WO2018224516A1

WO2018224516A1 - Detection and treatment of pmcv infection in cleaner fish

Info

Publication number: WO2018224516A1
Application number: PCT/EP2018/064806
Authority: WO
Inventors: Hamish RODGER; Felix Scholz
Original assignee: Fish Vet Group Norge As
Priority date: 2017-06-06
Filing date: 2018-06-05
Publication date: 2018-12-13

Abstract

A method for determining the presence or absence of a piscine myocarditis virus infection, in a biological sample obtained from a cleaner fish using an isolated or synthetic nucleic acid encoding a piscine myocarditis virus polypeptide, an isolated polypeptide encoded by the nucleic acid or an isolated antibody that specifically binds the polypeptide, optionally for use in the treatment or prophylaxis against a piscine myocarditis virus in a cleaner fish, an immunogenic composition comprising a piscine myocarditis virus nucleic acid and/or a piscine myocarditis virus polypeptide, and a kit for screening for a piscine myocarditis virus infection in a cleaner fish. The cleaner fish may be from the family Cyclopteroidea or Labridae.

Description

_ _

Detection and Treatment of PMCV Infection in Cleaner Fish

The present invention relates to an isolated or synthetic nucleic acid from piscine myocarditis virus (PMCV) isolated from a cleaner fish, a complementary nucleic acid, an isolated polypeptide encoded by the nucleic acid, an isolated antibody that specifically binds to a polypeptide encoded by the nucleic acid or polypeptide, an immunogenic composition comprising a PMCV nucleic acid, related treatments, diagnostics, methods for testing an agent for the prevention and/or treatment of a piscine myocarditis virus infection, and kits for screening for a piscine myocarditis virus infection in a cleaner fish.

Cleaner fish species are used as a biological control method for the parasitic copepod

Lepeophtheirus salmonis (Kroyer) and are widely used as part of an integrated pest management control programme. Sea lice, L. salmonis and Caligus spp, are the most widespread pathogenic marine parasite in the Atlantic salmon {Salmo salar, L.) farming industry and those with the greatest economic impact. Sea lice treatments are a significant cost to the industry, may have an impact on the local environment and can negatively influence the public perception of aquaculture. Furthermore, tolerance has developed in some lice populations to specific medicines. The acquisition and stocking of cleaner fish such as wild-caught wrasse species has been reported to cost less than one medical treatment against sea lice. Lumpfish {Cyclopterus lumpus, L.) is an effective alternative to wrasse species, with the advantage of being resistant to and effective at low water temperatures. Wrasse species and lumpfish are used as a biological control for sea lice on, for example, Atlantic salmon farms.

Infectious diseases are currently one of the biggest challenges and have caused high levels of mortality. To ensure sustainability and biosecurity, the long-term aim is to provide the aquaculture industry with farmed, certified specific pathogen- free cleaner fish to take pressure off wild stocks and avoid potential disease transmission to fish.

Cardiomyopathy syndrome (CMS) is an infectious disease of fish and is caused by the piscine myocarditis virus (PMCV), a double stranded RNA virus of the family Totiviridae. The disease typically affects farmed Atlantic salmon {Salmo salar L.) in their second year of sea _ _ culture, but recently cases in younger fish have been reported. Mortality is usually chronic and the economic impact high. CMS was first described in farmed Atlantic salmon in Norway, where it has been one of the significant diseases in Atlantic salmon aquaculture for the last decade. It has since been described in Scotland, the Faroe Islands and Ireland. PMCV and CMS -like pathology have also been described in wild Atlantic salmon in Norway.

Symptoms associated with CMS in Atlantic salmon include exophthalmia, skin oedema, an enlarged and swollen or ruptured atrium with subsequent haemopericardium, blood tinged ascites, petechial haemorrhaging and diphtheritic fibrinous membranes on the liver.

Histopathology of CMS is characterised by inflammation, necrosis and cardiomyocyte degeneration in the spongiform myocardium and atrium, often associated with liver necrosis.

It is highly desirable that any cleaner fish are free of PMCV (and other diseases) if they are to be used to treat sea lice infections in aquaculture. Potential infection of cleaner fish is a biosecurity concern in respect of infection of salmon populations. However, progress in developing methods of detection and treatment of PMCV in cleaner fish has been prevented by the lack of reports of PMCV and CMS-like pathology in cleaner fish.

The present invention relates to a novel infection of cleaner fish by a piscine myocarditis virus. There is a need for reagents and methods for the diagnosis, prevention and/or treatment of such infections.

The finding of piscine myocarditis virus infection in cleaner fish is surprising as the infection has not been previously found in any related species despite considerable interest.

Accordingly, a first aspect of the invention provides a method for determining the presence or absence of a piscine myocarditis virus infection, in a biological sample obtained from a cleaner fish, the method comprising: a) contacting nucleic acid from the biological sample with primers having a sequence defined in any one of SEQ ID NOs: 1, 2, 10, 11, 12 or 13, a sequence comprising at least 10 consecutive nucleotides having a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 10, 11, 12 or 13, or a sequence comprising at least 10 consecutive nucleotides having a complementary sequence to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 10, 11, 12 or 13, homologues thereof or a sequence - - having at least about 60%, about 75%, about 80%>, about 85%, about 90%, about 95%, about 96%), about 97%), about 98%>, about 99%, or about 99.5% sequence identity thereto; b) subjecting the nucleic acid and the primer to amplification conditions; and c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of nucleic acid associated with the piscine myocarditis virus infection in the sample.

In embodiments of the invention, the cleaner fish is from the family Cyclopteroidea or Labridae. Preferably the cleaner fish is a wrasse or lump fish.

In embodiments of the invention, the one or more primer sequences comprise at least 5, at least 7, at least 10, at least 15 consecutive nucleotides having a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 10, 11, 12 or 13. A second aspect of the inventions provides a kit for screening for a piscine myocarditis virus infection in a cleaner fish, comprising: a) one or more primers having a sequence defined in any one of SEQ ID NOs: 1, 2, 10, 11, 12 or 13; and optionally b) primers or adapters suitable to enable sequencing of the amplified nucleic acid and determination of the presence of the piscine myocarditis virus.

A particular embodiment of the invention provides kit according to claim 38, comprising: a) primers having a sequence defined in SEQ ID NOs: 1 and 2; and optionally b) a probe having a sequence defined in SEQ ID NO:3. In an embodiment of the invention, the kit comprises primers having a sequence defined in SEQ ID NOs: 10 and 11. In another embodiment of the invention, the kit comprises primers having a sequence defined in SEQ ID NOs: 12 and 13. In a further embodiment of the invention, the kit comprises primers having a sequence defined in SEQ ID NOs: 10, 11, 12 and 13.

A third aspect of the invention provides an isolated or synthetic nucleic acid encoding a piscine myocarditis virus polypeptide for use in the treatment or prophylaxis against a piscine myocarditis virus in a cleaner fish. - -

In all embodiments of the present invention described herein, the term "cleaner fish" refers to species of fish that provide a service to other fish species by removing undesirable matter such as dead skin and/or ectoparasites. In any embodiment of the invention, the cleaner fish may be one or more selected from the group consisting of: lumpfish/lumpsucker {Cyclopterus lumpus); wrasse of the family Labridae; cunner (Tautogolabrus adspersus); and patagonian blennie (Eleginops maclovinus). The wrasse of the family Labridae may be one or more selected from the group consisting of: ballan wrasse (Labrus bergylta); corkwing wrasse (Symphodus melops); rock cook wrasse (Centrolabrus exoletus); goldsinny wrasse

(Ctenolabrus rupestris); and cuckoo wrasse (Labrus mixtus).

A particular embodiment provides a nucleic acid for use according the invention having a sequence selected from the group consisting of SEQ ID NOs: 1-13 and 41-67, homologues thereof or a sequence having at least about 60%, about 75%, about 80%>, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs: 1-13 and 41-67.

Amongst other things, these nucleic acid sequences may be useful for expression of piscine myocarditis virus-encoded polypeptides, proteins or fragments, variants, or derivatives thereof, generation of antibodies against piscine myocarditis virus proteins, generation of primers and probes for detecting piscine myocarditis virus and/or for diagnosing piscine myocarditis virus infection, generating immunogenic compositions against piscine myocarditis virus, and screening for drugs effective against piscine myocarditis virus. "Identity" in the context of two or more nucleic acids or polypeptide sequences, refers to the percentage of nucleotides or amino acids that two or more sequences or subsequences contain which are the same. A specified percentage of nucleotides can be referred to such as: 60%> identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms known to the skilled person or by manual alignment and visual inspection. Preferably, identity is assessed over regions of contiguous nucleic acids or polypeptides two or more sequences or subsequences. - -

Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the nucleic acid sequences disclosed herein, and fragments thereof under various conditions of stringency

Polynucleotides homologous to the sequences described herein, can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. The term "nucleic acid hybridization" refers to anti-parallel hydrogen bonding between two single- stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide in the hybridization and washing solutions, as well as other parameters.

Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid). Hybridization conditions for various stringencies are known in the art and are disclosed in detail in at least Sambrook et al. (2001).

In embodiments of the invention, the nucleic acid has a sequence comprising at least 5, at least 7, at least 10, at least 15, at least 18, at least 19, at least 20, at least 21, at least 25, at least 50, at least 100, at least 250, at least 500, at least 600, at least 700, or at least 800 consecutive nucleotides having a sequence selected from the group consisting of SEQ ID NOs: 1-13 and 41-67. In specific embodiments of the invention, the nucleic acid for use according to the invention has a sequence selected from the group consisting of SEQ ID NOs:50-55 and 58-67, homologues thereof or a sequence having at least about 60%, about 75%, about 80%>, about - -

85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs:50-55 and 58-67.

In embodiments of the invention, the nucleic acid for use according to the invention has a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-13 and 41-67, homologues thereof or a sequence having at least about 60%>, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs: 1-13 and 41-67. In embodiments of the invention, the sequence comprises at least 5, at least 7, at least 10, at least 15, at least 18, at least 19, at least 20, at least 21, at least 25, at least 50, at least 100, at least 250, at least 500, at least 600, at least 700, or at least 800 consecutive nucleotides having a complementary sequence to a sequence selected from the group consisting of SEQ ID NOs: 1-13 and 41-67.

In specific embodiments of the invention, the nucleic acid for use according to the invention has a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs:50-55 and 58-67, homologues thereof or a sequence having at least about 60%>, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs:50-55 and 58-67.

In embodiments of the invention, the isolated nucleic acid of the invention is DNA, cDNA, RNA, or a combination of two or more thereof. In embodiments of the invention, the nucleotide sequence has an additional sequence at the 5'- and/or 3 '-end that would not be found in that position relative to the sequence found in nature. In embodiments of the invention, each or the additional sequence comprises 1 or more, 2 or more, 3 or more, 5 or more, 6 or more nucleotides. In embodiments of the invention, this sequence is not TAA, RAG or TGA, and/or the complimentary sequences thereof. - -

A fourth aspect of the invention provides an isolated polypeptide encoded by the nucleic acid of the invention, for use in the treatment or prophylaxis against a piscine myocarditis virus in a cleaner fish. It will be understood that, for the particular PMCV polypeptides described here, natural variations can exist between individual PMCV isolates. These variations may be

demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence.

Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al. (1979) in The Proteins, Academic Press New York. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, for example, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D. (1978), Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., vol. 5, suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/ Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val and Ala/ Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science (1985) 227: 1435) and determining the functional similarity between homologous proteins. Such amino acid substitutions of the exemplary embodiments of this invention, as well as variations having deletions and/or insertions are within the scope of the invention as long as the resulting proteins retain their immune reactivity. It is know that polypeptide sequences having one or more amino acid sequence variations as compared to a reference polypeptide may still be useful for generating antibodies that bind the reference polypeptide. These polypeptides may be useful for multiple applications, including, but not limited to, generation of antibodies and generation of immunogenic compositions. A peptide of at least 8 amino acid residues in length can be recognized by an antibody (MacKenzie et al. (1984) Biochemistry 23: 6544-6549). In certain embodiments, the invention is directed to fragments of the polypeptides described herein, which can, for example, be used to generate antibodies.

In embodiments of the invention, the isolated polypeptide has the sequence selected from the group consisting of SEQ ID NOs: 14-40, homologues thereof or a sequence having at least - - about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs: 14-40.

In specific embodiments of the invention, the isolated polypeptide has the sequence selected from the group consisting of SEQ ID NOs:23-28 and 31-40, homologues thereof or a sequence having at least about 60%>, about 75%, about 80%>, about 85%, about 90%>, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs: 23-28 and 31-40. In embodiments of the invention, the isolated polypeptide comprises at least 5, at least 6, at least 7, or at least 8, or at least 9, or at least 10, at least 15, at least 20, at least 30, at least 50, at least 100, at least 150, at least 200, at least 250, or at least 260 amino acids.

In embodiments of the invention, the isolated polypeptide is a recombinant protein.

A fifth aspect of the invention provides an isolated antibody that specifically binds to a polypeptide encoded by the nucleic acid according to the invention, or a polypeptide defined according to the invention, for use in the treatment or prophylaxis against a piscine myocarditis virus in a cleaner fish.

The antibodies may be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-human immunoglobulin). Species specific antibodies avoid certain of the problems associated with antibodies that possess variable and/or constant regions from other species. The presence of such protein sequences from other species can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by an antibody.

An antibody described in this application can include or be derived from any mammal, such as but not limited to, a bird, a dog, a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof and includes isolated avian, human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted or CDR-adapted antibodies, immunoglobulins, cleavage products and other portions and variants thereof. - -

Any method known in the art for producing antibodies can be used to generate the antibodies described herein. Exemplary methods include animal inoculation, phage display, transgenic mouse technology and hybridoma technology. The antibodies of the present invention can be used to modulate the activity of any polypeptide of the invention, variants or fragments thereof. In certain embodiments, the invention is directed to a method for treating a fish, the method comprising administering to the fish an antibody of the invention. In embodiments of the invention, the antibody of the invention may interfere or inhibit the function of the polypeptide, thus providing a method to inhibit pathogen propagation and spreading. In other embodiments, the antibody does not interfere or inhibit the function of the polypeptide. In embodiments of the invention, the antibodies of the invention can be used to purify a polypeptide of the invention. In other embodiments, the antibodies of the invention can be used to identify expression and localization of the polypeptide of the invention.

In embodiments of the invention, the antibodies of the invention can be used to analyse the expression and localization of a polypeptide of the invention, which may be useful in determining potential role of the polypeptide.

In other embodiments, the antibodies of the present invention can be used in various immunoassays to identify fish exposed to and/or samples that comprise polypeptides, in particular antigens, from PMCV.

Any suitable immunoassay which can lead to formation of polypeptide-antibody complex can also be used. For example, suitable methods known in the art include ELISA, lateral flow assays for detection of analytes in samples and immunoprecipitation. In various

embodiments, the polypeptide and/or the antibody can be labelled by any suitable label or method known in the art. For example, enzymatic immunoassays may use solid supports, or immunoprecipitation. Immunoassays which amplify the signal from the polypeptide-antibody immune complex can also be used. - -

Thus, in embodiments of the invention, the isolated antibody a binds to PMCV. Typically, the antibody binds to a PMCV polypeptide. In embodiments of the invention, the isolated antibody inhibits, neutralizes or reduces the function or activity of the PMCV or PMCV polypeptide.

In embodiments of the invention, the antibody is a polyclonal antibody, or a monoclonal antibody. In embodiments of the invention, the antibody is purified.

A sixth aspect of the invention provides an immunogenic composition comprising a piscine myocarditis virus nucleic acid, wherein the nucleic acid comprises the nucleic acid having the sequence according to the invention. The immunogenic compositions are capable of inducing an immune response against PMCV.

The immunogenic composition comprising a nucleic acid is administered to the fish, and the immunogenic proteins or peptides encoded by the nucleic acid are expressed in the fish, such that an immune response against the proteins or peptides is generated in the fish.

To make the nucleic acid immunogenic compositions of the invention, nucleic acid sequences of the invention may be incorporated into a plasmid or expression vector containing the nucleic acid. Any suitable plasmid or expression vector capable of driving expression of the protein or peptide in the fish may be used. Such plasmids and expression vectors should include a suitable promoter for directing transcription of the nucleic acid. The nucleic acid sequence(s) that encodes the protein or peptide may also be incorporated into a suitable recombinant virus for administration to the fish. Examples of suitable viruses include, but are not limited to, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses. The skilled person could readily select a suitable plasmid, expression vector, or recombinant virus for delivery of the nucleic acid sequences of the invention.

A seventh aspect of the invention provides an immunogenic composition comprising a PMCV polypeptide according to the invention. - -

To produce the immunogenic compositions comprising a polypeptide of the invention, nucleic acid sequences of the invention are delivered to cultured cells, for example by transfecting cultured cells with plasmids or expression vectors containing the nucleic acid sequences, or by infecting cultured cells with recombinant viruses containing the nucleic acid sequences. The polypeptides of the invention may then be expressed in the cultured cells and purified. The purified proteins can then be incorporated into compositions suitable for administration to fish. Methods and techniques for expression and purification of

recombinant proteins are well known in the art, and any such suitable methods may be used.

In embodiments of the invention, the immunogenic composition further comprises at least one excipient, additive or adjuvant.

In embodiments of the invention, the immunogenic composition further comprises at least one other polypeptide .

In embodiments of the invention, the at least one other polypeptide is selected from the group consisting of: one or more polypeptides from a different micro-organism; one or more polypeptides from the same micro-organism; and one or more promiscuous T-cell epitopes.

In embodiments of the invention, the polypeptides of the immunogenic composition are in admixture, or form a fusion protein.

In embodiments of the invention, the isolated nucleic acid, the isolated polypeptide, or the immunogenic composition for use according to the invention induces an immune response in the fish.

In embodiments of the invention, the isolated nucleic acid, the isolated polypeptide, or the immunogenic composition for use according to the invention prevents or reduces a PMCV infection in the fish.

In embodiments of the invention, the isolated polypeptide or the immunogenic composition for use according to the invention is administered orally, by immersion or by injection. - -

In embodiments of the invention, the cleaner fish is from the family Cyclopteroidea or Labridae. Preferably the cleaner fish is a wrasse or lump fish. In embodiments of the invention, the cleaner fish is a wrasse or lumpfish {Cyclopterus lumpus, L.). In embodiment of the invention the wrasse is a corkwing wrasse (Symphodus melops L.). In embodiment of the invention the wrasse is a ballan wrasse (Labrus bergylta Ascanius).

An eighth aspect of the invention provides a method of inducing an immune response in a cleaner fish, the method comprising administering the nucleic acid, the polypeptide, or the immunogenic composition according to the invention.

In embodiments of the invention, the method prevents or reduces a piscine myocarditis virus infection in the fish. In embodiments of the invention, the cleaner fish is from the family Cyclopteroidea or Labridae. In embodiments of the invention, the cleaner fish is a wrasse or lumpfish

{Cyclopterus lumpus, L.). In embodiment of the invention the wrasse is a corkwing wrasse {Symphodus melops L.). In embodiment of the invention the wrasse is a ballan wrasse {Labrus bergylta Ascanius).

A ninth aspect of the invention provides a method for determining the presence or absence of a piscine myocarditis virus infection in a biological sample obtained from a cleaner fish, the method comprising: a) contacting a biological sample with an antibody of the invention; and b) determining whether or not the antibody binds to an antigen in the biological sample, wherein binding indicates the presence of the piscine myocarditis virus infection in the biological sample.

A tenth aspect of the invention provides a method for determining the presence or absence of a piscine myocarditis virus infection in a biological sample obtained from a cleaner fish, the method comprising determining whether a biological sample contains antibodies that specifically bind to a polypeptide according to the invention. - -

An eleventh aspect of the invention provides a method of testing an agent for the prevention and/or treatment of a piscine myocarditis virus infection in a cleaner fish, comprising: a) contacting cells with the agent; b) contacting cells with piscine myocarditis virus; and c) measuring the number of cells infected with piscine myocarditis virus, wherein if the number of cells infected with the piscine myocarditis virus is decreased as a result of contact with the agent, the agent is a preventative and/or therapeutic agent for piscine myocarditis virus infection.

The present invention will now be described by way of example with reference to the accompanying drawings in which:

Figure 1 is a photograph of a recent corkwing wrasse {Symphodus melops) mortality showing a distended, blood engorged atrium (A), mildly congested liver (L) and ascites (arrow);

Figure 2 is a photomicrograph of the spongiform myocardium of a corkwing wrasse, {Symphodus melops), in which PMCV was the only pathogen detected, showing mild focal inflammation (circle) and necrosis (arrow); and Figure 3 is a condensed phylogenetic tree showing relationships between two wrasse (IRE- F208-16 #10 & #13) PMCV isolates and farmed Atlantic salmon isolates from Ireland and Norway, based on nucleotide sequence comparisons of a 759 bp fragment of the ORFl gene. Only bootstrap values > 60 are indicated. Examples

Example 1 - Fish Population and Sampling

The wrasse were wild caught and stocked with salmon as cleaner fish. CMS was diagnosed in the salmon population. Following this, the wrasse were sampled for molecular screening of PMCV and diagnosis of CMS. - -

The sample group consisted of 28 wrasse (five dead and 17 live corkwing; three dead and three live ballan). Live wrasse were caught with baited traps deployed overnight at a depth of six metres in one pen, and mortalities from the day of examination from the same and an additional pen. Five and three dead wrasse respectively were recovered with salmon mortalities from the two pens. Wrasse were transported off site for examination and sampling under laboratory conditions. The average size and weight was 18.6 ± 2.9 cm and 383.3 ± 172.2 g for ballan and 27.5 ± 1.7 cm and 96.3 ± 21.1 g for corkwing, which is within the normal range for wrasse in this area. Live wrasse were euthanized through an overdose of anaesthetic (450 mg/L of tricaine methanesulphonate; Tricaine 1,000 mg/g, Pharmaq).

Example 2 - Clinical and Post-Mortem Examination

All examined fish were subjected to a clinical and post-mortem examination. One recent corkwing mortality had a blood engorged, distended atrium comparable to gross pathology descriptions of CMS in salmon (Fig. 1; Table 1, fish 9). PMCV was the only pathogen detected in this wrasse by PCR, bacterial culture was negative for any significant fish pathogens and there were no indications of other aetiology associated with heart pathology observed on histology or parasitology. Other gross pathology observed included congested, mildly distended atria (two fish), petechial haemorrhaging around the mouth, abdomen and fin bases (three fish), ascites (three fish), congestion of liver (three fish) and congestion of spleen (two fish). Mild to moderate amoebic gill disease (AGD) was seen macroscopically on 10 fish, confirmed through histology and parasitology (see table 1 for details). The majority of wrasse had not been feeding. Post mortem changes complicated the interpretation of findings in some dead wrasse, which was consistent with reports from other sites at this time of year.

Table 1 - Overview of wrasse sampling, clinical and laboratory results.

Fish Species Clinical Diagnostic samples Histology no. (status) examination qRT- PCR Para Bact Hist results

& necropsy PMCV SAV PRV

1 B (M) 4,6,7 29.5 neg neg - + - -

2 B (M) 1, 2 29.7 neg neg + - + 1,2,4,6

3 B (M) 5 32.3 neg neg - - - - - -

- -

The status of ballan wrasse (B; Labrus bergylta) and corkwing wrasse (CW; Symphodus melops) at the time of sampling is given as alive (L) or mortality (M). Positive qRT-PCR results are given as Cq values, negative qRT-PCR results are given as "neg". Parasitology (para.), bacteriology (bact.) and histology (hist.) samples are marked as "+" where taken and "-" where not taken. Positive bacteriology samples are indicated as "+pos". Results of clinical examination (clin. exam.), necropsy (necr.) and histology are described. Pathogens/diseases found are listed. No significant findings are indicated as "nsf. Clinical examination and necropsy results: 1 : external petechial haemorrhaging; 2: ascites; 3: heart pathology; 4:

pathology of other internal organs; 5: post mortem changes; 6: AGD; 7: other. Histology results: 1 : heart pathology in spongiform myocardium; 2: heart pathology in compact myocardium; 3: hyaline cardiomyocyte degeneration; 4: epicarditis; 5: AGD; 6: other

Samples for histology (gills, heart, skin, skeletal muscle, liver, kidney, spleen and hind gut fixed in 10% neutral buffered formalin) were taken from six corkwing, four of which were PMCV positive, and four ballan, three of which were PMCV positive. Samples were embedded in paraffin wax blocks, sectioned at a thickness of 4 μιη and stained with haematoxylin and eosin. Histopathology revealed cardiac pathology in five of seven PMCV positive fish and included mild multifocal inflammation of compact (two fish) and

spongiform myocardium (four fish), mild multifocal hyaline degeneration (four fish), focal necrosis of cardiomyocytes (one fish; Fig. 2) and epicarditis (one fish). No heart pathology was seen on the three PMCV negative fish.

With the exception of AGD, no clinically significant parasitic infections were observed. - -

Inoculates from the head kidney from five dead and 20 live fish were swabbed onto tryptone soya agar (TSA), TSA + 2% NaCl, thiosulfate-citrate-bile salts-sucrose agar (TCBS), Columbia blood agar + 2% NaCl (CBA + 2% NaCl) and marine agar (MA) and incubated at 20-22°C. Aeromonas salmonicida A-layer type V (Gulla et al. 2016) was isolated from two corkwing mortalities on TSA.

Example 3 - Molecular Analysis

Molecular screening of the wrasse sampled according to Example 1 for PMCV was undertaken.

Heart samples from all fish were stored in RNAlater (Ambion) at 4°C for PCR analysis. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and one-step real-time RT-PCR assays were performed using the Precision OneStepPLUS qRT-PCR mastermix

(PrimerDesign) on a LightCycler 96 (Roche).

Samples were screened for salmonid alphavirus (SAV; Hodneland & Endresen 2006), piscine reovirus (PRV; Palacios et al. 2010) and PMCV (Haugland et al. 2011) following published molecular protocols, and using the primers and probes as set out in Table 2.

Table 2 - RT-PCR primers and probes.

While all wrasse samples tested negative for SAV and PRV, PMCV results were positive 20 of the 28 wrasse (71.4% prevalence). - -

All dead wrasse were positive (corkwing quantification cycle [Cq] range: 29.3 to 31.3; ballan Cq range: 29.5 to 32.3; table 1). Prevalence of PMCV in live wrasse was 58.8% in corkwing (Cq range 29.3 to 33) and 66.7% in ballan (Cq range 30.2 to 31.2). Partial sequences of the ORF1 gene (759 bp) of two corkwing PMCV isolates (Table 1, fish 10 and 13; SEQ ID NO:54 and SEQ ID NO:55 respectively), and the ORF3 gene (662 bp) from one corkwing (Table 1, fish 10; SEQ ID NO:66 ) and one ballan wrasse (Table 1, fish 1; SEQ ID NO:67), were obtained using primers described by Garseth et al. (2012, Table 3).

Table 3 - ORF1 and ORF3 primers

PCRs were performed using a one-step RT-PCR kit (Qiagen) and the products were separated by electrophoresis in a 2.5% (w/v) agarose gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 2 mM ethyl enediaminetetraacetic acid) stained with ethidium bromide and visualised using the Quantity One, 1-D Analysis System software on a UV Transilluminator (Bio-Rad). PCR products were purified and sequenced commercially (Sequiserve).

Multiple sequence alignments were performed by Clustal W analysis (Thompson, Higgins & Gibson 1994). The evolutionary history was inferred using the neighbour-joining method (Saitou & Nei 1987), and evolutionary distances computed using the Kimura 2-parameter method (Kimura 1980). One thousand bootstrap replicates were performed for the analysis and all phylogenetic analyses were conducted using MEGA 6 (Tamura et al. 2013).

Wrasse PMCV sequences were compared to Atlantic salmon sequences from Norway (AL V708, Haugland et al. 2011 ; 36-VA/l 0, Wiik-Nielsen et al. 2013) and Ireland (Rodger et al. 2014). In addition, sequences IRE-F196-16#33 and IRE-F196-16#3 were from Atlantic salmon with clinical CMS on the same site as the wrasse, sampled one month previously. Sequence IRE F187-16#23 was from Atlantic salmon from a separate site diagnosed with CMS in October 2016. Phylogenetic analysis of the ORF1 sequences (Fig. 3) indicates that both corkwing wrasse sequences (IRE-F208-16#10 and #13) are very similar to PMCV - - sequences previously detected in salmon in Ireland including IRE-F196-16#33 from the same site. The second salmon sequence from this site (IRE-F196-16#3) is in a separate cluster with the Norwegian and other Irish isolates supporting the observation by Wiik-Nielsen et al. (2013) of significant nucleotide sequence variation of PMC V within individual farms. Amino acid sequences of both partial ORF1 and ORF3 genes were compared to evaluate the significance of the nucleotide variations.

The amino acid sequences of ORF1 (Table 4) differed at position 587, with M₅87 in the reference strain AL V-708 being replaced by T₅87 in both corkwing sequences, 5 Irish salmon sequences and Norwegian sequence 36-VA/10. In addition to this, the Atlantic salmon sequence IRE-F196-16#3 differed from all other sequences at position 486 (P₄86 replacing A₄86), the significance of which is unknown.

Table 4 - Amino acid substitutions in the ORF1 sequences (see Figure 3) from Irish corkwing wrasse (IRE-F208-16#10 and #13) and farmed Atlantic salmon (IRE-F187-16#23 and 51, IRE-F196-16#3 and 33) relative to the Norwegian isolates AL V-708 and 36-VA/10 and previously published sequences from Irish farmed Atlantic salmon.

Amino acids: A (alanine); M (methionine); T (threonine); P (proline). - -

*: A and M are present at positions 486 and 587, respectively, of isolate of reference strain AL V-708. Differences in other isolates with respect to reference strain at equivalent positions are indicated, with no difference being indicated with A higher degree of variation was seen in the amino acid sequences of the partial ORF3 gene (Table 5). The amino acid sequences show wrasse isolates to be identical to motif

V84Q87Q97, one of two common salmon motifs described by Wiik-Nielsen et al. (2013) and Rodger et al. (2014). IRE-F208-16#1 was isolated from a ballan wrasse from which ORF 1 could not be sequenced. Wrasse sequences also show I₄₄ and T222, a trait differentiating Irish from Norwegian VQQ motifs (Rodger et al 2014).

Table 5 - Amino acid substitutions in the ORF3 sequences from Irish corkwing wrasse (IRE- F208-16#10), ballan wrasse (IRE-F208-16#1) and farmed Atlantic salmon (IRE-F187-16#23, IRE-F196-16#3) relative to the Norwegian isolates AL V-708 and 36-VA/10 and previously published sequences from Irish farmed Atlantic salmon.

Amino acids: E (glutamic acid); I (isoleucine); K (lysine); R (arginine); V (valine);

P (proline); Q (glutamine); T (threonine); L (leucine).

*: E, E, I, K, R, K, V and P are present at positions 44, 78, 84, 87, 97, 103, 222 and 241, respectively, of isolate of reference strain AL V-708. Differences in other isolates with respect to reference strain at equivalent positions are indicated, with no difference being indicated with - -

Example 4 - PMCV in Lumpfish

Lumpfish were caught from a population stocked with salmon in net pens, and placed in a bucket with PMCV positive salmon. PCR screening of 6 lumpfish hearts for PMCV was undertaken as described in Example 3.

All samples were positive for PMCV with a Cq range of 28.4 to 31.7.

References

Haugland 0., Mikalsen A. B., Nilsen P., Lindmo K., Thu B. J., Eliassen T. M., Roos N., Rode M. & Evensen 0. (2011) Cardiomyopathy syndrome of Atlantic salmon {Salmo salar L.) is caused by a double-stranded RNA virus of the Totiviridae family. Journal of Virology 85, 5275-5286.

Hodneland K. & Endresen C. (2006) Sensitive and specific detection of salmonid alphavirus using real-time PCR (TaqMan^®). Journal of Virological Methods 131, 184-192.

Kimura M. (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16, 111- 120.

Palacios G., Lovoll M., Tengs T., Hornig M., Hutchison S., Hui J., Kongtorp R.T., Savij N., Bussetti A.V., Solovyov A., Kristoffersen A.B., Celone C, Street C, Trifonov V., Hirschberg D.L., Rabadan R., Egholm M., Rimstad E. & Lipkin W.I. (2010) Heart and skeletal muscle inflammation of farmed salmon is associated with infection with a novel reovirus. PLoS ONE 5, el 1487.

Rodger H. D., McCleary S. J., & Ruane N. M. (2014) Clinical cardiomyopathy syndrome in Atlantic salmon, Salmo salar L. Journal of Fish Diseases 37, 935-939.

Saitou N. & Nei M. (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406-425. - -

Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. Tamura K., Stecher G., Peterson D., Filipski A. & Kumar S. (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30, 2725-2729.

Thompson J.D., Higgins D.G. & Gibson T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673-4680.

Wiik-Nielsen J., Alarcon M., Fineid B., Rode M. & Haugland 0. (2013) Genetic variation in Norwegian piscine myocarditis virus in Atlantic salmon, Salmo salar L. Journal of Fish Diseases 36, 129-139.

Listing of sequenced ORF1 amino acid sequences from various isolates

Amino acids at positions 486 and 587 are highlighted in SEQ ID NO: 14 (ORF1 AL V-708), and in equivalent positions in other ORF1 amino acid sequences.

SEQ ID NO: 14 (ORF1 AL V-708):

MEPNTSVIATEQQQAAMREVEAEAAARDEVVEKIAFAEGAMMVQTRRLPSGKSSVGGFLGE LAQ IRAMNRSLH DTNMLTEGAMVDRARAKVHKI IREGNLDSRVFSNTGSNTMLSLWVPA VPGPPAVPEHWDVAPSWFVCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNA FSMNAVLGALRAGGFNTEHSLVSFVEPLIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLV FTTSGNFFPGGPNLRVWGANDTVARIVNVEDYMREAAGEGRFDAGWGPEFWGGTGDDAVAV VPIRAVEAGLGEVNAGWTLAHMEYPVKVRLLDVDDRTIGPGGSLPLNANREYTAAGATHVP GPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYVIGGADLGMLPLIQWSVGLGAED MAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGVAVEGGFWAQPAAGAAP FPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYRERGVRPGSVANWQYVRFDPTVA VGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQAMGEDVINGQMGQPESMMRGVALNE NQGLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEGGTMRKRIP AIEMRENGVEGDLMNDDLYS IGTAAGYLAVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNT - -

VGAMGGWTRAVTPVDNVATMRDNGVEGEPCGIVMSLPTSGTAVVDRLANFGLPPARAELRE VPFGGYQRSVTNTNHRVKVSVSGGRAVVQKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMT TADFLDI SEQ ID NO: 15 (ORF1 36-VA/10):

MEPNTSVIATEQQQAAMREVEAEAAARDEVVEKIAFVEGAMMVQTRRLPSGKSSVGGFLGE LAQ IRAMNRSLH DTNMLTEGAMVDRARAKVHKI IREGNLDSRVFSNTGSNTMLSLWVPA VPGPPAVPEHWDVAPSWFVCRPGKKGGIKITQSASMAALNPLFRGADVGPIGTAVRADVNA FSMNAVLGALRAGGFNTEHSLVSFVEPLIRILLMGVQTQDRGTSPWDWVGGMSSRIVNPLV FTTSGNFFPGGPNLRVWGANDTVARIVNVEDYMREAAGEGRFDAGWGPEFWGGTGDDAVAV VPIRAVEAGLGEVNAGWTLAHMEYPVKVRLLDVDDRTIGPGGSLPLNANREYTAAGATHVP GPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYVIGGADLGMLPLIQWSVGLGAED MAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPAFRRSGVAVEGGFWAQPAAGAAP FPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYRERGVRPGSVANWQYVRFDPTVA VGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQATGEDVINGQMGQPESMMRGVALNE NQGLAAATVRRVVGLENESMQTTHWSTTEVAMNGYYGRAGATAHHAAFPLSEGGTMRKRIP AIEMRENGVEGDLMNDDLYS IGTAAGYLAVEGMAGAQGGIWDVVQYQLPGPDDEARGVMNT VGAMGGWTRAVTPVDNVATMRDNGVEGEPCGIVMSLPTSGAVVVDRLANFGLPPARAELRE VPFGGYQRSVTNTNHRVKVSVSGGRAVVQKGNKAEMNPVFVNRTPGQTTLGQPTTDTTGMT TADFLDI

SEQ ID NO: 16 (ORF1 IRE-F56-12#1):

PGGSLPLNVNREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQATG EDVINGQMG

SEQ ID NO: 17 (ORF1 IRE-F56-12#2):

PGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQAMG EDVINGQMG _ _

SEQ ID NO: 18 (ORF1 IRE-F56-12#3):

PGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQAMG EDVINGQMG

SEQ ID NO: 19 (ORF1 IRE-F58-12#1):

SEQ ID NO:20 (ORF1 IRE-F58-12#12):

SEQ ID NO:21 (ORF1 IRE-F58-12#13):

PGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQATG EDVINGQMG SEQ ID NO:22 (ORF1 IRE-F58-12#14):

PGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQATG - -

EDVINGQMG

SEQ ID NO:23 (ORF1 IRE-F187-16#23):

PGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQATG EDVINGQMG SEQ ID NO:24 (ORF1 IRE-F187-16#51):

PGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQATG EDVINGQMG

SEQ ID NO:25 (ORF1 IRE-F196-16#3):

PGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGPAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQAMG EDVINGQMG

SEQ ID NO:26 (ORF1 IRE-F196-16#33):

SEQ ID NO:27 (ORF1 IRE-F208-16#10):

PGGSLPLNANREYTAAGATHVPGPYARVLYVVVDQNADRCVGVRVQGQGAVIDVDPALNYV IGGADLGMLPLIQWSVGLGAEDMAQGS IAQTQRWVRMYGNEDDWESAWHLVSSAYTVYSPA FRRSGVAVEGGFWAQPAAGAAPFPLGGLAGWVRYDNQARAAQVALCRERADMAECPWGGYR - -

ERGVRPGSVANWQYVRFDPTVAVGVAAHFWSVVKVMVAPVPDRAAALADMAWGKGKVQATG EDVINGQMG

SEQ ID NO:28 (ORF1 IRE-F208-16#13):

Listing of sequenced QRF3 amino acid sequences from various isolates

Amino acid at position 44 is highlighted in SEQ ID NO:29 (ORF3 AL V-708), and in an equivalent position in other ORF3 amino acid sequences.

SEQ ID NO:29 (ORF3 AL V-708):

MSNKMKSFLLVLLCLCVGEGIVPMFRREWCLCTAGNARVPLVGEGRAEKIELFNQSATCGK KELI ITWKGKRWCYDIESKRGKILVKTLSGGGHLEREGKGYKLVRNGFHLASFGGKKEEIQ DSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTEGGNLCGNVGFFDNTQCTYNSVINIGGG SVDNSQEAKNDKSN IDNLIDMLPLVVGIAGGCLIVIVVLYLTIKYCKCKKKRTNPEPAEP EEHEMRDLRRRLEPRPPYQRQMGVEFEINEALEFMGVEGSESPDSGCQSDEEGFRVGV

SEQ ID NO:30 (ORF3 36-VA/10):

MSNKMKSFLLVLLCLCVGEGIVPMFRREWCLCTAGNARVPLVGEGRAEKIELFNQSATCGK KELI ITWKGKRWCYDIESKRGKVLVQTLSGGGHLEQEGKGYRLVRNGFHLASFGGKKEEIQ DSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTEGGNLCGNVGFFDNTQCTYNSVINIGGG SVDNSQEAKNDKSNTIDNLIDMLPLVVGIAGGCLIVIVILYLTIKYCKCKKKRTNPEPAEP EEHEMRDLRRRLEPRPPYQRQMGVEFEINEALEFMGVEGSESPDSGCQSDEEGFRVGV SEQ ID NO:31 (ORF3 IRE-F56-12#1):

GNARVPLVGKGRAEKIELFNQSATCGKKELI ITWKGKRWCYDIESKRGKVLVQTLSGGGHL EQEGKGYKLVRNGFHLASFGGKKEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSNTIDNLIDMLPLVVGIAGGCL IVIVTLYLTIKYCKCKKKRTNPEPAEPEEHEMRDLRR - -

SEQ ID NO:32 (ORF3 IRE-F56-12#2):

GNARVPLVGKGRAEKIELFNQSATCGKKELI ITWKGKRWCYDIESKRGKVLVQTLSGGGHL EQEGKGYKLVRNGFHLASFGGKKEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSN IDNLIDMLPLVVGIAGGCL IVIVTLYLTIKYCKCKKKRTNPEPAEPEEHEMRDLRR

SEQ ID NO:33 (0RF3 IRE-F56-12#3):

GNARVPLVGEGRAEKIELFNQSATCGKKELI ITWKGKRWCYDIQSKRGKILVKTLSGGGHL EREGKGYKLVRNGFHLASFGGKKEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSN IDNLIDMLPLVVGIAGGCL IVIVVLYLTIKYCKCKKKRTNPEPAEPEEHEMRDLRR

SEQ ID NO:34 (ORF3 IRE-F58-12#12):

GNARVPLVGEGRAEKIELFNQSATCGKKELI ITWKGKRWCYDIQSKRGKILVKTLSGGGHL EREGKGYKLVRNGFHLASFGGKKEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSNTIDNLIDMLPLVVGIAGGCL IVIVVLYLTIKYCKCKKKRTNPEPAEPEEHEMRDLRR SEQ ID NO:35 (ORF3 IRE-F58-12#13):

GNARVPLVGEGRXEKIELFNQSATCGKKELI ITWKGKRWCYXIXSKRGKILVKTLSGGGHL EREGKGYKLVRNGFHLASFGGKXEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSNTIDNLIDMLPXVVGIAGGCL IVIVVLYLTIKYCKCKKKRTNPEPAEPEEHEMRDLRR

NB: "X" in amino acid sequence SEQ ID NO:35 indicates that alternative amino acids may be present as defined at corresponding positions in nucleotide sequence SEQ ID NO: 62.

SEQ ID NO:36 (ORF3 IRE-F58-12#14):

SEQ ID NO:37 (ORF3 IRE-F187-16#23):

GNARVPLVGKGRAEKIELFNQSATCGKKELI ITWKGKRWCYDIESKRGKVLVQTLSGGGHL EQEGKGYKLVRNGFHLASFGGKKEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSN IDNLIDMLPLVVGIAGGCL IVIVTLYLTIKYCKCKKKRTNPELAEPEEHEMRDLRR

SEQ ID NO:38 (0RF3 IRE-F196-16#3):

GNARVPLVGEGRAEKIELFNQSATCGKKELI ITWKGKRWCYDIESKRGKILVKTLSGGGHL EREGKGYKLVRNGFHLASFGGKKEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSN IDNLIDMLPLVVGIAGGCL IVIVVLYLTIKYCKCKKKRTNPEPAEPEEHEMRDLRR

SEQ ID NO:39 (ORF3 IRE-F208-16#10):

GNARVPLVGKGRAEKIELFNQSATCGKKELI ITWKGKRWCYDIESKRGKVLVQTLSGGGHL EQEGKGYKLVRNGFHLASFGGKKEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSNTIDNLIDMLPLVVGIAGGCL IVIVTLYLTIKYCKCKKKRTNPEPAEPEEHEMRDLRR

SEQ ID NO:40 (0RF3 IRE-F208-16#1):

GNARVPLVGKGRAEKIELFNQSATCGKKELI ITWKGKRWCYDIESKRGKVLVQTLSGGGHL EQEGKGYKLVRNGFHLASFGGKKEEIQDSSHIEKVNGKDAIVKKGQHVEHLPGGNDLIVTE GGNLCGNVGFFDNTQCTYNSVINIGGGSVDNSQEAKNDKSNTIDNLIDMLPLVVGIAGGCL IVIVTLYLTIKYCKCKKKRTNPEPAEPEEHEMRDLRR Listing of nucleotide sequences (SEQ NOs:41-67) encoding the amino acid sequences listed as SEQ NO: 14-40. respectively

NB: IUPAC notation is used. Thus, for example, Y may be C or T, S may be G or C, and R may be A or G.

SEQ ID NO:41 (ORF1 AL V-708 nucleotides):

atggaaccaaacacatctgtcattgcaacggagcagcagcaggctgccatgagagaggtgg aggccgaggcggcggccagagacgaagtggtggagaagatcgcattcgctgaaggagcgat gatggtacagacgaggaggttaccatcaggaaagtcgtcggtaggaggttttctcggcgaa - - ctggcacagaacatacgtgccatgaatcggtcattgcacacagataccaacatgctgaccg aaggggcgatggtggacagagcgagggcaaaagtacacaaaatcattagggaagggaattt ggactctagggtattttcaaacacggggagcaacactatgttgtcactgtgggtaccagca gtaccgggaccaccggcagtaccggagcattgggacgttgcgccgtcctggttcgtatgca gaccggggaaaaagggggggataaagatcacacaaagcgcatcaatggcagcattaaaccc actatttagaggcgcagacgtggggccaatcgggacagcagtcagggcggatgtaaacgca ttttcaatgaatgcagttctgggagcactaagagccgggggatttaacaccgaacattccc tggtgtcattcgttgaaccactaattcggatcttgctaatgggggtacaaacacaagacag ggggaccagcccatgggattgggttggagggatgagttcgcgaatagtcaatcccctagta ttcacaacaagcgggaacttcttcccagggggaccaaatttgagggtgtggggagccaacg atacagtggccaggatagtaaacgttgaggactacatgcgcgaggcggccggggaggggag gttcgacgctggatggggaccggaattctggggtgggacaggggacgacgcagtggcggtg gtaccgataagggcagtagaagcagggctaggagaagtaaacgcagggtggacattggcac acatggaatacccagtcaaggttagactacttgacgtcgacgaccgaacaattggaccagg ggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatgtaccc gggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagacaggtgtgtggggg tgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtgatagg gggagcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccgaggac atggcgcagggatcgattgcacagacgcagcgatgggtgaggatgtatggaaacgaggacg attgggaatcagcgtggcatctagtgtctagcgcgtacacagtgtacagcccggcattcag gagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcagcaccg tttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggcgcagg ttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagggagag aggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacagtggct gtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtcccagaca gagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccatgggtgagga tgtgatcaacgggcagatgggacaacctgagtccatgatgagaggggtggcgctgaacgag aaccagggactagcggcggctacagtcaggagggtggttgggctggagaacgagtcgatgc aaacaacgcactggagtacaacggaggtagcaatgaacgggtactacgggagagcaggagc aacagcacaccacgctgcatttccgttgtccgagggggggacaatgcgaaaacgaatacca gctatagagatgagggagaacggggtggagggggacctgatgaacgatgatctctattcaa ttggaacggcagcggggtacctggcggtagaggggatggcaggtgcgcaggggggtatctg ggacgtggtccagtaccagctgcctgggcctgacgatgaggcgaggggggtgatgaacacg gtgggggcgatggggggatggacgagggcggtgacaccagtagacaatgtggccaccatga - - gggacaacggggttgagggggaaccttgtggaatagtgatgtctctaccaacaagtgggac cgctgtggtggataggttagctaatttcggattaccaccagcgagggcggaattaagagaa gtaccatttggcgggtaccaaagatcagtcacaaacaccaaccacagagtcaaggtgagtg tgagtggggggcgagcagttgttcaaaaagggaacaaagccgagatgaatccagtctttgt caataggacaccaggacaaacgaccctaggccaaccaacaacagacactacagggatgaca actgcagattttttagatatatag

SEQ ID NO:42 (ORF1 36-VA/10 nucleotides):

atggaaccaaacacatctgtcattgcaacggagcagcagcaggctgccatgagggaggtgg aggccgaggcggcggccagagacgaagtggtggagaagatcgcattcgttgaaggagcgat gatggtacagacgaggaggttaccatcaggaaagtcgtcggtaggaggttttctcggcgaa ctggcacagaacatacgtgccatgaatcggtcattgcacacagataccaacatgctgaccg aaggggcgatggtggacagggcgagggcaaaagtacacaaaatcatcagggaagggaattt ggactctagggtattttcaaacacggggagcaacactatgttgtcactgtgggtaccagca gtaccgggaccaccggcggtaccggagcattgggacgttgcgccgtcctggttcgtatgta gaccggggaaaaagggggggataaagatcacacaaagcgcatcaatggcagcattaaaccc actatttagaggcgcagacgtggggccaatcgggacggcagtcagggcggatgtgaacgca ttttcaatgaatgcagttctgggagcactaagagccgggggatttaacaccgaacattccc tggtgtcattcgttgaaccactaattcggatcttgttaatgggggtacaaacacaagacag ggggaccagcccatgggattgggttggagggatgagttcgcgaatagtcaatcccctagta ttcacaacaagcgggaacttcttcccagggggaccaaatttgagggtgtggggagccaacg acacagtggccaggatagtaaatgttgaggactacatgcgcgaggcggccggggaggggag gttcgacgccggatggggaccggaattctggggtgggacaggggacgacgcagtagcggtg gtaccgataagggcagtagaagcagggctaggggaagtaaacgcagggtggacattggcac acatggaatacccagtcaaggttagactacttgacgttgacgaccgaacaattggaccagg ggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatgtaccc gggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagacaggtgtgtggggg tgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtgatagg gggagcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccgaggac atggcgcagggatcgattgcacagacgcagcgatgggtgaggatgtatggaaacgaggacg attgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggcattcag gagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcagcaccg tttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggcgcagg ttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagggagag - - aggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacagtggct gtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtcccagaca gagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccacgggtgagga tgtgatcaacgggcagatgggacaacctgagtccatgatgagaggggtggcgctgaacgag aaccagggactagcggcggctacagtcaggagggtggttgggctggagaacgagtcgatgc aaacaacgcactggagtacaacggaggtagcaatgaacgggtactacgggagagcaggagc aacagcacaccacgctgcatttccattgtccgagggggggacaatgcgaaaacgaatacca gctatagagatgagggagaacggggtggagggggacctgatgaacgatgatctctattcaa ttgggacggcagcggggtacctggcggtagaggggatggcaggtgcgcaggggggtatctg ggacgtggtccagtaccagctgcctgggcctgacgatgaggcgaggggggtgatgaacacg gtgggggcgatggggggatggacgagggcggtgacaccagtggacaatgtggccaccatga gggacaacggggttgagggggaaccttgtgggatagtgatgtctctaccaacaagtggggc cgttgtggtggataggttagctaatttcggattaccaccagcgagggcagaattaagagaa gtaccatttggtgggtaccaaagatcagtcacaaacacgaaccacagagtcaaggtgagtg tgagtggggggcgagcagttgttcaaaaagggaacaaagccgagatgaacccagtctttgt caataggacaccaggacaaacgaccctaggccaaccaacaacagacactacagggatgaca actgcagattttttagatatatag

SEQ ID NO:43 (ORF1 IRE-F56-12#1 nucleotides):

ccaggggggagcctgcccctaaacgtaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagataggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggggcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcacagggatcgattgcacagacgcagcggtgggtgaggatgtatggaaacga ggatgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccacgggt gaggatgtgatcaacgggcagatggga - -

SEQ ID NO:44 (ORF1 IRE-F56-12#2 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagacaggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggagcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcgatgggtgaggatgtatggaaacga ggacgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagtccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggagtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccatgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:45 (ORF1 IRE-F56-12#3 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg ttcccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagacaggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggagcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcgatgggtgaggatgtatggaaacga ggacgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccatgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:46 (ORF1 IRE-F58-12#1 nucleotides):

ccaggggggagcctgcccctaaacgcaaatagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagacaggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg - - atagggggagcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcgatgggtgaggatgtatggaaacga ggacgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagtccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggagtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccatgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:47 (ORF1 IRE-F58-12#12 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg ttcccgggccctatgccagggtgctgtacgtcgtcgtggaccaaaacgcagacaggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggagcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcgatgggtgaggatgtatggaaacga ggacgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccatgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:48 (ORF1 IRE-F58-12#13 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagataggtgtgt gggggtgagggtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggggcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcggtgggtgaggatgtatggaaacga ggatgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag - - caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccacgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:49 (ORF1 IRE-F58-12#14 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagataggtgtgt gggggtgagggtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggggcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcggtgggtgaggatgtatggaaacga ggatgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccacgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:50 (ORF1 IRE-F187-16#23 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagataggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggggcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcggtgggtgaggatgtatggaaacga ggatgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccagggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc - - agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccacgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:51 (ORF1 IRE-F187-16#51 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagataggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggggcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcggtgggtgaggatgtatggaaacga ggatgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccagggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccacgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:52 (ORF1 IRE-F196-16#3 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagacaggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccagcgttgaattacgtg ataggaggagcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcgatgggtgaggatgtatggaaacga ggacgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtttacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcagggccag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccatgggt gaggatgtgatcaacgggcagatggga - -

SEQ ID NO:53 (ORF1 IRE-F196-16#33 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtggaccaaaacgcagataggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggggcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcggtgggtgaggatgtatggaaacga ggatgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctagcggacatggcgtgggggaaggggaaggtgcaagccacgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:54 (ORF1 IRE-F208-16#10 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtagaccaaaacgcagataggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg atagggggggcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcggtgggtgaggatgtatggaaacga ggatgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccacgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:55 (ORF1 IRE-F208-16#13 nucleotides):

ccaggggggagcctgcccctaaacgcaaacagagaatacacggcggcaggagctacgcatg tacccgggccctatgccagggtactgtacgtcgtcgtagaccaaaacgcagataggtgtgt gggggtgagagtgcagggacagggtgctgtaattgacgtggatccggcgttgaattacgtg - - atagggggggcggatttggggatgttgccgttgatacagtggagtgtagggctgggggccg aggacatggcgcagggatcgattgcacagacgcagcggtgggtgaggatgtatggaaacga ggatgattgggaatcagcgtggcatctggtgtctagcgcgtacacagtgtacagcccggca ttcaggagatcgggtgtcgcagtggagggaggattctgggcgcaaccagctgcaggggcag caccgtttccactaggaggattggcagggtgggtgaggtacgacaatcaggcacgggcggc gcaggttgcactttgcagagagagggcggatatggcggagtgtccttggggggggtacagg gagagaggggtgagaccggggagtgtggcaaactggcagtacgtaaggttcgatcccacag tggctgtaggagtagctgctcacttctggtcggtagtgaaggtgatggtggctcccgtccc agacagagcggctgctctggcggacatggcgtgggggaaggggaaggtgcaagccacgggt gaggatgtgatcaacgggcagatggga

SEQ ID NO:56 (ORF3 AL V-708 nucleotides):

atgtcaaacaagatgaagagttttttactggtgcttttatgtttgtgtgtaggggaaggga ttgttccaatgttcaggcgagaatggtgtttgtgcactgcagggaatgcgagagtaccctt ggtgggagagggaagggctgagaagatcgagctattcaatcagagcgccacatgtgggaag aaagagctaataataacgtggaagggaaagaggtggtgctacgatattgagagcaagcgag ggaaaatactagtaaagacactgagcggagggggacatctggaacgagagggaaaggggta caagttagttaggaacgggttccatcttgcttcattcgggggtaaaaaagaggaaatacag gattcaagtcacatagagaaagtaaacgggaaagacgcgatagttaagaaagggcaacacg tagaacatcttccaggggggaacgatttaattgttacagagggcgggaacctgtgtgggaa cgtggggttcttcgataatacgcagtgtacatacaacagtgttataaatataggaggggga agtgttgacaattcacaagaagccaagaacgataaatcaaacacaattgacaatttaattg atatgttacctttagttgtaggaatagccgggggctgtctcatagtgatagtagtcttata cttaacaattaaatactgtaaatgcaagaagaagaggaccaacccagaaccagcggaaccg gaggaacacgagatgagggatctcaggagaaggttagagccacgccctccataccaaagac agatgggtgttgagtttgaaataaatgaagctctcgaattcatgggcgtcgaagggagtga aagcccagactcaggatgtcagtcagatgaagagggattcagagtaggagtctag

SEQ ID NO:57 (ORF3 36-VA/10 nucleotides):

atgtcaaacaagatgaagagttttttactggtgcttttatgtttgtgtgtaggggagggga ttgttccaatgttcaggcgagaatggtgtttgtgcactgcagggaatgcgagagtaccctt ggtgggagagggaagggctgagaagatcgagctattcaatcagagcgccacatgtgggaag aaagagctaataataacgtggaaggggaagaggtggtgctacgatattgagagcaagcgag ggaaagtactggtacagacactgagcggagggggacatctggaacaagagggaaaggggta - - cagattagttaggaacgggttccatcttgcttcattcgggggtaaaaaagaggaaatacag gattcaagtcacatagagaaagtaaacgggaaagacgcgatagttaagaaagggcaacacg tagaacatcttccaggggggaacgatttaattgttacagagggcgggaacctgtgtgggaa cgtggggttcttcgataatacgcagtgtacatacaacagtgttataaatataggaggggga agtgttgacaattcacaagaagccaagaacgataaatcaaacacaattgacaatttaattg atatgttacctttagttgtaggaatagccgggggctgtctcatagtgatagtaatcttata cttaacaatcaaatactgtaaatgcaagaagaagaggaccaacccagaaccagcggaaccg gaggaacacgaaatgagggatctcaggagaaggttagagccacgccctccataccaaagac agatgggtgttgagtttgaaataaatgaagctctcgaattcatgggcgtcgaagggagtga aagcccagactcaggatgtcagtcagatgaagagggattcagagtaggagtctag

SEQ ID NO:58 (ORF3 IRE-F56-12#1 nucleotides):

gggaatgcgagagtacccttggtgggaaagggaagggctgagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaaggggaagaggtggtgcta cgatattgagagcaagcgagggaaagtactagtacagacactgagcggagggggacatctg gaacaagagggaaaggggtacaaattagttaggaacgggttccatcttgcttcattcgggg gtaaaaaagaggaaatacaggattcaagtcacatagagaaagtaaacgggaaagacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttagttgtaggaatagccgggggctgtctc atagtgatagtaaccttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaaccagcggaaccggaggaacacgagatgagggatctcaggagaag SEQ ID NO:59 (ORF3 IRE-F56-12#2 nucleotides):

gggaatgcgagagtacccttggtgggaaagggaagggctgagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaaggggaagaggtggtgcta cgatattgagagcaagcgagggaaagtactagtacagacactgagcggagggggacatctg gaacaagagggaaaggggtacaaattagttaggaacgggttccatcttgcttcattcgggg gtaaaaaagaggaaatacaggattcaagtcacatagagaaagtaaacgggaaagacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttagttgtaggaatagccgggggctgtctc - - atagtgatagtaaccttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaaccagcggaaccggaggaacacgagatgagggatctcaggagaag

SEQ ID NO:60 (ORF3 IRE-F56-12#3 nucleotides):

gggaatgcgagagtacccttggtgggagagggaagggctgagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaagggaaagaggtggtgcta cgacattcagagcaagcgagggaaaatactagtaaagacactgagcggagggggacatctg gaacgagagggaaaggggtacaagttagttaggaacgggttccatcttgcttcattcgggg gtaaaaaggaggaaatacaggattcaagtcacatagagaaagtaaacgggaaagacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttagttgtaggaatagccgggggctgtctc atagtgatagtagtcttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaaccagcggaaccggaggaacacgagatgagggatctcaggagaag

SEQ ID NO:61 (ORF3 IRE-F58-12#12 nucleotides):

gggaatgcgagagtacccttggtgggagagggaagggctgagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaagggaaagaggtggtgcta cgacattcagagcaagcgagggaaaatactagtaaagacactgagcggagggggacatctg gaacgagagggaaaggggtacaagttagttaggaacgggttccatcttgcttcattcgggg gtaaaaaggaggaaatacaggattcaagtcacatagagaaagtaaacgggaaagacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttagttgtaggaatagccgggggctgtctc atagtgatagtagtcttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaaccagcggaaccggaggaacacgagatgagggatctcaggagaag SEQ ID NO:62 (ORF3 IRE-F58-12#13 nucleotides):

gggaatgcgagagtacccttggtgggagagggaagggcygagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaagggaaagaggtggtgcta cgayattsagagcaagcgagggaaaatactagtaaagacactgagcggagggggacatctg gaacgagagggaaaggggtacaagttagttaggaacgggttccatcttgcttcattcgggg - - gtaaaaargaggaaatacaggattcaagtcacatagagaaagtaaacgggaaagacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttrgttgtaggaatagccgggggctgtctc atagtgatagtagtcttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaaccagcggaaccggaggaacacgagatgagggatctcaggagaag

SEQ ID NO:63 (ORF3 IRE-F58-12#14 nucleotides):

gggaatgcgagagtacccttggtgggaaagggaagggctgagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaaggggaagaggtggtgcta cgatattgagagcaagcgagggaaagtactagtacagacactgagcggagggggacatctg gaacaagagggaaaggggtacaaattagttaggaacgggttccatcttgcttcattcgggg gtaaaaaagaggaaatacaggattcaagtcacatagagaaagtaaacgggaaagacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttagttgtaggaatagccgggggctgtctc atagtgatagtaaccttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaaccagcggaaccggaggaacacgagatgagggatctcaggagaag

SEQ ID NO:64 (ORF3 IRE-F187-16#23 nucleotides):

gggaatgcgagagtacccttggtgggaaagggaagggccgagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaaggggaagaggtggtgcta cgatattgagagcaagcgagggaaagtactagtacagacactgagcggagggggacatctg gaacaagagggaaaggggtacaaattagttaggaacgggttccatcttgcttcattcgggg gtaaaaaagaggaaatacaggattcaagtcacatagagaaagtaaacgggaaggacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttagttgtaggaatagccgggggctgtctc atagtgatagtaaccttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaactagcggaaccggaggaacacgagatgagggatctcaggagaag - -

SEQ ID NO:65 (ORF3 IRE-F196-16#3 nucleotides):

gggaatgcgagagtacccttggtgggagagggaagggctgagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaagggaaagaggtggtgcta cgatattgagagcaagcgagggaaaatactagtaaagacactgagcggagggggacatctg gaacgagagggaaaggggtacaagttagttaggaacgggttccatcttgcttcattcgggg gtaaaaaagaggaaatacaggattcaagtcacatagagaaagtaaacgggaaagacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttagttgtaggaatagccgggggctgtctc atagtgatagtagtcttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaaccagcggaaccggaggaacacgagatgagggatctcaggagaag

SEQ ID NO:66 (ORF3 IRE-F208-16#10 nucleotides):

SEQ ID NO:67 (ORF3 IRE-F208-16#1 nucleotides):

gggaatgcgagagtacccttggtgggaaagggaagggctgagaagatcgagctattcaatc agagcgccacatgtgggaagaaagagctaataataacgtggaaggggaagaggtggtgcta cgatattgagagcaagcgagggaaagtactagtacagacactgagcggagggggacatctg gaacaagagggaaaggggtacaaattagttaggaacgggttccatcttgcttcattcgggg gtaaaaaagaggaaatacaggattcaagtcacatagagaaagtaaacgggaaagacgcgat agttaagaaagggcaacacgtagaacatcttccaggggggaacgatttaattgttacagag ggcgggaacctgtgtgggaacgtggggttcttcgataatacgcagtgtacatacaacagtg - - ttataaatataggagggggaagtgttgacaattcacaagaagccaagaacgataaatcaaa cacaattgacaatttaattgatatgttacctttagttgtaggaatagccgggggctgtctc atagtgatagtaaccttatacttaacaattaaatactgtaaatgcaagaagaagaggacca acccagaaccagcggaaccggaggaacacgagatgagggatctcaggagaag

Claims

1. A method for determining the presence or absence of a piscine myocarditis virus infection, in a biological sample obtained from a cleaner fish, the method comprising: a) contacting nucleic acid from the biological sample with primers having a sequence defined in any one of SEQ ID NOs: l, 2, 10, 11, 12 or 13, a sequence comprising at least 10 consecutive nucleotides having a sequence selected from the group consisting of SEQ ID NOs: l, 2, 10, 11, 12 or 13, a sequence comprising at least 10 consecutive nucleotides having a complementary sequence to a sequence selected from the group consisting of SEQ ID NOs: l, 2, 10, 11, 12 or 13, homologues thereof or a sequence having at least about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%o, about 99%), or about 99.5%> sequence identity thereto; b) subjecting the nucleic acid and the primer to amplification conditions; and c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of nucleic acid associated with the piscine myocarditis virus infection in the sample.

2. The method according to claim 1 , wherein the cleaner fish is from the family

Cyclopteroidea or Labridae, preferably wherein the cleaner fish is a wrasse or lumpfish.

3. A kit for screening for a piscine myocarditis virus infection in a cleaner fish, comprising: a) one or more primers having a sequence defined in any one of SEQ ID NOs: 1, 2, 10, 11, 12 or 13; and optionally b) primers or adapters suitable to enable sequencing of the amplified nucleic acid and determination of the presence of the piscine myocarditis virus.

4. The kit according to claim 3, comprising: a) primers having a sequence defined in SEQ ID NOs: 1 and 2; and optionally b) a probe having a sequence defined in SEQ ID NO :3.

5. The kit according to claim 4, comprising primers having a sequence defined in SEQ ID NOs: 10 and 11.

6. The kit according to claim 4 or claim 5, comprising primers having a sequence defined in SEQ ID NOs: 12 and 13.

7. The kit according to any one of claims 4 to 6, wherein the cleaner fish is from the family Cyclopteroidea or Labridae, preferably wherein the cleaner fish is a wrasse or lumpfish.

8. An isolated or synthetic nucleic acid encoding a piscine myocarditis virus polypeptide for use in the treatment or prophylaxis against a piscine myocarditis virus in a cleaner fish.

9. The nucleic acid for use according to claim 8 having a sequence selected from the group consisting of SEQ ID NOs: 1-13 and 41-67, homologues thereof or a sequence having at least about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%), about 98%>, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs: l-13 and 41-67.

10. The nucleic acid for use according to claim 8 having a sequence selected from the group consisting of SEQ ID NOs:50-55 and 58-67, homologues thereof or a sequence having at least about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%), about 98%>, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs:50-55 and 58-67.

11. The nucleic acid for use according to claim 8 having a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-13 and 41-67, homologues thereof or a sequence having at least about 60%, about 75%, about 80%>, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs: 1-13 and 41-67.

12. The nucleic acid for use according to claim 8 having a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs:50-55 and 58-67, homologues thereof or a sequence having at least about 60%>, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs:50-55 and 58-67.

13. The nucleic acid for use according to any one of claims 8 to 12, wherein the nucleic acid is DNA, cDNA, RNA, or a combination of two or more thereof.

14. An isolated polypeptide encoded by the nucleic acid having the sequence defined in any one of claims 8 to 12, for use in the treatment or prophylaxis against a piscine myocarditis virus in a cleaner fish.

15. The isolated polypeptide according to claim 14, wherein the polypeptide has the sequence selected from the group consisting of SEQ ID NOs: 14-40, homologues thereof or a sequence having at least about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs: 14-40.

16. The isolated polypeptide according to claim 14, wherein the polypeptide has the sequence selected from the group consisting of SEQ ID NOs:23-28 and 31-40, homologues thereof or a sequence having at least about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to any of SEQ ID NOs: 23-28 and 31-40.

17. An isolated antibody that specifically binds to a polypeptide encoded by the nucleic acid having the sequence defined in any one of claims 8 to 13, or the polypeptide as defined in claim 8 or 9, for use in the treatment or prophylaxis against a piscine myocarditis virus in a cleaner fish.

18. The antibody for use according to claim 17, wherein the antibody binds to piscine myocarditis virus.

19. The antibody for use according to claim 17, wherein the antibody binds to piscine myocarditis virus polypeptide.

20. The antibody for use according to any one of claims 10 to 12, wherein the antibody inhibits, neutralizes or reduces the function or activity of the piscine myocarditis virus, or the piscine myocarditis virus polypeptide.

21. The antibody for use according to any one of claims 17 to 20, wherein the antibody is a polyclonal antibody, or a monoclonal antibody.

22. An immunogenic composition comprising a piscine myocarditis virus nucleic acid, wherein the nucleic acid comprises the nucleic acid having the sequence as defined in any one of claims 8 to 13.

23. The immunogenic composition according to claim 22, further comprising at least one other nucleic acid.

24. The immunogenic composition according to claim 22 or claim 23, further comprising at least one immunogenic polypeptide.

25. An immunogenic composition comprising a piscine myocarditis virus polypeptide, wherein the polypeptide is as defined in any one of claims 14 to 16.

26. The immunogenic composition according to claim 25, further comprising at least one other polypeptide.

27. The immunogenic composition according to claim 26, wherein the at least one other polypeptide is selected from the group consisting of: one or more polypeptides from a different micro-organism; one or more polypeptides from the same micro-organism; and one or more promiscuous T-cell epitopes.

28. The immunogenic composition according to claim 26 or claim 27, wherein the polypeptides are in admixture, or form a fusion protein.

29. The immunogenic composition according to any one of claims 25 to 28, further comprising at least one immunogenic nucleic acid.

30. The immunogenic composition according to any one of claims 22 to 29, further comprising at least one excipient, additive or adjuvant.

31. The immunogenic composition according to any one or claims 22 to 30, for use in the treatment or prophylaxis against a piscine myocarditis virus in a cleaner fish.

32. The nucleic acid, polypeptide, antibody or immunogenic compositions for use according to any one of claims 8 to 21, or 31, wherein the cleaner fish is from the family Cyclopteroidea or Labridae, preferably wherein the cleaner fish is a wrasse or lumpfish.

33. The nucleic acid, polypeptide or immunogenic composition for use according to any one of claims 8 to 16, 31 or 32, wherein the treatment induces an immune response in the fish.

34. The nucleic acid, polypeptide or immunogenic composition for use according to any one of claims 8 to 16, or 31 to 33, wherein the treatment prevents or reduces a piscine myocarditis virus infection in the fish.

35. The polypeptide or immunogenic composition for use according to any one of claims 14 to 16 or 31 to 34, wherein the polypeptide or immunogenic composition is administered orally, by immersion or by injection.

36. A method of inducing an immune response in a cleaner fish, the method comprising administering the nucleic acid defined in any one of claims 8 to 13, the polypeptide defined in any one of claims 14 to 16, or the immunogenic composition according to any one of claims 22 to 31.

37. The method according to claim 36, wherein the cleaner fish is from the family Cyclopteroidea or Labridae, preferably wherein the cleaner fish is a wrasse or lumpfish.

38. The method according to claim 36 or claim 37, wherein the method prevents or reduces a piscine myocarditis virus infection in the fish.

39. The method according to any one of claims 36 to 37, wherein the administration is orally, by immersion or by injection.

40. A method for determining the presence or absence of a piscine myocarditis virus infection in a biological sample obtained from a cleaner fish, the method comprising: a) contacting a biological sample with an antibody defined in any one of claims 17 to

21; and b) determining whether or not the antibody binds to an antigen in the biological sample, wherein binding indicates the presence of the piscine myocarditis virus infection in the biological sample.

41. A method for determining the presence or absence of a piscine myocarditis virus infection in a biological sample obtained from a cleaner fish, the method comprising determining whether a biological sample contains antibodies that specifically bind to a polypeptide defined in any one of claims 14 to 16.

42. A method of testing an agent for the prevention and/or treatment of a piscine myocarditis virus infection in a cleaner fish, comprising: a) contacting cells with the agent; b) contacting cells with piscine myocarditis virus; and c) measuring the number of cells infected with piscine myocarditis virus, wherein if the number of cells infected with the piscine myocarditis virus is decreased as a result of contact with the agent, the agent is a preventative and/or therapeutic agent for piscine myocarditis virus infection.

43. The method according to any one of claims 36 to 42, wherein the cleaner fish is from the family Cyclopteroidea or Labridae, preferably wherein the cleaner fish is a wrasse or lumpfish.