaa60 ggtatgacttattcttttgatgtcagagacaacaccttggttgtaagaagatctaccgct120 actaaaagtggcattaagatctcctacagagaggatcgaggaacatcacttctccaaaag180 gcattcgccgggacagaagatgaattctgggtggagttagatcaagatgtctacgttgac240 aaaaagattagagaattcctggtagaagagaaaatgaaggacatgagcacaagagtgtct300 ggggcagtggcagcagcaattgaaagatcagttgaatttgacaatttctcaaaagaagca360 gcagctaacattgaaatggctggtgtagatgatgaagaagctggaggaagtggtctggta420 gacaacagaaggaagaacaaaggggtctcaaacatggcctacaatctgtctctattcata480 gggatggtgtttcctgctctcactactttcttcagtgctatcctatcagaaggtgaaatg540 agcatctggcaaaatggacaagcaatcatgagaattctggcactggcagatgaagacgga600 aagagacaaacaagaacaggaggacagagggtggacatggctgatgtaaccaagctgaac660 gtagtcacggctaacggaaaagtcaagcaagttgaagtaaacttgaacgatctcaaagca720 gcattcaggcagagtagacctaaaagatcggactacagaaaagggcaaggttccaaggct780 acagaatcaagcatctccaaccaatgtatggcactgattatgaaatctgtgctgtcagca840 gaccaactttttgctccgggagtgaagatgatgaggacgaacggtttcaatgcgtcgtac900 acaacactggcagaaggggcaaacattccgagcaagtacctaagacacatgaggaactgc960 ggaggagtagctctggacctgatgggaatgaagaggatcaaaaactcacctgaaggagcc1020 aagtctaagatcttttccatcatccagaagaaagtaagaggaagatgtcgcacagaggag1080 caacgcctcctgactagcgcactgaaaatcagcgacggtgaaaacaagttccagagaatc1140 atggacactctatgtacaagcttcctgattgaccctccaagaactaccaaatgcttcatt1200 ccacctatttccagtctcatgatgtacatccaagaaggcaactctgtactggcaatggat1260 ttcatgaaaaacggagaggacgcctgcaagatctgcagagaagccaaactgaaagtgggg1320 gtaaacagtacgttcacaatgtcagtagctagaacatgcgttgcagtgtcaatggttgca1380 acagctttttgttctgcagatatcatcgagaatgcagtgcctggttccgaaaggtacaga1440 tccaacatcaaggctaacacaaccaaaccaaaaaaggactccacttacacaattcaagga1500 cttagattgtctaacgtgaggtatgaagcaagacctgaaacatcacaaagcaacacagac1560 agaagttggcaagtgaacgtgactgacagcttcggaggacttgctgtgttcaaccaaggg1620 gcaattagagaaatgctaggagacggaacatcagagacaactagtgtgaacgtcagagcc1680 ctggtgaagagaattctgaaatcagcttcagagaggagtgcaagagctgtaaagacattt1740 atggtgggagaacaagggaaatcagctattgttatctctggtgtgggactgttctctatt1800 gactttgaaggggtagaggaagcggaaagaataactgacatgacacctgaaattgagttt1860 gacgaggacgacgaggaagaggaagacattgacatttagagtgacaattatgtaacttcc1920 taattaccctatattgtttgaatatataatgaaactattgtgtgttaaaggttgtgggtt1980 tgattattaa atttaaattg aaacggtatt gacgatattt acaaaaaaaa aaaaaaaaaa 2040 aaaaaa 2046 <210> 6 <211> 616 <212> PRT
<213> synthetic construct <400> 6 Met Ala Asp Lys Gly Met Thr Tyr Ser Phe Asp Val.Arg Asp Asn Thr Leu Val Va1 Arg Arg Ser Thr Ala Thr Lys Ser Gly Tle Lys Ile Ser Tyr Arg Glu Asp Arg Gly Thr Ser Leu Leu Gln Lys Ala Phe Ala Gly Thr Glu Asp G1u Phe Trp Val G1u Leu Asp Gln Asp Val Tyr Val Asp Lys Lys Ile Arg Glu Phe Leu Val Glu Glu Lys Met Lys Asp Met Ser Thr Arg Val Ser Gly Ala Val Ala Ala Ala Ile Glu Arg Ser Val Glu Phe Asp Asn Phe Ser Lys Glu Ala Ala Ala Asn Ile Glu Met Ala Gly Val Asp Asp Glu Glu Ala Gly Gly Ser Gly Leu Val Asp Asn Arg Arg Lys Asn Lys Gly Val Ser Asn Met Ala Tyr Asn Leu Ser Leu Phe Ile Gly Met Val Phe Pro Ala Leu Thr Thr Phe Phe Ser Ala Ile Leu Ser Glu Gly Glu Met Ser Ile Trp Gln Asn Gly Gln Ala Ile Met Arg Ile Leu Ala Leu Ala Asp Glu Asp Gly Lys Arg Gln Thr Arg Thr Gly Gly Gln Arg Val Asp Met Ala Asp Val Thr Lys Leu Asn Val Val Thr Ala Asn G1y Lys Val Lys Gln Val Glu Val Asn Leu Asn Asp Leu Lys A1a Ala Phe Arg Gln Ser Arg Pro Lys Arg Ser Asp Tyr Arg Lys Gly Gln Gly Ser Lys Ala Thr Glu Ser Ser Ile Ser Asn Gln Cys Met Ala Leu Ile Met Lys Ser Val Leu Ser Ala Asp Gln Leu Phe Ala Pro G1y Val Lys Met Met Arg Thr Asn Gly Phe Asn Ala Ser Tyr Thr Thr Leu Ala Glu Gly Ala Asn Ile Pro Ser Lys Tyr Leu Arg His Met Arg Asn Cys Gly Gly Val A1a Leu Asp Leu Met Gly Met Lys Arg Ile Lys Asn Ser Pro Glu Gly Ala Lys Ser Lys Ile Phe Ser Ile Ile Gln Lys Lys Val Arg Gly Arg Cys Arg Thr Glu Glu Gln Arg Leu Leu Thr Ser Ala Leu Lys Ile Ser Asp Gly Glu Asn Lys Phe Gln Arg Ile Met Asp Thr Leu Cys Thr Ser Phe Leu Ile Asp Pro Pro Arg Thr Thr Lys Cys Phe Ile Pro Pro Ile Ser Ser Leu Met Met Tyr I1e Gln Glu Gly Asn Ser Val Leu Ala Met Asp Phe Met Lys Asn Gly Glu Asp Ala Cys Lys Ile Cys Arg Glu Ala Lys Leu Lys Val Gly Val Asn Ser Thr Phe Thr Met Ser Val Ala Arg Thr Cys Val Ala Val Ser Met Val Ala Thr Ala Phe Cys Ser Ala Asp Ile Ile Glu Asn Ala Val Pro Gly Ser Glu Arg Tyr Arg Ser Asn Ile Lys Ala Asn Thr Thr Lys Pro Lys Lys Asp Ser Thr Tyr Thr Ile Gln Gly Leu Arg Leu Ser Asn Val Arg Tyr Glu Ala Arg Pro Glu Thr Ser G1n Ser Asn Thr Asp Arg Ser Trp Gln Val Asn Val Thr Asp Ser Phe Gly Gly Leu Ala Val Phe Asn Gln Gly Ala Ile Arg Glu Met Leu G1y Asp G1y Thr Ser G1u Thr Thr Ser Val Asn Val Arg Ala Leu Val Lys Arg Ile Leu Lys Ser Ala Ser Glu Arg Ser Ala Arg Ala Val Lys Thr Phe Met Val Gly Glu Gln Gly Lys Ser Ala Ile Val Ile Ser G1y Val Gly Leu Phe Ser Ile Asp Phe Glu Gly Val Glu Glu Ala Glu Arg I1e Thr Asp Met Thr Pro Glu Ile Glu Phe Asp Glu Asp Asp Glu Glu Glu Glu Asp Ile Asp Ile <210>

<211>

<212>
DNA

<213>
synthetic construct <400>

caagatggataacctccgtgaatgcataaaccgcaaaagaagactacttgccttaccaga60 tgttcctgaaacttcggatgcctttctaagtgatttgagacatctatacatgtgtgttgc120 tttctgtgatcaacacaaaaccactggagacgaatcaagattcaccaacctggaattact180 tgaccaagatgaagcactaggtgcccaaagagcttttgaagccaaacatggaataaaagg240 aggttctttaggagacgttcttgaccatgaactgaaaaaggtcattgaatttacttttac300 ttctggaagtttgtatattgccgaacaaagaaaaagaaagactcaagcagactcaataat360 tgtgtgcgtttcagaaggacttaacgacttcagcgtatcacacggagtgctagacatggg420 acttgtggaaacaggggtgaatgcagtaagagatttctgcacacaaaacggaataccaat480 gaagataaatcaggtaggatccacgagaacaccaacaccgatcagcacatgcaaaatctc540 tgaacaaataacacgacarataaacagtacaattactgaaaggaaaatggaaacagtact600 ggcagcaatcgcaattaaaccagaactcaaayyaactcagaaaggatgcagmmcttgtaa660 agaactagaagatgaaaatattctgtggatggaccctcaattctgtgaaattgatgaaag720 ttttccttacagaggagggccatacgggaacttcctgcaagaattgctgcttacaaccaa780 cgacgtagagaccaacgggaaagacagagaagaagtagtaaagaasatactggataacaa840 ggcgttcaccgttgaaagtggtgaatgcataataacacttccagacaaaatgacttgttt900 cggagaaccrgagaagaagagaccagcaacaatagacgaagtgagaaccgcaggagaaag960 gtttgaacagagtgttaaaccgaaaacccaaagatatggaaggttatcagacaaatggat1020 ggagcttgaaaagtttatctttactgcaagcaaaacagaagtggatactttcctttctgt1080 agggaccgaaagacttgagtcggttggagtgtgtgtcggagctttacacagagcgaccac1140 aaccaggataattagacctatgattcaaggagggaaatgttgggggatgatgttcaaaac1200 aaagtccaaaatgggagacacgaggaaggaaggatactgtcacgcaatcattttcggaaa1260 aggggaagataaatcaggacaaaacaagatgacaatgatggggaaaacagtacattggca1320 tctaagagtagttaagtctaaaggagactggatggcgcaacaactctgtgcaaacaaaag1380 cagaatatggcaacatgaccctgagctagtaacagaaggagtgacagttctaatgacgcc1440 tttttctcagaaaattgcaaccattagtagatggagggcaatgaggttagacagcatgtt1500 tcatgtttctagtgcctggcatcattcacctgcgtgtgaagctgcatcggcaatgctgag1560 aaagtttgtggagatagtacatgccatcaaccagaaaagagattggggtgttgtggggag1620 tatggaggacatggtgaaggaagtggaggaaataggggagcacttgcagacggcatgtga1680 ytttagagtttacaacatktgcaaagccttgattcagaaaattgcagtcagtacccaatg1740 agtggttatttacttgtaaattgttgtgtgtttgacgatatgtattt 1787 <210> 8 <211> 578 <212> PRT
<213> synthetic construct <220>
<221> misc_feature <222> (210)..(210) <223> X = any residue <220>
<221> misc_feature <222> (216)..(216) <223> X = any residue <220>
<221> misc_feature <222> (217)..(217) <223> X = any residue <220>
<221> misc_feature <222> (274)..(274) <223> X = any residue <220>
<221> misc_feature <222> (565)..(565) <223> X = any residue <400> 8 Met Asp Asn Leu Arg G1u Cys I1e Asn Arg Lys Arg Arg Leu Leu Ala Leu Pro Asp Val Pro G1u Thr Ser Asp Ala Phe Leu Ser Asp Leu Arg His Leu Tyr Met Cys Val Ala Phe Cys Asp Gln His Lys Thr Thr Gly Asp Glu Ser Arg Phe Thr Asn Leu Glu Leu Leu Asp Gln Asp Glu Ala Leu Gly Ala Gln Arg Ala Phe Glu Ala Lys His Gly Ile Lys Gly Gly Ser Leu Gly Asp Val Leu Asp His Glu Leu Lys Lys Val Ile Glu Phe Thr Phe Thr Ser Gly Ser Leu Tyr Ile Ala Glu Gln Arg Lys Arg Lys Thr Gln Ala Asp Ser Ile Ile Va1 Cys Va1 Ser Glu Gly Leu Asn Asp Phe Ser Val Ser His Gly Val Leu Asp Met Gly Leu Val Glu Thr Gly Val Asn Ala Val Arg Asp Phe Cys Thr Gln Asn Gly Ile Pro Met Lys Tle Asn Gln Val Gly Ser Thr Arg Thr Pro Thr Pro Ile Ser Thr Cys Lys I1e Ser Glu Gln Ile Thr Arg Gln Ile Asn Ser Thr Ile Thr Glu Arg Lys Met Glu Thr Val Leu Ala Ala Ile Ala Ile Lys Pro Glu Leu Lys Xaa Thr Gln Lys G1y Cys Xaa Xaa Cys Lys Glu Leu Glu Asp G1u Asn Ile Leu Trp Met Asp Pro Gln Phe Cys Glu Ile Asp Glu Ser Phe Pro Tyr Arg Gly G1y Pro Tyr Gly Asn Phe Leu G1n Glu Leu Leu Leu Thr Thr Asn Asp Val G1u Thr Asn Gly Lys Asp Arg Glu Glu Val Val Lys Xaa Ile Leu Asp Asn Lys Ala Phe Thr Val Glu Ser Gly G1u Cys Ile Ile Thr Leu Pro Asp Lys Met Thr Cys Phe Gly Glu Gln Glu Lys Lys Arg Pro Ala Thr Ile Asp Glu Va1 Arg Thr Ala Gly Glu Arg Phe 305 37.0 315 320 G1u Gln Ser Val Lys Pro Lys Thr Gln Arg Tyr Gly Arg Leu Ser Asp Lys Trp Met Glu Leu Glu Lys Phe Ile Phe Thr Ala Ser Lys Thr Glu Val Asp Thr Phe Leu Ser Val Gly Thr Glu Arg Leu Glu Ser Val Gly Val Cys Val Gly Ala Leu His Arg Ala Thr Thr Thr Arg Ile Ile Arg Pro Met Ile Gln Gly Gly Lys Cys Trp Gly Met Met Phe Lys Thr Lys Ser Lys Met Gly Asp Thr Arg Lys Glu Gly Tyr Cys His Ala Ile Ile 405 41.0 415 Phe Gly Lys Gly Glu Asp Lys Ser Gly Gln Asn Lys Met Thr Met Met Gly Lys Thr Val His Trp His Leu Arg Val Val Lys Ser Lys Gly Asp Trp Met Ala Gln Gln Leu Cys A1a Asn Lys Ser Arg Ile Trp G1n His Asp Pro Glu Leu Val Thr Glu Gly Va1 Thr Val Leu Met Thr Pro Phe Ser Gln Lys Ile Ala Thr Ile Ser Arg Trp Arg A1a Met Arg Leu Asp Ser Met Phe His Val Ser Ser Ala Trp His His Ser Pro Ala Cys G1u Ala A1a Ser A1a Met Leu Arg Lys Phe Val Glu Ile Val His Ala Ile Asn Gln Lys Arg Asp Trp Gly Val Val Gly Ser Met Glu Asp Met Val Lys Glu Val Glu Glu Ile Gly Glu His Leu Gln Thr Ala Cys Asp Phe Arg Val Tyr Asn Xaa Cys Lys Ala Leu Ile Gln Lys Ile Ala Val Ser Thr Gln <210>

<211>

<212>
DNA

<213>
synthetic construct <400>

agttaaagatggcttttctaacaattttagtcttgttcctttttaaagaggttctttgtg60 aaccttgtatttgtgagaacccaacatgtctaggaataacaatcccacaggcaggtttcg120 taagaagcgctccaggaggtgtacttctaactgagacaatcacggaaagaccacaactaa180 cagagtggacaacctccagaccgaagcttgaagaaactctctggttagatggggaaacaa240 agaacggaaaagtatctcagacactattcgaagccatccaaggtacacagatggagaact300 gtgcagtgaaagctgtgttagacacaacatttgtcaacctaaccaaacaagacattgtgc360 taggaaaaatcaaggtgtctgagtttggtggagacagtgacatttccaaatgtggaagaa420 aaggactaaaggttttcatctgtggaggtactgttggatacgtgacaagaggatgcccac480 ctgaggagtgcaaaggaaagaaagggagaatgatggctctcgaacccactacggattgtg540 gtgtcgaaaaaggacttacaactgacagaatcaaaacaggaatgttggacatcacaagtt600 gctgtacacaacatggatgcacaaagggaatcagagtagaggttccttcaccagtacttg660 tatcttcaaaatgtcaagaagtcactttcagagtggttccattccattcagtacctgaca720 agctagggtttgcacgcacaagctcattcacactaaaagctaacttcgtgaacaaacatg780 ggtggtccaagtataatttcaacctaagaggatttcctggagaagagttcattaagtgtt840 gtggatttacgttgggagtcggaggagcgtggtttcaagcctacttaaatggaatggttc900 aaggtgacggtgccgcatctgcagacgacgtgaaagagaaactcaacggaataatcgacc960 agataaacaaagcgaacacacttcttgaaggagaaattgaagcagtgaggaggattgcct1020 atatgaaccaagcatcaagtcttcagaaccaagtggaaatcggactaataggtgaatatt1080 tgaacattagcagttggttggagactactacattaactaaaacagaagaaggcttgatga1140 agaatggctggtgtcagtctaacacgcactgctggtgtccacctaaacctacaattgttc1200 ccaccattggatatgttgacagtataaaagaagtaacgggtacaagttggtggatggtta1260 tgatacattacattattgtggggttaatagttattgtggtggtggtgtttggtttaaaac1320 tatggggatgtcttagaaggtgaaatgtcggtctaaaaattctttttctgtacattacta1380 aagggtagcttaaccaaggtgtttatgtatatagactattattggataagttagaaattt1440 gtatctgattatgcattattaattgtataaatagaatcactagtgcggccgcctgcaggt1500 cgac 1504 <210> 10 <211> 497 <212> PRT
<213> synthetic construct <400> 10 Leu Lys Met Ala Phe Leu Thr Ile Leu Val Leu Phe Leu Phe Lys Glu Val Leu Cys Glu Pro Cys Ile Cys Glu Asn Pro Thr Cys Leu Gly Ile Thr Ile Pro Gln Ala Gly Phe Val Arg Ser Ala Pro Gly Gly Val Leu Leu Thr Glu Thr Ile Thr Glu Arg Pro G1n Leu Thr Glu Trp Thr Thr Sex Arg Pro Lys Leu Glu Glu Thr Leu Trp Leu Asp Gly Glu Thr Lys Asn Gly Lys Val Ser Gln Thr Leu Phe Glu Ala Ile Gln Gly Thr Gln Met Glu Asn Cys Ala Val Lys Ala Val Leu Asp Thr Thr Phe Val Asn Leu Thr Lys Gln Asp Ile Val Leu Gly Lys Ile Lys Val Ser Glu Phe Gly Gly Asp Ser Asp Ile Ser Lys Cys Gly Arg Lys Gly Leu Lys Val Phe Ile Cys Gly Gly Thr Val Gly Tyr Val Thr Arg Gly Cys Pro Pro Glu Glu Cys Lys Gly Lys Lys Gly Arg Met Met A1a Leu Glu Pro Thr Thr Asp Cys Gly Val Glu Lys Gly Leu Thr Thr Asp Arg Ile Lys Thr Gly Met Leu Asp Ile Thr Ser Cys Cys Thr Gln His Gly Cys Thr Lys Gly Tle Arg Val Glu Val Pro Ser Pro Val Leu Val Ser Ser Lys Cys Gln Glu Val Thr Phe Arg Val Val Pro Phe His Ser Val Pro Asp Lys Leu Gly Phe Ala Arg Thr Ser Ser Phe Thr Leu Lys Ala Asn Phe Val Asn Lys His Gly Trp Ser Lys Tyr Asn Phe Asn Leu Arg Gly Phe Pro Gly Glu Glu Phe Ile Lys Cys Cys Gly Phe Thr Leu Gly Val Gly Gly Ala Trp Phe G1n Ala Tyr Leu Asn Gly Met Va1 G1n Gly Asp Gly Ala Ala Ser Ala Asp Asp Val Lys Glu Lys Leu Asn Gly Ile Ile Asp Gln Ile Asn Lys Ala Asn Thr Leu Leu Glu Gly Glu Ile Glu Ala Va1 Arg Arg Ile Ala Tyr Met Asn Gln Ala Ser Ser Leu G1n Asn Gln Val Glu Ile Gly Leu Tle Gly Glu Tyr Leu Asn I1e Ser Ser Trp Leu Glu Thr Thr Thr Leu Thr Lys Thr Glu Glu Gly Leu Met Lys Asn Gly Trp Cys Gln Ser Asn Thr His Cys Trp Cys Pro Pro Lys Pro Thr Ile Val Pro 385 390 ' 395 400 Thr Ile Gly Tyr Val Asp Ser Ile Lys Glu Val Thr Gly Thr Ser Trp Trp Met Val Met Ile His Tyr Ile Ile Val Gly Leu Ile Val Ile Val Val Val Val Phe Gly Leu Lys Leu Trp Gly Cys Leu Arg Arg Asn Val G1y Leu Lys Ile Leu Phe Leu Tyr Ile Thr Lys Gly Leu Asn Gln Gly Val Tyr Val Tyr Arg Leu Leu Leu Asp Lys Leu Glu Ile Cys Ile Leu Cys Ile Ile Asn Cys Ile Asn Arg Ile Thr Ser Ala Ala Ala Cys Arg Ser <210>

<211>

<212>
DNA

<213> hetic synt construct <400>

agcaaagatggcacgattcataattttattcctactgttggcgcctgtttacagtcgtct60 atgtcttagaaaccatcctgacaccacctggataggtgactcccgaagcgatcaatcaag120 ggtgaaccaacagtctcttgatctggttacaaacttcaagggaattctacaagccaagaa180 cgggaatggtctcatgaagcagatgagcggaaggttcccaagtgattggtaccaacctac240 tacaaagtataggattctatacattggtacaaacgactgcactgagggccctaacgacgt300 gatcataccgacgtcaatgacactagacaatgtggcaagggacctgtacctgggagcatg360 tcgaggagatgtaagagtgacaccaaccttcgtgggagcagctgagcttggactgattgg420 gagaacagatgccttaacagaattttctgtaaaggtgctgactttcaacaaccctactat480 tgtagtagttggactaaatggaatgtcaggaatctacaaggtctgcattgctgcctcttc540 tggaaacgtaggcggagtcaacttggtgaacggatgcggatacttcagcgctcctctgag600 attcgacaacttcaaaggacagatctacgtgtcagacacctttgaagtcagaggaacaaa660 gaacaaatgtgtcatacttagatcttctagcaatgctcctttgtgtacacatatcaaaag720 aaacattgagttggatgagtacgttgacacaccaaacactgggggcgtatatccttctga780 tgggtttgattctcttcacggctctgcttcgattagaacttttttaacagaggcactgac840 atgtccaggtgtagattgggacagaattgatgcagcttcatgcgagtatgacagttgtcc900 taaacttgtgaaagaatttgaccaaacagggctcggaaacacagatactcaaataatgag960 agagctagaagcacaaaaggagatgattggtaaacttggcagaaacattacagacgtaaa1020 caacagagtagatgctattccaccacagcttagcaacatcttcatctctatgggagtggc1080 aggttttgggatagcactgtttctagcagggtggaaggcttgtgtttggatagcagcttt1140 catgtataagtctagaggtagaaacccacctgcaaatctgtctgttgcttgatactaaga1200 caaacaaagttttcaaataatcaaatgttttctaatgtaatgtaaaattcaaatcgtatg1260 tgatattattattttgaagacgttcttgatgttgtacgtttacagaaaagtgcatttttt1320 act 1323 <210> 12 <211> 394 <212> PRT
<213> synthetic construct <400> 12 Met Ala Arg Phe Ile Ile Leu Phe Leu Leu Leu Ala Pro Val Tyr Ser Arg Leu Cys Leu Arg Asn His Pro Asp Thr Thr Trp Ile Gly Asp Ser Arg Ser Asp Gln Ser Arg Va1 Asn G1n Gln Ser Leu Asp Leu Val Thr Asn Phe Lys Gly Ile Leu Gln Ala Lys Asn Gly Asn Gly Leu Met Lys Gln Met Ser Gly Arg Phe Pro Ser Asp Trp Tyr Gln Pro Thr Thr Lys Tyr Arg Ile Leu Tyr Ile Gly Thr Asn Asp Cys Thr Glu Gly Pro Asn Asp Val Ile Ile Pro Thr Ser Met Thr Leu Asp Asn Va1 Ala Arg Asp Leu Tyr Leu Gly Ala Cys Arg Gly Asp Va1 Arg Val Thr Pro Thr Phe Val Gly A1a Ala Glu Leu Gly Leu Tle Gly Arg Thr Asp Ala Leu Thr Glu Phe Ser Val Lys Val Leu Thr Phe Asn Asn Pro Thr Ile Val Val Val Gly Leu Asn Gly Met Ser Gly Ile Tyr Lys Val Cys Ile Ala A1a 165 l70 175 Ser Ser Gly Asn Val Gly Gly Val Asn Leu Va1 Asn Gly Cys Gly Tyr Phe Ser Ala Pro Leu Arg Phe Asp Asn Phe Lys Gly Gln Tle Tyr Val Ser Asp Thr Phe Glu Val Arg Gly Thr Lys Asn Lys Cys Val Ile Leu Arg Ser Sex Ser Asn Ala Pro Leu Cys Thr His Ile Lys Arg Asn Ile G1u Leu Asp Glu Tyr Val Asp Thr Pro Asn Thr Gly Gly Val Tyr Pro Ser Asp Gly Phe Asp Sex Leu His Gly Ser Ala Ser Ile Arg Thr Phe Leu Thr Glu Ala Leu Thr Cys Pro Gly Val Asp Trp Asp Arg Ile Asp Ala Ala Ser Cys Glu Tyr Asp Ser Cys Pro Lys Leu Val Lys Glu Phe Asp Gln Thr Gly Leu Gly Asn Thr Asp Thr Gln I1e Met Arg Glu Leu 305 310 ~ 315 320 Glu Ala Gln Lys Glu Met Ile Gly Lys Leu Gly Arg Asn I1e Thr Asp Val Asn Asn Arg Val Asp Ala Ile Pro Pro Gln Leu Ser Asn Ile Phe Ile Ser Met Gly Val Ala Gly Phe Gly Ile Ala Leu Phe Leu A1a Gly Trp Lys Ala Cys Val Trp Ile A1a Ala Phe Met Tyr Lys Ser Arg Gly Arg Asn Pro Pro Ala Asn Leu Ser Val Ala <210>

<211>

<212>
DNA

<213>
synthetic construct <400>

tacaaagaaaatgttcagaacatgtctggatttaacttcgaggtaatggtgccggaacaa60 ggaggaaaagtggtcttcagccttactgaaacggggtcatgtgtctcgttttacggagat120 gatgaaccaggtgaagggtcctgcgaacttgcctctgaaaacatggattttccaagttgt180 cctctggggaatggagatgacttctgtctgtcgctggcgctaagcacaatgagatggtct240 gggatgaccaagagaaacaacttcatggacagattcattggaagttttgttcattgtaca300 ccagtgatgatctggtcgtatggaaatttgtccaagaaaagccatcacaaaatggtttgc360 cacacttgcccagacgagtacaagttcagtgacaaggacgagatgcagggatactatgag420 gaatgtctagaggcttctactgacattttccttgatgaacttgctactgttgttacaggt480 ggcttctttcctgtcggactcaaaggttcctggggaggatggtacctcaagtacgtcagg540 tatgctggacctcttgcgggatcaagtggattcattgtcaatcaacgattctacgacaga600 gcccaaaacaagactggatccagggttgtatccatggttgaaatggacggagacggctta660 tcgttcatctacgagaagcctagcgtctaccatagtgatgggtgcactggttcagcagcg720 aggttctggaaacgggatcacaatgagagagctggagttgagcttagggctggacttcac780 ttcagaatgtgattggttgaaaacttgttatgtaaacaagaattttgtgtttttgtcaga840 aaaagaaattgctgtaaacatggaagttgaaaaattcatttgtaatgagaactaaagatg900 tctttgtgttcaaattttaactaatgacaatatatgaaatatgtcgtacatggtgttgat960 gataat 966 <210> 14 <211> 256 <212> PRT
<213> synthetic construct <400> 14 Met Ser Gly Phe Asn Phe Glu Val Met Val Pro Glu Gln Gly Gly Lys Val Val Phe Ser Leu Thr Glu Thr G1y Ser Cys Val Ser Phe Tyr G1y Asp Asp Glu Pro Gly Glu Gly Ser Cys Glu Leu Ala Ser Glu Asn Met Asp Phe Pro Ser Cys Pro Leu Gly Asn Gly Asp Asp Phe Cys Leu Ser Leu Ala Leu Ser Thr Met Arg Trp Ser Gly Met Thr Lys Arg Asn Asn Phe Met Asp Arg Phe I1e Gly Ser Phe Val His Cys Thr Pro Val Met Ile Trp Ser Tyr Gly Asn Leu Ser Lys Lys Ser His His Lys Met Val Cys His Thr Cys Pro Asp Glu Tyr Lys Phe Ser Asp Lys Asp G1u Met Gln Gly Tyr Tyr Glu Glu Cys Leu Glu Ala Ser Thr Asp Ile Phe Leu . 130 135 140 Asp Glu Leu Ala Thr Val Val Thr Gly Gly Phe Phe Pro Val Gly Leu Lys Gly Ser Trp Gly Gly Trp Tyr Leu Lys Tyr Val Arg Tyr Ala Gly Pro Leu Ala Gly Ser Ser Gly Phe Ile Val Asn Gln Arg Phe Tyr Asp Arg Ala Gln Asn Lys Thr Gly Ser Arg Val Val Ser Met Val Glu Met Asp Gly Asp Gly Leu Ser Phe Ile Tyr Glu Lys Pro Ser Val Tyr His Ser Asp Gly Cys Thr Gly Ser Ala Ala Arg Phe Trp Lys Arg Asp His Asn Glu Arg Ala Gly Val Glu Leu Arg Ala Gly Leu His Phe Arg Met <210> 15 <211> 146 <212> PRT
<213> synthetic construct <400> 15 Met Asn Leu Leu Leu Leu Leu Gln Val Ala Ser Phe Leu Ser Asp Ser l 5 10 15 Lys Val Pro Gly Glu Asp Gly Thr Ser Ser Thr Ser Gly Met Leu Asp Leu Leu Arg Asp Gln Val Asp Ser Leu Ser Ile Asn Asp Ser Thr Thr Glu Pro Lys Thr Arg Leu Asp Pro Gly Leu Tyr Pro Trp Leu Lys Trp Thr Glu Thr Ala Tyr Arg Ser Ser Thr Arg Ser Leu Ala Ser Thr Ile Val Met Gly Ala Leu Va1 Gln Gln Arg Gly Ser Gly Asn Gly Ile Thr Met Arg Glu Leu Glu Leu Ser Leu Gly Leu Asp Phe Thr Ser Glu Cys Asp Trp Leu Lys Thr Cys Tyr Val Asn Lys Asn Phe Val Phe Leu Ser Glu Lys Glu I1e Ala Val Asn Met Glu Val G1u Lys Phe Ile Cys Asn Glu Asn <210>

<211>

<212>
DNA

<213>
synthetic construct <400>

tgcaaagattggctatctaccatgcatgagagaagcaaacccaaaaccacgggagctgat60 cagacatgccttgaagaagaaaaagagaccagaggtggtttacgcaatgggagttcttct120 gacactggggggagagagcggactgaccgtggagtttcctgttccagaaggaaaaactgt180 gaaggtcaaaaccttgaaccaattggtgaacgggatgatcagtcgagcgacgatgaccct240 ctactgtgtgatgaaagatccaccatcgggaggcatggcaacgctgatgagagaccacat300 caggaactggctgaaggaggaatcaggatgccaggacgcggatggtggagaggaaaaatg360 ggcaatggtgtatggtatgatttcacccgacatggcagaggagaagacgatgctgaagga420 gctgaaaacaatgctacacagcaggatgcagatgtatgctctgggtgcaagttcgaaagc480 cctagagaatttagaaaaggccatcgtcgctgcagttcatcgacttccggcatcctgctc540 gacagagaagatggtgcttctggggtacctgaagtaagcttcaaagaaagaatggaagcg600 gagaagaagaaactgaaagagctggacgacaagatctacaagctaaggagaagattgagg660 aagatggagtacaagaaaatggggatcaaccgagaaatcgacaaattggaagactctgta720 caataaaatcactagt 736 <210> 17 <211> 234 <212> PRT
<213> synthetic construct <400> 17 Met His Glu Arg Ser Lys Pro Lys Thr Thr Gly Ala Asp Gln Thr Cys Leu Glu Glu Glu Lys Glu Thr Arg Gly Gly Leu Arg Asn Gly Ser Ser Ser Asp Thr Gly Gly Arg Glu Arg Thr Asp Arg Gly Val Ser Cys Ser Arg Arg Lys Asn Cys Glu Gly Gln Asn Leu Glu Pro Ile G1y Glu Arg Asp Asp Gln Ser Ser Asp Asp Asp Pro Leu Leu Cys Asp Glu Arg Ser Thr Ile Gly Arg His Gly Asn Ala Asp Glu Arg Pro His Gln Glu Leu Ala Glu G1y Gly Ile Arg Met Pro Gly Arg Gly Trp Trp Arg Gly Lys Met Gly Asn Gly Val Trp Tyr Asp Phe Thr Arg His Gly Arg Gly Glu Asp Asp Ala Glu Gly Ala G1u Asn Asn A1a Thr Gln Gln Asp Ala Asp Val Cys Ser Gly Cys Lys Phe Glu Ser Pro Arg Glu Phe Arg Lys Gly His Arg Arg Cys Ser Ser Ser Thr Ser Gly Ile Leu Leu Asp Arg Glu Asp Gly Ala Ser Gly Val Pro Glu Val Ser Phe Lys Glu Arg Met Glu Ala Glu Lys Lys Lys Leu Lys Glu Leu Asp Asp Lys Ile Tyr Lys Leu Arg Arg Arg Leu Arg Lys Met Glu Tyr Lys Lys Met Gly Tle Asn Arg Glu Ile Asp Lys Leu Glu Asp Ser Val Gln <210> 18 <211> 183 <212> PRT
<213> synthetic construct <400> 18 Met Arg Glu Ala Asn Pro Lys Pro Arg Glu Leu Ile Arg His Ala Leu Lys Lys Lys Lys Arg Pro Glu Val Va1 Tyr Ala Met Gly Val Leu Leu Thr Leu Gly Gly G1u Ser Gly Leu Thr Val Glu Phe Pro Val Pro Glu Gly Lys Thr Val Lys Val Lys Thr Leu Asn Gln Leu Val Asn Gly Met Ile Ser Arg Ala Thr Met Thr Leu Tyr Cys Val Met Lys Asp Pro Pro Ser Gly G1y Met Ala Thr Leu Met Arg Asp His Ile Arg Asn Trp Leu Lys Glu Glu Ser Gly Cys Gln Asp Ala Asp Gly Gly Glu Glu Lys Trp Ala Met Val Tyr Gly Met Ile Ser Pro Asp Met Ala Glu Glu Lys Thr Met Leu Lys Glu Leu Lys Thr Met Leu His Ser Arg Met Gln Met Tyr Ala Leu Gly Ala Ser Ser Lys Ala Leu Glu Asn Leu Glu Lys Ala Ile Val Ala Ala Val His Arg Leu Pro Ala Ser Cys Ser Thr Glu Lys Met Val Leu Leu Gly Tyr Leu Lys 1so <210> 19 <211> 31 <212> DNA
<213> Primer <400> 19 aagcagtggt aacaacgcag agtagcaaag a 31 <210> 20 <211> 2l <212> DNA
<213> Primer <400> 20 gaacgctctt taataaccat g 2l <210> 21 <211> 19 <212> DNA
<213> Primer <400> 21 tcaaacatgc tttttcttc 19 <210> 22 <211> 19 <212> DNA
<213> Primer <400> 22 agcaaagatg gcacgattc 19 <210> 23 <211> 24 <212> DNA
<213> Primer <400> 23 tgcacttttc tgtaaacgta caac 24 <210> 24 <211> 35 <212> DNA
<213> Primer <400> 24 aagcagtggt aacaacgcag agtctatcta ccatg 35 <210> 25 <211> 19 <212> DNA
<213> Primer <400> 25 ttattgtaca gagtcttcc 19

Claims (11)

1. A nucleic acid molecule, comprising a nucleic acid sequence at least 70% identical to SEQ ID NO: 1;
a nucleic acid sequence at least 85% identical to SEQ ID NO: 3; or a nucleic acid sequence at least 85% identical to SEQ ID NO: 11.

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence is at least 80% identical to SEQ ID NO: 1.

3. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ
ID
NO: 11.

4. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ
ID
NO: 11.

5. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence consists essentially of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID
NO:
11.

6. The nucleic acid molecule according to claim 1, operably linked to a heterologous nucleic acid comprising an expression control sequence.

7. The nucleic acid molecule according to claim 6, wherein the nucleic acid molecule encodes an antigenic epitope.

8. A vector comprising the nucleic acid molecule according to claim 6.

9. A host cell, comprising the nucleic acid according to claim 6.

10. The host cell according to claim 9, wherein the cell is a fish cell.
I 1. The host cell according to claim 10, wherein the fish cell is from rainbow trout, coho salmon, Chinook salmon, amago salmon, chum salmon, sockeye salmon, Atlantic salmon, arctic char, brown trout, cutthroat trout, brook trout, catfish, tilapia, sea bream, seabass, flounder, or sturgeon.
12. A nucleic acid comprising at least 100 consecutive nucleotides of SEQ ID NO: 1.
13. A transgenic animal, a nucleated cell of which comprises:
an expression control sequence operably linked to a nucleic acid sequence at least 70% identical to SEQ ID NO: 1, a nucleic acid sequence at least 85%
identical to SEQ ID NO: 3, or a nucleic acid sequence at least 85% identical to SEQ ID
NO:

11;
wherein the nucleic acid sequence at least 70% identical to SEQ ID NO: 1, the nucleic acid sequence at least 85% identical to SEQ ID NO: 3, or the nucleic acid sequence at least 85% identical to SEQ ID NO: 11 encodes an antigenic epitope.
14. The transgenic animal according to claim 13, wherein the animal exhibits an increased resistance to infection by infectious salmon anemia virus as compared to a non-transformed animal of the same species.
15. The transgenic animal according to claim 13, wherein the animal is an aquaculture animal.
16. The transgenic animal according to claim 15, wherein the animal is a fish.

17. The transgenic animal according to claim 16, wherein the fish is rainbow trout, coho salmon, Chinook salmon, amago salmon, chum salmon, sockeye salmon, Atlantic salmon, arctic char, brown trout, cutthroat trout, brook trout, catfish, tilapia, sea bream, seabass, flounder, or sturgeon.
18. A method of eliciting an immune response against infections salmon anemia virus in a fish, comprising introducing into the fish a therapeutically effective amount of the nucleic acid molecule according to claim 6, wherein the nucleic acid molecule encodes an antigenic epitope of infectious salmon anemia virus, thereby eliciting an immune response against infectious salmon anemia virus in the fish.
19. The method according to claim 18, wherein the nucleic acid molecule has a nucleic acid sequence at least 80% identical to SEQ ID NO: 1.
20. The method according to claim 19, wherein the nucleic acid molecule has a nucleic acid sequence at least 85% identical to SEQ ID NO: 1.
21. The method according to claim 20, wherein the nucleic acid molecule has a nucleic acid sequence at least 90% identical to SEQ ID NO: 1.
22. The method according to claim 21, wherein the nucleic acid molecule has a nucleic acid sequence at least 95% identical to SEQ ID NO: 1.
23. The method according to claim 22, wherein the nucleic acid molecule has a nucleic acid sequence consisting essentially of SEQ ID NO: 1.
24. The method according to claim 18, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 11.

25. The method according to claim 24, wherein the nucleic acid sequence is at least 95% identical to SEQ ID NO: 3 or SEQ ID NO: 11.
26. The method according to claim 25, wherein the nucleic acid sequence is at least 95% identical to SEQ ID NO: 3 or SEQ ID NO: 11.
27. The method according to claim 26, wherein the nucleic acid sequence consists essentially of SEQ ID NO: 3 or SEQ ID NO: 11.
28. A method of producing a transgenic fish, comprising contacting a nucleated cell of the fish with an amount of the nucleic acid molecule according to claim 6, wherein the amount of the nucleic acid molecule is sufficient to introduce the nucleic acid molecule into the cell, thereby producing a transgenic fish.
29. The method according to claim 28, wherein the fish is rainbow trout, coho salmon, Chinook salmon, amago salmon, chum salmon, sockeye salmon, Atlantic salmon, arctic char, brown trout, cutthroat trout, brook trout, catfish, tilapia, sea bream, seabass, flounder, or sturgeon.
30. A polypeptide encoded by the nucleic acid molecule according to claim l, or a conservative variant thereof.
31. A method of inducing an immune response in a fish, comprising:
delivering to the fish a therapeutically effective amount of a composition comprising a polypeptide having an amino acid sequence as set forth as SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 18, an antigenic fragment thereof, or a conservative variant thereof; and wherein the polypeptide is an antigenic epitope of infectious salmon anemia virus, thereby eliciting an immune response against infectious salmon anemia virus in the fish.

32. The method according to claim 31 wherein the polypeptide has an amino acid sequence consisting essentially of SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 18 33. The method according to claim 31 wherein the polypeptide comprises a fusion protein.