WO2016075277A1

WO2016075277A1 - Novel fish virus and method for detection

Info

Publication number: WO2016075277A1
Application number: PCT/EP2015/076527
Authority: WO
Inventors: Magnus Andreas DEVOLD; Vidar Teis ASPEHAUG
Original assignee: Patogen Analyse As
Priority date: 2014-11-14
Filing date: 2015-11-13
Publication date: 2016-05-19

Abstract

The present invention relates to the field of aquaculture. Particularly, the invention relates to a novel pathological agent, a virus identified in teleosts. The invention further relates to isolated nucleic acids, useful in diagnosis of the disease, as well as diagnostic kits, primers, probes and methods for determining the presences or absence of virus in a biological sample.

Description

Novel fish virus and method for detection.

The present invention relates to a novel ethological agent, a virus identified in teleosts. The invention further relates to an isolated nucleic acid sequence transcribed from the viral genome, useful in diagnosis of the disease. The present invention furthermore provides kits and reagents useful for the detection of the disease.

Background of the invention

Fish are an increasingly important source of food and income; global annual consumption projected to rise from 1 10 million tons in 2010 to more than 200 million tons in 2030. However, the emergence of infectious diseases in aquaculture threatens production and may also impact wild fish populations.

Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999 (Kongtorp et al., J Fish Dis 27, 351-358 (2004)), HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom (Ferguson et al., J Fish Dis 28, 1 19-123 (2005)). Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful for a long time. Today it is believed that Piscine reovirus (PRV), a newly discovered fish reovirus belongs to the family Reoviridae, subfamily Spinareovirinae, is the likely, causative agent of heart and skeletal muscle inflammation (HSMI). (Kibenge et al., Virol J. 10: 230 (2013)). HSMI is an increasingly economically significant disease in Atlantic salmon (Salmo salar) farms.

PRV and diagnostic compositions, for detecting the presence or absence of PRV, are disclosed in US 2013072542 Al .

In late august 2013 it was discovered a new disease in freshwater rainbow trout in Norway. Sick fish showed signs of circulatory failure and had pale viscera due to anaemia, there were even observed ascites. Histopathological findings were inflammation in heart- and red muscle tissue and cellular necrosis in the liver, signs typically for the HSMI of Atlantic salmon. However, when tissue from diseased fish was analyzed by PCR for different fish pathogenic viruses, no PRV or other viruses were found.

Summary of invention

The present invention is based on the need for specific diagnostic tools for determining the causative pathogenic agents in sick fish. More particularly, the present invention is based on the identification of a novel ethological agent, a virus isolated from freshwater rainbow trout. Specifically, the inventors are the first to provide the nucleic acid sequence, SEQ ID No. 1 , useful for establishing a PCR method for specific detection of the novel virus in salmonides.

Thus the present invention provides the first in vitro assay useful for the diagnosis of the novel fish virus. The PCR-assay, based on the isolated nucleic acid sequence, provided by the invention is highly sensible and is therefor particularly useful for detection of the new virus identified in teleost, e.g. halibut or trout, as for instant brown trout or sea trout, most preferably rainbow trout. .

According to one aspect of the invention, an isolated pathogenic agent is provided wherein the agent is a virus comprising a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID No. 1 and variants thereof being at least 85 % identical with SEQ ID No. 1 based on the entire sequence length of the nucleic acid molecule. According to one embodiment of this aspect, the virus is a novel Trout Piscine reovirus (TPRV) causing heart and skeleton inflammation (HSMI) in salmonids such as rainbow trout. The invention further provides an isolated nucleic acid molecule wherein the molecule comprises a sequence selected from the group consisting of SEQ ID No. 1 and variants thereof having at least 85 % identity to SEQ ID No. 1 based on the entire sequence length of the nucleic acid molecule. In one embodiment of this aspect, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of SEQ ID No. 1. In another embodiment of this aspect, the invention provides the isolated nucleic acid or an oligonucleotide primer or probe comprising a sequence of at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 for diagnosing HSMI in salmonids.

An oligonucleotide primer and/or probe wherein the primer and/or probe comprise a sequence of at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 is provided for detection of nucleic acids present in a biological sample wherein said primer hybridize to a nucleic acid molecule originating from a virus, comprising a sequence selected from the group consisting of SEQ ID No. 1 , and variants thereof being at least 85 % identical.

In one embodiment of this aspect, an oligonucleotide primer and/or probe is provided wherein the primer acting together with another primer constitute an oligonucleotide primer pair suitable for amplification of a region of the virus. In another embodiment, the oligonucleotide primer and/or probe is a primer or primer pair selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 1 1 , SEQ ID No. 12, SEQ ID No. 13 and/or SEQ ID No. 14.

An oligonucleotide primer and /or probe according to the invention may be labelled by any suitable molecule and/or markers, such as a detectable non-isotopic label selected from the group consisting of: a fluorescent molecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzyme substrate, and a hapten.

Further the invention provides a diagnostic kit for specific detection of a virus, comprising a nucleic acid molecule wherein the molecule comprises a sequence selected from the group consisting of SEQ ID No. 1 and variants thereof having at least 85 % identity to SEQ ID No. 1 based on the entire sequence length of the nucleic acid molecule, and/or a fragment or an oligonucleotide primer and/or probe comprising a sequence of at least 8 contiguous nucleotides of the sequence SEQ ID No. 1. According to one embodiment of the invention such a diagnostic kit is a realtime PCR-assay. According to another embodiment the kit comprises an

oligonucleotide primer and/or probe which is able to detect a virus in a biological sample upon hybridization of the oligonucleotide probe to complementary sequence(s) of the said target sequence to be analyzed.

In another aspect of the invention it is provided a method for determining the presence or absence of the fish virus in a biological sample, the method comprising: a) contacting nucleic acid from a biological sample with at least one primer which has an oligonucleotide sequence comprising at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 or a complementary

oligonucleotide thereof,

b) subjecting the nucleic acid and the primer to amplification conditions, and c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of RNA associated with the virus in the sample. In one embodiment of this aspect, the at least one primer used in step a) is selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 1 1 , SEQ ID No. 12, SEQ ID No. 13 and/or SEQ ID No. 14. In another aspect of the invention it is provide a primer set for determining the presence or absence of the fish virus in a biological sample, wherein the primer set comprises at least one synthetic oligonucleotide sequence selected from the group consisting of:

a) a synthetic oligonucleotide which has a sequence comprising at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 , and

b) a synthetic oligonucleotide sequence which has a sequence comprising at least 8 contiguous nucleotides which is complementary to a oligonucleotide sequence as depicted in SEQ ID No. 1.

According to one embodiment the primer set comprises at least one primer selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 1 1 , SEQ ID No. 12, SEQ ID No. 13 and/or SEQ ID No. 14.

In one aspect, the invention provides an isolated oligonucleotide sequence having a sequence as depicted in SEQ ID No. 1.

The present invention furthermore provides an isolated oligonucleotide sequence which is a variant of SEQ ID No. 1 and has at least about 85% identity to SEQ ID No. 1. In one embodiment, the variant has at least about 90%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of SEQ ID No. 1.

According to another embodiment, an isolated oligonucleotide sequence of the present invention comprises consecutive nucleotides having a sequence selected from a variant of SEQ ID No. 1 or a fragment thereof.

The present invention also provides for oligonucleotide sequences, such as primers and probes, useful in the detection of the virus in salmonids according to the present invention.

Furthermore, the present invention provides a kit for detection of the virus in salmonides comprising at least one oligonucleotide according to the present invention. In still another aspect, the present invention provides a method for determining the presence or absence of the fish virus in a biological sample, the method comprising: a) contacting nucleic acid from a biological sample with at least one primer which has an oligonucleotide sequence comprising at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 or a complementary oligonucleotide thereof, b) subjecting the nucleic acid and the primer to amplification conditions, and c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of RNA associated with the virus in the sample.

In still a further aspect, the invention provides a primer set for determining the presence or absence of the fish virus in a biological sample, wherein the primer set comprises at least one synthetic oligonucleotide sequence selected from the group consisting of: a) a synthetic oligonucleotide which has a sequence comprising at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 , and b) a synthetic oligonucleotide sequence which has a sequence comprising at least 8 contiguous nucleotides which is complementary to a oligonucleotide sequence as depicted in SEQ ID No. 1.

Finally, the present invention provides the use of one or more isolated

oligonucleotide sequence(s) comprising at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 or a complementary oligonucleotide thereof for the determination of the virus in salmonides.

The present invention and its various embodiments will be described in more detail in the following.

Figures

Figure 1 shows the percentage similarity of the new virus compared to known isolates.

Figure 2 shows the alignments of the new virus and other known virus isolates.

Detailed description of the invention

The present invention provides a new ethological agent, a virus isolated from freshwater rainbow trout in Norway. Specifically, the present invention provides an isolated nucleic acid sequence transcribed from the viral genome. The new nucleic acid shares some sequence homology with the RNA polymerase from PRV, the agent associated with HSMI in Atlantic salmon. The new virus will thus probably constitute a new strain of reovirus, primarily affecting trout, here termed Trout Piscine reovirus (TPRV). The present invention provides a method for diagnosis of a new disease in teleosts, in particular halibut or trout, as for instant brown trout, sea trout or rainbow trout, based on the identification of the pathogenic agent causing the disease. The invention provides the nucleic acid sequence as depicted in SEQ ID No. 1 , particular useful in diagnostic purposes. The sequence derives from the novel virus isolated from sick rainbow trout.

As used herein, an "oligonucleotide sequence" or "nucleic acid sequence" is to be understood to mean an oligonucleotide sequence or a nucleic acid sequence useful in a diagnosis tool for detection of virus in salmonids.

The term "sequence identity" indicates a quantitative measure of the degree of homology between two nucleic acid sequences. If the two sequences to be compared are not of equal length, they must be aligned to give the best possible fit, allowing the insertion of gaps or alternatively, truncation at the ends of the polypeptide sequences or nucleotide sequences.

The invention provides a diagnostic kit comprising a nucleic acid, a nucleic acid fragment or a nucleic acid variant, a nucleic acid substantially identical to a nucleic acid as depicted by SEQ ID No. 1.

In one embodiment, the invention provides an in vitro assay, with particular high sensitivity, for detection of the novel virus.

In a particular embodiment, the invention provides a Real-time PCR-assay with primers as shown in table 5.

One skill in the art will recognize that when nucleic acids are used for diagnostic purposes, it is not necessary to use the entire nucleic acid to detect the novel virus in an animal or in a sample. Thus, in one aspect, the invention relates to diagnostic kits for detecting virus infection or the presence of the novel virus in a sample, that comprise a nucleic acid as depicted in SEQ ID No. 1 , or a fragment thereof.

The present invention provides oligonucleotide probes and oligonucleotide primers that may be used for detection of the presence of virus in a biological sample, and thus for diagnosis of infected fish. The detection of nucleic acids present in a biological sample is widely applied in both human and veterinary diagnosis, wherein nucleic acids from e.g. pathogens present in biological samples are isolated and hybridized to one or more hybridizing probes or primers are used in order to amplify a target sequence.

One or more oligonucleotide probes may be constructed based on the teaching herein and used in hybridization based detection methods where upon the binding of the oligonucleotides to the target sequence enables detection of the presence of the virus described herein if present in the sample to be tested. The invention is also directed to primer and/or probes which can be labelled by any suitable molecule and/or markers known in the art, for example but not limited to fluorescent tags suitable for use in Real Time PCR amplification, for example TaqMan, cybergreen, TAMRA and/or FAM probes; radiolabels, and so forth. In certain embodiments, the oligonucleotide primers and/or probe further comprises a detectable non-isotopic label selected from the group consisting of: a fluorescent molecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzyme substrate, and a hapten.

Such primers and/or probes may be useful for detecting the presence of the virus of the invention, for example in samples of bodily fluids such as blood, saliva, or urine from an animal, and thus may be useful in the diagnosis of virus infection. Such probes can detect polynucleotides as SEQ ID No. 1 in samples which comprise virus represented by SEQ ID No. 1. The isolated nucleic acids which can be used as primer and/probes are of sufficient length to allow hybridization with, i.e. formation of duplex with a corresponding target nucleic acid sequence, a nucleic acid sequences as in SEQ ID No. l , or a variant thereof. The skilled person will acknowledge that an oligonucleotide probe according to the present invention may be a fragment of DNA or RNA of variable length used herein in order to detect a virus in a biological sample, e.g. RNA, upon hybridization of the oligonucleotide probe to complementary sequence(s) of the said target sequence to be analyzed.

A oligonucleotide probe according to the present invention is able to hybridize to another nucleic acid molecule, such as RNA originating from a virus to be analysed, under appropriate conditions of temperature and solution ionic strength, cf. e.g. Sambrook et al., Molecular Cloning: A laboratory Manual (third edition), 2001 , CSHL Press, (ISBN 978-087969577-4). The condition of temperature and ionic strength determine what the skilled person will recognise as the "stringency" of the hybridization. The suitable stringency for hybridisation of a probe to target nucleic acids depends on inter alia the length of the probe and the degree of

complementation, variables well known to the skilled person. As the skilled person will appreciate, high stringency conditions may comprise hybridization in a buffer consisting of 6 X SSC, 50 mM Tris-HCL (pH=7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 100 pg/ml denatured salmon sperm DNA, for 48 hours at 65°C, washing in a buffer consisting of 2 X SSC, 0.01% PVP, 0.01% Ficoll, 0.01% BSA for 45 minutes at 37°C, and washing in a buffer consisting of 0.1 X SSC, for 45 minutes at 50 °C

A oligonucleotide probe according to the present invention typically comprises a nucleotide sequence which under stringent conditions hybridize to at least 8, 10, 12, 16, 20, 22, 25, 30, 40, 50 (or any other number in-between) or more consecutive nucleotides in a target nucleic acid molecule, e.g. RNA isolated from the virus to be analyzed according to the present invention. According to one embodiment, the oligonucleotide probe according to the present invention comprises about 13 to 25 consecutive nucleotides. It is to be understood that the oligonucleotide probe according one embodiment comprise one of the fragments described herein or the complement thereof. New technology like specific Locked Nucleic Acid (LNA) hybridization probes allows for the use of extremely short oligonucleotide probes (You Y.; Moreira B.G.; Behlke M.A. and Owczarzy R. (2006), "Design of LNA probes that improve mismatch discrimination, Nucleic Acids Res. 34 (8): e60) The present invention furthermore provides oligonucleotide primers useful for amplification of any given region of a nucleotide sequence. An oligonucleotide primer according to the present invention typically comprises a nucleotide sequence at least 8, 10, 12, 16, 20, 22, 25, 30, 40, 50 (or any other number in-between) or more consecutive nucleotides. According to one embodiment, the oligonucleotide primer according to the present invention comprises about 18 - 25 consecutive nucleotides, more preferably about 20 nucleotides.

As used herein, the term "oligonucleotide primer" is to be understood to refer to a nucleic acid sequence suitable for directing an activity to a region of a nucleic acid, e.g. for amplification of a target nucleic acid sequence by polymerase chain reaction (PCR). According to one embodiment of the present invention, "oligonucleotide primer pairs" is provided suitable for amplification of a region of the virus according to the present invention.

The skilled person will acknowledge that an oligonucleotide primer according to the present invention may be a fragment of RNA of variable length used herein in order to detect the virus, e.g. RNA, upon alignment of the oligonucleotide probe to complementary sequence(s) of the said target sequence to be analyzed. An oligonucleotide primer according to the present invention may furthermore be labeled with a molecular marker in order to enable visualization of the results obtained. Various molecular markers or labels are available. An oligonucleotide primer according to the present invention typically comprises the appropriate number of nucleotides allowing that said primer align with the target sequence to be analyzed. It is to be understood that the oligonucleotide primer according to the present invention according to one embodiment comprises sequences described herein or the complement thereof.

According to one embodiment of the present invention, primer pairs are provided selected as depicted in table 5.

Oligonucleotide probes and oligonucleotide primers according to the present invention may be synthesized according to methods well known to the skilled person. EXAMPLES

SEQUENSING OF THE VIRUS

Rainbow trout with clinical signs not consistent with any known disease were received in PatoGens laboratory, and samples were collected from several tissues. Heart samples were analyzed for the presence of PRV, and positive results were obtained, but with Ct-values indicating low amount of virus particles, or a mismatch in a primer or a probe that could affect the sensitivity of the assay.

The Ct-values obtained were too high to explain the clinical signs observed in the fish, but we decided to perform a genome sequencing to find if the reason for this was mutations reducing the sensitivity of the Real-Time PCR-assay used.

MATERIALS AND METHODS

RNA from Salmon hearts was provided by PatoGen Analyse AS (Aalesund, Norway) (Table 2).

Table 2: RNA samples from PatoGen Analyse AS

The RNA was converted to cDNA by the cDNA synthesis kit Superscript III (Invitrogen, 18080-051), according to the manufacturer's protocol, using random hexamer, and 1 μΐ total RNA from the samples in table 1. The cDNA was then amplified by PCR with Red'Y'Gold Mix (Eurogentec, PK-0064-02R) with degenerated primers using an annealing temperature of 48°C. The degenerated primers were diluted to a concentration of 10 μΜ prior to the reaction and used in combinations illustrated in Table 3. Table 3 : Primers used in the PCR

5μ1 PCR products and 2μ1 250 bp ladder were loaded on to a 1% agarose gel, stained with GelRed (Biotium, 41003), and run at 100 V for 45 minutes. The gel was illuminated with UV-light and photographed. None of the PCRs where the primer combination 2 was used produced PCR product. And due to several unspecific bands on the gel, bands of interest were cut out of the gel and purified using the QlAquick gel extraction kit according to the manufacturer's protocol (Qiagen, 28704). The purified products were run on a 1% agarose gel and the DNA concentration in each sample were estimated by comparing band intensity towards a low mass ladder (Invitrogen, 10068-013).

5-20 ng of the purified DNA was used in sequencing reactions with BigDye 3.1. The sequencing reaction products were added 10 μΐ deionized water and shipped to Sequencing Facility, MBI (Thormohlensgt. 55, 5008 Bergen, Norway). Fluorescently labeled fragments were extracted at the Sequencing Lab and detected using the Applied Biosystems 3730XL Analyzer. Sequences were obtained from the samples listed in table 4.

Table 4: Overview of RNA samples and primers which produced readable sequences

RESULTS Sequences were obtained for one primer set only, and we obtained a part of the polymerase sequence of 735 nucleotides (SEQ ID No. 1 , see below). Surprisingly this sequence had only 85% similarity to the previously known PRV-polymerase sequences (Figure 1. Sequence analysis performed using Gene Doc ver. 2.7.000). Figure 2 shows an alignment generated in Vector NTI comparing the new

polymerase sequence to known PRV-sequences. A Real-Time PCR-assay was designed using Primer express 3.0.1 specific for the new virus (Table. 5). Table 5 also shows all primers designed initially to perform the sequencing of this new virus.

Claims

CLAIMS 1. An isolated pathogenic agent wherein the agent is a virus comprising a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID No. 1 and variants thereof being at least 85 % identical with SEQ ID No. 1 based on the entire sequence length of the nucleic acid molecule. 2. An virus according to claim 1 wherein the virus is a novel Trout Piscine

reovirus (TPRV) causing heart and skeleton inflammation (HSMI) in salmonids such as rainbow trout. 3. An isolated nucleic acid molecule wherein the molecule comprises a sequence selected from the group consisting of SEQ ID No. 1 and variants thereof having at least 85 % identity to SEQ ID No. 1 based on the entire sequence length of the nucleic acid molecule. 4. An isolated nucleic acid molecule according to claim 3 wherein the nucleic acid has a sequence with at least about 90%, about 95.5%, about 96%>, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of SEQ ID No. 1 based on the entire sequence length of the said isolated nucleic acid sequence. 5. An isolated nucleic acid according to claim 3 or 4 or an oligonucleotide primer or probe comprising a sequence of at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 for diagnosing HSMI in salmonids. 6. An oligonucleotide primer and/or probe wherein the primer and/or probe

comprise a sequence of at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 for detection of nucleic acids present in a biological sample wherein said primer hybridize to a nucleic acid molecule originating from a virus, comprising a sequence selected from the group consisting of SEQ ID No. 1 , and variants thereof being at least 85 % identical. 7. An oligonucleotide primer and/or probe according to claim 6 wherein the

primer acting together with another primer constitute an oligonucleotide primer pair suitable for amplification of a region of the virus. 8. An oligonucleotide primer and/or probe according to anyone of the claims 6 or 7 wherein the primer or primer pair are selected from the group consisting of SEQ ID No.

2, SEQ ID No.

3, SEQ ID No.

4, SEQ ID No.

5, SEQ ID No.

6, SEQ ID No.

7, SEQ ID No.

8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 1 1 , SEQ ID No. 12, SEQ ID No. 13 and/or SEQ ID No. 14.

9. An oligonucleotide primer and /or probe according to any one of the claims 6-8 wherein the primer and/or probes are labelled by any suitable molecule and/or markers.

10. An oligonucleotide primer and /or probe according to claim 9 wherein the suitable molecule and/or marker is a detectable non-isotopic label selected from the group consisting of: a fluorescent molecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzyme substrate, and a hapten.

1 1. A diagnostic kit for specific detection of a virus, comprising a nucleic acid according to any one of the claims 3 or 4, or an oligonucleotide primer and/or probe according to any one of the claims 6-10.

12. A diagnostic kit according to claim 1 1 , wherein the kit is a real-time PCR- assay.

13. A diagnostic kit according to any one of the claims 1 1-12, wherein the

oligonucleotide primer and/or probe is able to detect a virus in a biological sample upon hybridization of the oligonucleotide probe to complementary sequence(s) of the said target sequence to be analyzed.

14. A method for determining the presence or absence of the fish virus in a

biological sample, the method comprising:

a) contacting nucleic acid from a biological sample with at least one primer which has an oligonucleotide sequence comprising at least 8 contiguous nucleotides of the sequence SEQ ID No. 1 or a complementary

oligonucleotide thereof,

b) subjecting the nucleic acid and the primer to amplification conditions, and c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of RNA associated with the virus in the sample.

15. A method according to claim 14, wherein the at least one primer is selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 1 1 , SEQ ID No. 12, SEQ ID No. 13 and/or SEQ ID No. 14.

16. A primer set for determining the presence or absence of the fish virus in a biological sample, wherein the primer set comprises at least one synthetic oligonucleotide sequence selected from the group consisting of: