WO2002026784A2

WO2002026784A2 - Matrix proteins m1 and m2 of infections salmon anaemia virus

Info

Publication number: WO2002026784A2
Application number: PCT/EP2001/011129
Authority: WO
Inventors: Eirik Biering; Bjørn KROSSØY
Original assignee: Akzo Nobel N.V.
Priority date: 2000-09-28
Filing date: 2001-09-25
Publication date: 2002-04-04
Also published as: AU2001295584A1; WO2002026784A3

Abstract

The present invention relates to a nucleic acid encoding proteins M1 and M2 of Infections Salmon Anaemia Virus (ISAV), to proteins encoded by these sequences, to vaccines comprising these proteins, to antibodies reactive with these proteins, to the use of said nucleic acids, proteins or antibodies for diagnostic or vaccination purposes and to diagnostic kits comprising these nucleic acids, proteins or antibodies.

Description

DNA encoding proteins of Infectious Salmon Anaemia Virus and uses thereof.

The present invention relates to a nucleic acid encoding a viral structural protein, to proteins encoded by these sequences, to vaccines comprising these proteins, to antibodies reactive with these proteins, to the use of said nucleic acids, proteins or antibodies for diagnostic or vaccination purposes and to diagnostic kits comprising these nucleic acids, proteins or antibodies.

Infectious Salmon Anaemia (ISA) is a disease caused by a virus (ISAV) that belongs to the family Orthomyxoviridae. The disease is characterised by severe anaemia, leucopenia, ascites, haemorrhagic liver necrosis and petecchia of the vicera. The gills are pale, and petecchia of the skin is also common. The spleen is dark and swollen (Speilberg et al, 1995; Veterinary Pathology, 32, pp. 466-478). The virus replicates in endothelial cells, both in blood vessels and in the heart, and in polymorphonuclear leukocytes. Budding of the virus from pillar cells in the gills has been observed, indicating that gills are probably an important portal of entrance for ISAV.

ISA was observed for the first time in Norway (Thorud et al., 1988; Bull. Eur. Ass. Fish Pathol., 8 (5), pp. 109-111) and severe outbreaks have recently been diagnosed also in Scotland, the Shetland Islands and Canada. Mortality during outbreaks varies between 10 and 100% and younger individuals appear to be more susceptible than older. However, high mortality has also been observed among market size fish. Clinical outbreaks have been observed so far in Atlantic salmon, but rainbow trout and brown trout may act as carriers of the agent without developing clinical signs. Despite stamping out strategies, new outbreaks occur regularly and result in significant losses.

Control of the disease therefore has a high priority, and the present invention provides novel means to carry out such control. The present invention provides for a nucleic acid sequence encoding protein M1 and M2 and fragments of said protein. The cloned nucleotide sequence comprises 1006 nucleotides and contains two overlapping open reading frames. The first open reading frame from nucleotide 1 to nucleotide 903 codes for a protein of 300 amino acid residues (M1); the second from nucleotide 1 to 1006 codes for a protein of 159 amino acid residues (M2), due to the presence of an intron extending from position 67 to position 591. The cloning and characterisation of the nucleotide sequence according to the invention provides for the production of a protein of ISAV according to the invention using recombinant technology (Sambrook et al., Molecular cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989). Cloning techniques and subsequent protein expression using in vitro expression systems are well known in the art. In this way, recombinant protein can be obtained, that is substantially free from other ISAV proteins. The proteins M1 and M2 were found to be specific for ISA virus, which makes these proteins very suitable for use in vaccination and diagnostics. The isolated M1 and/or M2 protein can be used in the manufacture of a vaccine to protect fish against infectious salmon anaemia caused by infection with ISAV. Said vaccines may be used as marker vaccine to distinguish vaccination from field infections with ISAV. Alternatively the nucleotide sequences encoding the proteins according to the invention can be used to manufacture DNA vaccines or vector vaccines to protect fish against infectious salmon anaemia caused by infection with ISAV. The nucleotide sequence and recombinant protein of the present invention can furthermore be used for diagnostic purposes, for instance to detect the presence of the ISAV or anti-ISAV antibodies in fish. Additionally, a recombinant protein of the present invention can be used to produce ISAV specific antibodies. These antibodies can also be used for diagnostic purposes such as the detection of ISAV in fish.

Thus, in a first aspect the present invention provides for a nucleic acid sequence that has a homology of at least 70% with the nucleotide sequence as depicted in SEQ ID NO 1 , or a primer fragment of said nucleic acid sequence. A primer fragment is defined below. A homology of 80%, 85%, 90%, 95%, 98% and 100% is more preferred in that order.

Preferred nucleic acid sequences or primer fragments according to the invention comprise a stretch of at least 12 nucleotides selected from the nucleotides 1-1006 of the nucleotide sequence depicted in SEQ ID NO 1. More preferably, a nucleic acid sequence or primer fragment according to the invention consists of nucleotides 1-1006 of SEQ ID NO 1. Nucleic acid sequences that comprise tandem arrays of the sequences according to the invention or fragments of said sequences are also within the scope of this invention. Nucleic acid sequences that are complementary to the nucleotide sequence depicted in SEQ ID NO 1 are within the scope of the invention, as well as nucleic acid sequences that hybridise with the nucleotide sequence depicted in SEQ ID NO 1. The hybridisation conditions for this purpose are stringent, preferably highly stringent. According to the present invention the term "stringent" means washing conditions of 1 x SSC, 0.1% SDS at a temperature of 65°C; highly stringent conditions refer to a reduction in SSC concentration towards 0.3 x SSC.

Nucleic acid sequences that hybridise with the nucleotide sequence shown in SEQ ID NO 1 are understood to be nucleic acid sequences that have a sequence homology of at least 70%, preferably 80%, 85%, 90%, 95%, 98% or even 100% in that order of preference, with the corresponding matching part of the nucleotide sequence depicted in SEQ ID NO 1. According to the present invention the sequence homology is determined by comparing the nucleotide sequence with the corresponding part of the nucleotide sequence depicted in SEQ ID NO 1. The level of nucleotide homology can be determined with the computer program "BLAST 2 SEQUENCES" by selecting sub-program: "BLASTN" that can be found at www.ncbi.nlm.nih.qov/blast/bl2seq/bl2.html.

A reference for this program is Tatiana A. Tatusova, Thomas L. Madden FEMS Microbiol. Letters 174: 247-250 (1999). Parameters used are the default parameters: Reward for a match: +1. Penalty for a mismatch: -2. Open gap: 5. Extension gap: 2. Gap x_dropoff: 50.

Homologous sequences can easily be isolated with the nucleotide sequence depicted in SEQ ID NO 1 or fragments of this sequence from closely related ISAV strains using routine cloning and hybridisation techniques (Sambrook et al., supra).

The nucleotide sequences of the invention can also be used in the preparation of a DNA vaccine to vaccinate fish against Infectious Salmon Anaemia caused by infection with ISAV. DNA vaccination refers to the induction of an immune response to one or more antigens that are expressed in vivo from a gene inserted in a DNA plasmid that has been inoculated directly into the vaccinated fish.

Thus in a second aspect of the invention there is provided for a DNA vaccine comprising a pharmaceutical acceptable carrier and a DNA plasmid in which one or more nucleic acid sequences according to the invention are operably linked to a transcriptional regulatory sequence.

Preferably the nucleic acid sequence to be used in said DNA plasmid is a nucleic acid sequence comprising nucleotide sequence depicted in SEQ ID NO 1 such as the sequence stretching nucleotides 1-900 of SEQ ID NO 1 or fragments of said nucleotide sequence encoding an immunogenic fragment of a protein according to the invention. An immunogenic fragment will be defined below. Also suitable for use in said DNA plasmid are nucleic acid sequences that are complementary to the nucleotide sequence of SEQ ID NO 1 or the sequence stretching nucleotides 1-900 of SEQ ID NO 1 , as well as nucleic acid sequences that hybridise with the sequence of SEQ ID NO 1 or the sequence depicted by nucleotides 1-900 of SEQ ID NO 1. The sequence homology between the nucleic acid sequences that hybridise with the nucleotide sequence of SEQ ID NO 1 or particular parts thereof is determined as described earlier.

Alternatively, the nucleic acid sequence to be used in said DNA plasmid is a nucleic acid sequence comprising the nucleotide sequence depicted in SEQ ID NO 1 such as the sequence stretching nucleotides 1-477 of SEQ ID NO 3 or fragments of said nucleotide sequence encoding an immunogenic fragment of a protein according to the invention. An immunogenic fragment will be defined below. Also suitable for use in said DNA plasmid are nucleic acid sequences that are complementary to the nucleotide sequence of SEQ ID NO 3 or the sequence stretching nucleotides 1-477 of SEQ ID NO 3, as well as nucleic acid sequences that hybridise with the sequence of SEQ ID NO 3 or the sequence depicted by nucleotides 1-477 of SEQ ID NO 3. The sequence homology between the nucleic acid sequences that hybridise with the nucleotide sequence of SEQ ID NO 3 or particular parts thereof is determined as described earlier. DNA plasmids that are suitable for use in a DNA vaccine according to the invention are conventional cloning or expression plasmids for bacterial, eukaryotic and yeast host cells, many of said plasmids being commercially available. Well-known examples of such plasmids are pBR322 and pcDNA3 (Invitrogen). The DNA plasmids according to the invention should be able to induce protein expression of the nucleotide sequences. The DNA plasmid can comprise one or more nucleotide sequences according to the invention. In addition, the DNA plasmid can comprise other nucleotide sequences such as the immune-stimulating oligonucleotides having unmethylated CpG dinucleotides, or nucleotide sequences that code for other antigenic proteins or adjuvating cytokines.

Transcriptional regulatory sequences that are suitable for use in a DNA plasmid according to the invention comprise promoters such as the (human) cytomegalovirus immediate early promoter (Seed, B. et al., Nature 329, 840-842, 1987; Fynan, E.F. et al., PNAS 90, 11478-11482,1993; Ulmer, J.B. et al., Science 259, 1745-1748, 1993), Rous sarcoma virus LTR (RSV, Gorman, CM. et al., PNAS 79, 6777-6781 , 1982; Fynan et al., supra; Ulmer et al., supra), the MPSV LTR (Stacey et al., J. Virology 50, 725-732, 1984), SV40 immediate early promoter (Sprague J. et al., J. Virology 45, 773 ,1983), the metallothionein promoter (Brinster, R.L. et al., Nature 296, 39-42, 1982), the major late promoter of Ad2, the β-actin promoter (Tang et al., Nature 356, 152-154, 1992).The regulatory sequences may also include terminator and polyadenylation sequences. Amongst the sequences that can be used are the well known bovine growth hormone polyadenylation sequence, the SV40 polyadenylation sequence, the human cytomegalovirus (hCMV) terminator and polyadenylation sequences.

The DNA plasmid comprising a nucleotide sequence according to the present invention operably linked to a transcriptional regulatory sequence for use in the vaccine according to the invention can be naked or can be packaged in a delivery system. Suitable delivery systems are lipid vesicles, Iscoms, dendromers, niosomes, polysaccharide matrices, and the like. Also very suitable as delivery system are attenuated live bacteria such as Salmonella.

The nucleotide sequences according to the invention can additionally be used in the production of a vector vaccine to vaccinate fish against ISA virus infection. A vector vaccine is understood to be a vaccine in which a live, attenuated bacteria or virus has been modified so that it contains one or more heterologous nucleotide sequences inserted into its genetic material. These so called vector bacteria or viruses are capable of coexpressing the heterologous proteins encoded by the inserted nucleotides.

As an example of bacterial LRCs, bacteria such as Vibrio anguillarum known in the art can attractively be used. (Singer, J.T. et al., New Developments in Marine Biotechnology, p. 303-306, Eds. Le Gal and Halvorson, Plenum Press, New York, 1998).

Also, LRC viruses may be used as a way of transporting the nucleic acid sequence into a target cell. Viruses suitable for this task are e.g. alphavirus-vectors. A review on alphavirus-vectors is given by Sondra Schlesinger and Thomas W. Dubensky Jr. (1999) Alphavirus vectors for gene expression and vaccines. Current opinion in Biotechnology, 10:434-439.

Thus in a third aspect the invention provides for a vaccine comprising a live attenuated bacterial or viral vector which comprises in its genetic material one or more of the nucleic acid sequences of the present invention.

Preferably the nucleotide sequence to be used in said vector vaccine is a nucleic acid sequence comprising the nucleotide sequence depicted in SEQ ID NO 1 or SEQ ID NO 3, or fragments of said nucleotide sequences encoding an immunogenic fragment of a protein according to the invention such as the sequence stretching nucleotides 1-900 of SEQ ID NO 1 or nucleotides 1-477 of SEQ ID NO 3. Also suitable for use in said vector vaccine are nucleic acid sequences that are complementary to the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 3 or the sequence depicted by nucleotides 1- 900 of SEQ ID NO 1 or 1-477 of SEQ ID NO 3, as well as nucleic acid sequences that hybridise with the nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 3 or the sequence depicted by nucleotides 1-900 SEQ ID NO 1 or 1-477 of SEQ ID NO 3. The sequence homology between the nucleic acid sequences that hybridise with the sequence of SEQ ID NO 1 or SEQ ID NO 3 or particular parts thereof is determined as described earlier.

In a fourth aspect, the nucleotide sequences according to the invention can be used for the recombinant production of protein M1 or M2, substantially free from other ISAV proteins. Thus, the invention provides for a protein or an immunogenic fragment thereof encoded by a nucleic acid sequence according to the present invention. More specifically the invention provides for a protein having a homology level of at least 70% with the amino acid sequence as depicted in SEQ ID NO 2 or 4 or a derivative of said amino acid sequence, or an immunogenic fragment of said protein. A homology of 80%, 85%, 90%, 95%, 98% and 100% is more preferred in that order.

Proteins comprising an amino acid sequence that is a derivative of the sequence depicted in SEQ ID NO 2 or 4 are understood to be proteins which have alterations in their amino acid sequence with respect to the amino acid sequence depicted in SEQ ID NO 2 or 4, respectively, which do not affect the antigenic or immunogenic characteristics of said protein.

The level of protein homology can be determined with the computer program "BLAST 2 SEQUENCES" by selecting sub-program: "BLASTP", that can be found at www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html. A reference for this program is Tatiana A. Tatusova, Thomas L. Madden FEMS

Microbiol. Letters 174: 247-250 (1999). Matrix used: "blosum62". Parameters used are the default parameters:

Open gap: 11. Extension gap: 1. Gap x_dropoff: 50.

For the purpose of this invention, antigenic characteristics of a protein according to the invention are understood to be the ability to induce production of antibodies that recognise and (cross)-react with the ISA virus. Immunogenic characteristics of a protein according to the invention are understood to be the ability to induce an immune response in fish that protects against infectious salmon anaemia caused by infection with ISA virus.

The alterations that can occur in a sequence according to the present invention could for instance result from conservative amino acid substitutions, deletions, insertions, inversions or additions of (an) amino acid(s) in the overall sequence. Amino acid substitutions that are expected not to alter the immunological properties have been described. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, lle/Val (see Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.0, 1978, vol. 5, suppl. 3). Based on this information Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science, 1985, vol. 227, 1435-1441) and determining the functional similarity between proteins and peptides having sequence homology. The derivative proteins according to the invention are still capable to function as the native protein M1 or M2, respectively, and/or induce production of antibodies that recognise and/or (cross)-react with the ISA virus. Other derivatives according to the present invention are protein fragments that are still capable of inducing production of antibodies that recognise and/or (cross )-react with ISA virus or of inducing an immune response in fish that protects against infectious salmon anaemia caused by infection with ISA virus.

When a protein is used for e.g. vaccination purposes or for raising antibodies, it is not necessary to use the whole protein. It is also possible to use a fragment of that protein that is capable, as such or coupled to a carrier such as e.g. KLH, of inducing an immune response against that protein, a so-called immunogenic fragment. An "immunogenic fragment" is understood to be a fragment of the full-length protein that still has retained its capability to induce an immune response in a vertebrate host, e.g. comprises a B- or T-cell epitope. Antibodies raised in a vertebrate host are very suitable as passive means of vaccination in fish. At this moment, a variety of techniques is available to easily identify DNA fragments encoding antigenic fragments (determinants). The method described by Geysen et al (Patent Application WO 84/03564, Patent Application WO 86/06487, US Patent NR. 4,833,092, Proc. Natl Acad. Sci. 81: 3998- 4002 (1984), J. Imm. Meth. 102, 259-274 (1987), the so-called PEPSCAN method is an easy to perform, quick and well-established method for the detection of epitopes; the immunologicaliy important regions of the protein. The method is used world-wide and as such well-known to man skilled in the art. This (empirical) method is especially suitable for the detection of B-cell epitopes. Also, given the sequence of the gene encoding any protein, computer algorithms are able to designate specific protein fragments as the immunologicaliy important epitopes on the basis of their sequential and/or structural agreement with epitopes that are now known. The determination of these regions is based on a combination of the hydrophilicity criteria according to Hopp and Woods (Proc. Natl. Acad. Sci. 78: 38248-3828 (1981)), and the secondary structure aspects according to Chou and Fasman (Advances in Enzymology 47: 45-148 (1987) and US Patent 4,554,101). T-cell epitopes can likewise be predicted from the sequence by computer with the aid of Berzofsky's amphiphilicity criterion (Science 235, 1059-1062 (1987) and US Patent application NTIS US 07/005,885). A condensed overview is found in: Shan Lu on common principles: Tibtech 9: 238-242 (1991), Good et al on Malaria epitopes; Science 235: 1059-1062 (1987), Lu for a review; Vaccine 10: 3-7 (1992), Berzowsky for HIV-epitopes; The FASEB Journal 5:2412-2418 (1991).

The proteins or immunogenic fragments thereof according to the invention can be prepared via standard recombinant protein expression techniques. For this purpose a nucleic acid sequence according to the invention encoding protein M1 or M2, a part thereof encoding an immunogenic fragment of a protein according to the invention, a derivative of said proteins or a multimere of said proteins is inserted into an expression vector. Preferably the nucleic acid sequence is the nucleotide sequence depicted in SEQ ID NO 1 or SEQ ID NO 3 or one or more parts thereof encoding immunogenic fragments, such as nucleotides 1-900 of SEQ ID NO 1 or nucleotides 1-477 of SEQ ID NO 3. Also suitable are nucleic acid sequences that are complementary to the (particular part of the) sequence of SEQ ID NO 1 or SEQ ID NO 3 or nucleic acid sequences of which the sequence homology with the (particular part of the) sequence depicted in SEQ ID NO 1 or SEQ ID NO 3 is at least 70%, preferably 80%, 85%, 90%, 95%, 98% and 100% in that order of preference.

Suitable expression vectors are, amongst others, plasmids, cosmids, viruses and YAC's (Yeast Artificial Chromosomes) which comprise the necessary control regions for replication and expression. The expression vector can be brought to expression in a host cell. Suitable host cells include but are not limited to bacteria, yeast cells, insect cells and mammalian cells. Such expression techniques are well known in the art (Sambrooke et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989). Following expression, the expressed proteins can be isolated and purified from the medium.

In a further aspect the invention provides for a vaccine comprising at least one protein according to the present invention or an immunogenic fragment thereof and a pharmaceutical acceptable carrier. More specifically, a vaccine according to the invention comprises at least a protein having an amino acid sequence depicted in SEQ ID NO 2 or 4 or a derivative or an immunogenic fragment of one of said amino acid sequences.

Vaccines based upon a protein according to the invention are normally used for active protection, i.e. the administered protein induces an immune response by the host, e.g. the production of antibodies against the protein. Alternatively, one can produce antibodies against the proteins according to the invention in e.g. rabbits, collect these antibodies and use them in vaccine preparations. Such so-called passive vaccines have the advantage that the protective element, i.e. the antibody is already present in the vaccine, so immediate protection against ISAV is provided. Therefore, another embodiment of the invention relates to vaccine comprising antibodies against a protein according to the invention or immunogenic fragments thereof, and a pharmaceutically acceptable carrier.

Vaccines according to the invention can be prepared according to techniques well known to the skilled practitioner. Vaccines according to the invention comprise an effective amount of an immunogen or antibodies according to the invention and a pharmaceutical acceptable carrier. The term "effective " as used herein is defined as the amount sufficient to induce an immune response in the target fish. An immunogen according to the invention is either a DNA plasmid in which one or more nucleotide sequences according to the invention are operably linked to a transcriptional regulatory sequence, or a vaccine vector comprising one or more nucleic acids according to the invention, one or more proteins according to the invention, or antibodies against a protein according to the invention. The amount of plasmid, vector or protein will depend on the type of plasmid or vector, the route of administration, the time of administration, the species of the fish as well as age, general health and diet.

In general, a dosage of 0.01 to 1000 μg protein per kg body weight, preferably 0.5 to 500, more preferably 0.1 to 100 μg protein can be used. With respect to the DNA vaccines, generally a minimum dosage of 10 pg. up to dosages of 1000 μg have been described to be sufficient for a suitable expression of the antigens in vivo. Pharmaceutical acceptable carriers that are suitable for use in a vaccine according to the invention are sterile water, saline, aqueous buffers such as PBS and the like. In addition a vaccine according to the invention may comprise other additives such as adjuvants, stabilisers, anti-oxidants and others. Suitable adjuvants include, amongst others, aluminium hydroxide, aluminium phosphate, amphigen, tocophenols, monophosphenyl lipid A, muramyl dipeptide, oil emulsions, glucans, cytokines and saponins such as Quill A. The amount of adjuvant added depends on the nature of the adjuvant itself.

Suitable stabilisers for use in a vaccine according to the invention are for example carbohydrates including sorbitol, mannitol, starch, sucrose, dextrin, and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.

The vaccines according to the invention are administered to the fish via injection, spray, immersion or per oral. The administration protocol can be optimised in accordance with standard vaccination practice.

The nucleic acid sequences, the proteins and the antibodies according to the invention are also suitable for use in diagnostics.

Therefore, another embodiment of the invention relates to nucleic acid sequences, proteins and antibodies according to the invention for use in diagnostics.

The nucleic acid sequences or fragments thereof can be used to detect the presence of ISAV in fish. A sample of fish infected with ISAV will comprise nucleic acid material derived from said virus, including nucleic acid sequences encoding for the protein according to the invention. These protein-encoding nucleic acid sequences will hybridise with a nucleic acid sequence according to the invention. Suitable methods for the detection of nucleic acid sequences that are reactive with the nucleic acid sequences of the present invention include hybridisation techniques including but not limited to PCR techniques and NASBA techniques. Thus the nucleic acid sequences according to the invention, in particular the sequences depicted by nucleotides 1-900 of SEQ ID NO 1 or 1-477 of SEQ ID NO 3 can be used to prepare probes and primers for use in PCR and or NASBA techniques. A diagnostic test for the detection of ISAV is e.g. based upon the reaction of viral nucleic acid isolated from the fish to be tested, with specific probes or (PCR-) primers, also referred to as primer fragments, based upon the nucleic acid sequences according to the invention. If genetic material of ISAV is present in the animal, this will e.g. specifically bind to specific PCR-primers and, e.g. after cDNA synthesis, will subsequently become amplified in PCR-reaction. The PCR-reaction product can then easily be detected in DNA gel electrophoresis. The genetic material to be tested can most easily be isolated from the endothelial cells of leukocytes of the fish to be tested. Standard PCR-textbooks give methods for determining the length of the primers for selective PCR-reactions with ISAV DNA. Primer fragments with a nucleotide sequence of at least 12 nucleotides are frequently used, but primers of more than 15, more preferably 18 nucleotides are somewhat more selective. Especially primers with a length of at least 20, preferably at least 30 nucleotides are very generally applicable. PCR-techniques are extensively described in

(Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN 0-87969-447-5

(1995)).

Nucleic acid sequences according to the invention or parts of those nucleic acid sequences having a length of at least 12, preferably 15, more preferably 18, even more preferably 20, 22, 25, 30, 35 or 40 nucleotides in that order of preference, wherein the nucleic acid sequences or parts hereof have at least 70 % homology with the nucleic acid sequence as depicted in SEQ ID NO: 1 or SEQ ID NO: 3 are therefore also part of the invention. Such nucleic acid sequences can be used as primer fragments in PCR- reactions in order to enhance the amount of DNA that they encode or in hybridisation reactions. This allows the quick amplification or detection on blots of specific nucleotide sequences for use as a diagnostic tool for e.g. the detection of ISAV as indicated above.

Another test on genetic material is based upon growth of viral material obtained from the swab, followed by classical RNA purification followed (after optional cDNA synthesis) by classical hybridisation with radioactively or colour-labelled primer fragments. Colour- labelled and radioactively labelled fragments are generally called detection means. Both PCR-reactions and hybridisation reactions are well-known in the art and are i.a. described in Maniatis/Sambrook (Sambrook, J. et al. Molecular cloning: a laboratory manual. ISBN 0-87969-309-6). Thus, one embodiment of the invention relates to a diagnostic test kit for the detection of ISAV nucleic acid sequences. Such a test comprises a nucleic acid sequence according to the invention or a primer fragment thereof.

The proteins according to the present invention can be used to detect the presence of anti-M1 or M2-protein antibodies in the fish. Since proteins M1 and M2 are characteristic for the ISA virus, the presence of antibodies against at least one of the proteins of ISAV according to the invention in fish is an indication that said fish is infected by ISAV. In general, these antibodies can be detected by an immunoassay comprising the steps of:

(i) incubating a sample suspected of containing antibodies against ISAV with a protein according to the invention,

(ii) allowing the formation of antibody-protein complex , and

(ii) detecting the presence of the antibody-protein complex. The design of this immunoassay may vary. For example, the immunoassay may be based upon competition or direct reaction. Furthermore, protocols may use solid supports or may use cellular material. The detection of the antibody-antigen complex may involve the use of labelled antibodies; the labels may be, for example, enzymes, fluorescent-, chemiluminescent-, radio-active- or dye molecules. Suitable methods for the detection of antibodies reactive with a protein according to the present invention in the sample include the enzyme-linked immunosorbent assay (ELISA), immunofluorescent test (IFT) and Western blot analysis.

The proteins according to the invention can additionally be used for the production of antibodies, using the general techniques available to the practitioner in the field. Antibodies that are produced with a protein according to the invention have the advantage of specifically reacting with the M1 or M2 protein of ISA virus. Preferably the proteins are used to produce specific monoclonal antibodies. The obtained antibodies may be utilised in diagnostics, to detect the presence of ISAV in the fish, and as mentioned above, for passive vaccination purposes. Thus another embodiment of the invention relates to antibodies reactive with a protein according to the invention or an immunogenic fragment thereof.

Still another aspect of the present invention provides for a diagnostic kit comprising a suitable means for detection and one or more nucleic acid sequences according to the invention, or one or more proteins according to the invention, or antibodies specifically reactive with said proteins, respectively.

Antibodies according to the invention can be prepared according to standard techniques. Procedures for immunising animals, e.g. mice with proteins and selection of hybridomas producing immunogen specific monoclonal antibodies are well known in the art (see for example Coligan et al. (eds), Current protocols in Immunology, 1992; Kohler and Milstein, Nature 256:495-497, 1975; Steenbakkers et al., Mol. Biol. Rep. J9:125- 134, 1994). The antibody is preferably a monoclonal antibody.

Methods for large-scale production of antibodies according to the invention are also known in the art. Such methods rely on the cloning of (fragments of) the genetic information encoding the protein according to the invention in a filamentous phage for phage display. Such techniques are described i.a. at the "Antibody Engineering Page" under "filamentous phage display" at http://aximt1.imt.uni- marburq.de/~rek/aepphaqe.html.. and in review papers by Cortese, R. et al., (1994) in Trends Biotechn. 12: 262-267., by Clackson, T. & Weils, J.A. (1994) in Trends Biotechn. 12: 173-183, by Marks, J.D. et al., (1992) in J. Biol. Chem. 267: 16007-16010, by Winter, G. et al., (1994) in Annu. Rev. Immunol. 12: 433-455, and by Little, M. et al., (1994) Biotechn. Adv. 12: 539-555. The phages are subsequently used to screen camelid expression libraries expressing camelid heavy chain antibodies. (Muyldermans, S. and Lauwereys, M., Journ. Molec. Recogn. 12: 131-140 (1999) and Ghahroudi, M.A. et al., FEBS Letters 414: 512-526 (1997)). Cells from the library that express the desired antibodies can be replicated and subsequently be used for large scale expression of antibodies.

EXAMPLES

Example 1:

Virus isolation and construction of a cDNA library Kidney samples were taken from ISAN-infected Atlantic salmon (Salmo salar). Virus was isolated and propagated in Atlantic salmon kidney (ASK) cells as described by Devoid et al. in Diseases of Aquatic Organisms 40: 9-18 (2000). R A was isolated from ISAN-infected ASK cells using Trizol reagent (Life Technologies). RΝA was isolated from these cells on days 2, 3 and 4 post-infection. RΝA was pooled and mRΝA was isolated using the Dynabeads mRΝA Purification kit (Dynal). A sample of 2 μg mRΝA was used for cDΝA synthesis with the cDΝA Synthesis Kit (Stratagene). A unidirectional bacteriophage Lambda cDΝA library from IS AN infected ASK-cells was then constructed using the Uni-ZAP XR vector and Gigapack III Gold packaging extract (Stratagene).

Example 2:

Cloning and characterisation of an ISAV specific cDΝA

Probe screening was used to eliminate known viral genes and sequencing of remaining clones identified a clone comprising an ISAV genetic segment with two open reading frames (ORFs) of 900 and 477 bases respectively. The clone was designated 9AI, and database searches revealed no significant homology to other sequences. The viral origin of the cDΝA sequence was demonstrated using PCR and hybridisation reactions. 9AI specific PCR primers (5'-tggtgtgctggttgaccaactaaa-3' and 5'-ccatctcattgtgctcagggccag-3') amplified a product from ISAV infected ASK cells and not from uninfected cells, and a DIG labelled 9AI specific DΝA probe prepared using the same primers hybridised to total RΝA from ISAN infected ASK cells and not to RΝA from uninfected cells. Also, 9AI derived primers amplified a specific product from tissue of ISAV infected salmon whereas uninfected individuals were negative. To obtain a full-length cDΝA sequence, 5' RACE was performed with the 5'RACE

System, Version 2.0 (Life Technologies). RACE products were cloned into the pCR 2.1- TOPO vector using the TOPO TA Cloning Kit (Invitrogen) and sequenced. The 9AI sequence contains two large open reading frames, and is thus expected to encode at least two different polypeptides. In order to confirm the theoretical MW of the protein encoded by the smaller ORF, one DIG-labelled DΝA probe complementary to both reading frames was prepared using primers 5'-tgtctggaagcctctactga-3' and 5'- gaatgatacgccgtctctgt-3'. PCR products were then generated from positive clones using T7 and T3 vector primers and sequencing revealed the presence of two partly overlapping mRNA species in infected cells. A sequence analysis of the two mRNAs showed that they had identical 5' and 3' ends (except for the 5' heterogeneous region of cellular origin), while a 526 nucleotide region was missing in the smaller (9AI-2) compared to the larger (9AI-1) mRNA. To demonstrate that this deletion was due to a splicing event, the 5' and 3' sequences in the splice junctions were compared with the consensus sequences deduced from a large number of cellular and viral mRNAs. The consensus sequences are found at the 5 ' and 3 ' splice sites at the junctions of exons and introns, and in introns 18 to 40 nucleotides upstream of the 3' splice site. A comparison of the ISAV sequence and the consensus sequence is shown in Fig. la. The ISAV sequence shows distinct similarities with the consensus sequence, and it is deduced that the donor site in the splicing reaction is the G at nucleotide 63 and that the acceptor site is the G at nucleotide 590. The predicted 9AI- 1 and 9AI-2 proteins share the same AUG codon for initiation of protein synthesis and 21 subsequent amino acids before the intron. The splicing event then removes a 526 nucleotide intron and the 9AI-2 mRNA continues in the +1 reading frame, encoding a predicted protein of 159 amino acids with a calculated molecular weight of 17.5 kilodalton (kDa). Figure lb shows the arrangement of the 9AI-1 and 9AI-2 mRNAs and their open reading frames (ORFs). 9AI encodes two proteins with predicted molecular weights of 34.2 and 17.5 kDa.

The largest 9AI ORF was amplified using primers 5'-ataagaatgcggccgccagccaatcacatt c- 3' and 5'-gcgatatcattcggcacgagtctacaa-3.' The product was then cloned into the Eco RV and Not I sites of the pET 30a vector (Novagen), generating a construct fused to a poly- histidine tag. The construct was transformed into TOP 10 cells (Invitrogen) and the isolated plasmid was used to transform E. coli BL21(DE3)pLys S (Invitrogen). Expression and isolation of protein inclusion bodies were performed according to the pET System Manual (Novagen) (Fig. 2). The identity of the protein was confirmed by sequencing and detection in Western blotting using an antiserum raised against a mix of the synthetic peptides YGDDEPDEGSCELAS, QRFYDRAQNRAGSRV and SRVWRRDHNERAGVE derived from the 9AI sequence (Fig. 2). A polyclonal antiserum prepared against purified ISAV virions reacted specifically with the 4 major ISAV polypeptides in purified virus preparation (Fig. 3). This antiserum did not react with the recombinant protein in immunoblotting, although some weak, unspecific bands were observed. (Fig. 3). As the recombinant protein has a molecular mass of approximately 45 kDa in SDS-PAGE/Western blotting, due to the tag attached to the protein, it co-localises with the protein band of purified virus known to be the haemagglutinin.

Example 3

Use of ISAV segment for RT-PCR diagnostic

A primer set (S7F1 and S7R1) targeted against the ISA virus genome segment according to the invention was constructed of which the sequences are given in Table 1.

Table . Sequence of the primers tested for use in RT-PCR amplification of the segment.

The RNA was extracted from Kidney tissues with Trizol (Life Technologies). About 1.5 μg total RNA + 1.5 μl (1.5 μg) random hexamers (pd(N)₆) in a total volume of 10 μl were incubated at 70 °C for 5 minutes and then cooled to 4°C. RT-mix containing 5.0 μl 5 x RT buffer + 1.2 μl 200 mM DL-dithiothreitol (DTT) + 2.5 μl 10 mM dNTP + 0.5 μl RNasin (20 units/μl) + 4.3 μl dH2O + 1.5 μl (20 units/μl) Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), were added at room temperature making a total of 25 μl. This solution was incubated at 37 °C for 60 minutes. The PCR consisted of 2 μl of cDNA-solution that was added to 23 μl of reaction mixture consisting of 14.2 μl ddIΪ2θ, 2.5 μl (xlO) running buffer, 2.0 μl (25 mM MgCl), 2 μl dNTP (10 mM), 1.0 μl of each primer (20 μM), 0.3 μl (5 units/μl) Taq DNA polymerase (Pharmacia Biotech). The mixture was denatured at 94 °C for 3 minutes and amplification was performed with 35 cycles of 94°C for 30 seconds, 65°C for 45 seconds, and 72 °C for 90 seconds, followed by extension at 72°C for 10 minutes. The tubes were then held at 4 °C. Amplification and reverse transcription were performed in a thermal cycler with heated lid (TECHNE, Progene).

Aliquots (lOμl) of the PCR reaction mixture were electrophoresed in 1% agarose 1 x TBE gel containing ethidium bromide stain (Gibco-BRL) and photographed under UV transillumination. DNA molecular weight marker type 250 bp DNA ladder was applied to identify the size of the PCR products. In addition to RNA from fish that were not infected with ISA virus, negative controls containing H2O instead of RNA or cDNA were used to test each reaction mixture.

Specificity.

The products from the RT-PCR gave the predicted size of 293 base pairs. The specificity of the primer set was determined by sequencing the PCR products using the BigDye

Sequencing Kit and an ABI Prism 6700 sequencing machine (PE Biosystems).

In the specificity tests of the ISA virus RT-PCR assay, using the sense and antisense primers, non-infected rainbow trout gave, as expected, no PCR amplification products.

Sequencing of the PCR products from ISA-positive rainbow trout gave sequences that were, again as expected, similar to the target ISA virus sequence.

Example 4

Diagnostic RT-PCR on rainbow trout samples

The RT-PCR assay S7F1/S7R was tested on rainbow trout challenged with ISA virus and non-infected rainbow trout. Kidney tissues were collected from the rainbow trout during a

242 days period, frozen in nitrogen and stored at -80 °C for later use. Materials from all sampling dates were screened by RT-PCR

Table 2 gives the results of the RT-PCR on kidney tissues from challenged and control rainbow trout. In this challenge there were tested 258 samples with the RT-PCR assay, 53 of the samples tested positive for ISA virus. A few control trout were found to be positive for the ISA virus in the beginning and at the end of the experimental period (Table 2). An explanation for the positive samples in the control group could be contamination with ISAV, from the tanks with the challenged groups.

Table 2. The results of the RT-PCR screening of the control and ISAV challenged rainbow trout using the S7F1/S7R1 primer set. The columns show the fraction of ISAV positive fish in the RT-PCR screening. OM = rainbow trout groups challenged by ISA virus. KT = groups of control rainbow trout. % = the total percentage of ISAV challenged fish found positive.

Diagnostic RT-PCR on Atlantic salmon samples

This RT-PCR assay was also tested for detecting ISA virus on Atlantic salmon (Salmo salar) from a fish farm on the Norwegian west cost, with known history of ISAV infection. The samples from this farm had been frozen in liquid nitrogen and were tested with the RT-PCR assay (S7F1/S7R1) described above. A total of 120 fish was tested and 3 ISAV positive samples were found with this assay. The low prevalence of ISAV infected salmon was expected; because there were no clinical signs or higher motility rate in the net pens from which we collected the samples. This shows that this assay can detect ISA virus in fish farms with a low prevalence of fish infected with ISAV. LEGENDS TO THE FIGURES Fig. 1

The ISAV segment encodes two mRNAs due to splicing, (a) Comparison of the ISAV sequences in the splice junctions with the conserved sequences found in cellular and viral pre-mRNAs. The 5' and 3' splice sites are indicated with arrows. Nucleotide numbers in the ISAV sequence is relative to the initiation codon. (b) The largest ORF (9AI-1) is co- linear and predicted to encode 300 amino acids (aa) (black area). The spliced transcript (9AI-2), which shares the first 22 aa with 9AI-1 (black area), has an 526 nucleotide intron (V-shaped line) removed by splicing. The mRNA then continues in the +1 reading frame (gray area) encoding a predicted 159 aa protein. Thin lanes before and after the colored areas represent untranslated regions. Scale bar represents total coding region in the co- linear transcript.

Fig. 2

Expression of 9AI fusion protein in E. coli: Molecular weight markers (lane a),

Coomassie brilliant blue stained 9AI fusion protein (lane b), Western blot of 9AI fusion protein detected with polyclonal antiserum raised against a mixture of the peptides

YGDDEPDEGSCELAS, QRFYDRAQNRAGSRV and SRVWRRDHNERAGVE (lane c).

Fig. 3

Immunoblots of ISAV purified by gradient centrifugation (lane a) and recombinant 9AI fusion protein (lane b). The polyclonal antiserum used for detection was prepared against purified ISAV. The polyclonal antiserum used for detection was prepared against purified ISAV and reacts specifically with the 4 major structural ISAV proteins (lane a), but not against the recombinant protein (lane b).

Claims

Claims:

1. Nucleic acid sequence or a primer fragment thereof, characterised in that said nucleic acid sequence or primer fragment has a homology of at least 70% with the nucleotide sequence as depicted in SEQ ID NO 1 or SEQ ID NO 3.

2. Nucleic acid sequence or a primer fragment thereof according to claim 1 , characterised in that said nucleic acid sequence or primer fragment comprises a stretch of at least 12 nucleotides of the nucleotide sequence depicted in SEQ ID NO 1 or SEQ ID NO 3.

3. Protein encoded by a nucleic acid sequence according to claim 1 or 2, or an immunogenic fragment of said protein.

4. Protein according to claim 3 or an immunogenic fragment of said protein characterised in that said protein or immunogenic fragment thereof comprises an amino acid sequence having a homology of at least 70 % with the amino acid sequence as depicted in SEQ ID NO 2 or 4 or a derivative of said amino acid sequence or immunogenic fragment thereof.

5. Protein according to claim 3 or 4 or an immunogenic fragment of said protein, characterised in that said protein or an immunogenic fragment thereof has an amino acid sequence depicted in SEQ ID NO 2 or 4.

6. Vaccine comprising a live attenuated bacterial or viral vector which comprises in its genetic material one or more of the nucleic acid sequences according to claim 1 or 2.

7. Vaccine comprising a pharmaceutically acceptable carrier and a DNA plasmid, said DNA plasmid comprising the nucleotide sequence stretching nucleotides 1-900 of SEQ ID NO 1 or fragments of said nucleotide sequence encoding an immunogenic fragment or the nucleotide sequence stretching nucleotides 1-477 of SEQ ID NO 3, or fragments of said nucleotide sequence encoding an immunogenic fragment, operably linked to a transcriptional regulatory sequence.

8. Vaccine comprising at least a protein or immunogenic fragment thereof according to claims 3-5 or antibodies against said protein or immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

9. Antibodies that are specifically reactive with a protein or immunogenic fragment thereof according to claims 3-5.

10. A nucleic acid according to claim 1 or 2 or a protein according to claims 3-5 or an antibody that is reactive with a protein according to claims 3-5 for use in diagnostics.

11. A diagnostic kit comprising suitable detection means and a nucleic acid sequence or primer fragment according to claims 1 or 2, or a protein or immunogenic fragment according to claims 3-5, or antibodies that are reactive with a protein according to claims 3-5.