WO2015185605A1

WO2015185605A1 - New reovirus infecting rainbow trout

Info

Publication number: WO2015185605A1
Application number: PCT/EP2015/062346
Authority: WO
Inventors: Monika JANKOWSKA HJORTAAS; Øystein W. FINSTAD; Maria K. DAHLE; Espen Rimstad
Original assignee: Veterinærinstituttet
Priority date: 2014-06-03
Filing date: 2015-06-03
Publication date: 2015-12-10
Also published as: NO346439B1; NO20161983A1

Abstract

The present document is directed to a novel virus infecting fish, herein denoted virus Y and genomic nucleic acid sequences thereof. Further disclosed are methods and means for detecting the presence of virus Y in a sample and methods for treating and/or preventing virus Y infection.

Description

NEW REOVIRUS INFECTING RAINBOW TROUT

TECHNICAL FIELD

The present document is directed to a new double-stranded RNA virus herein denoted virus Y. The present document is further directed to methods and means for detection and diagnosis of said virus as well as the prevention and/or treatment of a viral infection caused by said virus.

BACKGROUND Salmonidae is a family of ray-finned fish, which is the only living family currently placed in the order Salmoniformes. These are also referred to as salmonids. Salmonidae includes salmon, trout, chars, freshwater whitefishes and graylings.

Rainbow trout Oncorhynchus mykiss (Walbaum) is native on the north pacific coast of America, and has long been a well-known farmed fish in most parts of the world, both in fresh and salt water. In Norway, the main production is in salt water in open net pens. The yearly production of approximately 70.000 tons is small compared to the large production of Norwegian Atlantic salmon which has past 1 million tons. According to FAO-FishStat, the global production of rainbow trout reached 850.000 tons in 2012, and is a growing industry in many parts of the world.

Rainbow trout is in general a robust fish with few diseases, and important disease problems have been solved with vaccination or eradication. In the fresh water stage in Norway there are cases with infectious pancreatic necrosis (IPN) and farms have experienced serious mortalities due to Flavobacterium psychrophilum. F. psychrophilum also causes problems after sea transfer to brackish locations. In salt water locations, the losses of rainbow trout to infectious disease problems are sparse compared to Atlantic salmon, even though eight outbreaks of pancreas disease (PD) were recorded on rainbow trout in Norway 2013.

Heart inflammation, as described here, has earlier not been observed on rainbow trout in the diagnostic services at the Norwegian Veterinary Laboratory (NVI). Findings of heart inflammation have though been published from rainbow trout in Canada that was used in an infectious trial with infectious salmon anaemia virus (ISAV) (MacWillians et al. 2007). Immunhistochemistry examination for ISAV did not reveal the cause of the heart inflammation in Canada and this heart pathology has not been observed in other ISA cases. Similar hearth inflammation is often seen in Atlantic salmon Salmo salar with the disease heart and skeletal muscle inflammation (HSMI) (Kongtorp et al. 2004) which has been associated with piscine orthoreovirus (PRV) (Palacios et al. 2010, Lovoll et al. 2012, Finstad et al. 2012). Piscine orthoreovirus has also been detected in other salmonids like rainbow trout, chum salmon (Oncorhynchus keta) and cutthroat trout (O. clarkia) in Canada, but the virus has not been associated with disease in these species.

The Reoviridae family are non-enveloped, icosahedral shaped, with 9-12 segments of linear dsRNA and are found in a wide range of hosts including insects, plants, birds, mammals and fish. Most reoviruses from fish are in the aquareovirus genus and their effects on fish health are not well documented. Some strains have been isolated from diseased fish in combination with other disease problems, and it is discussed whether reoviruses suppress the immune system, thereby making the fish more susceptible to other diseases or if they are the main cause of the disease. Diseases associated with reoviruses are not recorded from rainbow trout.

SUMMARY

The present inventors have found a new disease in Salmonidae, such as rainbow trout and salmon, and an associated virus, herein denoted virus Y. The present document describes this new disease in three rainbow trout smolt farms and in salmon. Gene sequences from a novel virus were detected in diseased fish using a PRV-primer set, and two new PCR methods were established for specific detection of the novel virus. The new methods have e.g. been used for mapping the distribution of the novel virus in the affected farms and some of the contact farms including the two brood fish farms.

The present document is thus directed to an isolated nucleic acid molecule comprising or consisting of at least one of a nucleic acid sequence according to any one of SEQ ID NO: 1 -55 or a variant thereof having at least 85% identity to any one of SEQ ID NO: 1 -55, or a sequence complementary to any one of SEQ ID NO: 1 -55 or a sequence having at least 85% identity thereto. The variant of the isolated nucleic acid molecule may e.g. have at least 90% identity to a sequence according to SEQ ID NO: 1 -55. The isolated nucleic acid sequence may e.g. be a nucleic acid sequence according to any one of SEQ ID NO: 1 -49 or 55, or SEQ ID NO: 38-49 or 55. The present document is also directed to a (isolated) nucleic acid fragment of an isolated nucleic acid molecule as defined above, said fragment comprising or consisting of at least 5 contiguous nucleic acid bases of a nucleic acid sequence as defined above. Such a nucleic acid fragment may be a nucleic acid primer or nucleic acid probe capable of detecting virus Y in a sample. Such a primer or probe may thus detect virus Y in a sample. A nucleic acid probe may in addition to a nucleic acid sequence comprise one or more label(s) for detection of said probe or a target sequence to which said probe is bound. An example of a nucleic acid probe comprises or consists of a nucleic acid sequence according to SEQ ID NO: 52 as the nucleic acid sequence part of the probe. The label in a nucleic acid probe may be a fluorescent label, such as MGBNFQ (Minor Groove Binding Non-Fluorescence Quencher), TAMRA (tetramethylrhodamine) and/or FAM 6-carboxyfluorescein.

The present document is also directed to an isolated RNA molecule having a sequence corresponding to the sequence of a nucleic acid molecule or fragment as defined herein, such as anyone of SEQ ID NO: 1 -55.

The present document is also directed to an isolated double-stranded RNA molecule comprising or consisting of an RNA sequence corresponding to a nucleic acid sequence having at least 85% identity, such as 95-100% identity, to any one of nucleic acid sequence(s) SEQ ID NO: 1 -55 and an RNA sequence complementary thereto.

The present document is also directed to a double-stranded RNA virus characterized in that it comprises an RNA sequence corresponding to a nucleic acid sequence having at least 85% identity, such as 95-100% identity, to one or more nucleic acid sequence(s) selected from SEQ ID NO: 1 -49 and 55, such as SEQ ID NO: 1 -37. The virus typically comprises an RNA sequence corresponding to a nucleic acid sequence having at least 85% identity to the sequence of nucleotide bases of all of the corresponding DNA sequences of SEQ ID NO: 1 -37 taken together, even if some of the sequences may be overlapping. Such a virus thus comprises RNA sequences corresponding to of all of SEQ ID NO: 1 -37 or sequences having at least 85% identity thereto. This virus is herein denoted virus Y. The virus may be isolated.

The present document is also directed to a vector comprising one or more nucleic acid molecule(s) and/or fragment(s) as defined herein, or one or more isolated RNA molecule(s) as defined herein. Also disclosed herein is a host cell comprising one or more nucleic acid molecule(s) and/or fragment(s) as defined herein, one or more isolated RNA molecule(s) as defined herein, or a vector as defined herein. The present document is also directed to a polypeptide encoded by a consecutive string of at least 12 nucleic acid bases of a nucleic acid molecule or fragment as defined herein or an isolated RNA molecule as defined herein, or a nucleic acid reverse complementary thereto. Also disclosed herein is an antigen comprising such a polypeptide. Also disclosed is an antibody specifically directed to such an antigen.

The present document is also directed to the use of a nucleic acid molecule or fragment thereof as defined herein as a probe for detecting the presence of a virus, such as virus Y, or diagnosing a viral infection, such as virus Y infection, in a sample, such as a sample from a salmonid, such as rainbow trout or salmon. In situ hybridization or polymerase chain reaction may e.g. be used for such detection or diagnosis.

The present document is also directed to a method for detecting a virus, such as virus Y, said method comprising detecting at least 5 consecutive nucleic acid bases of a nucleic acid sequence according to any one of SEQ ID NO: 1 -55, or a sequence having at least 85% identity thereto, or a sequence complementary to any one of SEQ ID NO: 1 -55 or a sequence having at least 85% identity thereto. Such a method may comprise performing a polymerase chain reaction or in situ hybridisation.

The present document is also directed to a method for detecting the presence of a virus, such as virus Y, and/or diagnosing a viral infection, such as virus Y infection, in a sample, said method comprising the steps of:

a) contacting the sample with a nucleic acid fragment as defined herein or an antibody as defined herein;

b) detecting the formation of a complex between a virus Y specific nucleic acid or polypeptide, respectively, and said nucleic acid fragment or antibody, respectively,

wherein the presence of a complex indicates the presence of a virus and/or a viral infection in said sample.

For all aspects of the present document, the methods and means for detecting virus Y in a sample, the sample may be a biological sample, such as a tissue sample from fish, such as a salmonid, such as rainbow trout or salmon. A biological sample may e.g. be a blood sample and/or a tissue sample from internal organs such as heart, liver, kidney, spleen, pancreas, pylorus or skeletal musculature. The sample may also be a non-biological sample, such as a water sample. A method for detecting the presence of a virus, such as virus Y, and/or diagnosing a viral infection, such as virus Y infection, in a sample, may be performed ex vivo.

The present document is also directed to a diagnostic kit for diagnosing a viral infection, such as virus Y infection, in a subject, said kit comprising one or more nucleic acid fragment(s) as defined herein, a polypeptide as defined herein, an antigen as defined herein and/or an antibody as defined herein and reagents for performing a diagnosis, and/or optionally instructions for use.

The present document is also directed to a pharmaceutical composition comprising one or more of 1 ) a nucleic acid molecule as defined herein, 2) a nucleic acid fragment as defined herein, 3) an RNA molecule as defined herein, 4) a virus as defined herein, 5) a vector as defined herein, 6) a host cell as defined herein, 7) a polypeptide as defined herein, 8) an antigen as defined herein, and/or 9) an antibody as defined herein.

The present document is also directed to 1 ) a nucleic acid molecule as defined herein, 2) a nucleic acid fragment as defined herein, 3) an RNA molecule as defined herein, 4) a virus as defined herein, 5) a vector as defined herein, 6) a host cell as defined herein, 7) a polypeptide as defined herein, 8) an antigen as defined herein, and/or 9) an antibody as defined herein for medical use.

The present document is also directed to a pharmaceutical composition as defined herein for use in the prevention and/or treatment of a viral infection, such as a virus Y infection, such as a virus Y infection in a salmonid, such as rainbow trout or salmon. The present document is also directed to such a pharmaceutical composition as defined herein for use as a vaccine.

The present document is also directed to 1 ) a nucleic acid molecule as defined herein, 2) a nucleic acid fragment as defined herein, 3) an RNA molecule as defined herein, 4) a virus as defined herein, 5) a vector as defined herein, 6) a host cell as defined herein, 7) a polypeptide as defined herein, 8) an antigen as defined herein, and/or 9) an antibody as defined herein for use in the prevention and/or treatment of a viral infection, such as a virus Y infection, such as a virus Y infection in a salmonid, such as rainbow trout or salmon.

The present document is also directed to the use of 1 ) a nucleic acid molecule as defined herein, 2) a nucleic acid fragment as defined herein, 3) an RNA molecule as defined herein, 4) a virus as defined herein, 5) a vector as defined herein, 6) a host cell as defined herein, 7) a polypeptide as defined herein, 8) an antigen as defined herein, and/or 9) an antibody as defined herein for the preparation of a medicament for the prevention and/or treatment a viral infection, such as a virus Y infection.

The present document is also directed to a method for preventing and/or treating a viral infection, such as a virus Y infection, in a subject, such as a fish, such as a salmonid, such as rainbow trout or salmon, said method comprising administering a pharmaceutically effective amount of 1 ) a nucleic acid molecule as defined herein, 2) a nucleic acid fragment as defined herein, 3) an RNA molecule as defined herein, 4) a virus as defined herein, 5) a vector as defined herein, 6) a host cell as defined herein, 7) a polypeptide as defined herein, 8) an antigen as defined herein, 9) an antibody as defined herein and/or 10) a pharmaceutical composition as defined herein, to said subject. The administration may take place via intraperitoneal injection, dip vaccination, bath vaccination and/or by oral vaccination.

Other features and advantages of the invention will be apparent from the following detailed description, drawings, examples, and from the claims.

DEFINITIONS

As used herein, the term "nucleic acid sequence", "nucleic acid molecule", "nucleic acid" and the like refers to a polynucleotide molecule (DNA - deoxyribonucleic acid, or RNA - ribonucleic acid) comprising a string of nucleic acid bases. These nucleic acid bases are "A" (adenine), "T" (thymidine)/"U" (uracil), "C" (cytidine) and "G" (guanidine). In RNA, "T" is replaced with "U". DNA or RNA may be single-stranded or double-stranded. By an RNA sequence "corresponding to" a nucleic acid sequence expressed herein as a DNA sequence, the same nucleic acid sequence but wherein "T" is replaced by "U" to get the corresponding RNA sequence is intended. The term, "nucleic acid" may comprise both DNA and/or RNA sequences unless one or the other is specifically referred to.

cDNA (complementary DNA) can be produced by reverse transcription of RNA.

The genomic nucleic acid sequences (sequences based thereupon) disclosed in this document are genomic RNA sequences of virus Y written using the DNA code. These genomic nucleic acid sequences have been obtained by reverse transcription of the genomic RNA and next generation sequencing. Thus, these sequences, which have been produced by reverse transcription of virus RNA are not naturally occurring. The same is true for any primer or probe sequences based upon these genomic RNA sequences written using the DNA code. As used herein in connection with nucleic acid molecules (DNA and RNA molecules) and polypeptides, the term "isolated" means that the molecule or polypeptide has been removed from its original environment. This means that a nucleic acid molecule or polypeptide when present in a living organism is not "isolated". Breaking of chemical bonds and/or by other means separating the sequence from its natural environment means that the nucleic acid molecule or polypeptide is "isolated".

The term "identity" is in the context of the present document intended to describe the extent to which two (nucleic or amino acid) sequences have the same residues at the same positions in an alignment, expressed as a percentage. A local algorithm program may be used to determine sequence identity. Local algorithm programs, (such as Smith Waterman) compare a subsequence in one sequence with a subsequence in a second sequence, and find the combination of subsequences and the alignment of those subsequences, which yields the highest overall similarity score. Internal gaps, if allowed, are penalized. Local algorithms work well for comparing two multidomain proteins, which have a single domain or just a binding site in common. Methods to determine identity and similarity are codified in publicly available programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J et al. (1994)) BLASTP, BLASTN, and FASTA (Altschul, S.F. et al (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S.F. et al. (1990)). Each sequence analysis program has a default scoring matrix and default gap penalties. In general, a molecular biologist would be expected to use the default settings established by the software program used. By e.g. a sequence having 95 % identity it is intended that the amino acid or nucleotide sequence is identical to the reference sequence, except that the amino acid/nucleotide sequence may include up to 5 point mutations per each 100 amino acids or nucleotides of the reference amino acid/nucleotide sequence. In other words, to obtain an amino acid/nucleotide sequence having at least 95% identity to a reference sequence up to 5% of the amino acids/nucleotides in the reference sequence may be deleted or substituted with another amino acid/nucleotide, or a number of amino acids/nucleotides up to 5% of the total number of amino acids/nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the terminal positions of the reference amino acid or nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids or nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. By "variant thereof" or "variants thereof" and the like , as used in the present document, a nucleic acid or polypeptide sequence(s) is intended, having an identity to a specified nucleic acid or polypeptide sequence of at least 85% or at least 90%, such as 85-100%, 86-100%, 87-100%, 89-100%, 90-100%, 91 -100%, 92-100%, 93-100%, 94-100%, 95- 5 100%, 96-100%, 97-100%, 98-100%, 99-100% or about 100%.

A "probe", such as a DNA probe, refers to an isolated nucleic acid sequence capable of hybridizing to an, at least partially, complementary nucleic acid sequence. A probe often contains a label allowing detection of the complex formed between the probe and the target nucleic acid sequence. Examples of such probes include, but are not limited to0 radioactive probes, fluorescent agents, chemiluminescent agents, enzyme substrates and enzymes. Further information regarding the use and choice of labels can e.g. be found in Sambrook et al. Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al. Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-lntersciences (1987).

5 A double-stranded nucleic acid molecule (DNA or RNA molecule) consists of two "complementary" nucleic acid strands. Generally, "A" (adenine) is complementary to "T" (thymidine) in a DNA molecule and "U" (uracil) in an RNA molecule, while "C" (cytidine) is complementary to "G" (guanidine). A thus binds to T and G to C via hydrogen bonds. If in a double-stranded DNA molecule one strand reads "5^'-ACGCT-3" its "complementary"0 strand reads '"3^'-TGCGA^'-5". As used herein, the terms "complement" "complementarity", "complementary" and the like, are thus used to describe single-stranded polynucleotides related by the rules of antiparallel base-pairing. A "reverse complementary" strand and the like expressions refers to a DNA sequence read in the reverse direction on the opposite strand, e.g. the reverse complementary strain to the sequence 5'-ATGC-3' is 5'-GCAT-5 3'. Complementarity may be "partial" where the base pairing is less than 100%, or it may be "complete" or "total," implying perfect 100% antiparallel complementation between the two polynucleotides. By convention in the art, single-stranded nucleic acid molecules are written with their 5' ends to the left, and their 3' ends to the right.

A "vector" is a DNA or RNA molecule used to carry foreign material to a cell. A vector0 typically contains sequences for its replication in a host cell and one or more transgene(s).

It may also contain one or more promoter sequence for the expression of inserted genes and/or sequence regulating transcription and/or translation. Vectors are typically inserted into their target cells (host cells) by transformation (for bacterial cells, transfection (for eukaryotic cells) or transduction (often used terminology when a viral vector is inserted into a host cell). Viral vectors generally have a modified viral DNA or RNA rendering them non-infectious.

As used herein, a "host cell" includes an individual cell or cell culture which can be or has been a recipient of any vector of this document. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected with a vector comprising a nucleic acid of the present document. Host cells may be prokaryotic or eukaryotic cells.

By "polypeptide" is herein intended a string of amino acid bases linked by a covalent peptide (amide) bond between the carboxyl group of one amino acid to an amino group on the adjacent amino acid. Amino acid sequences are usually expressed with their N- terminal to the left and the carboxy-terminal on the right. A polypeptide is generally shorter than a "protein" which latter term is usually used for polypeptides being longer than 50 amino acids. Herein, these two terms may be used interchangeably independently of the length of the amino acid string.

The term "antigen," as used herein, refers to any agent that is recognized by an antibody, while the term "immunogen" refers to any agent that can elicit an immunological response in a subject. The terms "antigen" and "immunogen" both encompass, but are not limited to, polypeptides. In most, but not all cases, antigens are also immunogens.

The term "antibody" is directed to an immunoglobulin molecule and immunologically active parts (fragments) of such immunoglobulin molecules. An antibody is capable of binding an antigen. Natural antibodies are Y-shaped protein molecules containing two each of a heavy chain and a light chain connected with each other by disulfide bonds. Although the overall structure of different antibodies is very similar, the tip of the antibody is highly variable allowing different antibodies to recognize different kinds of antigens. Antibodies are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. Antibodies may be coupled to labels, such as fluorescent, chemiluminescent or enzymatic labels, which allow their use for detection of certain antigens in situ and ex situ and diagnosis of disease.

A vaccine or a vaccine composition, as mentioned herein, is intended to refer to a composition resulting in immunological prophylaxis and/or stimulation in a subject to which the vaccine is administered. A vaccine composition induces an immune response and thus a long-acting immunity, to a specific antigen. In the present context, an antigen is mainly intended to refer to an inactivated form of virus Y, or parts or fragments thereof, or DNA sequences obtained from virus Y by reverse transcription, or parts or fragments thereof which are still capable of generating an immune response in a subject.

PCR (polymerase chain reaction) is a method for amplification of nucleic acid molecules. The PCR reaction is well-known to the person skilled in the art and involves contacting a sample with a pair of so called oligonucleotide primers (one forward and one reverse primer) under conditions allowing the hybridization between the primers and a target (template) sequence having complementarity to the primers and which target sequence possibly is present in the sample in order to amplify the target sequence.

BRIEF DESCRIPTION OF DRAWING

Fig. 1. Overview of infected farms. The novel virus was detected in all seven farms. Disease was seen in farms A-E. Arrows show movement of eggs or fish. DETAILED DESCRIPTION

The present document is related to a new disease in Salmonidae (salmonids) such as rainbow trout or salmon, and infection by a novel virus (herein referred to as virus Y). Different observations indicate the new disease as an infectious disease. It spreads between tanks, and from fish to fish in the tank. Clinical signs last some days before the mortality starts, and the pathological findings show inflammation in several organs pointing towards a viremia. Liver necrosis is often seen in fish with circulatory failure and is possibly secondary due to heart and circulatory failure. In this case, the degenerative changes seemed to travel along the sinusoids, which also could indicate a haematogenious spread of an agent. The pathological examination revealed an inflammatory reaction involving neutrophils.

The disease was observed in three fresh water (salinity < 1%₀) smolt farms on the west coast of Norway in the fall 2013. The fish of 30-100 g showed clinical signs correlated to circulation failure, anemia and ascites. The unique histopathological changes were dominated by inflammation of the heart and necrosis in the liver. The disease is therefore characterized by circulation failure, anemia, ascites, liver necrosis and/or heart inflammation. Moderate to high mortalities were observed. All three farms received eggs of fry from the same brood fish farms. Mortality and disease was also observed up to four months after sea water transfer. Extended microbiological examination did not reveal presence of any known pathogens. Gene sequences from a novel virus was detected using a primer set aimed for detection of piscine orthoreovirus (PRV) and the obtained sequence showed 85% identity to a part of segment S1 of PRV. Two PCR methods were developed for specific detection of the novel virus.

The present document is thus directed to a new reovirus herein denoted virus Y. Virus Y has a linear double-stranded (ds) RNA genome comprising at least 10 segments denoted L1 -L3, M1 -M3 and S1 -S4. Genomic RNA has been reverse transcribed from these segments, and thereafter sequenced. The genomic nucleic acid sequences which are recited in SEQ ID NO: 1 -37 and 55 as DNA sequences thus correspond to the genomic RNA sequences but written using the DNA code. Thus, the sequences presented in the sequence listing are not naturally occurring. Sequence fragments of L1 are listed in SEQ ID NO: 1 -6, sequence fragments of L2 in SEQ ID NO: 7-12, sequence fragments of L3 in SEQ ID NO: 13-17, sequence fragments of M1 in SEQ ID NO: 18-21 , sequence fragments of M2 in SEQ ID NO: 22-24, sequence fragments of M3 in SEQ ID NO: 25-26, sequence fragments of S1 in SEQ ID NO: 27-32 and 55, sequence fragments of S2 in SEQ ID NO: 33-34, sequence fragments of S3 in SEQ ID NO: 35-36, and sequence fragments of S4 in SEQ ID NO: 37. Altogether this corresponds to approximately 50% of the virus Y genome being sequenced. SEQ ID NO: 38-49 represent parts of the virus Y genome (again written using the DNA code of the corresponding RNA sequences of the virus) which have a lower identity to its closest relative PRV (piscine reovirus) and which are therefore more unique for the novel virus Y. SEQ ID NO: 50-54 discloses exemplary primer and probe sequences based on sequence parts of SEQ ID NO: 1 -37. In the below, the virus Y genome may be referred to by referring to SEQ ID NO: 1 -55. When the genomic sequences are referred to, it is in the present context intended to refer to the corresponding RNA sequences, even if this is not always explicitly mentioned. It is to be noted that some of the sequences presented in SEQ ID NO: 1 -37 may partially overlap with each other.

Virus Y typically infects fish, such as salmonids, such as rainbow trout or salmon, but the substances (such as nucleic acids and polypeptides etc.) and methods disclosed herein may be used for analysing the presence or absence of virus Y in any kind of sample. Typically, the sample is a biological sample, even though it may also e.g. be a water sample. The sequences of virus Y provided in the present document may e.g. be used in methods for detecting the presence of virus Y in a sample or for the diagnosis of a virus Y infection, e.g. by the construction of primers, probes, antigens, and/or antibodies based on these sequences. It also allows for the development of vaccines (such as DNA or polypeptide based vaccines) for the prevention and/or treatment of virus Y infection. Further, the isolated virus Y genetic material may be used as a vector for insertion of genetic material into a subject.

The present document is therefore directed to an isolated nucleic acid molecule 5 comprising or consisting of a nucleic acid sequence according to any one of SEQ ID NO:

1 -55 or a variant thereof having at least 85% or at least 90% identity, such as 85-100%, 86-100%, 87-100%, 89-100%, 90-100%, 91 -100%, 92-100%, 93-100%, 94-100%, 95- 100%, 96-100%, 97-100%, 98-100%, 99-100% or about 100% identity, to a nucleic acid sequence of SEQ ID NO: 1 -55, or a sequence complementary to any of these sequences.

10 Unless something else is explicitly mentioned herein, whenever a nucleic acid sequence or fragment or part thereof is referred to in this document, this is also intended to include a nucleic acid variant having at least 85% or at least 90% identity, such as 85-100%, 86- 100%, 87-100%, 89-100%, 90-100%, 91 -100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%, 99-100% or about 100% identity, such as about 85%, 86%,

15 87% 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to such a nucleic acid sequence.

The present document is also directed to a nucleic acid fragment of an isolated nucleic acid molecule as disclosed herein, wherein said fragment comprises or consists of at least 5, such as about 5-782, 5-500, 5-400, 5-300, 5-200 or 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 20 5-40, 5-35, 5-30, 5-25, 5-20, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 15-100, 15-80, 15- 70, 15-60, 15-50, 15-40, 15-30, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, or 30- 40 contiguous nucleic acid bases of a nucleic acid sequence of any one of SEQ ID NO: 1 - 55 or a variant thereof, or a sequence complementary thereto.

A nucleic acid fragment of a sequence according to SEQ ID NO: 1 -55 as defined herein 25 may e.g. be used as a primer or a probe for detecting virus Y in a sample. The present document is therefore also directed to a primer or a probe comprising or consisting of at least 5, such as about 5-782, 5-500, 5-400, 5-300, 5-200 or 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 15-100, 15- 80, 15-70, 15-60, 15-50, 15-40, 15-35, 15-30, 18-30, 20-100, 20-80, 20-70, 20-60, 20-50, 30 20-40, 20-30, or 30-40, such as such as 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35, contiguous nucleic acid bases of a nucleic acid sequence of SEQ ID NO: 1 -55 or a variant thereof, or a sequence complementary thereto. Such a primer or probe is capable of (specifically) detecting virus Y in a sample. By "specifically detecting" is intended that the primer or probe allows the detection of virus Y without any cross- reactivity with other known piscine viruses, such as PRV.

The present document is thus also directed to a nucleic acid probe (a DNA or an RNA probe), the nucleic acid of which probe comprises or consists of at least 5, at least 7, at least 10, at least 12, at least 15, such as 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 15-100, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, or 30-40, such as 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 contiguous nucleic acid bases of a nucleic acid sequence according to SEQ ID NO: 1 -55 or a sequence complementary thereto (or an RNA sequence corresponding thereto) or a variant thereof. A specific example of a probe sequence suitable for detection of virus Y is SEQ ID NO: 52. A probe is generally at least 15 nucleic acid bases long, such as about 15-30 nucleic acids long, but it may be shorter. DNA and RNA probes are herein collectively denoted probes or nucleic acid probes. A probe in accordance with the present document may be constructed having high specificity, i.e. a large sequence identity, to a target sequence to allow for a specific detection of a target sequence, or it may be constructed with a lower sequence identity to a target sequence to allow for detection of a target sequence having a lower sequence identity to the probe. The specificity of a probe may be affected by the length of the probe. Probe specificity may also be affected by the conditions used for hybridization, such as salt concentration, temperature and pH. A person skilled in the art knows how to elaborate with these so called stringency conditions used for hybridization to affect probe-target sequence complex formation. By increasing the pH and/ temperature and/or lowering the salt (sodium ion) concentration, the hybridization conditions provide for a higher stringency, i.e. rendering the formation between a probe and a target sequence more difficult. The person skilled in the art also knows how to elaborate with sequence specificity to a target sequence in order to obtain a primer and/or probe having the desired sequence specificity.

A probe according to the present document generally contains one or more labels which allow the detection of the probe and optionally one or more label(s) for detection of said probe. Examples of such labels include, but are not limited to radioactive labels, fluorescent agents, chemiluminescent agents, enzyme substrates and enzymes. Further information regarding the use and choice of labels can e.g. be found in Sambrook et al. Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al. Current Protocols in Molecular Biology, Green Publishing Associates and Wiley-lntersciences (1987). A radioactive label may e.g. be ¹⁴C, ³²P, ³⁵S, ³H or ¹⁵0, which may be detected using suitable radiation detection means. A fluorescent label may e.g. be a fluorescent dye, such as rhodamine, SYBR Green, fluorescein, thiazole orange, FAM, FAM 6-carboxyfluorescein, fluorescein istothiocyanate (FITC), or TAMRA (tetramethylrhodamine). MGBNFQ (minor groove binding non-fluorescence quencher) may also be used in combination with a fluorophore to increase the specificity of a shorter probe. The label may also be a chemiluminescent agent, an enzyme substrate and/or an enzyme, such as b-galactosidase, horseradish peroxidase, streptavidin, biotin or digoxigenin.

Examples of methods where probes may be used include, but are not limited to, in situ hybridization, analysis of nucleic acid fragments on gels, real time PCR, digital PCR etc.

Methods where probes are used generally involve denaturing double-stranded nucleic acids (i.e. separating the two nucleic acid strands from each other) in a sample, allowing the probe to bind, wash off any unbound probe and detecting the formation of a probe- target sequence complex. However, this is not always the case. For example, during real time PCR, the probe binds to the target sequence, and is then fragmented by the Taq polymerase during the elongation step. Thus, in real time PCR there is no washing off of the unbound probe.

The nucleic acid molecules disclosed herein or variants thereof may also be used for the construction of primers for a PCR reaction. The person skilled in the art is well acquainted with how such primers are to be prepared. A primer is typically about 15-35 bases long, such as about 18-30, 18-25 or 18-22 bases, e.g. 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases. It is generally considered that a primer has an optimal length of about 18-30 bases. Such a length ensures an adequate specificity of the primer while the primer is still sufficiently short to easily bind to the template nucleic acid. Examples of primers suitable for the detection and/or diagnosis of virus Y include, but are not limited to a primer according to SEQ ID NO: 50, 51 , 53 and 54. A primer may be constructed based on the sequence of any one of SEQ ID NO: 1 -55 or a sequence complementary thereto.

PCR (polymerase chain reaction) is a method for amplification of nucleic acid molecules, well-known to the person skilled in the art. In short, PCR involves contacting a sample with a pair of so called oligonucleotide primers (one forward and one reverse primer) under conditions allowing the hybridization between the primers and a target (template) nucleic acid sequence having complementarity to the primers (i.e. the formation of a complex between the respective primers and the target sequence). The target (template) nucleic acid may e.g. be virus Y genomic RNA. The primers are constructed to bind on the 3' side of the sense and antisense strands of the target sequence, respectively. Thereafter the primers are extended by using a polymerase, dissociated from the template, re-annealed, extended, dissociated in a number of cycles. The number of cycles may be adjusted depending on the amount of target sequenced present in the sample and the amount of copies needed but is typically 20-40 although it may be both higher and lower. If the target sequence was present in the sample, the PCR reaction will allow for the provision of a number of copies of it (the amplification product).

A PCR reaction may e.g. be used to amplify a nucleic acid sequence e.g. for its subsequent use in a cloning reaction wherein the amplified nucleic acid sequence is inserted into another nucleic acid molecule, such as a vector, or for its sequencing. A PCR reaction may also be used for analyzing a sample for the presence of a specific target sequence, as amplification of the sequence will only occur if the target sequence is present in the sample. The amplification product can be analyzed e.g. by electrophoresis, probe hybridization and/or sequencing.

PCR may also be made quantitative, so that the initial amount of a target nucleic acid in a sample, and consequently e.g. a virus containing this target sequence, can be quantified. PCR can be made quantitative (qPCR) and allow for real time measurement of the amplified product by the use of fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan.

Reverse Transcription PCR (RT-PCR) can be used for amplifying DNA from RNA. Reverse transcriptase reverses RNA into (c)DNA, which is then amplified by PCR. RT- PCR is e.g. used for studying expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript. It may also be used for sequencing RNA genomes. The genomic sequences of virus Y disclosed in the present document are prepared by reverse transcription of genomic RNA and written using the DNA code.

Only for exemplary purposes, the different steps of a PCR reaction may be performed in accordance with the below. Total RNA from a sample, such as a tissue sample possibly containing virus Y genomic RNA, is extracted, e.g. using QIAsymphony SP (QIAGEN, http://www.qiagen.com) in accordance with manufacturer's instructions. An appropriate set of primers is constructed based on the target sequence. PCR reactions may be conducted in a final volume of about 20 μΙ, e.g. with OneStep RT-PCR kit (QIAGEN). E.g. about five hundred ng total RNA and about 0.75 μΜ of each primer may be denatured (95°C/5 minutes) and mixed with 1 x RT-PCR buffer, 400 μΜ dNTPs, 4 U RNaseOUT and 1 μΙ OneStep RT-PCR enzyme mix. The cycling parameters in a PCR reaction may be as follows: cDNA synthesis 50 °C/30 min followed by 95°C/15 min and PCR: 40 cycles of 94°C/30s, 55°C/30s and 72°C/60 s. The final PCR products may be visualized by gel electrophoresis. PCR products of approximately right size may then be sequenced, e.g. by using the amplification primers and the ABI Prism Big Dye Terminator Cycle sequencing kit on ABI Genetic Analyser.

For example, conventional RT-PCR may be performed by amplification of a 423 bp fragment of the virus Y genome with primers 6F 5'-GAC-CAA-CAT-AAC-GTT-TCA-GGC- 3' (SEQ ID NO: 53) 6R 5'-ATC-CAA-CCA-CTA-AAA-CCG-AGA- 3' (SEQ ID NO: 54) using the protocol for RT-PCR as described above without the RNA denaturation step. One example of an rRT-PCR based method that may be used to analyse the presence of virus Y in a sample is a reaction wherein the forward primer is 5'- TCG-TGG-TTC-CAA- TGA-CAG-3' (SEQ ID NO: 50), the reverse primer is 5'- CCA-ACC-ACT-AAA-ACC-GAG- 3' (SEQ ID NO: 51 ) and the probe is 6-FAM-5'-ACG-CCT-TAG-AGA-CAA-CAT-GCG- AAG-3'-BHQ-1® (SEQ ID NO: 52-BHQ ®) for amplification and detection of a 121 bp fragment of the virus Y genome. In order to perform the method, the QIAGEN OneStep kit may be used. Reactions may be conducted in a final volume of about 20 μΙ with ROX passive reference dye RT-RARE 03 (Eurogentec http://www.eurogentec.com) and RNaseOUT (Invitrogen, www.lifetechnologies.com). About 500 ng of total RNA from fish displaying clinical signs of the novel disease may be mixed with 1 x RT-PCR buffer, 30 nM ROX, 400 μΜ dNTPs, 0.5 μΜ of each primer, 0.3 μΜ probe , 1 .25 mM MgCI₂, 4 U RNaseOUT and 0.8 μΙ OneStep RT-PCR enzyme mix. Amplifications may e.g. be performed on a Stratagene Mx3005P real-time machine (Agilent Technologies Inc, http://www.agilent.com) with the following conditions: 30 min at 50°C (reverse transcription), followed by 15 min at 95°C (RT inactivation and activation of Taq polymerase, 40 cycles of 94°C/30s, 55°C/30s and 72°C/30s. This method does not provide for false positives due to the presence of a selection of viruses infecting salmonids including PRV, ISAV, SAV, PMCV, VHSV, IHNV and IPNV.

The present document is also directed to an isolated RNA molecule corresponding to a nucleic acid molecule or fragment, or a variant thereof, as disclosed herein, or an RNA sequence complementary thereto. Such an isolated RNA sequence may be obtained by replacing the "T" residues with "U" residues in a nucleic acid sequence of the present document.

The present document is also directed to an isolated double-stranded RNA molecule comprising or consisting of an RNA sequence corresponding to a nucleic acid sequence having at least 85% or at least 90% identity, such as 85-100%, 86-100%, 87-100%, 89- 100%, 90-100%, 91 -100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%,

98- 100%, 99-100% or about 100% identity, to a nucleic acid sequence of any one of SEQ ID NO: 1 -55 and the RNA sequence complementary thereto.

The present document is further directed to a double-stranded RNA virus characterized in that it comprises an RNA sequence corresponding to a nucleic acid sequence having at least 85% or at least 90% identity, such as 85-100%, 86-100%, 87-100%, 89-100%, 90- 100%, 91 -100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%,

99- 100% or about 100% identity, to one or more of a nucleic acid sequence of SEQ ID NO: 1 -55, such as SEQ ID NO: 1 -37 or SEQ ID NO: 38-49. Such an RNA virus may comprise a nucleic acid sequence which has at least 85% or at least 90%, such as 85- 100%, 86-100%, 87-100%, 89-100%, 90-100%, 91 -100%, 92-100%, 93-100%, 94-100%, 95-100%, 96-100%, 97-100%, 98-100%, 99-100% or about 100% identity to the sequence of nucleotide bases of the corresponding DNA sequences of all of SEQ ID NO: 1 -37 taken together. As disclosed elsewhere herein, SEQ ID NO: 1 -37 may have sequence parts overlapping with each other. All of the RNA sequences corresponding to SEQ ID NO: 1 - 37 or a sequence having at least 85% identity to SEQ ID NO: 1 -37 may thus be found in the viral genome, even of some of the sequences are overlapping. The virus is herein denoted virus Y.

A nucleic acid molecule according to SEQ ID NO: 1 -49 or 55 or a fragment thereof, or variant(s) thereof, as disclosed herein, or an RNA sequence corresponding thereto, or sequences complementary thereto, may also be used as a vector for inserting foreign material in a cell. The present document is therefore directed to the use of a nucleic acid molecule according to SEQ ID NO: 1 -49 or 55 or a fragment thereof, or variant(s) thereof, as disclosed herein, or an RNA sequence corresponding thereto, or sequences complementary thereto, as a vector, such as a vector for inserting foreign material into. The present document is therefore also directed to a vector comprising a nucleic acid molecule or a fragment thereof, or variant thereof, as defined herein. For this purpose, one or more isolated part(s) or the whole of one or more segment(s) of virus Y or the whole of the isolated virus Y genome, or a corresponding DNA sequence thereof may be used. Also, it is possible to recombinantly assemble different parts of the isolated virus Y genome (or the corresponding DNA sequence) to construct a recombinant vector. Importantly, such a vector should not contain any infectiously harmful sequence(s). The vector may be used for carrying foreign material into a cell by inserting genetic material of interest into the vector. To accomplish this, the vector is preferably constructed to contain one or more multiple cloning sites allowing for specific opening of the vector to insert the genetic material of interest. The vector may also be constructed to contain sequences for expression of inserted genetic material, such as promoter sequences, ribosome binding sites and/or sequences regulating the translation of the inserted genetic material.

The present document is also directed to a host cell comprising one or more nucleic acid molecule(s) or fragment(s) thereof, or variant(s) thereof, as disclosed herein or a vector as disclosed herein. The host cell may be a prokaryotic or eukaryotic host cell. A host cell typically allows for the amplification and/or replication of the genetic material inserted (e.g. nucleic acid(s) and/or vector(s)) therein.

The nucleic acid molecule(s) or fragment(s) thereof, or variant(s) thereof, or an RNA molecule(s) or variant(s) thereof, as disclosed herein may also be used for the expression of polypeptide(s). The present document is therefore also directed to a polypeptide encoded by a consecutive string of at least 12 nucleic acid bases, such as about 12-782, 12-600, 12-500, 12-400, 12-300, or 12-200 nucleic acid bases, of a nucleic acid sequence according to SEQ ID NO: 1 -55 or a fragment thereof, or a variant thereof, or a sequence reverse complementary thereto. Such a polypeptide may e.g. be used as an antigen in order to prepare an antibody and/or it may be used to elicit an immune response in an organism. When a variant of a nucleic acid molecule or fragment thereof is used for producing a polypeptide in accordance with the present document, such a polypeptide may have a substantially the same biological activity as a polypeptide encoded by a sequence identical to SEQ ID NO: 1 -55 or a fragment thereof.

Also disclosed herein is an antigen comprising a polypeptide as defined herein. Such an antigen may be used for the preparation of an antibody capable of binding specifically to said antigen. Such an antibody may e.g. be used for the detection of a virus Y specific peptide in a sample. The antibody may in addition or alternatively also neutralize or reduce the function or activity the antigen (polypeptide). The present document is therefore also directed to an antibody specifically directed to an antigen as defined herein. The antibody may be e.g. a polyclonal antibody or a monoclonal antibody. The antibody may e.g. be a teleost antibody or a chimeric antibody. An antigen may also be used for the preparation of a vaccine composition used for eliciting an immune response to such an antigen. Such a vaccine composition may be used for the prevention and/or treatment of a viral infection, such as a virus Y infection.

As mentioned above, the nucleic acid molecule(s) disclosed in the present document or fragments thereof may be used as primers or probe(s), e.g. for detecting the presence of a virus Y nucleic acid in a sample and/or for diagnosing a viral infection in a subject, such as a virus Y infection. In particular in situ hybridization or polymerase chain reaction may be used for such detection and/or diagnosis. Also, an antibody as disclosed herein may be used for detecting the presence of a virus, such as virus Y, in a sample and/or diagnosing a viral infection, such as virus Y infection, in a subject. Such a detection of an antibody may be effected by labelling the antibody with a label, such as an enzymatic or fluorescent label. Commonly used labels for antibodies include, but are not limited to horseradish peroxidase, alkaline phosphatase and biotin.

In situ hybridization involves detecting a specific DNA or RNA nucleic acid in a sample, such as a tissue sample. The method generally comprise the steps of fixating the sample, allowing a probe to hybridize to complementary DNA or RNA in the sample, washing off unbound probe and thereafter detecting the formation of a complex between the probe and a target nucleic acid possibly present in the sample. A probe for use in an in situ hybridization reaction is preferably longer than a probe used for detection of a PCR amplified nucleic acid fragment, and is typically about 35 nucleic acid bases long.

Disclosed herein is also a method for detecting a virus, such as virus Y, wherein the method comprises detecting at least 5 consecutive nucleic acid bases of a nucleic acid sequence according to any one of SEQ ID NO: 1 -55, or a variant thereof, or a sequence complementary thereto. For example at least 5, at least 7, at least 10, at least 12, at least 15, such as 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 10-80, 10- 70, 10-60, 10-50, 10-40, 10-30, 15-100, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, or 20-40, such as 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 consecutive nucleic acid bases may be detected. Methods suitable for use in such a method include, but are not limited to, polymerase chain reaction (PCR) or in situ hybridisation (both being disclosed in further detail elsewhere herein.

The present document is also directed to a method for detecting the presence of a virus, such as virus Y, and/or diagnosing a viral infection, such as virus Y infection, in a sample, such as a biological sample, said method comprising the steps of:

a) contacting the sample with a nucleic acid molecule according to any one of

SEQ ID NO: 1 -55 or a nucleic acid fragment thereof, or a variant thereof, as defined herein, a nucleic acid probe as defined herein, or an antibody as defined herein; b) detecting the formation of a complex between a virus Y specific nucleic acid sequence or polypeptide, respectively, and said nucleic acid molecule or fragment thereof, nucleic acid probe or antibody, respectively.

The sample may contain virus Y specific DNA, RNA and/or polypeptides/proteins.

It is herein demonstrated that blood is suitable for detection of the virus Y. In all aspects of the present document, a biological sample may thus be a blood sample. However, a biological sample may also be a tissue a sample from any tissue including, but not limited to internal organs such as heart, liver, kidney, spleen, pancreas, pylorus or skeletal musculature. In all aspects of the present document a tissue or blood sample may e.g. be from fish, such as a salmonid, such as rainbow trout or salmon. A method according to the present document for detecting virus Y or diagnosing a virus Y infection is typically performed ex vivo. In an ex vivo method, the biological sample is first isolated from the organism to be tested before the analyzing the presence or absence of a virus Y. The sample to be tested for the presence of virus Y may also be a non-biological sample, such as a water sample.

The present document is also directed to a diagnostic kit for diagnosing a viral infection, such as virus Y infection, in a subject, such as a fish, such as a salmonid, such as rainbow trout or salmon, said kit comprising one or more nucleic acid molecule(s) according to SEQ ID NO: 1 -55 and/or a nucleic acid fragment(s) thereof as defined herein, or variant(s) thereof, a nucleic acid probe as defined herein, a polypeptide as defined herein, a primer as defined herein, an antigen as defined herein and/or an antibody as defined herein and reagents for performing a diagnosis, and/or optionally instructions for use.

The methods provided in the present document may also be useful in epidemiological studies to reveal both the geographical distribution and investigate material from earlier years to possibly detect the source of the novel virus.

Also disclosed herein is a pharmaceutical composition comprising a nucleic acid molecule or a variant thereof, a nucleic acid fragment or variant thereof, a polypeptide, antigen, vector, host cell and/or antibody as disclosed herein. Such a pharmaceutical composition may be used as a medicine, such as a vaccine composition, e.g. for the prevention and/or treatment of a viral infection, such as a virus Y infection, such as a virus Y infection in a salmonid, such as rainbow trout or salmon. The present document is therefore also directed to a nucleic acid molecule or variant thereof, a nucleic acid fragment or variant thereof, a polypeptide, an antigen, a vector, a host cell, an antibody and/or a pharmaceutical composition as disclosed herein for use in (for) the prevention and/or treatment a viral infection, such as a virus Y infection, such as a virus Y infection in a salmonid, such as rainbow trout or salmon.

A pharmaceutical composition according to the present document may also comprise one or more adjuvant(s) (such as a mineral oil, muramyldipeptides, lipopolysaccharides, glucans and Carbopol®), pharmaceutically acceptable excipients, carrier(s), emulgator(s) etc. Liquid carriers include, but are not limited to water, petroleum, plant and animal oils, such as peanut oil, mineral oil, soybean oil, or sesame oil, and synthetic oils. A liquid compositions may also comprise physiological saline solution, saccharide solutions (e.g. dextrose), glycols (e.g. ethylene glycol, propylene glycol, or polyethylene glycol. The active component of a pharmaceutical composition as disclosed herein may constitute about 0.5 to 90% by weight of the pharmaceutical composition. Methods and means for preparing a vaccine composition suitable for storage are well known for the skilled practitioner within this field.

Vaccine components may be in liquid form both as hydrophilic and lipophilic, which phased may often then be mixed in emulsions that need to be stabilized for storage. Examples of vaccine preparations suitable for vaccination of fish may be found in Roar Gudding (Editor) et al. "Fish Vaccinology", Developments in Biological Standardization, 484 pages.

In addition, dry vaccines may also be prepared which are dissolved before use. Such vaccines are particularly useful for dip, bath or oral vaccines that are not using oil adjuvants or the like.

Further disclosed herein is the use of a nucleic acid molecule or a variant thereof, a nucleic acid fragment or variant thereof, polypeptide, antigen, vector, host cell and/or antibody as disclosed herein in the preparation of a medicament for the prevention and/or treatment a viral infection, such as a virus Y infection, such as a virus Y infection in a salmonid, such as rainbow trout or salmon.

The present document is also directed to a method for preventing and/or treating a viral infection, such as a virus Y infection, in a subject, such as a fish, such as a salmonid, such as rainbow trout or salmon, said method comprising administering a pharmaceutically effective amount of a nucleic acid molecule or a variant thereof, a nucleic acid fragment or variant thereof, polypeptide, antigen, vector, host cell, antibody and/or a pharmaceutical composition as defined herein to said subject. Said subject may be a fish, such as a salmonid, such as rainbow trout or salmon. The administration of such a composition may take place by any suitable means such as by intraperitoneal injection, immersion vaccination (bath vaccination or dip vaccination) and/or by oral vaccination.

The nucleic acid, polypeptide sequences and viruses disclosed herein may be isolated.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXPERIMENTAL SECTION

Example 1 : Identification and pathology of virus Y

Materials and methods

Study material

Samples from seven farms are included in this study and Figure 1 provides an overview of movement of eggs and fish between the farms. Fish and eggs from the brood fish and smolt farms have also been moved to other locations not included in this study. The first samples came from both diseased and clinically healthy fish at the three smolt farms (A, B, C) in the fall 2013. Later samples were taken from clinically healthy fish at the brood fish farms. After sea transfer in the spring 2014 samples were provided from diseased and healthy fish at the sea farms D and E. Number of samplings per farm were 3 to 5 and total number of fish sampled per farm varied from 20 to 100. Samples were delivered to the NVI, either as whole fish on ice or as tissue samples on formalin, RNA/ater ^® or transport medium (Eagle's Minimum Essential Medium with 10% new-born bovine serum and 100 ml^"1 gentamicin). In some cases blood samples were received either on EDTA or heparin containers. From farm C hearth samples were also fixated in a glutaraldehyd solution for electron microscopic examination.

Histopathology. Gill, heart, liver, spleen, mid kidney, pancreatic tissue, pylorus and skeletal musculature were sampled and fixed in 10% neutral buffered formalin, embedded in paraffin and routinely processed. The sections, 3-4 μηι, were stained by haematoxylin and eosin (H&E) and studied by light microscopy. Immunohistochemistry. A selection of heart, kidney and spleen with lesions from fish in all three smolt farms were tested for Flavbacterium psychrophilum antigen with the enzyme-labelled (alkaline phosphatase) streptavidin procedure, with fast red as the chromogen. A polyclonal rabbit antiserum raised against F. psychrophilum serovar Th was used (Evensen a & Lorenzen 1996).

Electron microscopy. Hearth tissues from affected fish at farm C were fixated in a 1 .25% glutaraldehyd and 2% formaldehyde PBS solution for a minimum of 24 hours. Tissue were then washed several times in PBS and cacodylate buffer (0.1 M, pH6.8) previous to post fixation with Osmium (Os04) in 1 hour and washed again in cacodylate buffer. Dehydration in an ethanol series (70, 90, 96, 100%) was then performed before embedding in LR-White resin (Electron Microscopy Sciences) and ultra sections were made. Sections were stained with 4% aqueous uranyl acetate and 1 % potassium permanganate (KMN04) for 10 min and then washed in distilled water before examined and photographed in the transmission electron microscopy (TEM, FEI MORGAGNI 268). Bacteriology. Four to 20 samples from kidney of diseased fish from farms A, B and C were inoculated onto blood agar as well as Anacker and Ordal's medium and incubated at 22^°C and 15^°C respectively for 7 d.

Real time RT-PCR (rRT-PCR). Samples were screened by rRT-PCR for the detection of a range of RNA viruses capable of infecting salmonids. A minimum of three diseased fish from farms with clinical disease (farm A, B, C, E, F) were tested for piscine reovirus (PRV), infectious salmon anaemia virus (ISAV), salmonid alphavirus (SAV), piscine myocarditis virus (PMCV), viral heamorrhagic septicaemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) using primers and probe as described elsewhere (Palacios et al. 2010, Snow et al. 2006, Hodneland & Endresen 2006, Lovoll et al. 2010, Jonstrup et al. 2013, Liu et al. 2008, 0rpetveit et al. 2010).

Identification of a new virus by RT-PCR and Sanger sequencing. As the pathological findings could resemble HSMI, which is associated with PRV, investigations to detect a virus within the family Reoviridae were initiated. Total RNA from tissue samples from smolt farm A was extracted using QIAsymphony SP (QIAGEN) in accordance with manufacturer's instructions using the QIA symphony RNA kit. Amplification of PCR products was performed by one-step RT-PCR using primer sets targeting parts of the PRV genome as described by Garseth and co-workers (Garset et al. 2013). Reactions were conducted in a final volume of 20 μΙ with OneStep RT-PCR kit (QIAGEN). Five hundred ng total RNA and 0.75 μΜ of each primer was denatured (95°C/5 minutes) and mixed with 1 x RT-PCR buffer, 400 μΜ dNTPs, 4 U RNaseOUT and 1 μΙ OneStep RT- PCR enzyme mix. The cycling parameters were as follows: cDNA synthesis 50 °C/30 min followed by 95°C/15 min and PCR: 40 cycles of 94°C/30s, 55°C/30s and 72°C/60 s. The final PCR products were visualized by gelelectrophoresis. All PCR products of approximately right size were sequenced using the amplification primers and theABI Prism Big Dye Terminator Cycle sequencing kit on ABI Genetic Analyser. Obtained sequences were assembled using the Sequencher 4.5 software from GeneCode and blasted against GenBank. Primers S1_39F and S1_621 R (Garseth et al. 2013) provided a 562 bp nucleotide sequence displaying 85% identity to parts of S1 segment of PRV with accession number GU994022. This sequence (SEQ ID NO: 55) was used to establish the RT-PCR methods described below.

rRT-PCR. Forward primer 5'- TCG-TGG-TTC-CAA-TGA-CAG-3', reverse primer 5'- CCA- ACC-ACT-AAA-ACC-GAG- 3' and probe 6-FAM-5'- ACG-CCT-TAG-AGA-CAA-CAT- GCG-AAG-3'- BHQ-1® were designed for amplification and detection of a 121 bp fragment of the genome of the new virus by rRT-PCR.

The rRT-PCR for the new virus was performed using the QIAGEN OneStep kit. Reactions were conducted in a final volume of 20 μΙ with ROX passive reference dye RT-RARE 03 (Eurogentec) and RNaseOUT (Invitrogen). 500 ng of total RNA from fish displaying clinical signs of the novel disease was mixed with 1 x RT-PCR buffer, 30 nM ROX, 400 μΜ dNTPs, 0.5 μΜ of each primer, 0.3 μΜ probe , 1 .25 mM MgCI₂, 4 U RNaseOUT and 0.8 μΙ OneStep RT-PCR enzyme mix. Amplifications were done on a Stratagene Mx3005P realtime machine (Stratagene) with following conditions: 30 min at 50°C (reverse transcription), followed by 15 min at 95°C (RT inactivation and activation of Taq polymerase, 40 cycles of 94°C/30s, 55°C/30s and 72°C/30s. Specificity of the rRT-PCR assay was determined by blasting of the nucleotide sequences of forward and reverse primers and the probe against GenBank using BLAST X to identify any known organism with which they might cross-react. Additionally, the newly established rRT-PCR was tested against nucleic acid extracted from a selection of other RNA viruses capable of infecting salmonids including PRV, ISAV, SAV, PMCV, VHSV, IHNVand IPNV.

Conventional RT-PCR. Amplification of a 423 bp fragment of the genome of the new virus by conventional RT-PCR was performed with primers 6F 5'-GAC-CAA-CAT-AAC- GTT-TCA-GGC-3' 6R 5'-ATC-CAA-CCA-CTA-AAA-CCG-AGA- 3' using the protocol for RT-PCR as described above without the RNA denaturation step. Screening of the farms for the novel virus. From each of the seven farms included in this study (Fig. 1 ), 20-50 fish were analysed with the newly established rRT-PCR method for detection of the new virus. In the first cases from 2013, blood and several tissues including heart and kidney were tested. As the initial screening results showed highest amount of virus in blood at the acute stage of the disease, mainly blood were tested from the subsequent cases.

Total nucleic acid from organ samples on RNA/ate/⁹ was extracted as described elsewhere (Jansen et at. 2010). The following protocol was established for extraction of nucleic acid from blood: 100 μΙ of blood on EDTA or heparin was mixed with 750 μΙ of lysis buffer (BioMerieux) and homogenized using Mixer Mill MM 400 (Retsch). Total nucleic acid was extracted on automated NucleotideliSens®easyMAGTM (BioMerieux, Norge AS, Oslo, Norway) in accordance with the manufacturer's protocol for whole blood and on board lysis.

Analyses were performed with QIAGEN OneStep kit and 500 ng nucleic acid using the rRT-PCR protocol for detection of the new virus as described above. Positive findings at each site were confirmed by conventional RT-PCR and sequencing of the obtained products with reverse primers from the rRT-PCR assay and 6F&6R.

Results

Epidemiology

Figure 1 shows the connection between the farms. The first disease outbreak was recorded in farm B in August 2013. Farm A and C were diagnosed with the same condition in October and November respectively. The fish were sized 25 - 100 g and held in fresh water with none or minimal addition of salt water (<1 %₀). Water temperature at the start of clinical disease was 7-10^°C. For farm A the disease was observed mainly in two tanks, mortalities were only moderately increased and no clinical disease has been observed after sea transfer, except for one group which experienced some mortalities the first few days after transfer. Farm B reported low mortalities in some tanks (0.1 -0.75% per week), but at sea transfer Autumn 2013 high mortalities were observed the first days in sea water. No clinical disease or mortalities have been recorded later on at this sea farm. Farm C experienced clinical disease in several tanks and mortalities were observed for approx. six months up to 10-12 000 fish died per week. During late winter 2014 fish from diseased groups in freshwater farms B and C were transferred to sea farm D, and from farm C also to sea farm E. In both sea farms the disease was diagnosed until about four months after transfer. Total mortalities were < 4%.

Clinical signs and gross pathology

Typical clinical signs were loss of appetite and lethargy. Fish seemed to stay along the wall of the tank and rather high in the water column. The lethargic stage could last for days before mortalities occurred.

The skin and gills were pale and bilateral exophthalmos was often observed. Cutaneous haemorrhages, particularly on the abdomen and at the bases of the pectoral and abdominal fins, were frequent findings. Autopsy revealed pale heart and sometimes dilated, blood filled atrium. Haemopericardium due to rupture of the heart was also seen. Liver was pale or yellowish, the kidney and spleen swollen, although in some fish a very small spleen was reported. Serohaemorrhagic ascites was a common finding. The gastrointestinal tractus was usually empty.

Histopathological findings

Histological lesions as described below were found in the 80 clinically diseased fish. Also 80% of the 33 apparently healthy fish from affected tanks or cages had similar finding although of a more moderate degree. Main organs affected were heart, red skeletal muscle and liver, but only 20 of the 103 fish had lesions in all three organs.

Heart

The consistent organ affected was the heart. All 103 fish with histopathological findings suffered from pancarditis and for 34 fish this was the only histopathological finding. Increased cellularity was seen in all parts of the heart, but often the spongious layer of the ventricle was most severely affected. In mild cases the inflammatory response was focally to multifocally distributed whereas severely affected hearts had an extensive and generalized reaction. The cellular reaction seen in both the spongious layer of the ventricle and the atrium comprised circulatory, subendocardial and intramuscular neutrophils and monocytes/macrophages. Endocardial hypertrophy and proliferation was also seen. In moderate and severe cases cardiomyocyt degeneration was evident and especially in the spongious ventricle, multiple, strong eosinophilic, hyalinic cardiomyocytes, consistent with necrosis, were present. In mild cases only minor, if any, increased cellularity was found in the compact, outer muscle layer of the ventricle. When present, cellular infiltrates were localized perivascularly to the coronary vessels and in more advanced cases the cellularity was also distributed into the myocardium. Cardiomyocyt necrosis in compact layer was also observed.

Mild to severe endo-and myocarditis was accompanied by a similar degree of epicarditis. The subepicardial cellularity comprised mono- and polymorph nucleated cells.

Skeletal muscle

Lesions in red skeletal muscle were seen in 59 of the 103 fish with hearth inflammation. Focal, multifocal to diffuse distribution of degenerated and necrotic red fibers often infiltrated in sarcoplasm by marcrophages along with endomysial cell infiltration and proliferation were observed. In severe cases the red fibers were to a large extent replaced by hypercellularity including fibrosis. Evidence of regeneration of red muscle fibers were seen in a few fish. Fifteen fish also had degeneration of white muscle fibers. These findings were usually moderate and affecting the dorsal white fibers below the red muscle layer and was only observed when red muscle was severely affected.

Liver

Liver was affected in 35 fish. Acute focal to multifocal and partly confluating vacuolization and necrosis of hepatocytes were observed. The degenerative and necrotic changes seemed to travel along sinusoids. In severe cases sometimes livers were massively affected with lesions covering large areas. Subacute to chronic stages were recognized by the infiltration of inflammatory cells and mobilization of fibroblasts.

Haematology

Haematocrit (her) was measured in 65 fish from farms A, B and C. The median hematocrit for the 17 fish with no histopathological findings observed was 48 (range 12-58) and for the 48 fish with typical findings the median was 20 (range 4-58). About one third of the fish in this group had a her of 15 or below.

Immunohisochemistry

Flavobacterium psychrophilum antigen was not found in any of the samples tested by immunohistochemistry.

Electron microscopy

High amounts of inflammatory cells where present both in the tissue and lumen of the hearth, but no virus particles where detected. Bacteriological examination

No specific bacteria were isolated.

rRT-PCR and virus cultivation

PRV, ISAV, SAV, PMCV, VHSV or IHNV were not detected at any of the farms when 5 examined by rRT-PCRs for detection of RNA viruses capable to infect salmonids. Low amounts of IPNV (Ct 33 to 35) were found at farm A in October 2013. Attempts to cultivate virus from diseased fish were unsuccessful.

Screening of the farms for the novel virus

The rRT-PCR assay demonstrated efficient amplification and detection of the novel virus.

10 No detectable products were obtained when the assay was tested against RNA derived from viruses other than the novel virus. As a constant amount of nucleic acid was used in our analyses and the PCR efficiency of the rRT-PCR assay was 99.7% (data not shown), the Ct values provide an indication of the virus amounts in each sample. For the purpose of this study we have correlated the Ct values and amounts of virus as follows: Ct below

15 25 high amounts, Ct 25-30 medium amounts and Ct over 31 low amounts of virus.

The novel virus was found at all seven farms. At farms A, B, C, D and E, where clinical signs and mortality were observed, high amounts of virus were detected in moribund fish. Fish in chronic stage (looser fish) had medium, low or none amounts of virus. Healthy fish from tanks or cages with no disease detected had no virus, whereas apparently healthy 20 fish, from diseased groups had high amount of virus. This was the case whether these fish had typical pathological findings or no pathology observed. In one sampling from farm A no virus sequence was amplified from clinically healthy fish with typical histopathology.

At the brood fish farms (Farms 1 & 2), 3/20 and 1 1/46 respectively, were positive with low amounts of virus. No disease was observed.

25 The rRT-PCR results from all farms were confirmed by the conventional PCR and sequencing showing that the gene sequences detected at all seven farms were 100 % identical.

Discussion

The present document provides the first description of a new disease in rainbow trout 30 observed in three rainbow trout fresh water farms on the west coast of Norway in 2013- 14. All three farms had received eggs and/or fish from the same brood stock source. A novel virus was also detected in the affected farms, as well as in the brood stock. In two cases, fish developed the disease and carried the virus after sea transfer. The PCR methods established in the present study will be useful in future epidemiological studies to reveal both the geographical distribution and investigate material from earlier years to possibly detect the source of the novel virus.

It is herein described a new disease in rainbow trout with inflammation of the heart and red skeletal muscle, liver necrosis, anaemia and moderate to high mortality rates. Except for the anaemia the pathological findings resembles HSMI which is a well-known disease in salmon in Norway. Heart and skeletal muscle inflammation has not been described in rainbow trout. HSMI is diagnosed in freshwater salmon farms, but it is mainly a problem 5- 9 months after sea water transfer. In the present study the new disease occurred primarily in the fresh water stage and only a few farms were affected after sea transfer. So even if clinical signs and pathology resembles HSMI, the course of the disease seems to be quite different.

Diseases involving the heart are also cardiomypathi syndrome (CMS), which is only seen in adult salmon in Norway, and pancreas disease (PD) affecting both species. For the CMS there is no involvement of the red muscle and for PD also exocrine pancreas is affected. In addition the causative viruses for these two diseases, PMCV and SAV respectiveley, were not detected.

Heart inflammation in rainbow trout is described by MacWilliams et al. (2007), and was seen in some fish used in an infectious trial with ISAV. The main finding was epicarditis which subsequently seemed to affect the compact myocardium in severely affected fish. This is in contrast to the present findings, were the spongious layer seemed to be most severely affected. In their case also no reaction in the red muscle was noted and liver necrosis was seen in only a few fish. Although the cause of the inflammatory reaction in the ISAV trial was unclear, in the present case all fish tested for ISAV were negative.

Different observations indicate the new disease as an infectious disease. It spread between tanks, and from fish to fish in the tank. Clinical signs lasted some days before the mortality started, and the pathological findings show inflammation in several organs pointing towards a septicemia. Liver necrosis is often seen in fish with circulatory failure and is possibly secondary due to heart and circulatory failure. In this case the degenerative changes seemed to travel along the sinusoids, which also could indicate a haematogenious spread of an agent. The pathological examination revealed an inflammatory reaction involving neutrophils. Although being a feature in fish also with viral infections, it is typical when bacteria are involved. No bacteria were, however, found, neither by culture nor by special staining of histological sections examined by light microscopy. As Flavobacterium psychrophilum infectons in rainbow trout may cause an inflammatory reaction in the heart in chronic cases, specific tests for this bacterium were performed, with negative results. Thus, bacteria does not appear to be the cause of this new disease, but the cause of the disease is most likely a virus.

The virus detected showed 85% identity with PRV in the part of the investigated genome. The genome of reoviruses is segmented and it is so far detected partial sequence corresponding to one of the segments. Full genome characterization of the new virus is therefore necessary to confirm that it belongs to the Reoviridea family and its genius affiliation. We did not find any virus particles in the electron microscopic studies in hearts and in blood samples in which the novel virus has been detected with low Ct by rRT-PCR. Also in earlier studies of HSMI in Atlantic salmon virus particles were difficult to detect in inflamed tissues (Kongtorp 2008).

The diseased fish in the present study had in many cases a severe anaemia. Anaemia in rainbow trout and other salmonids has earlier been linked to erythrocytic inclusion body syndrome (EIBS) (Rodger et al. 2007). Electron microscopic images of the EIBS- inclusions reveal virus particles that resemble PRV (Finstad et al. 2014). The TEM studies did not reveal any viral inclusions in the red blood cells even if large amounts of the novel virus were detected in blood.

The results provided herein indicate an association between the disease described and the virus detected, as clinically diseased fish had high amounts of virus whereas fish in a chronic phase, so-called loosers, had lower viral loads and in some cases virus was not found. This is expected at later stages of disease if the immune system manages to fight the virus. In affected farms large amounts of the novel virus was detected also in some apparently healthy fish in diseased groups. This is a typical observation for other viral diseases due to a stage where large amounts of virus precede the clinical disease stage.

In the present studies it was found that blood is suitable for detection of the novel virus. Internal organs may also be suitable for virus detection at the chronic stage of the disease. Blood was used for detection of the novel virus in the clinical healthy brood fish. The prevalence and amount of virus in these fish where low, pointing to a possible carrier state. Example 2: Infection in salmon

The aim of this study was to study the long term effects of a virus Y cohabitation challenge on Atlantic salmon focusing on:

- Viral kinetics and pathogenesis

5 - Pathology

- Haematology

- Immune responses

Design summary

Shedder fish (90) were injected i.p. (intraperitoneal^) with a virus Y challenge material 10 (lysed blood cell material from diseased fish), and marked by cutting off of the adipose fin.

The 90 shedders were mixed in tank 1 in a 1 :1 relationship with 90 unchallenged fish (cohabitants). 50 untreated fish (controls) were added to tank 2. Fish were sampled every 2nd week from week 4 as described in Table 1 below. A stress test (4 hours at high density and oxygen levels measured down to 40%) was performed for 30 cohabitants and 15 15 control fish in week 10. From week 1 2-16 stressed fish will also be sampled at the same time points. The study will be terminated after 16 weeks.

Table 1 :

Activity Study week

Sampling of 8 untreated fish 0

Challenge: i.p injection and labelling of 90 shedderfish (AF) - added to 90 0

untreated fish (cohabitants) in tank 1 .

50 untreated fish (controls) added to tank 2.

Sampling: 20 (8 shedders (AF), 8 cohabitants, 4 controls) 4

Sampling: 20 (8 shedders (AF), 8 cohabitants, 4 controls) 6

Sampling: 20 ( 8 shedders (AF), 8 cohabitants, 4 controls) 8

Sampling: 20 ( 8 shedders (AF), 8 cohabitants, 4 controls) 10

Stress test: 30 cohabitants and 15 controls exposed to stress^* in separate 10 tanks. Marked in the left maxilla (LM) and returned to tanks 1 and 2.

Sampling: 32 (8 shedders (AF), 8 cohabitants, 4 controls, 8 stress-treated 12 cohabitants (LM), 4 stress-treated controls (LM))

Sampling: 32 (8 shedders (AF), 8 cohabitants, 4 controls, 8 stress-treated 14 cohabitants (LM), 4 stress-treated controls (LM))

Sampling: 32 (8 shedders (AF), 8 cohabitants, 4 controls, 8 stress-treated 16 cohabitants (LM), 4 stress-treated controls (LM))

Terminate experiment 16

Results

Blood and heart samples of shedders and cohabitants have been analysed for virus Y in blood and hearts until study week 10 by realtime qPCR (see Table 2):

Table 2:

-Study week 4: all shedders are positive for virus Y in blood and hearts.

-Study week 8: four of eight cohabitant fish are positive for virus Y in blood and the two individuals with the most virus in blood (Ct -21 ) are also positive in hearts (Ct -23). All shedders are positive.

-Study week 10: three of eight cohabitant fish are positive for virus Y in blood and eight of eight fish are positive in hearts. All shedders are positive in blood and three of eight fish are positive in heart.

The results show that the salmon is successfully infected by the i.p.-injected challenge material and that virus Y shed from salmon to the environment is able to infect cohabitant salmon. Atlantic salmon is thus susceptible to virus Y infection. It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Unless expressly described to the contrary, each of the preferred features described herein can be used in combination with any and all of the other herein described preferred features.

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Jansen, M.D., Gjerset, B., Modahl, I. & Bohlin, J. (2010a) Molecular epidemiology of salmonid alphavirus (SAV) subtype 3 in Norway. Virology Journal, 7, 188.

20 Jonstrup S. P., Kahns S., Skall H. F., Boutrup T. S. & Olesen N. J. (2013) Development and validation of a novel Taqman-based real-time RT-PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus. Journal of fish diseases, 36(1 ), 9-23

25 Kongtorp R.T., Koppang E.O., Bjerkas I., Falk K. & Taksdal T. (2008) Features of the pathogenesis of heart and skeletal muscle inflammation in Atlantic salmon , Salmo salar L. in Kongtorp RT, Thesis PhD, Heart and skeletal muscle inflammation (HSMI) in Atlantic salmon, Salmo salar. pathology, pathogenesis and experimental infection, Norwegian School of Veterinary Medicine, paper IV

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(2012) Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production. Dis Aquat Organ, 99, 7-12. Lovoll M., Wiik-Nielsen J., Grove S., Wiik-Nielsen C. R., Kristoffersen, A. B., Faller R. Poppe, T., Jung, J., Pedamallu, C. S., Nederbragt, A. J., Meyerson, M., Rimstad, E & Tengs, T. (2010). A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS). Virology Journal, 7, 309.

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MacWilliams C, Johnson G., Groman, D. & Kibenge, F. S. B. (2007) Morphologic description of infectious salmon anaemia virus (ISAV)-induced lesions in rainbow trout Oncorhynchus mykiss compared to Atlantic salmon Salmo salar. Diseases of Aquatic 10 Organisms, 76(1 ), 1 -12. doi:10.3354/dao01866

Palacios G., Lovoll M., Tengs T., Hornig M. & Hutchison, S (2010) Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus. PLoS ONE 5(7): e1 1487. doi:10.1371 /journal.pone.001 1487

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Claims

1 . An isolated nucleic acid molecule comprising or consisting of a nucleic acid sequence according to any one of SEQ ID NO: 1 -55, or a variant thereof having at least 85% identity to any one of SEQ ID NO: 1 -55, or a sequence complementary to any one of SEQ ID NO: 1 -55 or a variant thereof having at least 85% identity to any one of SEQ ID NO: 1 -55.

2. The isolated nucleic acid molecule according to claim 1 , wherein said variant has at least 90% identity to a sequence according to SEQ ID NO: 1 -55.

3. The isolated nucleic acid molecule according to claim 1 or 2, wherein said nucleic acid sequence is selected from SEQ ID NO: 1 -49 and 55.

4. A nucleic acid fragment of an isolated nucleic acid molecule according to any one of claims 1 -3, said fragment comprising or consisting of at least 5 contiguous nucleic acid bases of a nucleic acid sequence as defined in any one of claims 1 -3.

5. The nucleic acid fragment according to claim 4, wherein said nucleic acid fragment is a nucleic acid primer or nucleic acid probe capable of detecting virus Y in a sample, said probe optionally further comprising one or more label(s) for detection of said probe.

6. The nucleic acid fragment according to claim 5, wherein said nucleic acid probe comprises a nucleic acid sequence according to SEQ ID NO: 52.

7. An isolated RNA molecule corresponding to a nucleic acid molecule or fragment as defined in any one of claims 1 -4.

8. An isolated double-stranded RNA molecule comprising or consisting of an RNA sequence as defined in claim 7 and an RNA sequence complementary thereto.

9. A double-stranded RNA virus characterized in that it comprises an RNA sequence corresponding to a nucleic acid sequence having at least 85% identity to a nucleic acid sequence selected from any one of SEQ ID NO: 1 -49 and 55, such as from SEQ ID NO: 1 -37.

10. The double-stranded RNA virus according to claim 9, wherein said virus comprises RNA sequences corresponding to all of SEQ ID NO: 1 -37 or sequences having at least 85% identity thereto.

1 1 . A vector comprising one or more nucleic acid molecule(s) and/or fragment(s), as defined in any one of claims 1 -5, or one or more isolated RNA molecule(s) as defined in claim 7 or 8.

12. A host cell comprising one or more nucleic acid molecule(s) and/or fragment(s) as defined in any one of claims 1 -5, one or more isolated RNA molecule(s) as defined in claim 7 or 8 and/or a vector as defined in claim 1 1 .

13. A polypeptide encoded by a consecutive string of at least 12 nucleic acid bases of a nucleic acid molecule or fragment as defined in any one of claims 1 -4, or an isolated RNA molecule as defined in claim 7 or 8, or a nucleic acid reverse complementary thereto.

14. An antigen comprising a polypeptide as defined in claim 13.

15. An antibody specifically directed to the antigen of claim 14.

16. Use of a nucleic acid fragment as defined in any one of claims 4-6 as a probe for detecting the presence of a virus, such as virus Y, or diagnosing a viral infection, such as virus Y infection, in a sample.

17. The use according to claim 16, wherein said sample is from rainbow trout or

salmon.

18. A method for detecting the presence of a virus, such as virus Y, and/or diagnosing a viral infection, such as virus Y infection, in a sample, said method comprising the steps of:

a) contacting the sample with a nucleic acid fragment as defined in any one of claims 4-6 or an antibody as defined in claim 15;

19. The method according to claim 18, wherein said method is performed ex vivo.

20. The method according to claim 18 or 19, wherein said sample is a blood sample and/or a tissue sample from internal organs such as heart, liver, kidney, spleen, pancreas, pylorus or skeletal musculature.

21 . The method according to any one of claims 18-20, wherein said sample is from rainbow trout or salmon.

22. A diagnostic kit for diagnosing a viral infection, such as virus Y infection, in a subject said kit comprising one or more nucleic acid fragment(s) as defined in any one of claims 4-6, a polypeptide as defined in claim 13, an antigen as defined in claim 14 and/or an antibody as defined in claim 15 and reagents for performing a diagnosis, and/or optionally instructions for use.

23. A pharmaceutical composition comprising one or more of 1 ) a nucleic acid molecule as defined in any one of claims 1 -3, 2) a nucleic acid fragment as defined in claim 4, 3) an RNA molecule as defined in claim 7 or 8, 4) a virus as defined in claim 9 or 10, 5) a vector as defined in claim 1 1 , 6) a host cell as defined in claim 12, 7) a polypeptide as defined in claim 13, 8) an antigen as defined in claim 14, and/or 9) an antibody as defined in claim 15.

24. A nucleic acid molecule as defined in any one of claims 1 -3, a nucleic acid fragment as defined in claim 4, an RNA molecule as defined in claim 7 or 8, a virus as defined in claim 9 or 10, a vector as defined in claim 1 1 , a host cell as defined in claim 12, a polypeptide as defined in claim 13, an antigen as defined in claim

14, and/or an antibody as defined in claim 15 for medical use.

25. A pharmaceutical composition as defined in claim 21 for use in the prevention and/or treatment of a viral infection, such as a virus Y infection.

26. Use of 1 ) a nucleic acid molecule as defined in any one of claims 1 -3, 2) a nucleic acid fragment as defined in claim 4, 3) an RNA molecule as defined in claim 7 or

8, 4) a virus as defined in claim 9 or 10, 5) a vector as defined in claim 1 1 , 6) a host cell as defined in claim 12, 7) a polypeptide as defined in claim 13, 8) an antigen as defined in claim 14, and/or 9) an antibody as defined in claim 15 for the preparation of a medicament for the prevention and/or treatment of a viral infection, such as a virus Y infection.

27. A method the treating and/or prevention a viral infection, such as a virus Y infection, said method comprising the administration of a pharmaceutically effective amount of 1 ) a nucleic acid molecule as defined in any one of claims 1 -3, 2) a nucleic acid fragment as defined in claim 4, 3) an RNA molecule as defined in claim 7 or 8, 4) a virus as defined in claim 9 or 10, 5) a vector as defined in claim

1 1 , 6) a host cell as defined in claim 12, 7) a polypeptide as defined in claim 13, 8) an antigen as defined in claim 14, 9) an antibody as defined in claim 15, and/or a pharmaceutical composition as defined in claim to a subject in need thereof.

28. The method according to claim 27, wherein said subject is rainbow trout or salmon.