NO20201187A1

NO20201187A1 - Novel fish virus

Info

Publication number: NO20201187A1
Application number: NO20201187A
Authority: NO
Inventors: Linda Ramsevik Teigene; Magnus Andreas Devold; Håvard Aanes; Morten Lund; Jostein Grip; Henriette Glosvik; Espen Grøtan
Original assignee: Patogen As
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2022-05-02
Also published as: NO347379B1

Description

The invention

The present invention relates to an isolated nucleic acid sequence comprising a sequence selected from the group consisting of SEQ ID No 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants thereof being at least 70 % identical thereof. The invention further relates to nucleic acid sequences encoding an amino acid sequence according to SEQ ID NO 3, 5 and/or 8, isolated recombinant proteins or amino acids sequences, antibodies, immunogenic compositions, diagnostic methods and kits. The invention also relates to use of the virus, isolated nucleic acid sequences and isolated or recombinant amino acid sequences for manufacturing a substance for treating or prophylaxis of infection in an animal.

Background

In Norwegian salmonid farming the use of cleaner fish has become more and more popular to prevent infections by sea lice (Lepeophtheirus salmonis and Caligus spp.). Sea lice are one of the major pathogens affecting global salmonid farming industry, with an estimated loss of €300 million. The most common species of cleaner fish are the wrasse species Ctenolabrus rupestris, Symphodus melops and Labrus bergylta, but also lumpsucker (Cyclopterus lumpus) are widely used.

Ballan wrasse (Labrus bergylta) has demonstrated to be an effective sea lice eater, as it catches lice at lower temperature than the other species and is the species most frequently farmed among the cleaner fish. In 201949,1 mill cleaner fish were transferred to salmon sea cages in Norway with the purpose of removing sea lice from the salmon (Fiskehelserapporten 2019).

By the expression "cleaner fish" is meant any species selected from the whole group of cleaner fish used in salmonid farming, preferably wrasse, more preferred family of wrasse Labriformes and Scorpaneniformes, more preferred the family of wrasse Labridae. The most preferred species is Ballan wrasse Labrus bergylta.

By the expression “fish” is meant any species within sub class of bony fishes Actinoperterygii. More preferable marine fish species within Actinoperterygii.

As cleaner fish, including wrasse, are keeping the salmonids healthy, they are very valuable to the fish farming industry, even if they are not used for consumption themselves. Therefore, it is important to keep them healthy. Known diseases in wrasse are typically of bacterial origin, furunculosis caused by atypical Aeromonas salmonicida, Vibrio anguillarum, Pseudomonas anguilliseptica and Amoebic gill disease (AGD).

Among viral disease has Cyclopterus lumpus virus (CLuV) or lumpfish flavivirus been frequently reported in farmed lumpsucker since 2016. There are reports of other viruses from lumpfish, as ranavirus from lumpsucker in Ireland, Scotland, and Faeroes. In 2018 it was described two new viruses from diseases from lumpfish juveniles, with liquid in the intestine, the disease is called Cyclopterus lumpus totivirus (CLutV) and Cyclopterus lumpus Coronavirus (CLuCV). It is also demonstrated that Lumpfish can be infected by nodavirus, and that wrasse and lumpfish can be affected by Infectious Pancreatic Necrosis virus (IPNV). Viral Haemorrhages Septicaemia virus (VHSV) has been isolated in wrasse and lumpfish in Scotland and Island respectively. Viruses normally causing disease in salmon like Salmonid Alfavirus (SAV), Infectious Salmon Anaemia Virus (ISAV), Piscine Myocarditis Virus (PMCV) and Piscine Orthoreovirus (PRV) have been detected sporadically, from wrasse in salmon farms where the salmon have had disease problems with the respective diseases. Clinically the impact of these diseases on wrasse is unclear.

It is well known that there is high mortality during larval rearing of many species of fish. Little has been described on mortality and or disease of larval rearing for the wrasse. During several years it was discovered particularly high mortality in several tanks in a hatchery producing wrasse juveniles in Norway. A novel fish virus was characterized from samples of the fish. The samples were taken from wrasse, more precisely Labrideae, in a farm with high losses caused by disease.

The novel virus is different to all known Birnaviruses. The virus shows unique characters and 67% similarity to an unassigned Birnavirus, that has been isolated from rotifers. The rotifers are used as start feed for marine fish larvae. The virus is herein called Ballan Wrasse Birna Virus (BWBV).

Fish vaccines against birnaviruses can be based on attenuated live virus or inactivated virus produced in cell lines. Virus production in cell lines is expensive and labour consuming, but such vaccines are commonly used in salmon aquaculture. Live vaccines are not often used because of the risk of mutating back to virulence.

According to Evensen et al 2016, birnaviruses of aquatic organisms constitute small, nonenveloped icosahedral viruses with bisegmented, double-stranded RNA (dsRNA) genomes, and currently, two genera infect aquatic organisms; Aquabirnavirus and Blosnarvirus. Among aquabirnaviruses, the infectious pancreatic necrosis virus (IPNV) is the type species and causes severe diseases in salmonids, while there are a number of other aquabirnaviruses that infect other aquatic organisms. Blotched snakehead virus is the type strain of the Blosnarvirus genus, infecting blotched snakehead (Channa maculata). Vaccination and selective breeding in Atlantic salmon have contributed to reducing the impact of IPNV infection in salmonid aquaculture worldwide.

The current virus does genetically belong to the birnavirus family, the closest relation is blosnavirus, but with a similarity to blosnaviruses of 58% (polmerase) the new virus is considered a new virus in fish.

In order to reduce the high mortality and disease during larval rearing, it is a need for a method to identify the virus, and a tool to monitor the production of virus and avoid outbreak in fish. Further there is a need to develop a vaccine and possibly a functional feed or additive for functional feed, whereby the feed or additive may be used for the treatment or prophylaxis of infection in an animal. Another object of the invention is to develop further treatment to treat infected fish.

The present invention as described below, characterizes the novel virus from a sample of a fish, and describes among others methods for identification, detection and vaccine development.

The invention also relates to challenge methods using a virus according to the invention for testing susceptibility of animals, with the purpose of testing for instance efficacy of a vaccine or functional feed, or resistance towards disease. The invention also relates to use of the virus in breeding programs to obtain animals that are resistant to the virus as defined above.

The invention

The invention relates to the new virus isolated from wrasse, more precisely Labrideae. The virus comprises a ribonucleic acid genome selected from the group including SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants and fragments of the sequences being at least 70 % identical.

In one embodiment, the virus of the invention further comprises a ribonucleic acid genome having at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO 1, 2, 4, 6 and/or 7, or sequences being complementary to SEQ ID NO 1, 2, 4, 6 and/or 7.

According to another aspect of the invention, it relates to an isolated nucleic acid sequence originating from the virus. The sequence comprises a sequence selected from the group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences complementary to any of SEQ ID NO 1, 2, 4, 6 and 7, and variants thereof being at least 70% identical.

The isolated nucleic acid sequence may further comprise a sequence being at least 80%, preferably 90%, more preferably 95 % or 99% identical with any of the sequences SEQ ID NO 1, 2, 4, 6 and/or 7, or any sequences being complementary to SEQ ID NO 1, 2, 4, 6 and/or 7.

By "comprising a sequence" it is herein meant that a sequence according to the invention may be longer than the stated sequence, by having nucleotides before and/or after the stated sequence. The expression should also be understood to include sequences identical to the stated sequence, and including more than one sequence.

In another aspect of the invention, it relates to a nucleic acid sequence comprising a sequence encoding an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO 3, 5 and 8, and variants of these sequences being at least 70 % identical.

In a preferred embodiment, the nucleic acid sequence comprises a sequence encoding an amino acid sequence being at least 80 %, preferably 90%, more preferably 95 %, 99 % or 100 % identical with any of the sequences SEQ ID No 3, 5 and/or 8.

In another aspect of the invention, it relates to a nucleic acid sequence comprising a sequence of at least 10 consecutive nucleotides selected from the group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants being at least 70 % identical with any of the sequences SEQ ID NO 1, 2, 4, 6 and 7 and sequences being complementary to the variants of SEQ ID NO 1, 2, 4, 6 and 7.

In a more preferred embodiment of the invention, it relates to a nucleic acid sequence comprising a sequence of at least 10 consecutive nucleotides selected from the group consisting of SEQ ID NO 9-17, sequences being complementary to SEQ ID NO 9-17, and variants of these sequences being at least 70% identical.

I another embodiment, the above mentioned nucleic acids comprises a sequence of at least 15 consecutive nucleotides.

In a more preferred embodiment of the invention, it relates to a nucleic acid sequence comprising a sequence according to SEQ ID NO 9-17.

The shorter nucleic acids mentioned above may be used as primers and probes for instance for PCR and/or for identification of a nucleic acid sequence or gene in general.

According to another aspect of the invention, a method for detection of a virus in a biological sample is provided. The method comprises the following steps:

a) preparing a sample comprising nucleic acid sequences isolated from the biological sample for a reverse transcription reaction,

b) subjecting the sample of step a) to a polymerase chain reaction with a primer pair, wherein each primer of said primer pair comprises at least 10 consecutive nucleotides selected from the group consisting of SEQ ID No 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID No 1, 2, 4, 6 and 7, and variants being at least 70 % identical with any of the sequences,

c) determining whether binding of the primers to nucleotide sequences in the biological sample and amplification of the sequence between them have occurred, indicating the presence of virus in the sample tested.

In a preferred embodiment, in step b) of the method above, the primers are selected from a group consisting of sequences being at least 80%, preferably 90%, more preferred 95 % or 99 % identical with the sequences SEQ ID No 1, 2, 4, 6 and 7, or any sequences being complementary to such sequences.

In a preferred embodiment, the primers are selected from a group consisting for SEQ ID No 12-17. In a more preferred embodiment the primer pairs are selected from a group consisting of the following primer pairs:

primers according to SEQ ID NO 12 and 15,

primers according to SEQ ID NO 13 and 16,

primers according to SEQ ID NO 14 and 17.

Any primers comprising SEQ ID NO 12-17 should also be included in this embodiment.

In a preferred embodiment of the method above, step c) is performed by using a probe comprising at least 10 consecutive nucleotides selected from the group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants of these sequences being at least 70 % identical.

In a more preferred embodiment of the method above, the probe is selected from a group consisting of sequences being at least 80%, preferably 90%, more preferred 95 % or 99 % identical with the sequences SEQ ID No 1, 2, 4, 6 and/or 7, or any sequences being complementary to such sequences.

In a more preferred embodiment, the probes are selected from a group consisting of SEQ ID NO 9-11. Any probes comprising SEQ ID NO 19-11 should also be included in this embodiment.

In a preferred embodiment of the method above, primers of step b) and probes of step c) are selected from a group consisting of the following primer pairs and probes: primers according to SEQ ID NO 12 and 15, and probe according to SEQ ID NO 9, primers according to SEQ ID NO 13 and 16, and probe according to SEQ ID NO 10, primers according to SEQ ID NO 14 and 17, and probe according to SEQ ID NO 11. Any primers and probes comprising SEQ ID NO 12-17 should also be included in this embodiment.

In a preferred embodiment, the primers and/or probes of the method above comprise at least 15 consecutive nucleotides.

According to another aspect of the invention, another method for detection of a virus in a biological sample is provided. The method comprises the following steps: a) preparing a sample comprising nucleic acid sequences isolated from the biological sample for a reverse transcription reaction,

b) sequencing the sample of step a), and

c) comparing the resulting sequence with a sequence selected of the group consisting of SEQ ID No 1, 2, 4, 6 and 7 and sequences being complementary to SEQ ID No 1, 2, 4, 6 and 7, wherein at least 70 % identity indicates the presence of virus in the biological sample tested.

In a preferred embodiment of step c) of the method above, 80 %, 90%, 95%, 99% or 100 % identity is required to confirm the presence of virus in the biological sample.

In a preferred embodiment, the sequencing of the sample in the method above is performed by a method selected from the group consisting of Next Generation Sequencing, preferably Illumina (Solexa) sequencing, Roche 454 sequencing, Ion Torrent or SOLiD sequencing (Goodwin S, et al., (2016) Coming of age: Ten years of next-generation sequencing technologies. Nature reviews, Genetics, 17, 333-351).

Another aspect of the invention relates to use of a nucleic acid sequence comprising at the least 10 contiguous nucleotides of any of the sequences selected from the group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants being at least 70 % identical with any of these sequences, for confirming the presence of the virus in a biological sample. In another embodiment it is used a nucleic acid sequence having a sequence selected from the group of SEQ ID NO 9-17, and sequences being complementary to the any of the sequences SEQ ID NO 9-17, for confirming the presence of the virus in a biological sample.

The methods and uses above may be used to confirm that the virus is present in the biological samples, but it may also be used to confirm that the virus is not present in the biological samples. Further, it may be used to monitor a fish population, for instance for disease/sickness control. Information in this regard, desirable with methods for identifying other diseases and/or sicknesses, may be valuable information as to if or when the fish population should be treated. The biological samples to be analysed are preferably from dead fish, but may also be samples removed from live fish without harming the fish.

Another aspect of the invention relates to a vector comprising nucleic acid sequences according to the present invention, and host cells comprising said vectors.

According to yet another aspect of the invention, DNA vaccines are provided comprising a nucleic acid sequence selected from the group consisting of SEQ ID No 1, 2, 4, 6 and 7, sequences being complementary to sequences of SEQ ID No 1, 2, 4, 6 and 7, and variants of these sequences being at least 70% identical.

The nucleic acid sequence of the DNA vaccine may further be at least 80%, preferably 90%, more preferably 95 % or 99 % identical with any of the sequences SEQ ID No 1, 2, 4, 6 and/or 7, or any sequences being complementary to SEQ ID No 1, 2, 4, 6 and/or 7.

Another aspect of the invention relates to recombinant proteins encoded by a nucleic acid sequence selected from the group consisting of SEQ ID No 1, 2, 4, 6 and 7, and variants thereof being at least 70 %, 80 %, preferably 90%, more preferably 95 %, 99 % identical. In one aspect the amino acid sequence of the recombinant protein is given in SEQ ID No 3, 5 and/or 8.

The present invention also provides a recombinant vaccine comprising at least one of the recombinant proteins according to the present invention.

Another aspect of the invention relates to an immunogenic composition comprising a recombinant protein and/or a nucleic acid sequence having at least 70 % sequence identity with SEQ ID No 1, 2 ,4, 6 and/or 7.

The immunogenic composition may further comprise at least one excipient, additive or adjuvant, and may be administrated orally, by immersion or by intraperitoneal or intramuscular injection.

The immunogenic composition may be a monovalent vaccine or combined with other relevant antigens.

The invention also relates to use of the immunogenic composition described above in the manufacture of a vaccine for the treatment or prophylaxis of infection in an animal.

The invention also relates to use of the immunogenic composition described above in the manufacture of a functional feed or additive to a functional feed for the treatment or prophylaxis of infection in an animal.

Furthermore, according to another aspect of the invention, it is provided an antibody that recognises and binds to a recombinant protein according to the present invention.

The invention also relates to challenge methods using a virus as defined above for testing susceptibility of animals, with the purpose of testing for instance efficacy of a vaccine or functional feed, or resistance towards disease. The invention also relates to use of the virus in breeding programs to obtain animals that is resistant to the virus as defined above.

The invention also relates to a diagnostic kit, which may be used to decide whether a sample comes from an organism being infected by the virus according to the invention. The kit may comprise at least one primer or a pair of primers wherein the primer, or each primer of said primer pair comprises at least 10 consecutive nucleotides selected from a group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants of these sequences being at least 70 % identical.

In a preferred embodiment, the primer(s) of the kit are selected from a group consisting of sequences being at least 80%, preferably 90%, more preferred 95 % or 99% identical with the sequences SEQ ID No 1, 2, 4, 6 and 7, or any sequences being complementary to such sequences.

In a preferred embodiment, the primer sequences are selected from a group consisting of SEQ ID NO 12-17, sequences being complementary to SEQ ID NO 12-17, and variants being at least 90 % identical

In a preferred embodiment, the diagnostic kit further comprises a probe comprising at least 10 consecutive nucleotides selected from the group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants of these sequences being at least 70 % identical.

In a more preferred embodiment, the probe of the kit is selected from a group consisting of sequences being at least 80%, preferably 90%, more preferred 95 % or 99% identical with the sequences SEQ ID No 1, 2, 4, 6 and 7, or any sequences being complementary to such sequences.

In an even more preferred embodiment, the diagnostic kit comprises at least one primer pair and a probe selected from the group comprising the following primer pairs and probes:

primers according to SEQ ID NO 12 and 15, and a probe according to SEQ ID NO 9, primers according to SEQ ID NO 13 and 16, and a probe according to SEQ ID NO 10, and

primers according to SEQ ID NO 14 and 17, and a probe according to SEQ ID NO In another embodiment, the diagnostic kit may in addition or instead comprise an antibody or a recombinant protein.

Finally, the present invention relates to the use of the nucleic acid sequences having a sequences selected from the group consisting of SEQ ID No 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID No 1, 2, 4, 6 and 7, and variants thereof being at least 70 %, 80 %, preferably 90%, more preferably 95 %, or 99% identical with any of these sequences, for the preparation of DNA vaccine, recombinant vaccine or a live recombinant microorganism.

The present invention relates to isolated nucleic acid sequences and variants thereof being at least 70% identical with the isolated nucleic acid sequences. The term "% identity" is to be understood to refer to the percentage of nucleotides that two or more sequences or fragments thereof contains that are the same. The term "at least 70 % identical" thus means that at least 70 % of the nucleotides over the entire sequences which are compared, are identical. A specified percentage of nucleotides can be referred to as e.g. 70% identical, 80% identical, 85% identical, 90% identical, 95% identical, 99% identical or more over a specified region when compared and aligned for maximum correspondence. The skilled person will acknowledge that various means for comparing sequences are available.

The term " % identity" or "% identical" on "local alignment" is to be understood to refer to the percentage of nucleotides that two or more sequences contain that are the same, when the most similar regions are aligned for maximum correspondence. A sequence comprising another sequence would thus have 100% identity on local alignment, but not necessarily 100% identity on overall or global alignment. This is obvious to a skilled person.

The term "variants thereof" and "variants of these sequences" and similar expressions, as used in respect of the nucleic acid sequences and recombinant proteins according to the present invention, is to be understood to encompass nucleic acid sequences and recombinant proteins that only differs from the isolated sequences SEQ ID No.1-8 by way of some amino acid or nucleotide additions, deletions or alteration that have little effect, if any, on the functional activity of the claimed sequences. The skilled person will acknowledge that modifications of a nucleotide sequence encoding a protein may be introduced without altering the amino acid sequence, e.g. the substitution of a nucleotide resulting in that the triplett affected by the substitution still codes for the same amino acid. Such alterations may be introduced to adapt the nucleic acid sequence to the codons preferably used by a host cell and thus to enhance the expression of a desired recombinant protein.

Furthermore, the addition of nucleic acid sequences coding polypeptides which facilitates purification may be added without affecting the activity of the resulting recombinant protein.

The skilled person will further acknowledge that also alterations of the nucleic acid sequence resulting in modifications of the amino acid sequence of the recombinant protein it encodes, may have little, if any, effect on e.g. the proteins' ability to induce protection against the virus if the alteration does not have any impact on the resulting three dimensional structure of the recombinant protein. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a recombinant protein with substantially the same functional activity as the protein encoded by the nucleotide sequence SEQ ID No 1, 2, 4, 6 and/or 7, and are thus to be expected to constitute a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal or C-terminal portions of the protein molecule would also not be expected to alter the functional activity of the protein. Each of the proposed modifications is well within the routine skills in the art, as is determination of retention of biological activity of the encoded products. Therefore, where the terms "nucleic acid sequences of the invention" or "recombinant protein of the invention" are used in either the specification or the claims each will be understood to encompass all such modifications and variations which result in the production of a biologically equivalent protein.

Thus, the present invention encompasses recombinant proteins and variants thereof which differ in respect of amino acid substitutions, addition or deletions compared with the proteins of SEQ ID No 3, 5 and/or 8, and proteins being encoded by the sequence SEQ ID No.1, 2, 4, 6 and/or 7.

The primers and probes according to the present invention, will hybridize under stringent conditions with the sequence in question. The term “hybridizing under stringent conditions” refers to conditions of high stringency, i.e. in term of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acids having a high frequency of complementary base sequences. Stringent hybridization conditions are known to the skilled person (see e.g. Green M. R., Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th edition, 2012).

The precise conditions for stringent hybridization are typically sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

The term "antigen" when used in connection with the present invention is to be understood to refer to a recombinant protein or fragment thereof according to the invention being able to induce protection against the virus in fish or to be able to bind to an antibody which recognise and bind to the virus.

The term "vaccine" as used herein refers to a material that can produce an immune response that blocks the infectivity, either partially or fully, of an infectious agent, which in respect of the present invention is the virus affecting fish such as e.g. wrasses. Thus, when administering the vaccines of the invention to a fish, the fish becomes immunised against the disease caused by the virus. The immunising component of the vaccine may be e.g. DNA as in a DNA vaccine, RNA as in a RNA vaccine, a recombinant protein or fragment thereof according to the present invention, or a live recombinant microorganism. The vaccines may be administered by immersion, orally or by intraperitoneal or intramuscular injection. The vaccines may be monovalent vaccine to protect against the virus, or combined with other antigens to multivalent vaccines.

The present invention also provides short nucleotide sequences having a length of at least 10 nucleotides. These sequences may be primers or probes useful in polymerase chain reaction techniques to be used as diagnostic tools. The terms "primer" and "probes" as used herein refers to an oligonucleotide either naturally occurring or produced synthetically which is significantly complementary to a virus target sequence and thus capable of hybridizing to nucleic acid sequences of the present invention. When a "primer pair" or "primer set" is being used, it is generally one forward and one reverse primer, and the sequence between the primers will be multiplied during a PCR. This is well known to a skilled person, and he/she would know which primers constitute a suitable pair. The amplified sequence may be labelled to facilitate detection, e.g. using fluorescent labels on a probe, or other label means well known to the skilled person.

Reference throughout the description to "an embodiment" signifies that a particular feature, structure or property specified in connection with an embodiment is included in the least in one embodiment. The expressions "in one embodiment", "in a preferred embodiment" or "in an alternative embodiment" different places in the description does not necessarily point to the same embodiment. Further, the different features, structures or properties may be combined in any suitable way in one or more of the embodiments.

The sequences according to the invention is as follows:

SEQ ID NO 1 Nucleotide sequence of Segment A full length (3744 NT)

SEQ ID NO 2 Nucleotide sequence of reading frame 1 of segment A (342 NT) SEQ ID NO 3 Protein sequence of reading frame 1 of segment A (114 aa)

SEQ ID NO 4 Nucleotide sequence of reading frame 2 of segment A (3288 NT) SEQ ID NO 5 Protein sequence of reading frame 2 of segment A (1096 aa) SEQ ID NO 6 Nucleotide sequence of segment B full length (2607 NT)

SEQ ID NO 7 Nucleotide sequence of reading frame of segment B (2445 NT) SEQ ID NO 8 Protein sequence of reading frame of segment B (815 aa)

SEQ ID NO 9 Oligonucleotide probe BWBV-1-P for detecting segment B

SEQ ID NO 10 Oligonucleotide probe BWBV-2-P for detecting segment B

SEQ ID NO 11 Oligonucleotide probe BWBV-3-P for detecting segment B

SEQ ID NO 12 Oligonucleotide forward primer BWBV-1-F for detecting segment B SEQ ID NO 13 Oligonucleotide forward primer BWBV-2-F for detecting segment B SEQ ID NO 14 Oligonucleotide forward primer BWBV-3-F for detecting segment B SEQ ID NO 15 Oligonucleotide reverse primer BWBV-1-R for detecting segment B SEQ ID NO 16 Oligonucleotide reverse primer BWBV-2-R for detecting segment B SEQ ID NO 17 Oligonucleotide reverse primer BWBV-3-R for detecting segment B

The sequences 1-17 are enclosed as a sequence listings, the sequences 9-17 are also given in tables in the example below.

The invention will now be described in detail with reference to an example and the enclosed Figures

Figure 1 shows the count of dead fish per tank (K4-K8). Days post hatching is plotted on the X-axis.

Figure 2 shows percentage of dead fish with no feed in the intestine at each time point along with the count of moribund per tank (K5-8). Days post hatching is plotted on the X-axis, the percentage of dead fish with no feed is plotted on the primary Y-axis indicated and the count of dead fish is plotted on the secondary Y-axis (dotted line).

Figure 3 shows box plot showing the detections of the novel Birnavirus at different days post hatching in tanks at the site. Days post hatching is indicated at the x-axis and Ct-values is indicated at the y-axis. Negative samples are indicated by Ct 37.0. The number above each box indicates the count of samples tested at each time point in each tank. The lower and upper border of boxes indicates the 25th and 75th percentiles, respectively and the centerline indicates the 50th percentile. The upper and lower whiskers correspond to the highest and lowest value of the 1.5*IQR (interquartile range).

Figure 4 shows the genome organization of Segment A and B for birnavirus, and Figure 5 shows the CPE from CHSE-214 cell lines after inoculation with homogenate from the Ballan wrasse larvae.

Examples

Disease outbreak

Ballan wrasse eggs were hatched from the 2nd of April 2020 at a commercial hatchery. The facility has for the past years experienced significant mortality in Ballan wrasse around 20 days post hatching (dph). This year the mortality in all tanks was low until day 15 post hatching and increased significantly thereafter (Figure 1).

The only symptom is loss of appetite which is significantly reduced during the onset and duration of the disease. Figure 2 illustrates the increase in the percentage of dead fish with empty intestine along with the increase in mortality.

Samples were taken for PCR and histopathology using known assays and methods, but no known pathogens were detected. Metatranscriptomic sequencing (Meta seq) was therefore performed using Ion Torrent sequencing technology on samples collected from tank 5 (K5) at 1, 7 and at 14 dph and tank 8 at 21 dph. The Meta seq of the sample collected at peak mortality in tank K8 detected a genetic sequence of a novel virus grouping in the Birnaviridae virus family.

The Real Time-PCR assay described below (Assay BWBV-1) was developed, and analysis using the PCR assay confirmed presence of the virus in the samples collected from the diseased fish (Figure 3). In tank K8 the new virus was observed the first times in sample from day 21 which coincides with the peak of mortality and the symptoms of empty stomach. The virus was observed in the fish throughout the observation period of 80 days (Figure 3). The mortality at day 1 to 14 days post hatching was caused by mortality often observed in larvae rearing of marine fish at very early stages. In tank 5, the same were observed, no virus was found/detected in tank K5 before day 21, the mortality were more dragged out in time but the peak of symptoms were the highest at day 23. In tank K3, K6 and K7 only sporadic samples were taken, all confirmed the virus to be present, and all confirmed similar mortality and symptoms (Figure 2 and 3).

In order to verify that the virus was not originating from live feed, rotifers were used as live feed for the fish larvea in the farm. The relevant samples of rotifers used in tank K2 showed negative results to the new virus by using the assay BWBV-1 described below.

Tissue preparation, RNA isolation and analysis

RNA was extracted from samples of fish larvae using RNAeasy Universal kit (Qiagen) on an automated system at the PatoGen AS accredited commercial laboratory. The RNA quality and concentration of the samples were assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies) and the Qubit 3.0 Fluorometer (Thermo Fischer scientific) using the RNA 6000 Pico Kit (Agilent) and the Qubit<TM >RNA HS Assay kit (Thermo Fischer Scientific).

Library preparation and RNA sequencing with Ion Torrent S5

4,6 µg of RNA from larvae sample was used as starting input for the RNA sequencing library preparation. The RNA from the sample was treated with RiboMinus TM Eukaryote Kit v2 (Thermo Fischer Scientific) to remove ribosomal RNA. The rRNA-depleted RNA was fragmented and library was constructed using the Ion Total-RNA Seq Kit v2 (Thermo Fischer Scientific). The library was bar-coded and further quantified with Real Time-PCR.

Using the Ion Chef and the Ion 510 <TM >, Ion 520 <TM >and Ion 530 <TM >kit-Chef (Thermo Fischer Scientific), the library was clonally amplified on Ion Sphere Particles, loaded into one Ion 530 <TM >Chip and sequenced a Ion Torrent S5 sequencer (Thermo Fischer Scientific).

Description of bioinformatics analysis

Approximately 15 million reads were generated for the RNA sequencing experiment. BAM-files were converted to FASTQ files using the SAMTOOLS fastq command, and we applied our in-house pathogen detection pipeline using the reads. Our approach utilizes publicly available research tools, as described in the following.

First, we removed short reads (<100 bps) using VSEARCH (https://github.com/torognes/vsearch). Reads mapping to the Ballan wrasse genome were then removed using Kraken (Wood and Salzberg, 2014), before sequence construction with SPADES using the remaining reads were performed (Bankevich et al., 2012). The resulting scaffold sequences were then tested for complementarity with known sequences using blastn and blastx algorithms against the nucleotide (nt) and Swiss-Prot databases, respectively.

The obtained sequence was blasted and showed similarities to members of the Birnavirideae. Particularly it shows less than 58% similarity to the protein of segment B of a virus detected in Lates calcarifer.

The final genome is divided into segments as shown in Figure 4;

Segment A with full length of 3744 nucleotides (SEQ ID NO 1) with two reading frames. Reading frame 1 (SEQ ID NO 2) consists of 342 nucleotides with the related protein sequence (SEQ ID NO 3) of 114 amino acids. Reading frame 2 (SEQ ID NO 4) of 3288 nucleotides with the related protein sequence (SEQ ID NO 5) of 1096 amino acids.

Segment B with full length of 2607 nucleotides (SEQ ID NO 6) with one reading frame (SEQ ID NO 7) consists of 2445 nucleotides with the related protein sequence (SEQ ID NO 8) of 815 amino acids.

Real Time PCR

To validate if the tentative virus, in the following referred to as Ballan Wrasse Birnavirus (BWBV), could be disease causing we designed a Real Time PCR-assay in the sequence of segment B using forward and reverse primers (BWBV-1-F- and BWBV-1-R (Table 3 and 4), and a minor groove binding (MGB) probe (BWBV-1-P), the assay was named BWBV - 1 (Table 2-4). The primers were designed using the software Primer Express 3.0.1 (Thermo Fischer Scientific). Secondary structures and the possibility of primer dimers were tested using the online software IDT OligoAnalyzer 3.1. The specificity of the primers and probe target were checked using NCBI’s Blastn. The primers and the probe were found not to form secondary structures or primer dimers, nor to hybridize to any other known sequence. The primers and the probe were manufactured by Thermo Fischer Scientific.

The Real Time PCR assay was performed using TaqMan® Fast Virus 1-Step Master Mix.

Amplifications were done on a QuantStudio 5 Real Time PCR systemThermo Fischer Scientific) with the following conditions: 5 min at 50 °C, 20 sec at 95 °C followed by 45 cycles of 95 °C/3 sec and 60 °C/30 seconds.

Results of the qPCR

In addition to the farm where the virus was first identified (Disease Outbreak above), we analysed samples from a range of other farms producing Ballan Wrasse, with the purpose to see the spread of the virus in different farms in Norway and Scotland. Nine different farms was tested with the BWBV-1 assay. The result shows that 2 of the nine farms were positive for the Ballan Wrasse Birnavirus (BWBV). The prevalence in the two positive farms are 31% and 88% and Ct values range from 19 to 37 (table 1). The farms with positive identification of BWBV had related mortality.

Table 1: Farms in Norway and Scotland tested with the BWBV-1 Real Time PCR assay using whole fry. ND=Not detected

Additional qPCR assays

We have designed 3 quantitative Real Time TaqMan assays targeting various regions of the genome, as described above for Ballan Wrasse Birnavirus.

Table 2: Probes tar etin the Ballan Wrasse Birnavirus se uence

Table 3: Forward rimers tar etin the Ballan Wrasse Birnavirus se uence

Table 4: Reverse rimers tar etin the Ballan Wrasse Birnavirus se uence

Virus propagation

The whole fry were homogenised using combination of mechanical and stomacher, 2 fry/5 ml. The homogenate was added to Eppendorf tubes and spun at 10 000 rpm, 4<o>C for 10 minutes. The suspension was filtrated through 0,45 um filter. CHSE-214 cells were infected with 100 ul and 10 ul in CHSE, P24. The cells were observed for 9 days, and cytopathic effect (CPE) was observed, before transferred to second passage. For second passage (P2) 100 ul from each well of P1 were passed onto CHSE-214, the cells were observed for 4 weeks and CPE was observed . For third passage (P3) 100 ul from each well of P2 were passed onto CHSE-214, the cells were observed for 2 weeks and CPE was observed.

CPE was observed in homogenates taken from dead fish. The CPE were observed as syncytia or a clump of dead cells boxed in the pictures in Figure 5.

Virus stability

The virus was passed through cell lines (CHSE-214) as described 10 times, and the virus did not change.

Virulence of the virus

Juvenile Ballan Wrasse were challenged to a suspension of the virus, in seawater. High mortality was observed.

Claims

1. An isolated nucleic acid sequence originating from a virus in fish comprising a sequence selected from the group consisting of SEQ ID No 1, 2, 4, 6 and/or 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and/or 7, and variants thereof being at least 70 % identical.

2. An isolated nucleic acid sequence according to claim 1, characterized in that the sequence is at least 80 %, preferably 90%, more preferably 95 % or 99 % identical with any of the sequences SEQ ID No 1, 2, 4, 6 and/or 7, or any sequences being complementary to SEQ ID No 1, 2, 4, 6 and/or 7.

3. An isolated nucleic acid sequence comprising a sequence encoding an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO 3, 5 and 8, and variants of these sequences being at least 70 % identical thereof.

4. An isolated nucleic acid sequence according to claim 3, characterized in that the amino acid sequence is at least 80 %, preferably 90%, more preferably 95 % or 99 % identical with any of the sequences SEQ ID No 3, 5 and/or 8.

5. A method for detection of a virus in a biological sample, characterized by comprising the following steps:

b) subjecting the sample of step a) to a polymerase chain reaction with a pair of primers wherein each primer of said primer pair comprises at least 10 consecutive nucleotides selected from a group consisting of SEQ ID NO 1, 2, 4, 6 and/or 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and/or 7, and variants of these sequences being at least 70 % identical, and

c) determining whether binding of the primers to nucleotide sequences in the sample and amplification of the sequence between them have occurred indicating the presence of the virus in the sample tested.

6. A method according to claim 5, wherein the primers are selected from a group consisting of sequences being at least 80 %, preferably 90%, more preferably 95 % or 99 % identical with any of the sequences SEQ ID No 1, 2, 4, 6 and/or 7, or any sequences being complementary to SEQ ID No 1, 2, 4, 6 and/or 7.

7. A method according to claim 5 or 6, wherein the primers are selected from a group consisting of SEQ ID No 12-17.

8. A method according to any one of claims 5 - 7, wherein the primer pairs are selected from a group consisting of the following primer pairs

- primers according to SEQ ID NO 12 and 15,

- primers according to SEQ ID NO 13 and 16, and

- primers according to SEQ ID NO 14 and 17.

9. A method according to any one of claims 5 - 8, wherein step c) is performed by using a probe comprising at least 10 consecutive nucleotides selected from the group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants of these sequences being at least 70 % identical.

10. A method according to claim 9, wherein the probe is selected from a group consisting of SEQ ID NO 9-11.

11. A method according to claim 10, wherein the primers and probes are selected from a group consisting of the following primer pairs and probes:

- primers according to SEQ ID NO 12 and 15, and a probe according to SEQ ID NO 9,

- primers according to SEQ ID NO 13 and 16, and a probe according to SEQ ID NO 10, and

- primers according to SEQ ID NO 14 and 17, and a probe according to SEQ ID NO 11.

12. A method according to any one of the claims 5-11, wherein the primer and/or probe comprises at least 15 consecutive nucleotides.

13. A method for detection of a virus in a biological sample, characterized by comprising the following steps:

b) sequencing the mixture of a), and

c) comparing the resulting sequence with a sequence selected of the group consisting of SEQ ID No 1, 2, 4, 6 and 7 and sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, wherein at least 70 % identity on local alignment verifies the presence of the virus in the biological sample tested.

14. A method according to any one of claim 13, wherein the sequencing is performed by a method selected from the group consisting in Illumina (Solexa) sequencing, Roche 454 sequencing, Ion Torrent and SOLiD sequencing.

15. A method according to any one of claims 13-14, wherein the sequence is at least 80 %, preferably 90%, more preferably 95 %, 99 % or 100 % identical on local alignment with any of the sequences SEQ ID NO 1, 2, 4, 6 and/or 7 or sequences being complementary to SEQ ID No 1, 2, 4, 6 and/or 7.

16. A vector comprising a nucleic acid sequence according to any one of claims 1-4.

17. A host cell comprising a vector according to claim 16.

18. A DNA-vaccine comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO 1, 4, 6 and 7, and variants thereof being at least 70 % identical.

19. A recombinant protein encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO 1, 2 ,4, 6 and 7, and variants thereof being at least 70 % identical with any of the sequences of SEQ ID NO 1, 2 ,4, 6 and 7.

20. A recombinant protein according to claim 19, characterized by having an amino acid sequence according to SEQ ID No 3, 5 or 8.

21. A recombinant vaccine comprising at least one recombinant protein according to claim 19 or 20.

22. An antibody specifically recognizing and specifically binding to a recombinant protein according to claim 19 or 20.

23. Diagnostic kit comprising at least a pair of primers wherein each primer of said primer pair comprises at least 10 consecutive nucleotides selected from a group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants of these sequences being at least 70 % identical.

24. Diagnostic kit according to claim 23, wherein the primer is selected from a group consisting of sequences being at least 80%, preferably 90%, more preferred 95 % or 99% identical with the sequences SEQ ID No 1, 2, 4, 6 and 7, or any sequences being complementary to such sequences.

25. Diagnostic kit according to claim 23 or 24, wherein the kit further comprises a probe comprising at least 10 consecutive nucleotides selected from the group consisting of SEQ ID NO 1, 2, 4, 6 and 7, sequences being complementary to SEQ ID NO 1, 2, 4, 6 and 7, and variants of these sequences being at least 70 % identical.

26. Diagnostic kit according to claim 25, wherein the probe is selected from a group consisting of sequences being at least 80%, preferably 90%, more preferred 95 % or 99% identical with the sequences SEQ ID No 1, 2, 4, 6 and 7, or any sequences being complementary to such sequences.

27. Diagnostic kit according to claims 25 or 26, wherein the kit comprises at least one primer pair and a probe selected from the group comprising the following primer pairs and probes:

28. An immunogenic composition comprising at least one of the following

- a recombinant protein according to any one of claims 19-20,

- a nucleic acid sequence having at least 70 % sequence identity with SEQ ID No 1, 2 ,4, 6 and/or 7.

29. Use of the immunogenic composition according to claim 28, in the manufacture of a vaccine, functional feed or additive for functional feed, whereby the vaccine, feed or additive may be used for the treatment or prophylaxis of infection in an animal.

30. Use of a recombinant protein according to any one of claims 19-20, antibody according to claim 22 or an immunogenic composition according to claim 28, in a challenge model.