AU2014273183B2 - Scale drop disease (SDD) causative virus and derivatives thereof - Google Patents

Abstract

The present invention relates to an isolated virus causing Scale Drop Disease in fish, to cell cultures comprising said virus, to vaccines on the basis of said virus and methods for the preparation of such a vaccine, to antibodies reactive with said virus, to diagnostic test kits for the detection of that virus and to uses of said virus.

Description

PCT/EP2014/061014 WO 2014/191445 1

SCALE DROP DISEASE (SDD) CAUSATIVE VIRUS AND DERIVATIVES THEREOF

The present invention relates to an isolated virus causing Scale Drop Disease in fish, to cell cultures comprising said virus, to vaccines on the basis of said virus and methods for the preparation of such a vaccine, to antibodies reactive with said virus, to diagnostic test kits for the detection of that virus and to uses of said virus.

Over the last decades, world-wide a strong increase is seen in the consumption of fish. This equally regards the consumption of cold water fish such as salmon, turbot, halibut and cod, and tropical fish such as Asian seabass (barramundi), tilapia, milkfish, yellowtail, ambeijack, grouper and cobia.

As a consequence, an increase is seen in the number and the size of fish farms, in order to meet the increasing needs of the market.

As is known from e.g. animal husbandry, large numbers of animals living closely together are vulnerable to all kinds of diseases, even diseases hardly known or seen or even unknown before the days of large-scale commercial farming. This is equally the case in fish farming.

Recently, a new disease syndrome was found in farmed Asian seabass (Lates calcarifer). The disease is most prominently characterized by scale-loss, and became therefore commonly known as “Scale Drop Syndrome” (SDS). The disease was first reported in Asian seabass farmed in Penang, Malaysia. Outbreaks of the new disease were later found to occur also in Singapore in 2002, 2006 and 2009. More recent cases were reported in 2010 from fish farms in Bantam, Indonesia and again from Singapore, and in 2011 also from farms in the Straits of Malaka. The incidence of the disease is currently increasing.

The disease syndrome was recently described by Gibson-Kueh, S., et al., (Journal of Fish Diseases 35; 19-27 (2012)).

The disease is initially seen in adult cage reared fish, and is also seen in juveniles in nursery. The mortality is described as chronic, lingering and varies between 30% initially to 75% overall.

The main clinical signs of fish suffering from the syndrome are first of all scale-drop as mentioned above, lethargic behavior and sometimes swollen eyes. Fish also sometimes show neurological signs: some affected fish show spiral swimming possibly due to vascular damage in the brain resulting in multifocal encephalomalacia.

Major histological signs are i.a. a vascular endothelial degeneration (vasculitis) in all major organs including the skin. This vasculitis leads to tissue necrosis that also affects gastric PCT/EP2014/061014 WO 2014/191445 2 glands, spleen, kidneys and heart. As also described by Gibson-Kueh, the dermis overlaying the scale beds is often necrotic and is associated with the scale loss.

The cause of the disease could however not be found. Gibson-Kueh says that the histopathological changes in diseased fish and the observation of both large hexagonal virions and much smaller hexagonal virions in tissue may suggest the possibility of a viral aetiology, but she concludes that overall, virion numbers in tissues examined were low. Based on size and morphology, some of the observed virions resembled iridovirus, but immunohistochemistry using the anti-Red Seabream Iridovirus (RSIV) monoclonal antibody M10 (Nakajima, K. et al., Fish Pathology 30: 115-119 (1995)) gave negative results. PCR tests using RSIV primers known to target a large range of known Iridoviruses also gave negative results.

Attempts to isolate a virus by contacting fish tissue with Haemulon sciurus Shaw (GF) cells and Asian Seabass cells (Chong, S. et al., Singapore Vet. J. 1; 78-89 (1987)) were also not successful.

Given the very low number of virus-like particles observed, Gibson-Kueh suggests that the disease might, instead of being caused by a virus, be the result of an immune hypersensitivity reaction to viral antigens. Such reactions are e.g. suggested to be the cause of strawberry disease in salmonids.

Apart from this, the vasculitis and associated necrosis that are the hallmarks of SDS are not typical of Irido disease.

Moreover, the lesions seen in SDS differ in several ways from lesions seen in irido viral diseases.

The fact that in spite of the attempts mentioned above no viral origin could be found and even no proof of viral involvement was found, leads Gibson-Kueh to the following explanation for the presence of low numbers of different viruses: “Virus-like particles were relatively hard to find. Moreover, systematic iridoviral disease is now endemic in L. calcarifer farms, so their presence could be an incidental finding of a common pathogen”.

For these reasons, the causative agent of the disease until now remained totally in the dark.

It is an objective of the present invention to provide the causative agent of this disease as well as vaccines aiming at combating the disease. Moreover, it is an objective of the present invention to provide means to detect and identify the causative agent.

It has now been determined that the causative agent of this disease is an icosahedral virus of about 140 nm in diameter. PCT/EP2014/061014 WO 2014/191445 3

The virus was found to belong to the double-stranded DNA-viruses and a large part of the DNA sequence of this virus has now been determined.

Comparison of the sequence of this new virus with other sequences in a genome databank unexpectedly revealed that at nucleotide level the virus bears a certain albeit low, level of resemblance to viruses of the family Iridoviridae, a family of viruses that have an icosahedral shape, have a size of between 120 and 350 nm and possess a double-stranded genome.

Since the causative agent of the disease has now been identified, the disease will no longer be referred to as Scale Drop Syndrome, but as Scale Drop Disease (SDD), in the description (vide infra). A representative of the virus has been deposited with the Collection Nationale de Cultures de Microorganisms (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France, under accession number CNCM 1-4754.

On the basis of sequence comparison, a gene encoding a Major Capsid Protein and a gene encoding an ATPase of this virus could be identified, that bear some resemblance to known Iridoviridae.

An example of the DNA sequences of the gene encoding the Major Capsid Protein and the gene encoding the ATPase are depicted in SEQ ID NO: 1 and SEQ ID NO: 3 respectively. SEQ ID NO: 2 represents the amino acid sequence of the Major Capsid Protein. SEQ ID NO: 4 represents the amino acid sequence of the ATPase.

The family Iridoviridae currently comprises 5 genera: the Ranaviruses, the Megalocytiviruses, the Lymphocystiviruses, the Chloriridoviruses and the Iridoviruses (Jun Kurita and Kazuhiro Nakajima, Viruses 4; 521-538 (2012)).

The paper by Kurita and Nakajima i.a. shows an overview of the 5 genera in a phylogenetic tree of a total of 20 known species of the 5 genera (additionally three Ascovirus homologs were added as an out-group). This phylogenetic tree gives an indication of the mutual relatedness/distance of the various species and visualizes why each of these viruses is classified as a member of one of the 5 genera.

Based upon the MCP and ATPase encoding DNA sequences of the newly found causative agent of SDD according to the invention, new phylogenetic trees, based on the Neighbor-Joining method, could be made, and it was found that the MCP and ATPin coding sequences show a certain fit in the phylogenetic tree of the Iridoviridae.

These trees were made using the program MEGA, version 5, using standard settings. (MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Koichiro Tamura, Daniel PCT/EP2014/061014 WO 2014/191445 4

Peterson, Nicholas Peterson, Glen Stecher, Masatoshi Nei and Sudhir Kumar. Mol. Biol. Evol. 28(10): 2731-2739. 2011 doi:10.1093/molbev/msrl21 Advance Access publication May 4, 2011).

Based upon the icosahedral shape, the genomic size of between 120 and 350 nm and the double-stranded genome of the novel virus, and on the basis of the Major Capsid Protein neighbor-joining tree, obtained using MEGA5 with the statistical support indicating the robustness of the inferred branching pattern as assessed using the bootstrap test, the inventors consider the virus to be a member of the Famly Iridoviridae.

The tree on the basis of the MCP sequence is depicted in figure 8. The tree on the basis of the ATPase sequence is depicted in figure 9.

Quite surprisingly, based upon its distance to the 5 known genera the newly found causative agent of SDD does not seem to fit in any of the 5 genera, as can readily be seen from figure 8.

The virus can thus i.a. be distinguished from known members of the Iridoviridae on the basis of the coding DNA sequences of its Major Capsid Protein and its ATPase.

It turned out that the Major Capsid Protein of the virus according to the invention has a level of sequence identity with the MCP of even the nearest of the other species of the Iridoviridae of only 65%.

The ATPase has a level of sequence identity of only 68% with the nearest ATPase of the other species of the Iridoviridae.

Using the Major Capsid Protein and ATPase encoding DNA sequences primers were developed that are specific for the virus according to the invention. SEQ ID NO: 1 shows a typical example of the nucleotide sequence of a gene encoding the Major Capsid Protein of a virus according to the invention.

It will be understood that, for the particular proteins embraced herein, natural variations can exist between individual representatives of the causative agent. Genetic variations leading to minor changes in e.g. the Major Capsid Protein sequence do exist. This is equally true for the ATPase. First of all, there is the so-called “wobble in the second and third base” explaining that nucleotide changes may occur that remain unnoticed in the amino acid sequence they encode: e.g. triplets TTA, TTG, TCA, TCT, TCG and TCC all encode Leucine. In addition, minor variations between representatives of the SDD virus according to the invention may be seen in amino acid sequence. These variations can be reflected by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al in "The Proteins" Academic Press New York (1979). Amino acid replacements between PCT/EP2014/061014 WO 2014/191445 5 related amino acids or replacements which have occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,

Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val and Ala/Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science 227, 1435-1441, 1985) and determining the functional similarity between homologous proteins. Such amino acid substitutions of the exemplary embodiments of this invention, as well as variations having deletions and/or insertions are within the scope of the invention.

This explains why MCP and ATPase, when isolated from different representatives of an SDD virus according to the invention, may have homology levels that are significantly below 100%, while still representing the MCP or ATPase of SDD virus, the causative agent of Scale Drop Disease.

This is clearly reflected e.g. in figure 4 of the paper by Kurita and Nakajima, where it is shown that even within the group Lymphocystivirus consisting of highly related lymphocystis disease viruses (LCDV), all LCDVs nevertheless have significantly different MCP amino acid sequences.

Thus, a first embodiment of the present invention relates to an isolated virus which is a member of the family Iridoviridae comprising an MCP gene and an ATPase gene, characterized in that: a) the virus is the causative agent of Scale Drop Disease in fish and b) the nucleoti de sequence of the MCP gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 1

For the purpose of this invention, a level of identity is to be understood as the level of identity of the sequence of SEQ ID NO: 1 and the corresponding region of the Major Capsid Protein of the virus of which the level of identity has to be determined. A suitable program for the determination of a level of identity is the nucleotide blast program (blastn) of NCBI’s Basic Local Alignment Search Tool, using the “Align two or more sequences” option and standard settings (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

For the purpose of this invention, isolated means: set free from tissue with which the virus is associated in nature. An example of the isolated virus is the virus as present in cell culture. A preferred form of this embodiment relates to a virus that has a Major Capsid Protein (MCP) gene that has a level of identity of at least 82%, more preferably 84%, 86%, 88%, 90%, 91%, PCT/EP2014/061014 WO 2014/191445 6 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, in that order of preference, to the nucleotide sequence of the MCP as depicted in SEQ ID NO: 1.

Another, alternative way to characterize a virus according to the invention relates to the sequence of the ATPase of the virus. SEQ ID NO: 3 shows a typical example of the nucleotide sequence of the ATPase gene of a virus according to the invention. As explained above, natural variations leading to minor changes in the ATPase sequence are however found.

Thus, another form of this embodiment of the present invention relates to an isolated virus which is a member of the family Iridoviridae comprising an MCP gene and an ATPase gene, characterized in that: a) the virus is the causative agent of Scale Drop Disease in fish and b) the nucleotide sequence of the ATPase gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 3 A preferred form of this embodiment relates to a virus that has an ATPase gene that has a level of identity of at least 82%, more preferably 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, in that order of preference, to the nucleotide sequence of the ATPase gene as depicted in SEQ ID NO: 3. A more preferred form of this embodiment relates to a virus according to the invention wherein the nucleoti de sequence of the MCP gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 1 and the nucleotide sequence of the ATPase gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 3.

Still another, alternative, way to characterize the virus according to the invention depends on a PCR-test using primer sets that are specific for the Major Capsid Protein gene sequence or the ATPase gene sequence of a virus according to the invention. Three different primer sets of which the sequence is depicted in SEQ ID NO: 5-6, SEQ ID NO: 7-8 and SEQ ID NO: 9-10 were elected for their specificity for the virus. The PCR-test using the first primer set (SEQ ID NO: 5-6) that specifically reacts with the Major Capsid Protein gene of the virus uses the two primers SDD-50-FW:CAGTGCATTACAAGAAAG and SDD-213-REV:GCTGAAACAACAATTTAG.

The PCR-test using the second primer set (SEQ ID NO: 7-8) also specifically reacts with the Major Capsid Protein gene of the virus and uses the two primers sdd-mcp-277-fw : TCCTGTGCAGCTGTCTAAAC and SDD-MCP-1090-REV: ACTGGCAATGATGGGCGATG. PCT/EP2014/061014 WO 2014/191445 7

The PCR-test using the third primer set (SEQ ID NO: 9-10) specifically reacts with the ATPase gene of the virus and uses the two primers SDD-ATPase-65-FW: TCGGAGGGATGAAATTGG and SDD-ATPase-618-REV: AGCGTTGTCGATGTAGAG.

The tests, which are described in more detail in the Examples section, are standard PCR tests.

If analysis of the PCR-product of the first primer set reveals a PCR product of approximately 164 base pairs or if analysis of the PCR-product of the second primer set reveals a PCR product of approximately 814 base pairs or if analysis of the PCR-product of the third primer set reveals a PCR product of approximately 554 base pairs, and the virus is the causative agent of Scale Drop Disease, this unequivocally demonstrates that the analysed virus belongs to the virus according to the invention.

Merely as an example: a PCR product of approximately 164 base pairs is a PCR product with a length of between 164 + 10 and 164-10 base pairs. A PCR product of approximately 814 base pairs is a PCR product with a length of between 814 + 10 and 814 - 10 base pairs.

Thus again another form of this embodiment of the present invention relates to an isolated virus which is a member of the family Iridoviridae comprising an MCP gene and an ATPase gene, characterized in that: a) the virus is the causative agent of Scale Drop Disease in fish and b) the viral DNA reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 5 and 6 to give a PCR product of 164 +/- 10 base pairs or reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 7 and 8 to give a PCR product of 814 +/-10 base pairs or reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 9 and 10 to give a PCR product of 554 +/- 10 base pairs. A preferred form of this embodiment relates to a virus according to the invention wherein the viral DNA reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 5 and 6 to give a PCR product of 164 +/- 10 base pairs and reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 7 and 8 to give a PCR product of 814 +/-10 base pairs and reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 9 and 10 to give a PCR product of 554 +/- 10 base pairs. A more preferred form of this embodiment relates to a virus according to the invention wherein the nucleotide sequence of the MCP gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 1 and the nucleotide sequence of the ATPase gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 3 and wherein the viral DNA reacts in a PCR reaction with a primer set as PCT/EP2014/061014 WO 2014/191445 8 depicted in SEQ ID NO: 5 and 6 to give a PCR product of 164 +/-10 base pairs and reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 7 and 8 to give a PCR product of 814+/-10 base pairs and reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 9 and 10 to give a PCR product of 554 +/- 10 base pairs.

The viruas according to the invention can be in a live, a live attenuated or an inactivated form.

As indicated above, the DNA sequences of the genes encoding the MCP and the ATPase of the virus have now been characterized.

The identification of these genes is highly useful, since they can now be used in DNA-vaccines, for the expression of these proteins, and for diagnostic purposes, as will extensively be explained below.

Therefore, another embodiment of the present invention relates to a DNA fragment comprising a gene encoding a Major Capsid Protein characterized in that that gene has a level of identity of at least 80% to the nucleotide sequence of the MCP gene as depicted in SEQ ID NO: 1. A preferred form of this embodiment relates to such a DNA fragment comprising a gene having a level of identity of at least 82%, more preferably 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, in that order of preference, to the nucleotide sequence of the MCP as depicted in SEQ ID NO: 1.

Again another embodiment of the present invention relates to a DNA fragment comprising a gene encoding an ATPase characterized in that that gene has a level of identity of at least 80% to the nucleotide sequence of the ATPase gene as depicted in SEQ ID NO: 3. A preferred form of this embodiment relates to such a DNA fragment comprising a gene having a level of identity of at least 82%, more preferably 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, in that order of preference, to the nucleotide sequence of the ATPase as depicted in SEQ ID NO: 3.

Still another embodiment of the present invention relates to a Major Capsid Protein characterized in that this MCP is encoded by a DNA fragment encoding a Major Capsid Protein according to the invention.

Such MCPs of the virus according to the invention are highly suitable because they are suitable in vaccines and they make diagnostic tests possible, as explained below. WO 2014/191445 PCT/EP2014/061014 9 A preferred form of this embodiment relates to a MCP having the amino acid sequence as depicted in SEQ ID NO: 2.

Again another embodiment of the present invention relates to an ATPase, characterized in that that ATPases is encoded by a DNA fragment encoding an ATPase according to the invention.

Such ATPases of the virus according to the invention are highly suitable i.a. because they make diagnostic tests possible, as explained below. A preferred form of this embodiment relates to an ATPase having the amino acid sequence as depicted in SEQ ID NO: 4. A preferred form of this embodiment relates to an ATPase having the amino acid sequence as depicted in SEQ ID NO: 4.

Several fish cell lines have now been identified that are able to support the replication of a virus according to the invention.

An example of a cell line that can be used to grow the virus according to the invention is a cell line from brain cells of Asian sea bass. Methods for the isolation of such a cell line have been described i.a. by Hasoon et al., in In Vitro Cell. Dev. Biol. - Animal 47: 16-25 (2011). Another example of a cell line that can be used to grow the virus according to the invention is deposited with the Collection Nationale de Cultures de Microorganisms (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France, under accession number CNCM 1-4755.

Thus, again another embodiment of the present invention relates to a cell culture comprising a virus, wherein that cell culture comprises a virus according to the invention.

Now that the cause of the disease has been found and could be demonstrated to be of viral origin, the disease could deliberately be induced and the typical signs of the disease described above could indeed be induced in healthy fish, as is shown in detail in the Examples section.

It is again one of the merits of the present invention that, the causative agent being known now, the development of vaccines has become feasible. PCT/EP2014/061014 WO 2014/191445 10

Thus, another embodiment of the present invention relates to vaccines for combating Scale Drop Disease in fish, wherein such vaccines comprise a virus according to the invention and a pharmaceutically acceptable carrier.

Combating in this respect should be interpreted in a broad sense: combating Scale Drop Disease is considered to comprise vaccination in order to prevent the disease, vaccination to diminish the signs of the disease and therapeutic vaccination after the disease is diagnosed.

For vaccine purposes, the virus preferably reacts serologically with covalescent anti-SDD virus antiserum or with antiserum raised against the deposited virus. A serological reaction should be interpreted broadly: a serological reaction is considered to be a reaction in a standard serological test such as an ELISA test.

Examples of pharmaceutically acceptable carriers that are suitable for use in a vaccine for use according to the invention are sterile water, saline, aqueous buffers such as PBS and the like. In addition a vaccine according to the invention may comprise other additives such as adjuvants, stabilizers, anti-oxidants and others, as described below.

The vaccine according to the invention may comprise the virus according to the invention in attenuated live or inactivated form.

Attenuated live virus vaccines, i.e. vaccines comprising the virus according to the invention in a live attenuated form, have the advantage over inactivated vaccines that they best mimic the natural way of infection. In addition, their replicating abilities allow vaccination with low amounts of viruses; their number will automatically increase until it reaches the trigger level of the immune system. From that moment on, the immune system will be triggered and will finally eliminate the viruses. A minor disadvantage of the use of live attenuated viruses however might be that inherently there is a certain level of virulence left. This need not be a real disadvantage as long as the level of virulence is acceptable, i.e. as long as the vaccine at least prevents the fish from dying. Of course, the lower the rest virulence of the live attenuated vaccine is, the less influence the vaccination has on weight gain during/after vaccination. A live attenuated virus is a virus that has a decreased level of virulence when compared to virus isolated from the field. As mentioned above, the virulence of virus isolated from the field is relatively high: mortality typically exceeds 30% of all infected fish. A virus having a decreased level of virulence is considered a virus that only induces disease to the extent that mortality does not exceed 10%, and 90% or more of all infected fish survive the infection. Therefore, one preferred form of this embodiment of the invention relates to a vaccine comprising a virus according to the invention wherein said virus is in a live attenuated form. PCT/EP2014/061014 WO 2014/191445 11

Attenuated viruses can e.g. be obtained by growing the viruses according to the invention in the presence of a mutagenic agent, followed by selection of virus that shows a decrease in progeny level and/or in replication speed. Many such agents are known in the art.

Another very often used method is serial in vitro passage. Viruses then get adapted to the cell line used for the serial passage, so that they behave attenuated when transferred to the natural host again as a vaccine.

Still another way of obtaining attenuated viruses is to subject them to growth under temperatures deviating from the temperature of their natural habitat. Selection methods for temperature sensitive mutants (Ts-mutants) are well-known in the art. Such methods comprise growing viruses in the presence of a mutagen followed by growth at a sub-optimal temperature and at the optimal temperature, titration of progeny virus on cell layers and visual selection of those plaques that grow slower at the optimal temperature. Such small plaques comprise slow-growing and thus desired live attenuated viruses.

Inactivated vaccines are, in contrast to their live attenuated counterparts, inherently safe, because there is no rest virulence left. In spite of the fact that they usually comprise a somewhat higher dose of viruses compared to live attenuated vaccines, they may e.g. be the preferred form of vaccine in fish that are suffering already from other diseases. Fish that are kept under sub-optimal conditions, such as incomplete nutrition or sub-optimal temperatures, would also benefit from inactivated vaccines.

Therefore, another preferred form of this embodiment relates to a vaccine comprising a virus according to the invention wherein said virus is in an inactivated form.

Many physical and chemical methods of inactivation of viruses are nowadays known in the art. Examples of physical inactivation are UV-radiation, X-ray radiation, gamma-radiation and heating. Examples of inactivating chemicals are B-propiolactone, glutaraldehyde, ethylene-imine and formaldehyde. The skilled person knows how to apply these methods. Preferably the virus is inactivated with β-propiolactone, glutaraldehyde, ethylene-imine or formaldehyde. It is obvious that other ways of inactivating the virus are also embodied in the present invention.

In principle, the standard methods for the preparation of a vaccine on the basis of inactivated iridovirus are equally applicable to the virus according to the invention. Merely as an example: methods for the preparation of a vaccine on the basis of inactivated whole Singapore grouper iridovirus have been described i.a. by Zhengliang Ou-yang et al., in Developmental and Comparative Immunology 38: 254-261 (2012). PCT/EP2014/061014 WO 2014/191445 12

Furthermore, in the Examples section below, examples of methods for the preparation of vaccines on the basis of inactivated virus according to the invention are presented.

Another approach for combating SDD is the use of subunit vaccines. Such vaccines do not comprise the whole virus, but merely one or more antigenic components of the virus.

Subunit vaccines have the advantage that for their preparation no growth of virus is required. It suffices to express the subunit of choice by cloning the DNA encoding that subunit in an expression system.

It was found that the MCP of the virus according to the invention is a very relevant immunogenic protein.

It apparently shares this characteristic with the known members within the family Iridoviridae. The immunogenic relevance of the MCP of marine iridovirus from grouper was shown by Qi Wei Qin (J. Virological Methods 106: 89-96 (2002)). Recently, Xiaozhe Fu et al., showed protective immunity against iridovirus disease in mandarin fish, induced by recombinant MCP of infectious spleen and kidney necrosis virus (Fish and Shellfish Immunology 33: 880-885 (2012)). Zhengliang Ou-yang et al., (Veterinary Immunology and Immunopathology 149: 38-45 (2012)) describe vaccination on the basis of the MCP as expressed in E. coli. They obtained significant protection with the recombinant MCP protein (50 microgram of protein).

Vaccines on the basis of MCP for oral application in fish are i.a. described by Yutaka Tamara et al., (Biotechn. Prog. 22: 949-953 (2006)). They describe the expression of the MCP of Red Sea Bream Iridovirus on the surface of yeast cells as a basis for oral vaccination of fish against iridovirus.

In addition to this, more general guidance in the field of protein expression is given in many text books. Handbooks giving extensive information about expression in bacterial expression systems are e.g.: Manual of Industrial Microbiology and Biotechnology, 3rd Edition by Richard H. Baltz (Editor-in-Chief), Arnold L. Demain (Editor-in-Chief), Julian E. Davies (Editor-in-Chief) ISBN: 978-1-55581-512-7, E. coli gene expression protocols by Peter E. Vaillancourt in Methods in Molecular Biology 205 ISBN: 1-58829-008-5, Protein expression: a practical approach by S. J. Higgins and B. D. Hames ISBN: 0-19-963624-9, Protein expression technologies: current status and future trends, 2004 by Francois Baneyx, Baculovirus expression vectors; a laboratory manual, by O’Reilly, D et al., Oxford University Press 1994, ISBN 0-19-509131-0 and. Production of recombinant proteins: novel microbial and eukaryotic expression systems by Gerd Gellissen, ISBN: 3-527-31036-3.

Thus, a third preferred form of this embodiment of the present invention relates to a vaccine for combating Scale Drop Disease in fish, wherein said vaccine comprises a Major Capsid Protein according to the invention and a pharmaceutically acceptable carrier. WO 2014/191445 PCT/EP2014/061014 13

Finally, it turned out that DNA fragments comprising the sequence of the MCP gene as described in the present invention are very suitable for use in a DNA vaccine.

Such DNA fragments, comprising the DNA of the MCP-gene of Singapore grouper iridovirus in a eukaryotic expression vector, are i.a. described by Zhengliang Ou-yang (Veterinary Immunology and Immunopathology 149: 38-45 (2012)). They reached protection against Singapore grouper iridovirus infection with a prime-boost regime of vaccination with 30 microgram of DNA.

Robust protection of red Seabream against red Seabream iridovirus using a DNA vaccine was shown by Caipang, C.M.A. et al., (Fish and Shellfish Immunology 21: 130-138 (2006)). They used a DNA vaccine comprising the MHC-gene of RSI V under the control of the cytomegalovirus immediate/early enhancer promoter.

Thus, a fourth preferred form of this embodiment of the present invention relates to a vaccine for combating Scale Drop Disease in fish, wherein said vaccine comprises a DNA fragment comprising a gene encoding a Major Capsid Protein according to the invention and a pharmaceutically acceptable carrier.

If a virus according to the invention is used as a vaccine component for oral administration (e.g. through dipping or balneation), there will usually be no need for the administration of an adjuvant.

If however the vaccine preparation is injected directly into the fish, the use of an adjuvant is optional.

Especially when the viral component in an injection vaccine is in an inactivated form, the addition of adjuvants may be preferred.

In general, in order to boost the immune response, the vaccine may include a variety of adjuvants, particularly in the case of preparations that are intended for injection.

An adjuvant is an immune stimulatory substance boosting the immune response of the host in a non-specific manner. The adjuvant may be hydrophilic adjuvant, e.g. aluminum hydroxide or aluminum phosphate, or hydrophobic adjuvant, e.g. mineral oil based adjuvants. Adjuvants such as muramyl dipeptides, avidine, aluminum hydroxide, aluminum phosphate, oils, oil emulsions, saponins, dextran sulphate, glucans, cytokines, block co-polymers, immune stimulatory oligonucleotides and others known in the art may be admixed with the virus according to the invention. Examples of adjuvants frequently used in fish vaccines are muramyl dipeptides, lipopolysaccharides, several glucans and glycans and Carbopol® (a homopolymer). Suitable adjuvants are e.g. water in oil (w/o) emulsions, o/w emulsions and w/o/w double-emulsions. Oil adjuvants suitable for use in w/o emulsions are e.g. mineral oils PCT/EP2014/061014 WO 2014/191445 14 or metabolisable oils. Mineral oils are e.g. Bayol®, Marcol® and Drakeol®; metabolisable oils are e.g. vegetable oils, such as peanut oil and soybean oil, or animal oils such as the fish oils squalane and squalene. Alternatively a vitamin E (tocopherol) solubilisate as described in EP 382,271 may advantageously be used. Very suitable o/w emulsions are e.g. obtained starting from 5-50 % w/w water phase and 95-50 % w/w oil adjuvant, more preferably 20-50 % w/w water phase and 80-50 % w/w oil adjuvant are used. The amount of adjuvant added depends on the nature of the adjuvant itself, and information with respect to such amounts will be provided by the manufacturer.

Another example of a non-mineral oil adjuvant is e.g. Montanide-ISA-763-A.

An example of a water-based nano-particle adjuvant is e.g. Montanide-IMS-2212.

An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review paper by Jan Raa (Reviews in Fisheries Science 4(3): 229-288 (1996)).

Thus, a preferred form of a vaccine according to the invention relates to a vaccine that comprises an adjuvant.

Especially in case it comprises live attenuated virus, the vaccine according to the invention additionally comprises a stabilizer. A stabilizer can be added to a vaccine according to the invention e.g. to protect it from degradation, to enhance the shelf-life, or to improve freezedrying efficiency. Useful stabilizers are i.a. SPGA (Bovamik et al., 1950, J. Bacteriology, vol. 59, p. 509), skimmed milk, gelatin, bovine serum albumin, carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, lactoses, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates. To reconstitute a freeze-dried composition, it is suspended in a physiologically acceptable diluent. Such a diluent can e.g. be as simple as sterile water, or a physiological salt solution. In a more complex form the freeze-dried vaccine may be suspended in an emulsion e.g. as described in EP 1,140,152.

Antibiotics such as neomycin and streptomycin may be added to prevent the potential growth of germs.

In addition, the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span ® or Tween®. The vaccine may also comprise a so-called "vehicle". A vehicle is a compound to which the virus according to the invention adheres, without being covalently bound to it. Such vehicles are i.a. bio-microcapsules, micro-alginates, liposomes and macrosols, all known in the art. A special form of such a vehicle is an Iscom. It goes without saying that admixing other stabilizers, carriers, diluents, emulsions, and the like to vaccines according to the invention are also within the scope of the invention. Such additives PCT/EP2014/061014 WO 2014/191445 15 are for instance described in well-known handbooks such as: "Remington: the science and practice of pharmacy" (2000, Lippincot, USA, ISBN: 683306472), and: "Veterinary vaccinology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN: 0444819681 ).

The dosing scheme for the application of a vaccine according to the invention to the target organism can be the application of single or multiple doses, which may be given at the same time or sequentially, in a manner compatible with the dosage and formulation and in such an amount as will be immunologically effective.

What constitutes an "immunogenically effective amount" for a vaccine according to the invention that is based upon a virus according to the invention (or e.g. a subunit of said virus such as the MCP or a DNA vaccine encoding the MCP) is dependent on the desired effect and on the target organism. The term "immunogenically effective amount" as used herein relates to the amount of immunogenic virus according to the invention (or e.g. a subunit of said virus such as the MCP or a DNA vaccine encoding the MCP) that is necessary to induce an immune response in fish to the extent that it decreases the pathological effects caused by infection with a wild-type SDD virus (SDDV), when compared to the pathological effects caused by infection with a wild-type SDDV in non-immunized fish.

It is well within the capacity of the skilled person to determine whether a treatment is "immunologically effective", for instance by administering an experimental challenge infection to vaccinated animals and next determining a target animal’s clinical signs of disease, serological parameters or by measuring re-isolation of the pathogen, followed by comparison of these findings with those observed in field-infected fish.

The amount of virus administered will depend on the route of administration, the presence of an adjuvant and the moment of administration. A preferred amount of a live vaccine comprising virus according to the invention is expressed for instance as Tissue Culture Infectious Dose (TCID50). For instance for a live virus a dose range between 1 and 1010 TCID50 per animal dose may advantageously be used; preferably a range between 102 and 106 TCID50 is used.

Many ways of administration can be applied, all known in the art. The vaccines according to the invention are preferably administered to the fish via injection (intramuscular or via the intraperitoneal route), immersion, dipping or per os. The protocol for the administration can be optimized in accordance with standard vaccination practice.

If a vaccine comprises inactivated virus according to the invention, the dose would be expressed as the number of virus particles to be administered. The dose would usually be somewhat higher when compared to the administration of live virus particles, because live PCT/EP2014/061014 WO 2014/191445 16 virus particles replicate to a certain extent in the target animal, before they are removed by the immune system. For vaccines on the basis of inactivated virus, an amount of virus particles in the range of about 104 to 109 particles would usually be suitable, depending on the adjuvant used.

If a vaccine comprises subunits, e.g. the MCP according to the invention, the dose would be expressed in micrograms of protein. For vaccines on the basis of subunits, a suitable dose would usually be in the range between 5 and 500 micrograms of protein, depending on the adjuvant used.

If a vaccine comprises a DNA fragment comprising a gene encoding the Major Capsid Protein, the dose would be expressed in micrograms of DNA. For vaccines on the basis of subunits, a suitable dose would usually be in the range between 5 and 500 micrograms of DNA, i.a. depending on the efficiency of the expression plasmid used. In many cases an amount of between 20 and 50 micrograms of plasmid per fish would be sufficient for an effective vaccination. A vaccine according to the invention may take any form that is suitable for administration in the context of aqua-culture farming, and that matches the desired route of application and desired effect. Preparation of a vaccine according to the invention is carried out by means conventional for the skilled person.

Preferably the vaccine according to the invention is formulated in a form suitable for injection or for immersion vaccination, such as a suspension, solution, dispersion, emulsion, and the like.

Intraperitoneal application is an attractive way of administration in case an inactivated virus vaccine or a subunit vaccine is used. Especially in the case of intraperitoneal application, the presence of an adjuvant would be preferred. This route of vaccination is however more labor intensive than application routes such as immersion.

Oral and immersion vaccination routes are preferred when it comes to ease of administration of the vaccine.

For oral administration the vaccine is preferably mixed with a suitable carrier for oral administration i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin. Also an attractive method is administration of the vaccine to high concentrations of live-feed organisms, followed by feeding the live-feed organisms to the fish. Particularly preferred food carriers for oral delivery of the vaccine according to the invention are live-feed organisms which are able to encapsulate the vaccine. PCT/EP2014/061014 WO 2014/191445 17

Suitable live-feed organisms include plankton-like non-selective filter feeders, preferably members of Rotifera, Artemia, copepodites, algae and the like.

The age of the fish to be vaccinated is not critical, although clearly one would want to vaccinate fish against SDD virus infection in as early a stage as possible, i.e. prior to possible exposure to the pathogen.

Immersion vaccination would be the vaccination of choice especially when fish are still small, e.g. below 5 grams. Fish from 5 grams and up can, if necessary or desired, also be vaccinated by means of injection.

Otherwise, man skilled in the art finds sufficient guidance in the references mentioned above and in the information given below, especially in the Examples.

Review articles relating to fish vaccines and their manufacture are i.a. by Sommerset, 1.. Krossov, B·, Biering. E. and Frost. P. in Expert Review of Vaccines 4: 89-101 (2005), by Buchmann, K., Lindenstrom, T. and Bresciani, in J. Acta Parasitologica 46: 71-81 (2001), by Vinitnantharat, S., Gravningen, K. and Greger, E. in Advances in veterinary medicine 41: 539-550 (1999) and by Anderson, D.P. in Developments in Biological Standardization 90: 257-265 (1997).

Moreover, the skilled practitioner will find ample guidance in the Examples below.

It is clear that SDD virus is by far not the only fish pathogen: examples of commercially important warm water fish-pathogenic microorganisms and viruses are Vibrio anguillarum, Photobacterium damsela subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, Viral Nervous Necrosis virus, iridoviruses other than the virus according to the invention that shares a number of characteristics of the Iridoviridae, and Koi Herpesvirus.

Thus, it would be beneficial to combine a vaccine according to the invention with at least one other fish-pathogenic microorganism or virus and/or at least one immunogenic component and/or genetic material encoding said other immunogenic component of that other fish-pathogenic microorganism or virus: one single vaccination could then protect against both SDD virus infection and infection with that other fish-pathogenic microorganism or virus.

Therefore, a preferred form of this embodiment relates to a vaccine according to the invention, wherein that vaccine comprises at least one other immunogenic component and/or antigen or genetic material encoding said other immunogenic component, of a fish-pathogenic microorganism or virus. PCT/EP2014/061014 WO 2014/191445 18

Preferably the fish-pathogenic microorganism or fish-pathogenic virus would be selected from the group consisting of Vibrio anguillarum, Photobacterium damsela subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, Viral Nervous Necrosis virus, iridoviruses other than the virus according to the invention that shares a number of characteristics of the Iridoviridae, and Koi Herpesvirus.

Still another embodiment relates to a method for the preparation of a vaccine according to the invention, wherein the method comprises the mixing of a virus according to the invention and/or a MCP according to the invention and/or a DNA fragment encoding a MCP according to the invention, and a pharmaceutically acceptable carrier.

Again another embodiment of the present invention relates to a virus according to the invention and/or a MCP according to the invention and/or a DNA fragment encoding a MCP according to the invention, for use in a vaccine.

As mentioned above, lethality after SDD virus infection can easily be up to 30% and can easily reach 75%. In addition to this, disease strikes at a relatively high speed. Thus, for efficient protection against disease, a quick and correct diagnosis of SDD is important.

Therefore it is another objective of this invention to provide diagnostic tools suitable for the detection of SDD and SDD virus.

These tools partially rely on the availability of antibodies against the virus. Such antibodies can e.g. be used in diagnostic tests for SDD and SDD virus. A very suitable source of antibodies against the virus according to the invention is the blood or serum of e.g. sea bass that has been infected with viruses according to the invention.

Antibodies or antiserum against viruses according to the invention can also be obtained quickly and easily by vaccination of e.g. pigs, poultry or e.g. rabbits with the virus according to the invention in e.g. a water-in-oil suspension followed, after about four weeks, by bleeding, centrifugation of the coagulated blood and decanting of the sera. Such methods are well-known in the art.

Other methods for the preparation of antibodies, which may be polyclonal, monospecific or monoclonal (or derivatives thereof) are well-known in the art. If polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are well-known in the art PCT/EP2014/061014 WO 2014/191445 19 for decades (e.g. Mayer and Walter, eds. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987).

Monoclonal antibodies, reactive against the virus according to the invention can be prepared by immunizing inbred mice by techniques also long known in the art (Kohler and Milstein, Nature, 256, 495-497, 1975).

Thus, another embodiment of the present invention relates to antibodies or antiserum that are reactive with the virus according to the invention. A diagnostic test kit based upon the detection of a virus according to the invention or antigenic material of that virus and therefore suitable for the detection of SDD viral infection may e.g. comprise a standard ELISA test. In one example of such a test the walls of the wells of an ELISA plate are coated with antibodies directed against the virus. After incubation with the material to be tested, labeled antibodies reactive with the virus are added to the wells. If the material to be tested would indeed comprise SDD virus, this virus would bind to the antibodies coated to the wells of the ELISA. Labeled antibodies reactive with the virus that would subsequently be added to the wells would in turn bind to the virus and a color reaction would then reveal the presence of antigenic material of the virus.

Therefore, still another embodiment of the present invention relates to diagnostic test kits for the detection of a virus according to the invention or antigenic material of the virus, that comprise antibodies reactive with a virus according to the invention or with antigenic material thereof. Antigenic material of the virus is to be interpreted in a broad sense. It can be e.g. the virus in a disintegrated form, or viral envelope material comprising viral outer membrane proteins. As long as the material of the virus reacts with antiserum raised against the virus, the material is considered to be antigenic material. A diagnostic test kit based upon the detection in serum of antibodies reactive with the virus according to the invention or antigenic material of the virus and therefore suitable for the detection of SDD viral infection may also e.g. comprise a standard ELISA test. In such a test the walls of the wells of an ELISA plate can e.g. be coated with the virus according to the invention or antigenic material thereof. After incubation with the material to be tested, e.g. serum of a fish suspected from being infected with SDD virus, labeled antibodies reactive with the virus according to the invention are added to the wells. If anti-SDD virus antibodies would be present in the tested serum, these antibodies will bind to the viruses coated to the wells of the ELISA. As a consequence the later added labeled antibodies reactive with the virus would not bind and no color reaction would be found. A lack of color reaction would thus reveal the presence of antibodies reactive with the virus according to the invention. PCT/EP2014/061014 WO 2014/191445 20

Therefore, still another embodiment of the present invention relates to diagnostic test kits for the detection of antibodies reactive with the virus according to the invention or with antigenic material of the vims that comprise the virus according to the invention or antigenic material thereof.

The design of the immunoassay may vary. For example, the immunoassay may be based upon competition or direct reaction. Furthermore, protocols may use solid supports or may use cellular material. The detection of the antibody-antigen complex may involve the use of labeled antibodies; the labels may be, for example, enzymes, fluorescent-, chemoluminescent-, radio-active- or dye molecules.

Suitable methods for the detection of antibodies reactive with a vims according to the present invention in the sample include, in addition to the ELISA mentioned above, immunofluorescence test (IFT) and Western blot analysis.

An alternative but quick and easy diagnostic test for diagnosing the presence or absence of a vims according to the invention is a PCR test as described above, comprising a PCR primer set reactive with a specific region of e.g. the MCP or ATPase gene of SDD vims. Specific in this context means unique for e.g. the MCP or ATPase gene of SDD vims, i.e. not present in other members of the family Iridoviridae.

Preferably such a test would use the primer set (SEQ ID NO: 5-6) that specifically reacts with the Major Capsid Protein of the vims using the two primers sdd-50- FW:CAGTGCATTACAAGAAAG and SDD-213-REV:GCTGAAACAACAATTTAG ΟΓ the primer set (SEQ ID NO: 7-8) also specifically reactive with the Major Capsid Protein of the vims and using the two primers SDD-MCP-27 7-FW: TCCTGTGCAGCTGTCTAAAC and SDD-MCP-10 90-REV: actggcaatgatgggcgatg or the primer set (SEQ ID NO: 9-10) that specifically reacts with the ATPase of the vims and uses the two primers SDD-ATPase-65-FW: TCGGAGGGATGAAATTGG and SDD-ATPase-618-REV: AGCGTTGTCGATGTAGAG.

It goes without saying, that more primers can be used than the primers identified above. The present invention provides for the first time the unique sequence of the MCP and ATPase gene of SDD vims. This allows the skilled person to select without any additional efforts, other selective primers. By simple computer-analysis of the MCP or ATPase gene sequence provided by the present invention with the, known, MCP or ATPase gene sequence of other members of the family Iridoviridae, the skilled person is able to develop other specific PCR-primers for diagnostic tests for the detection of an SDD vims and/or the discrimination between an SDD vims and other viral (fish) pathogens. PCR-primers that specifically react with the MCP or ATPase gene of SDD vims are understood to be those primers that react only with the MCP or ATPase gene of SDD vims PCT/EP2014/061014 WO 2014/191445 21 and not with the MCP gene of another (fish) pathogenic vims, or group of (fish) pathogenic viruses.

Thus, another embodiment relates to a diagnostic test kit for the detection of a vims according to the invention, characterised in that said test kit comprises a PCR primer set that is reactive with a specific region of the MCP or ATPase gene of SDD vims. A preferred form of this embodiment relates to a diagnostic test kit for the detection of a vims according to the invention, characterised in that said test comprises the primer set as depicted in SEQ ID NO: 5-6 or the primer set as depicted in SEQ ID NO: 7-8 or the primer set as depicted in SEQ ID NO: 9-10. EXAMPLES:

Example 1: Isolation and in vitro culture of SDD virus

Collection of serum, heart, spleen, and kidney samples for isolation of the virus, PCR analysis and virus culture in vitro

Experiment 1:

Samples were collected at a Singaporean farm from fish with typical Scale Drop Disease signs and fish without symptoms of Scale Drop Disease. The samples consisted of one heart, two spleens, three kidneys and four semm samples from diseased animals. In addition, two spleens, four kidneys and two semm samples were collected from healthy seabass.

Experiment 2:

In this experiment, semm, kidney and spleen samples were collected from three groups of fish obtained from an Indonesian farm. Group 1 consisted of three control fish from a cage with no fish carrying symptoms of Scale Drop Disease (SDD). Group 2 consisted of five fish from a cage with early stage of SDD, where mortality had just started. Group 3 consisted of five fish from a cage with severe signs of SDD, when mortality reached a peak.

Experiment 3:

In this experiment, semm samples were collected from two groups of fish obtained from an Indonesian farm. Group 1 consisted of three control fish from a cage with no fish carrying symptoms of SDD. Group 2 consisted of 20 fish from a cage where the SDD outbreak was in an initial stage.The fish showed either no symptoms or subtle symptoms. PCT/EP2014/061014 WO 2014/191445 22

Sample preparation/tissue homogenization and DNA isolation

Tissue samples (spleen, kidney and heart) were homogenized to a 10% (w/v) homogenate in 0,01M PBS using glass beads.

Serum was obtained as follows: blood was collected by caudal venipuncture, allowed to cloth and serum was collected after spinning down the blood cells through standard centrifugation. DNA was isolated from fish serum and homogenized fish tissue samples using the Qiagen DNeasy Blood & Tissue kit using the manufacturer’s instructions.

The samples were treated as follows:

For serum: 50 μΐ of serum was added to 20 μΐ Proteinase K (Qiagen DNeasy Blood & Tissue kit Proteinase K “600 mAU/ml solution (or 40 mAU/mg protein)”) in a 1.5 mL Eppendorf tube and mixed well. To this mixture, 150 μΐ Phosphate-buffered Saline Solution (PBS) was added and mixed well. To this mixture, 20 μΐ RNAse A (20 mg/mL) was added, mixed well, and incubated for 2 minutes at room temperature. From here, the manufacturer’s instructions were followed.

For homogenized tissue samples: 50 μΐ of tissue homogenate was added to 20 μΐ Proteinase K in a 1.5 mL Eppendorf tube and mixed well. To this mixture, 130 μΐ of solution ATL (Qiagen) was added, mixed well, and incubated for 60 minutes at room temperature. To this mixture, 20 μΐ RNAse A (20 mg/mL) was added, mixed well, and incubated for 2 minutes at room temperature. From here, the manufacturer’s instructions were followed.

Detection of the virus using VIDISCA-454 and PCR and quantitation of virus load using qPCR

Serum samples of experiment 1, derived from Asian sea bass with and without symptoms of Scale Drop Disease were analyzed using the VIDISCA-454 virus discovery technique (De Vries et al. (2011) PLoS ONE 6(l):el6118). Multiple sequences were obtained that were suspected to originate from a new fish pathogen. These sequences were used to derive PCR primers for conventional PCR and qPCR (see Table 1). Blasting of the new sequences revealed that the pathogen detected in fish suffering from Scale Drop Disease bears a certain level of resemblance to viruses of the family Iridoviridae. PCRs using primers specific for SDD virus were performed on DNA extracted from heart, spleen, kidney and serum samples, collected both from fish with and without symptoms of Scale Drop Disease (Experiment 1). In addition, PCRs with a primer set specific for another PCT/EP2014/061014 WO 2014/191445 23 member of the family Iridoviridae: Red Sea Bream Iridovirus (Table 1 and SEQ ID NO’s: 12 and 13) were performed. The PCRs were conducted using standard methods with an annealing temperature of 50°C for the SDD virus primer set, and 57°C for Red Sea Bream Iridovirus primer set. PCR samples were run on agarose gels. PCR products were excised from agarose, purified using the QIAquick Gel Extraction Kit (Qiagen) and sequenced.

As is shown in Figure 1, an SDD virus PCR product was exclusively generated in the samples containing DNA from fish with Scale Drop Disease. In only one serum sample from a diseased animal (Scale Drop serum 3) SDD virus DNA could not be amplified. None of the samples derived from healthy animals yielded a PCR product. Sequencing of these PCR products confirmed that they originated from SDD virus. Using the primer set for SDD virus no PCR product for Red Sea Bream Iridovirus could be generated (Figure 1). Furthermore, the Red Sea Bream Iridovirus primer set did not show cross-reactivity with SDD virus (Figure 2).

In addition, a probe was designed for a SDD virus qPCR (table 1 and SEQ ID NO: 11). qPCR was performed applying standard methods and using the probe in combination with the SDD virus primer set at an annealing temperature of 50°C. Data was analyzed using Bio-Rad CFX Manager 2.0 software. A duplicate measurement of a dilution series of a cloned SDD virus PCR product in pCR4-TOPO (Invitrogen) functioned as a standard curve. Positive or negative classification of the samples and determination of the original quantity of SDD virus genomic material in each sample was based on the threshold cycle, as compared to the standard curve. The lower detection limit of this qPCR is approximately 102 copies/μ 1 (Figure 3). As is shown in Table 2, the qPCR results completely correspond with the PCR results (see column Results PCR and column Results Q-PCR).

Similarly, the starting quantity of SDD virus genome copies in serum samples derived from fish with no, early and late stage Scale Drop symptoms (Experiment 2) was determined using the qPCR (Table 3). Except for fish 8, all serum samples from fish with both early and late stage disease were positive, while all the samples from fish with a healthy appearance were negative.

In addition, the serum samples from experiment 3 were analysed by qPCR. Viral genome sequences were detected in 17 out of the 20 serum samples that were derived from fish suffering from Scale Drop Disease. None of the serum samples collected from healthy fish were positive for SDD virus. PCT/EP2014/061014 WO 2014/191445 24

In summary, 40 serum and tissue samples collected from fish with mild to severe symptoms of Scale Drop Disease were analysed. SDD viral genome sequences were detected in 35 of these samples. In stark contrast, no virus has been detected in 14 samples from healthy animals.

Cell culture of the Bluegill fry (BF-2) cell line established at MSD AH

The cell line BF-2 has originally been derived from a trypsinised suspension of pooled caudal portions of the trunk of 1 year old fingerlings (Bluegill fry, Lepomis macrochirus, see Science 1966; 151:1004, J Virol 1968; 2:393, J Infect Dis 1968; 11:253, In Vitro Cell Dev. Biol 1992; 28A:385). The cell line is commercially available through ATCC and ECACC, but the line used for experiments described below was cultured at MSD AH for +70 passages.

The BF-2 cell culture medium consists of 899 ml E-MEM supplemented with 2 mM L-glutamine and 110 mg/L sodium pyruvate, 100 ml FCS (10%) and 1 mL of a Neomycin Polymyxin antibiotics solution lOOOx stock. Cells were routinely grown at 28°C and 5% CO2 in a humidified incubator.

The culture medium was kept at 4°C prior to startup of a culture. One ampoule of frozen stock BF-2 was used to start a culture. Cells from liquid nitrogen were thawed fast in 20-28°C water. The cell suspension was pipetted into a 15 mL tube and diluted slowly with 7 mL culture medium. Subsequently, cells were counted. The suspension was further diluted with culture medium until the DMSO was diluted at least 50 times. Subsequently, the suspension was dispensed to the appropriate culture flask or roller bottle and incubated at 28°C and 5% CO2. After 6-24 hours or complete attachment of the cells, the culture medium was refreshed to remove the remaining DMSO (freeze medium consists of 80% culture medium plus 20% DMSO). The cells were further incubated for 3-7 days or until confluence was reached. For roller bottles, a roller speed of 0.2-0.5 rpm was required. The cells were inspected at regular intervals under an inverted microscope.

Once confluence was reached, cells were passaged. The passage can be performed every 3-4 days with an initial seeding density of 2.0 x 104 cells/cm2. Alternatively, shorter passage intervals can be obtained by seeding cells at a higher density up to 4.0 x 104 cells/cm2. The reagents for cell passage (medium, PBS, Trypsin/EDTA) were pre-warmed to 28°C. The culture medium was discarded and the confluent monolayer was washed once with an appropriate volume of PBS (3 mL for a T25). The PBS was subsequently discarded and the cells were incubated in an identical volume of PBS supplemented with 1% (vol/vol) of a 2.5% trypsin solution and 1% (vol/vol) of a 2% EDTA solution for 15 minutes at 28°C. An PCT/EP2014/061014 WO 2014/191445 25 identical volume of fresh culture medium was added and the cells were resuspended and counted. A new flask was set up at the desired cell density in a culture volume appropriate for the culture flask or roller bottle.

For freezing cells, culture medium and freeze medium (80% (vol/vol) culture medium plus 20% (vol/vol) DMSO) were kept at 4°C prior to the procedures. The confluent cell culture was treated as described above up to and including trypsinization. Cells were resuspended, counted, further resuspended in a suitable amount of culture medium, and an equal volume of 2 x freeze medium was added drop by drop while swirling the suspension. Ampoules for liquid nitrogen storage were filled with 5 x 106 cells per ampoule to start a T175 or with 7.5 x 105 cells per ampoule to start a T25.

Inoculation of BF-2 cells with SDD virus

The cells cultured from liquid nitrogen storage were passaged at least once before inoculation experiments were set up. The cells were passaged and cultured 24 hours before inoculation at 5.0 x 104 cells/cm2 in T25 culture flasks. The inoculum consisted of a 1:10 dilution of serum obtained from animals affected by SDD in culture medium (a pooled serum sample of fish 4-7 of experiment 2 was used for establishing the virus culture). The culture medium was removed from the flask. The flask was subsequently inoculated with 0.5 ml inoculum/T25 at 28°C / 5% CO2 for a minimum of 30 min.

It is equally possible to inoculate cells with a freeze-thawed harvest of a previous passage of SDD virus in cells diluted in culture medium.

Subsequently, the inoculum was removed (although this is not an absolute requirement), fresh culture medium was added and cells were cultured for up to 10 days, or until full CPE was observed using an inverted light microscope. Virus was harvested by one to three cycles of freeze-thawing (-70C0 to 4°C), and subsequently the harvest was cleared from cell debris by centrifugation at 1000 x g for 5 minutes at 4°C. Replication of the virus could be confirmed with qPCR analysis and/or titration of the harvest. DNA sequencing techniques were used to confirm the identity of the virus. DNA for qPCR was isolated from tissue culture medium and freeze-thawed cell harvests using the Qiagen DNeasy Blood & Tissue kit using, the manufacturer’s instructions.

For medium harvests: 200 pi of medium harvest was added to 20 μΐ Proteinase K in a 1.5 mL Eppendorf tube and mixed well. To this mixture, 20 μΐ RNAse A (20 mg/mL) was added, PCT/EP2014/061014 WO 2014/191445 26 mixed well, and incubated for 2 minutes at room temperature. From here, the manufacturer’s instructions were followed.

For cell lysates: 50 μΐ of cell lysate was added to 20 μΐ Proteinase K in a 1.5 mL Eppendorf tube and mixed well. To this mixture, 130 μΐ of solution ATL (Qiagen) was added, mixed well, and incubated for 60 minutes at room temperature. To this mixture, 20 μΐ RNAse A (20 mg/mL) was added, mixed well, and incubated for 2 minutes at room temperature. From here, the manufacturer’s instructions were followed.

No virus could be cultured from serum samples derived from fish with no symptoms of Scale Drop Disease.

Inactivation of SDD virus

The virus harvest was inactivated by adding lOx pre-diluted formalin (1 part formalin added to 9 parts H20). A final volumetric dilution of formalin of 1000 x was effective for SDD virus inactivation, so 1 volume of the 1 Ox pre-diluted formalin was added to 99 volumes of harvest (final formaldehyde content 0.037%). The contents of the vessel were gently stirred at 4°C. Directly after addition of formalin and stirring, the entire mixture was transferred to a new vessel to ensure that all virus has been in contact with the formalin. The contents of the vessel were continuously but gently stirred for three days and subsequently incubated for 11 days without stirring. The mixture was kept at 4°C during the whole inactivation period of 14 days.

Concentration of the inactivated virus

Cross flow filtration was applied to obtain concentrated inactivated virus. GE Healthcare Filter 56-4100-92, UFP-100-E-H22LA, 38cm2, 100.000 NMWC was used to concentrate the inactivated virus. The filter was washed with ultrapure water and sterilized with 2% (vol/vol) formalin in water. Subsequently, the filter was rinsed with PBS and with EMEM medium. The inactivated virus batch was added to the fiter en concentrated to 1/10 of the original volume.

Titration of SDD virus on BF-2 cells BF-2 cells were cultured as described above. One day prior to the test, a BF-2 cell suspension containing 3 x 104 cells/ml in cold (2-8 °C) culture medium was prepared. The 96 wells of a PCT/EP2014/061014 WO 2014/191445 27 microtiter plate were seeded with 100 μΐ/well of this cell suspension. The plates were incubated for 24 hours at 28 °C / 5% CO2 in a humid atmosphere. The monolayer should be approximately 30-50% confluent after this incubation period.

At the day of the test, 10-fold serial dilutions of each virus sample were prepared up to 10'8 by transferring 0.5 ml sample to a tube containing 4.5 ml of cold (0-20°C) titration medium (culture medium without FBS), mixing and transferring 0.5 ml in a next tube containing 4.5 ml titration medium, followed by careful mixing, transferring, etc. Column 1 and 12 and row A and Π served as negative control and were inoculated with 100 μΐ/well fresh titration medium. Microtiter plates were inoculated with 100 μΐ/well of the virus, dilutions 10"3, 10"4, 1 O'5, 1 O'6, 10'7, 10'8 being inoculated in rows B to G (10 wells/dilution). During handling, the temperature of the virus dilutions was kept between 0°C and 20°C. The plates were incubated at 28°C / 5% CO2 for 7 days. After the 7 day virus incubation period the plates were screened for SDD virus specific CPE with an inverted light microscope. SDD virus specific CPE was characterized by rounding-up of cells in the monolayer, followed by cell detachment. This phenomenon of rounding-up of cells followed by detachment can clearly be seen in Figure 4. Figure 5 shows a monolayer of non-infected control cells. The holes in the BF-2 monolayer were surrounded by rounded cells. Each well that shows SDD virus specific CPE was scored as a positive. TCID50 was determined according to the method and calculations described Reed and Muench, Am. J. Epidemiol. (1938) 27(3): 493-497. qPCR analysis of DNA samples isolated from positive wells in the titration assay confirmed presence of the virus.

Sequences and phylogenetic analysis of SDD virus major capsid protein and ATPase

Figure 6 and 7 show the full length DNA and protein sequences of the SDD virus major capsid protein (MCP) and the ATPase, respectively.

The SDD virus MCP and ATPase DNA sequences were used to create phylogenetic trees (Figures 8 and 9). Trees were created with MEGA5 software using the neighbor-joining method and applying standard settings. Only DNA sequences encoding continuous ORFs were included in the alignment. Bootstrap analysis (2000 replicates) was performed and the percentage bootstrap support is specified at the nodes. Distance bars indicate the number of nucleotide substitutions per site.

The SDD virus MCP DNA sequence was aligned with the iridoviral MCP DNA sequences that were included in the phylogenetic analysis that is described by Kurita and Nakajima (Viruses 2012, 4:521-538). As is shown in figure 8, SDD virus may be considered as the single member of a separate genus within the Iridovirus family. The SDD virus MCP sequence is most closely related to MCP sequences of members of the Megalocytivirus genus. However, the pairwise distance (the fraction of sites that have been substituted) is still PCT/EP2014/061014 WO 2014/191445 28 49%, compared to the most closely related MCP sequence (Red Sea Bream Iridovirus MCP ORF). (A blast comparison reveals 65% homology with RSIV MCP, the difference with the 49% “pairwise distance” is mainly due to repair substitutions such as a mutation from e.g. a nucleotide A to G and a second mutation back to A). 5

Figure 9 depicts the phylogenetic analysis of the SDD virus ATPase-coding DNA sequence. It is evident that the SDD virus ATPase sequence is a remote outlier, compared to the other Iridovirus ATPase sequences that were included in this analysis. 10

Table 1: Primer sequences for SDD virus and RSIV PCRs

Primer: Sequence (5’-3’): SDD-50-FW CAG TGC ATT ACA AGA AAG SDD-143-PROBE 6FAM-ATG CCG TCA TTG TAA CAC TG-BHQ1 SDD-213-REV GCT GAA ACA ACA ATT TAG IRIDO-FW-5 CGT GAG ACC GTG CGT AGT IDIDO-REV-5 AGG GTG ACG GTC GAT ATG SDD-ATPase-65-FW TCGGAGGGATGAAATTGG SDD-ATPase-618-REV AGCGTTGTCGATGTAGAG SDD-MCP-277-FW TCC TGT GCA GCT GTC TAA AC SDD-MCP-1090-REV ACT GGC AAT GAT GGG CGA TG 15

Table 2: SDD virus qPCR and PCR results (Experiment 1).

Sample Threshold Cycle (Cq) Starting Quantity (copies/μΐ) RESULTS Q-PCR RESULTS PCR Scale Drop spleen 1 25.55 3.06E+04 POS POS Seale Drop spleen 2 22.50 2.91 E+05 POS POS Healthy spleen 1 N/A N/A NEG NEG Healthy spleen 2 N/A N/A NEG NEG PCT/EP2014/061014 29

Seale Drop serum 1 23.97 9.81 E+04 POS POS Seale Drop serum 2 22.95 2.09E+05 POS POS Seale Drop serum 3 N/A N/A NIX i NEG Seale Drop serum 4 27.12 9.57E+03 POS POS Healthy serum 1 N/A N/A NEG NEG Healthy serum 2 N/A N/A NEG NEG Seale Drop heart 23.42 1.47E+05 POS POS Seale Drp kidne> 1 23.26 1.65E+05 POS POS Seale Drp kidne\ 2 23.81 1.11E+05 POS POS Seale Drp kitlne\ 3 21.60 5.65E+05 POS POS Healthy kidney 1 N/A N/A NEG NEG Healthy kidney 2 N/A N/A NEG NEG Healthy kidney 3 N/A N/A NEG NEG Healthy kidney 4 N/A N/A NEG NEG WO 2014/191445 N/A: not detectable POS: Positive NEG: Negative

Table 6: SDD virus qPCR and PCR results on SDD virus-suspected and control fish (Experiment 2)

Fish NC/Early/Late Starting quantity (copies/μΐ) Result q-PCR Fish 1 NC N/A NEG Fish 2 NC N/A NEG Fish 3 NC N/A NEG Fish 4 Early l,50E+04 POS Fish 5 Early 5,13E+04 POS Fish 6 Early 7,53E+04 POS Fish 7 Early l,23E+05 POS Fish 8 Early N/A NEG Fish 9 Late 9,08E+04 POS 30

Fish 10 Late 6,45E+02 POS Fish 11 Late 2,01E+04 POS Fish 12 Late l,27E+04 POS Fish 13 Late 4,83E+04 POS NC: negative control N/A: not detectable POS: Positive NEG: Negative

Example 2: SDD virus challenge experiments in fish

Experimental set-up in brief: (Abbreviation used: IM: intramuscular, IP: intraperitoneal, ppt: parts per thousand.)

This experiment was conducted with cell culture propagated SDD virus. Four hundred sixty (460) Asian seabass (20 g) were used in this study.

Ninety five (95) fish were challenged by 1) high dose intraperitoneal (IP) injection, 2) low dose IP injection, 3) low dose intramuscular (IM) injection, and 4) combination of high dose IP and low dose IM injection. Another 80 unchallenged fish were kept as negative control for sample comparison purpose.

Fifteen (15) fish each from the 5 groups were harvested at time point day 1, 3,7,10 and 14 postchallenge. The remaining 15 fish were observed until day 28 to evaluate mortality from each challenge method.

At each sampling time point, the kidneys of the harvested fish were sampled individually and the serum was pooled by group. The samples were checked for virus quantification to define the time period of highest virus titres in fish serum or kidney.

Challenge material

Cell culture propagated SDD virus was used as challenge material. The virus was originally isolated from Asian seabass in Indonesia and propagated in vitro. The virus titer was determined using the titration method described in Example 1.

Challenge material diluent

Standard Vaccine Dilution Buffer: PBS + 1.5% NaCl was used as a challenge material diluent. Animals

Species Av. weight on arrival Av. weight at start of Exp. WO 2014/191445 PCT/EP2014/061014 : Asian seabass (Lates calcarifer) : approximately 2 gram/fish (average) : 20 g PCT/EP2014/061014 WO 2014/191445 31

Number of the fish : 460 fish total

Husbandry

Water: Sea water (30 ppt) of 28°C ± 2°C post challenge Feed: the fish were fed ad libitum from the day after challenge.

Tanks: Fish were housed in four 250L tanks. A vertical net was installed in each tank, to create a partition in 1/3 of the tank. This 1/3 partition of the tank held 15 fish used for mortality observation. The other 2/3 tank held 80 fish used for time course harvesting (see Table 3). Unchallenged control fish were housed in half of a 500L tank. The water temperature of the tank for control fish was aligned with the tanks for challenged fish.

Grouping and Dosing:

Assignment of animals A total of 460 seabass were required for this experiment. Fish within the desired size (20 g) were randomly picked as they come at hand.

Challenge:

Fish preparation and fish body weight measurement

Fish were starved for at least 36 hours prior to the challenge. Before the challenge, 20 fish from each group were weighed together to obtain the average body weight for each group.

Table 3. Observation tank

Condition Fish for time course sampling Fish for mortality observation total tank # of fish tank # of fish 1 IP (high dose ') 3F01A 80 3F01B 15 95 2 IP (low dose2) 3F02B 80 3F02A 15 95 3 IM (low dose 2) 3F03A 80 3F03B 15 95 4 IP (high dose) + IM (low dose) 3F04B 80 3F04A 15 95 5 Control (no challenge) 5F08A'3 80 - - 80 Total 400 60 460 *'lligh dose = O.lml/fish of undiluted SDD virus. *2Low dose = O.lml/fish of lOx diluted SDD virus. *3Fish were taken from quarantine tank at each time period. IP challenge (High dose and low dose!

All the fish were anaesthetized using AQUI-S prior to the challenge procedure. Fish were IP injected ventrally with 0.1 ml of challenge material. Two different challenge conditions, 1) undiluted challenge material (high dose: 5.5 x 106 TCID50/fish), and 2) 10 times diluted PCT/EP2014/061014 WO 2014/191445 32 challenge with SVDB material (low dose: 5.5 x 105 TCID50/fish) were applied. Immediately after the challenge, the fish were transferred back to their allocated tank for recovery. Details of each treatment are as follows (see also Table 3). IM challenge (low doscl

The material for IM challenge was 10 times diluted with SVDB (low dose: 5.5 x 105 TCID50/fish), and 0.1ml of challenge material was injected into each fish intramuscularly. All the fish were anaesthetized using AQUI-S before IM challenge. IP challenge (high dose) + IM challenge flow dose)

Combined challenge was done by IP (high dose) challenge first, followed by IM (low dose) challenge. After the two injections, fish were housed to their allocated tank for recovery from anesthesia.

Samnling

Up to fifteen fish were collected from 5 fish groups at days 1,3,7, 10 and 14 post challenge (Tables 4, 5). All the harvested fish were bled first. After that, kidney tissue from each fish was individually sampled into sterile tubes. condition Sampling time points tank Day 1 Day 3 Day 7 Day 10 Day 14 1 IP (high) 3F01A # of fish sampling 15 15 15 15 15 # of remaininq fish 65 50 35 20 5 2 IP (low) 3F02B # of fish sampling 15 15 15 15 15 # of remaining fish 65 50 35 20 5 3 IM (low) 3F03A # of fish sampling 15 15 15 15 15 # of remaininq fish 65 50 35 20 5 4 IP (high) + IM (low) 3F04B # of fish sampling 15 15 15 15 15 # of remaining 65 50 3b 20 5 WO 2014/191445 PCT/EP2014/061014 33 fish 5 Negative control 5F08A # of fish sampling 15 15 15 15 15 # of remaining fish 65 50 35 20 5

Table 4. Sampling time plan condition organ Name of Ihr sample Day 1 Day 3 Day 7 Day 10 Day 14 IP (high dose) Serum 1 tube IPH-1 IPH-3 IPH-7 IPH-10 IPH-14 Kidney 15 tubes IPH-l(l) - IPH-1(15) IPH-3(1) -IPH-3(15) IPH-7(1) - IPH-7(15) IPH-10 (1) -IPH-10 (15) IPH-14(1) - IPH-14(15) IP flow dose) Serum 1 tube IPI 1 IPI 3 IPI 7 IPL-10 IPI 14 Kidney 15 tubes IPI 1(1) IPI 1(15) IPI 3(1) IPI 3(15) IPI 7(1) - IPL 7(15) IPI 10(1) IPI 10(15) IPI 14(1) - IPL-14( 15) IM (low dose) Serum 1 tube IML-1 IML-3 IML-7 IML-10 IML-14 Kidney 15 tubes IML-l(l) -IML-1(15) IML-3(1) -IML-3(15) IML-7(1) - IML-7(15) IML-10 (1) - IML-10 (15) IML-14(1) - IM L-14( 15) IP (high dose) +1M (low dose) Serum 1 tube IPIM-1 IPIM-3 IPIM-7 IPIM-10 IPIM-14 Kidney 15 tubes IPIM-l(l) -IPIM-1(15) IPIM-3(1) -IPIM-3(15) 1PIM-7(1) - 1PIM-7(15) IPIM-10 (1) -IPIM-10 (15) IP1M-14(1) - IPIM-14( 15} Negative control (no challenge) Serum 1 tube NC-1 NC-S-3 NC-S-7 NC-S-10 NC-S-14 Kidney 15 tubes NC-l(l) - NC-1(15) NC- 3(1) - NC-3(15) NC-7(1) - NC-7(15) NC-10(1) - NC-10 (15) NC-14(1) - NC-14(15)

5 Table 5. Sampling plan and sample tube ID

All fish were anaesthetized with AQUI-S and blood was collected by caudal vein puncture. The blood from the same fish group was pooled into 1 tube and allowed to clot at room temperature. The blood was processed to isolate serum on the same day or the next day. The serum was isolated by centrifugation of blood at 3.700 rpm for 20 minutes. Obtained sera 10 were transferred to sterile tubes and stored at <-50°C. WO 2014/191445 PCT/EP2014/061014 34

Observations and post-mortem examination

No mortality occurred during or within 5 days after challenge.

Fish mortalities in observation tanks 3F01B, 3F02A, 3F03B and 3F04A (15 fish/each tank half) were recorded daily after challenge. Mortalities were observed for 28 days.

Symptoms characteristic of Scale Drop Disease, including fin erosion and loss of scales, were observed amongst the challenged fish. Morover, in the groups with fish that received the high challenge dose (IP high or IP high + IM low) mortality was observed from 5 days postchallenge. Animals that received IP low dose or IM low dose challenges showed a slight delay of the onset of mortality. In the different groups, mortality accumulated to 60% (IP high), 47% (IP high + IM low), 20% (IP low) and 13% (IM low). No mortality was observed in unchallenged control fish (Figure 10).

The pooled serum samples, collected from up to 15 animals of each group at 1, 3, 7, 10 and 14 days after challenge, were analysed by qPCR for the presence of SDD virus DNA sequences. As is shown in figure 11, SDD virus DNA is detected in all groups, except for the unchallenged control group. The amount of viral DNA copies increases, peaks and decreases from 10 days post-challenge. The highest levels of viral DNA are detected at day 10 in the sera collected from the groups that received the high challenge dose (IP high or IP high + IM low). In these groups also the highest levels of mortality were observed (60% and 47%, respectively).

Finally, serum of positive fish was used to inoculate BF2 cells. CPE was observed and qPCR confirmed the presence of SDD virus DNA, which confirmed that SDD virus is the pathogen that causes Scale Drop Disease.

The following can thus be concluded:

The first postulate of Koch states that the microorganism must be detected in animals affected by the disease, but should not be found in healthy animals. This postulate was fulfilled for SDDV, as the VIDISCA-454 and the qPCR detected SDDV DNA only in SDS affected fish. Furthermore, no DNA was detected in a PCR for the megalocytivirus RSIV.

The second postulate, stating that the microorganism/virus has to be isolated from an organism with the disease and grown (preferably) on a cell line, was also fulfilled. Cytopathogenic effect was observed after BF-2 cell lines were inoculated with SDDV-positive serum. The virus titers increased over time, showing that the virus replicated on these PCT/EP2014/061014 WO 2014/191445 35 cells. Subsequent VIDISCA-454 and qPCR analysis verified that the replicating virus was indeed SDDV. The isolation was completed when titrations, differential centrifugation and cryo-Transmission Electron Microscopy confirmed the presence of infectious virus particles. The three passages of SDDV-negative serum on BF-2 cells remained free of CPE, which demonstrated that the virus was truly absent in healthy animals. The virus could be harvested and purified from the cell culture.

The third postulate of Koch was fulfilled when the inventors showed that the cultured SDDV virus induced mortality as well as the main symptom of SDD; Scale-drop, after infection in barramundi. What should be noted is that the collected samples from 15 fish at the five different days could result in measured DNA quantities that are an underestimation of the DNA content of the whole population. This is possible because the dead fish are not included in this sampling, while these are arguably the most affected fish and thus may have the highest virus titers.

Finally the last of Koch’s postulates was fulfilled with the re-isolation on BF-2 cells of SDDV from serum samples that were obtained from fish in which the virus had induced SDD.

The inventors believe that the detection and isolation of SDDV succeeded i.a. because of the very specific selection of the cells for growing the pathogen, the choice (against the art) for serum instead of diseased tissue as a source for the pathogen, the choice (against the art) to avoid the use of 0.22 micron filters during the isolation steps of the virus from serum of diseased fish and the choice of fish in a very early stage of disease as a source for the pathogen.

Legend to the figures:

Figure 1: Results of SDD virus PCR on SDD virus-suspected and control fish (Experiment 1)

Figure 2: Results of red sea bream iridovirus PCR on SDD virus-suspected and control fish (Experiment 1)

Figure 3: SDD virus qPCR standard curve (Experiment 1).

Figure 4: CPE in monolayer of BF-2 cells at day 5 post infection. Note the rounded-up cells. Scale bar 100 pm

Figure 5: BF-2 monolayer (control) at day 5 post infection. Scale bar 100 pm Figure 6: SDD virus major capsid protein DNA (a) and protein (b) sequences.

Figure 7: SDD virus ATPase DNA (a) and protein (b) sequences.

Figure 8: Phylogenetic tree of Iridovirus major capsid protein ORFs. For each sequence the species name, the genus and the accession number are shown. The percentage bootstrap PCT/EP2014/061014 WO 2014/191445 36 support of 2000 replicates is specified at the nodes. The distance bar indicates the number of nucleotide substitutions per site.

Figure 9: Phylogeny of Iridovirus ATPase ORFs. For each sequence the species name and the accession number are shown. The percentage bootstrap support of 2000 replicates is 5 specified at the nodes. The distance bar indicates the number of nucleotide substitutions per site.

Figure 10: Cumulative mortality (%) after SDD virus challenge as observed in the various groups. Each tank contained 15 fish that were injected (IP and / or IM) with different doses of cell culture propagated SDD virus.

10 Figure 11: SDD virus DNA copies in sea bass serum after challenge. SDD virus DNA copies/μΐ were measured by qPCR in pooled sera from 15 fish per group, sampled on day 1, 3, 7, 10 and 14.

Figure 12: Cryo-TEM picture of the concentrated cell &amp; medium harvest from SDDV passage 3 in tissue culture. 15

PCT 1 1-1 1-2 The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: page line 3 11-13 1-3 1-3-1 1-3-2 1-3-3 1-3-4 Identification of deposit Name of depositary institution Address of depositary institution Date of deposit Accession Number CNCM Collection nationale de cultures de microorganismes (CNCM) Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris Cedex 15, France 23 May 2013 (23.05.2013) CNCM 1-4754 1-5 Designated States for Which Indications are Made All designations 2 2-1 2-2 The indications made below relate to the deposited microorganism(s) or other biological material referred to in the description on: page line 9 24-26 2-3 2-3-1 2-3-2 2-3-3 2-3-4 Identification of deposit Name of depositary institution Address of depositary institution Date of deposit Accession Number CNCM Collection nationale de cultures de microorganismes (CNCM) Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris Cedex 15, France 23 May 2013 (23.05.2013) CNCM 1-4755 2-5 Designated States for Which Indications are Made All designations FOR RECEIVING OFFICE USE ONLY 0-4 This form was received with the international application: (yes or no) YES 0-4-1 Authorized officer Appelen, Annik FOR INTERNATIONAL BUREAU USE ONLY 0-5 This form was received by the international Bureau on: 0-5-1 Authorized officer

Print Out (Original in Electronic Form) 0-1 Form PCT/RO/134 (SAFE) Indications Relating to Deposited Microorganlsm(s) or Other Biological Material (PCT Rule 13bis) 0-1-1 Prepared Using PCT Online Filing Version 3.5.000.235 MT/FOP 20020701/0.20.5.20 0-2 International Application No. 0-3 Applicant's or agent's file reference 23503 WO WO 2014/191445 PCT/EP2014/061014 37 SUBSTITUTE SHEET (RULE 26)

Claims (21)

1. Claims: 1) An isolated virus which is a member of the family Iridoviridae comprising a Major Capsid Protein (MCP) gene and an ATPase gene, characterized in that: a) the virus is the causative agent of Scale Drop Disease in fish and b) the nucleotide sequence of the MCP gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 1 or the nucleotide sequence of the ATPase gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 3,
2. 2) An isolated virus according to claim 1, characterized in that the nucleotide sequence of the MCP gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 1 and the nucleotide sequence of the ATPase gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: Λ
3. 3) An isolated virus which is a member of the family Iridoviridae comprising an MCP gene and an ATPase gene, characterized in that: a) the virus is the causative agent of Scale Drop Disease in fish and b) the MCP gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 5 and 6 to give a PCR product of 164 +/- 10 base pairs or reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 7 and 8 to give a PCR product of 814 +/- 10 base pairs or the ATPase gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 9 and 10 to give a PCR product of 554 +/- 10 base pairs.
4. 4) An isolated virus according to claim 3, characterized in that the MCP gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 5 and 6 to give a PCR product of 164 +/- 10 base pairs and reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 7 and 8 to give a PCR product of 814 +/- 10 base pairs and the ATPase gene reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 9 and 10 to give a PCR product of 554 +/- 10 base pairs.
5. 5) An isolated virus according to any one of claims 1-4, characterized in that the nucleotide sequence of the MCP gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 1 and the nucleotide sequence of the ATPase gene has a level of identity of at least 80% to the nucleotide sequence as depicted in SEQ ID NO: 3 and in that the viral DNA reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 5 and 6 to give a PCR product of 164 +/- 10 base pairs and reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 7 and 8 to give a PCR product of 814 +/- 10 base pairs and reacts in a PCR reaction with a primer set as depicted in SEQ ID NO: 9 and 10 to give a PCR product of 554 +/- 10 base pairs.
6. 6) A cell culture comprising a virus, characterized in that said culture comprises the virus according to any one of claims 1-5.
7. 7) An isolated Major Capsid Protein (MCP) characterized in that said MCP is encoded by a DNA fragment comprising a gene which has a level of identity of at least 80% to the nucleotide sequence of the MCP gene as depicted in SEQ ID NO: 1.
8. 8) An isolated ATPase characterized in that said ATPase is encoded by a DNA fragment comprising a gene which has a level of identity of at least 80% to the nucleotide sequence of the ATPase gene as depicted in SEQ ID NO: 3.
9. 9) A vaccine for combating Scale Drop Disease in fish, characterized in that said vaccine comprises a virus according to any one of claims 1-5 and a pharmaceutically acceptable carrier.
10. 10) A vaccine according to claim 9, characterized in that said vims is in a live attenuated form.
11. 11) A vaccine according to claim 9, characterized in that said vims is in an inactivated form.
12. 12) A vaccine for combating Scale Drop Disease in fish, characterized in that said vaccine comprises a MCP according to claim 7 and a pharmaceutically acceptable carrier.
13. 13) A vaccine according to any one of claims 9-12, characterized in that said vaccine comprises at least one other fish-pathogenic microorganism or fish-pathogenic vims and/or at least one other immunogenic component and/or genetic material encoding said other immunogenic component of said fish-pathogenic microorganism or fish-pathogenic vims.
14. 14) A vaccine according to claim 13, characterized in that said fish-pathogenic vims or fish-pathogenic microorganism is selected from the group consisting of Vibrio anguillarum, Photobacterium damsela subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, Viral Nerous Necrosis vims, iridovimses other than the vims according to any one of claims 1-5, and Koi Herpesvirus.
15. 15) A vaccine according to any one of claims 9-14, characterized in that said vaccine comprises an adjuvant.
16. 16) An antibody or antiserum reactive with a virus according to any one of claims 1-5.
17. 17) A virus according to any one of claims 1-5 and/or a MCP according to claim 7, for use in a vaccine for combating Scale Drop Disease in fish.
18. 18) A method for the preparation of a vaccine according to any one of claims 9-15, characterized in that said method comprises the mixing of a vims according to any one of claims 1-5 and/or a MCP according to claim 7, and a pharmaceutically acceptable carrier.
19. 19) A diagnostic test kit for the detection of antibodies reactive with a virus according to any one of claims 1-5 or with antigenic material thereof, characterised in that said test kit comprises a vims according to any one of claims 1-5, or an MCP according to claim 7 or an antigenic fragment of said MCP.
20. 20) A diagnostic test kit for the detection of a vims according to any one of claims 1-5 or antigenic material thereof, characterised in that said test kit comprises antibodies reactive with a vims according to any one of claims 1-5 or with antigenic material thereof.
21. 21) A diagnostic test kit for the detection of a vims according to any one of claims 1-5, characterised in that said test kit comprises a PCR primer set that is reactive with a specific region of the MCP or ATPase gene of SDD vims.