WO2009153337A1

WO2009153337A1 - Infection inhibitor

Info

Publication number: WO2009153337A1
Application number: PCT/EP2009/057653
Authority: WO
Inventors: Justinus Maria Vlak
Original assignee: Wageningen Universiteit
Priority date: 2008-06-20
Filing date: 2009-06-19
Publication date: 2009-12-23
Also published as: BRPI0913246A2; CN102046650A; ECSP11010772A; MX2010013681A

Abstract

The present invention relates to a White Spot Syndrome virus protein and a White Spot Syndrome virus nucleic acid molecule encoding said protein. It also relates to compositions comprising said protein, its use in a vaccine, vaccines comprising said protein and diagnostic tests for the detection of White Spot Syndrome virus specific DNA or antigenic material. Finally it relates to antibodies against said protein.

Description

Infection inhibitor

White spot syndrome virus (WSSV) is a pathogen of major economic importance in cultured penaeid shrimp. The virus is not only present in shrimp but also occurs in other freshwater and marine crustaceans including crabs and crayfish. In cultured shrimp WSSV infection can reach a cumulative mortality of up to 100% within 3-10 days and can cause large economic losses to the shrimp culture industry. The virus was i.a. found in China in the early '90's, from where it quickly spread to other shrimp farming areas in Southeast Asia. WSSV initially appeared to be limited to Asia until it was found in Texas and South-Carolina in November 1995. In early 1999 WSSV was also reported from Central- and South-America and it has now also been detected in Europe. Intensive shrimp cultivation, inadequate sanitation and worldwide trade has increased the disease incidence in crustaceans and enhanced disease dissemination. As such WSS has become an epizootic disease and is not only a major threat to shrimp culture but also to marine ecology. The disease is O.I.E. notifiable.

WSSV is a large DNA virus. WSSV virions circulate ubiquitously in the haemolymph of infected shrimp. Electron microscopy studies revealed that WSSV virions are enveloped particles with a bacilliform to ovoid shape of about 275 nm in length and 120 nm in width. Most characteristic is the tail-like appendage at one end of the virion, in suspension.

The virus particle contains at least 6 major virion proteins, of which three (VP664, VP26, VP24 and VP 15) are present in the rod-shaped nucleocapsid and two (VP28 and VP 19) reside in the envelope, and about 40 minor proteins. The complete genome sequence of WSSV has been determined (Van Hulten et al., Virology 286; 7-22 (2001), Yang et al., J. Virol. 75; 11811-11820 (2001), Chen et al., Virology 293; 44-53 (2002)). The DNA has a size of about 300 kbp. It contains approximately 180 open reading frames. Most of the genes encode proteins that bear no resemblance to any known proteins or motifs (Van Hulten et al, Virology 286; 7-22 (2001), Yang et al, J. Virol. 75; 11811-11820 (2001)).

To date, the only possibility to protect shrimps specifically against WSSV infection is vaccination. Most experimental vaccines are based upon the WSSV envelope protein VP28 (Witteveldt et al., in Fish Shellfish Immunol 16: 571--579 (2004), Witteveldt et al.. in J. Virol. 78:2057-2061 (2004)).

A disadvantage of vaccines in general is that vaccination is a prophylactic action in order to protect against an infection that may or may not happen. Therefore, if infection pressure is low, a therapeutic method to solve the infection problem is more convenient, available on demand and cheaper. For therapeutic purposes however, a vaccine is not a suitable option, because it takes time to build up a prophylactic response.

It is an objective of the present invention to provide improved or alternative ways for the protection of shrimps against WSSV infection, more specifically against oral infection.

Surprisingly, a new gene was found in WSSV, encoding a novel protein that was shown to play a key role in the primary attachment of the virus to the shrimp's (or more generally; the crustacean's) epithelial cells. As such the gene turned out to encode the main initial factor in the oral infection process. This will be further explained below. A nucleotide sequence of the gene is presented in SEQ ID NO.: 1.

It is clear that, if only due to wobble in the second and third base of many codons, there may be some variation in nucleotide sequence between individual viruses. Therefore, nucleic acid molecules having at least 90 % nucleotide sequence identity with the nucleotide sequence presented in SEQ ID NO.: 1 are also considered to represent the new gene according to the invention.

The "% nucleotide sequence identity" of a nucleic acid molecule's nucleotide sequence with that of a nucleic acid molecule according to the invention can be determined by nucleotide sequence alignment to the whole of, or to the relevant part of the nucleotide sequence of SEQ ID NO: 1. The percentage of identity between a nucleic acid molecule and a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 can be determined with the computer program "BLAST 2 SEQUENCES" by selecting sub-program: "BlastN" (T. Tatusova & T. Madden, 1999, FEMS Microbiol. Letters, vol. 174, p. 247-250), that can be found at the internet address www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html. Parameters that are to be used are the default parameters: reward for a match: +1; penalty for a mismatch: -2; open gap penalty: 5; extension gap penalty: 2; and gap x dropoff: 50. The BlastN program does not list similarities, only identities: the percentage of nucleotides that are identical is indicated as "Identities".

Next to using computer algorithms for determining the level of identity/mismatch between a nucleic acid and a nucleic acid according to the invention, experimental techniques can also be used. Especially suitable is hybridisation under conditions of controlled stringency.

The definition of stringent hybridisation conditions, as a function of the identity between two nucleotide sequences, follows from the formula for the melting temperature Tm of Meinkoth and Wahl (1984, Anal. Biochem., vol. 138, p. 267-284):

Tm = [81.5°C + 16.6(log M) + 0.41(%GC) - 0.61(%formamide) - 500/L] - 1°C/1% mismatch

In this formula: M is the molarity of monovalent cations; %GC is the percentage of guanosine and cytosine nucleotides in the DNA; L is the length of the hybrid in base pairs; and "mismatch" is the lack of an identical match.

Washing conditions subsequent to the hybridization can also be made more or less stringent, thereby selecting for higher or lower percentages of identity respectively. In general, higher stringency is obtained by reducing the salt concentration, and increasing the incubation temperature. It is well within the capacity of the skilled person to select hybridisation conditions that match a certain percentage-level of identity as determined by computer analysis.

For the purpose of this patent, "stringent conditions" are those conditions under which a nucleic acid molecule hybridises if it has a mismatch of 10 % or less,; i.e. if it is at least 90 % identical to the nucleotide sequence depicted in SEQ ID NO: 1. Therefore, if a nucleic acid molecule hybridises under stringent conditions to the nucleotide sequence depicted in SEQ ID NO: 1 i.e. if it is at least 90 % identical to the nucleotide sequence depicted in SEQ ID NO: 1, it is considered as a nucleic acid molecule according to the invention. Thus, a first embodiment of the present invention relates to a White Spot Syndrome virus (WSSV) nucleic acid molecule having a nucleotide sequence according to SEQ ID NO.: 1 or a nucleic acid molecule capable of hybridizing under stringent conditions to said nucleic acid molecule having a nucleotide sequence according to SEQ ID NO.: 1.

Since the nucleic acid molecule according to the invention is specific for White Spot Syndrome virus, a diagnostic test on the basis of this nucleic acid molecule is very suitable to diagnose the presence or absence of WSSV in water, more specifically in the water of shrimp farm basins. Such a test can be a hybridization test as described above. It can also be a standard PCR-test.

Thus, another embodiment of the present invention relates to diagnostic tests for the detection of White Spot Syndrome virus specific DNA, characterized in that the test comprises a nucleic acid molecule according to the invention or PCR-primers based upon that nucleic acid molecule. Standard PCR-textbooks give methods for determining the length of the primers for selective PCR-reactions with the WSSV DNA according to the invention. Primer fragments with a nucleotide sequence of at least 12 nucleotides are frequently used, but primers of more than 15, more preferably 18 nucleotides are somewhat more selective. Especially primers with a length of at least 20, preferably at least 30 nucleotides are very generally applicable. PCR-techniques are extensively described in Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN 0- 87969-447-5 (1995). Merely as an example; such primers can e.g. comprise the 20 first and 20 last nucleotides of the nucleic acid molecule having a nucleotide sequence according to SEQ ID NO.: 1.

The protein encoded by the nucleic acid molecule having a nucleotide sequence according to SEQ ID NO.: 1 has an amino acid sequence as depicted in SEQ ID No.: 2. The protein encoded by said gene has a molecular weight of about 110 kD. Further analysis of the protein showed that surprisingly it fulfills a role in WSSV that is analogous to the role of the P74 protein in baculoviruses, although the P74 protein is much smaller: about 74 kD. In itself, the fact that these proteins differ so much is undoubtedly due to the fact that baculovirus and WSSV belong to totally different virus families and have a totally different host range. The novel protein according to the invention will, for reasons given below, be further referred to also as WSSV PAP, or briefly PAP (Primary Attachment Protein).

The huge difference in MW for the WSSV PAP on the one hand, and the analogous baculovirus protein P74 on the other hand follows clearly from table 1.

The evolutionary distance of WSSV PAP according to the invention and baculovirus P74 clearly shows from figure 1. WSSV PAP and baculovirus P74 do not share any significant sequence homology.

Table 1: comparison of the number of amino acids in baculo P74 and WSSV PAP. PAP was found to be the WSSV analogue of the baculovirus protein P74.

There are several ways in which the protein of the present invention can be used to mitigate or prevent WSSV infection in shrimp.

One approach is to use the protein or a fragment thereof capable of inhibiting the binding between WSSV and crustacean host cells as an infection inhibitor. The isolated protein is very suitable as an inhibitor of infection when administered to shrimps, due to the fact that its natural role is to target the virus to the host cell's virus receptor. The principle is easy: by adding isolated protein PAP according to the invention to e.g. the food or the water, the shrimp's cellular virus receptors will become occupied by the isolated PAP protein, thus blocking the host cell's virus receptors. As a consequence virus particles can no longer attach to the host cells; the receptor sites are blocked by PAP. PAP protein according to the invention can successfully be used in therapeutic treatments. Especially in regions where the infection pressure is relatively low, farmers can deliberately take the risk and only start treatment as soon as the first clinical signs become manifest. Immediate treatment with either the full-length isolated protein or a fragment thereof capable of inhibiting the attachment of the WSSV virus to the host's cells then prevents further infection. This has the advantage of saving cost: costs would only be made after infection actually occurred. As mentioned above, the protein according to the invention is attached to the viral envelope. In the in vivo situation, only the part of the protein that protrudes from the virus surface plays a role in the attachment. Therefore, for the purpose of blocking the cellular virus receptor, a small fragment of the viral protein, provided that it comprises the part of the protein that is capable of attaching to the shrimp's cellular virus receptor would already suffice.

Thus, another embodiment of the present invention relates to a White Spot Syndrome virus protein or a fragment of said protein capable of inhibiting the binding between WSSV and crustacean host cells, wherein that protein or fragment thereof is encoded by a nucleic acid molecule having a nucleotide sequence according to SEQ ID NO. : 1 or by a nucleic acid molecule capable of hybridizing under stringent conditions to said nucleic acid having a nucleotide sequence according to SEQ ID NO.: 1.

In a preferred form of this embodiment, the White Spot Syndrome virus protein according to the invention is a protein having an amino acid sequence according to SEQ ID NO.: 2 or a fragment of said protein capable of inhibiting the binding between WSSV and crustacean host cells.

As said before, in principle the isolated whole protein can be used for inhibiting. However a fragment of said protein that is capable of inhibiting the binding between WSSV and host cells can also be used. This has the advantage that it can often be produced somewhat easier, due to the general rule that shorter proteins can be made in expression systems in larger amounts.

Moreover, really short peptides of < 50 amino acids can very efficiently and cheaply be made synthetically. Another advantage of expressing a suitable fragment instead of the whole PAP protein is, that a fragment can be selected that has hydrophilic but no hydrophobic or membrane-spanning regions.

This avoids sticking of the protein fragment to the cell membrane after expression.

Hydrophilicity-predicting computer programs are extensively known in the art.

Preferably, a whole protein according to the invention is used. Nevertheless, if one prefers to use a fragment of the protein that is capable of inhibiting the binding between WSSV and host cells, its selection can very easily be done by making a fragment and checking in a blocking assay if it inhibits the binding of the virus to epithelial cells. Such an assay, in its basic form, comprises the steps of: a) preparing an epithelial tissue explant, e.g. from stomach or gills, b) incubating the epithelial tissue explant with either the full-length protein according to the invention (control) or the fragment thereof to be tested c) incubation of the epithelial tissue explant with WSSV, followed by the removal of excess WSSV d) incubation of the epithelial tissue explant with conjugated anti-WSSV or anti-VP28 antibodies followed by a coloring reaction.

If the protein fragment to be tested is not capable of inhibiting, WSSV will bind to the cells and an enzymatic color reaction with the conjugated anti-WSSV or anti-VP28 antiserum will reveal the presence of the virus on the cell surface. A lack of color reaction indicates a successful inhibiting activity by the fragment to be tested.

So, as mentioned above, it is clear that the PAP protein according to the invention can successfully be used in therapeutic treatments. Especially in regions where the infection pressure is relatively low, farmers can deliberately take the risk and only start treatment as soon as the first clinical signs become manifest. They thus would save significant cost: costs would only be made after infection actually occurred.

The way to administer the protein or a fragment thereof according to the invention can be very straightforward: the protein or fragment thereof can be given in a pharmaceutical composition comprising the protein or fragment thereof and a pharmaceutically acceptable carrier. Such a carrier can be water or a buffer solution such as PBS.

For oral administration the inhibitor is preferably mixed with a suitable carrier for oral administration. In that case, the carrier is preferably a vehicle to which the protein adheres, preferably without being covalently bound to it. Such vehicles are i.a. bio-microcapsules, micro- alginates, liposomes and macrosols, cellulose, food or a metabolisable substance such as alpha- cellulose or different oils or emulsions of vegetable or animal origin. These are all known in the art.

Also an attractive approach is administration of the protein to high concentrations of live-feed organisms, followed by feeding the live-feed organisms such as artemia to the target animal, e.g. the shrimps. Particularly preferred food carriers for oral delivery of the vaccine according to the invention are live-feed organisms which are able to encapsulate the protein. Another possibility is to express the protein or a fragment thereof according to the invention in yeast or algea, followed by top-dressing the yeast cells or algea on the food.

Thus, another embodiment of the present invention relates to a pharmaceutical composition comprising a White Spot Syndrome virus protein or a fragment of said protein according to the invention and a pharmaceutically acceptable carrier.

An alternative approach is also feasible: instead of blocking the host cell's PAP receptor, one might prefer to block the viral PAP protein itself. This requires antibodies capable of binding to White Spot Syndrome virus protein PAP and at least capable of binding to a fragment of that protein involved in inhibiting the binding between WSSV and crustacean host cells. The protein according to the invention or fragments of that protein capable of inhibiting the binding between WSSV and crustacean host cells can be used to produce antibodies, which may be polyclonal, monospecific or monoclonal (or derivatives thereof). If polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are well-known in the art (e.g. Mayer and Walter, eds. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987).

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

Methods for large-scale production of antibodies according to the invention are also known in the art. Such methods rely on the cloning of (fragments of) the genetic information encoding the protein according to the invention in a filamentous phage for phage display. Such techniques are described i.a. at the "Antibody Engineering Page" under "filamentous phage display" at http://aximtl.imt.uni-marburg.de/~rek/aepphage.html., and in review papers by Cortese, R. et al., (1994) in Trends Biotechn. 12: 262-267., by Clackson, T. & Wells, J.A. (1994) in Trends Biotechn. 12: 173-183, by Marks, J.D. et al., (1992) in J. Biol. Chem. 267: 16007-16010, by Winter, G. et al., (1994) in Annu. Rev. Immunol. 12: 433-455, and by Little, M. et al., (1994) Biotechn. Adv. 12: 539-555. The phages are subsequently used to screen camelid expression libraries expressing camelid heavy chain antibodies. (Muyldermans, S. and Lauwereys, M., Journ. Molec. Recogn. 12: 131-140 (1999) and Ghahroudi, M.A. et al., FEBS Letters 414: 512- 526 (1997)). Cells from the library that express the desired antibodies can be replicated and subsequently be used for large scale expression of antibodies.

A very simple and efficient alternative for producing antibodies is the production of avian (egg yolk) antibodies: IgY. Such methods have been described i.a. by Schade, R. et al., in ATLA 24; 925-934 (1996). Application of the antibodies thus produced is simple: the egg yolk is e.g. mixed with the food and fed to the shrimp.

Thus, another embodiment of the present invention relates to antibodies capable of binding to a White Spot Syndrome virus protein or a fragment thereof according to the invention, where these antibodies are at least capable of binding to a fragment of said protein involved in inhibiting the binding between WSSV and crustacean host cells.

With respect to antibodies capable of binding to with White Spot Syndrome virus protein PAP; as will be discussed below, such antibodies are also suitable for diagnostic tests. In such tests, there is no requirement for the antibodies to be at least capable of binding to a fragment of that protein capable involved in the binding between WSSV and crustacean host cells. For such diagnostic purposes, it suffices that antibodies are capable of binding to with White Spot Syndrome virus protein PAP as such.

Therefore, another embodiment of the present invention relates to antibodies capable of binding to with White Spot Syndrome virus protein PAP or a fragment thereof. In regions where the infection pressure is relatively high, it would be advisable to take prophylactic, rather than therapeutic, measures. This can be done by vaccinating shrimps with the protein or a fragment thereof according to the invention.

Basically, there are two approaches for vaccination: oral vaccination and vaccination through injection. Both ways of vaccination have been described for another WSSV-protein: VP28. Thus, vaccination can be done i.a. analogous to vaccination with the WSSV envelope protein VP28 as described by Van Hulten et al., in Virology 285, 228-233 (2001), Witteveldt et al., in Fish Shellfish Immunol 16: 571-579 (2004), Witteveldt et al., in J. Virol. 78:2057-2061 (2004) and Witteveldt et al., in Archives of Virology J 50; 1121 -1133 (2005).

Both oral vaccination and vaccination through injection require the production of the protein according to the invention, PAP.

For the expression of PAP or fragments thereof, an expression construct of PAP fused to a (HΙS)ό-tag using (he pET28a vector (Novagen) can be used. An empty pET28a vector can be used as a control. Both the (HIS)6-PAP and pET28a control vector can be overexpressed according to the manufacturer's instructions. A suitable expression strain is E. coli BL.21. When expression has reached the desired level, inaetivation of bacteria can be done in formalin for 15 minutes at 20 degrees Celsius.

For oral vaccination application, commercial food pellets of approximately 0.02 grams (Coppeus International, The Netherlands) can be coated with approximately 10^h of the inactivated bacteria. To this end, bacteria are washed twice in PBS, rεsuspended in PBS. mixed with the food pellets, incubated on ice for 15 minutes to allow absorption of the bacterial suspension and coated with cod liver oil to prevent dispersion of the inactivated bacteria in the water. Each shrimp can be fed 8 pellets, divided over two rations per day.

This approach is attractive since expression in bacteria is cheap.

Another attractive way of expressing the PAP protein is, by using a baculovirus expression system. This expression system has the advantage that it yields high amounts of protein that moreover are of invertebrate nature. Coating of the particles can be done in the same way, now however using crude cell preparations. The amount of PAP protein can easily be determined on gel, and amounts given can be equal to the amounts given in a bacterial expression system.

Vaccination through injection, analogous to the approach followed for VP28, is preferably done with amounts ranging between 1 and 10 μg of PAP protein in 330 mM NaCl at a final volume of 10 μl. A booster injection with a comparable amount of protein after 5 days enhances the level of protection.

Thus, another embodiment of the present invention relates to a vaccine for combating White Spot Syndrome virus infection where the vaccine comprises a White Spot Syndrome virus protein or a fragment thereof according to the invention and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier for oral vaccination has been described above. A pharmaceutically acceptable carrier for injection is e.g. a physiological salt solution or a buffer.

The WSSV protein VP28, mentioned above, is known to provide some level of protection, but this level of protection decreases after some time. Therefore, a vaccine comprising both WSSV PAP and WSSV VP28 benefits from the presence of an additional antigen. Therefore, a preferred form of this embodiment relates to a combination vaccine for combating White Spot Syndrome virus infection according to the invention, characterized in that it additionally comprises White Spot Syndrome virus protein VP28.

As stated above, the present invention describes for the first time the novel WSSV protein PAP. Two known WSSV proteins; VP 187 and VP 110, described earlier by respectively Hongyan Li et al., (Virus Res. 115; 76-84 (2006)), Deng-Feng Li et al., (Virology 368; 122-132 (2007)) and by Li Li et al., (Journ. Gen. Virol. 87; 1909-1915 (2006)) are also known to play a role, albeit a different one, in virus attachment.

It is thus very attractive to provide a combination vaccine that comprises both PAP and VP187, or PAP and VPl 10, or even PAP, VPl 10 and VPl 87. Such a vaccine interferes at an even broader level with the attachment of WSSV to host cells, since it raises an immune response against two or even three viral attachment proteins. Thus, another preferred form of this embodiment relates to a combination vaccine for combating White Spot Syndrome virus infection according to the invention, characterized in that it additionally comprises White Spot Syndrome virus protein VPl 10.

Still another preferred form of this embodiment relates to a combination vaccine for combating White Spot Syndrome virus infection according to the invention, characterized in that it additionally comprises White Spot Syndrome virus protein VPl 87.

Often, the vaccine is mixed with stabilizers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf- life of the vaccine, or to improve freeze-drying efficiency. Useful stabilizers are i.a. SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates. In addition, the vaccine may be suspended in a physiologically acceptable diluent.

It goes without saying, that other ways of adjuvating, adding vehicle compounds or diluents, emulsifying or stabilizing a protein are also embodied in the present invention.

Vaccines according to the invention that are based upon the protein according to the invention or immunogenic fragments thereof can very suitably be administered in amounts ranging between 1 and 100 micrograms of protein per animal, although smaller doses can in principle be used. A dose exceeding 100 micrograms will, although immunologically very suitable, be less attractive for commercial reasons.

Another embodiment relates to White Spot Syndrome virus protein or a fragment thereof according to the invention, for use in a vaccine.

Still another embodiment relates to the use of a White Spot Syndrome virus protein or a fragment thereof according to the invention for the manufacturing of a vaccine for combating White Spot Syndrome virus infection. The protein according to the invention is also very suitable as a tool in a marker-vaccine approach. It is possible to make WSSV-mutants that carry a deletion in the PAP -gene and therefore do not comprise the PAP protein according to the invention. This can be done using standard methods such as homologous recombination in crustacean cells after co-transfection with a plasmid carrying a PAP-deletion and a marker gene. Such mutants have been described for baculovirus protein P74 by Gutierrez et al, (J. Biotechnol. 116; 135-143 (2005)) with a background given by Faulkner et al., (J. Gen. Virol. 78; 3091-3100 (1997)) and Slack et al., (J. Gen. Virol. 82; 2279-2287 (2001)). Such a mutant can be propagated e.g. through injection in crayfish and can be used as a live attenuated vaccine, since it still produces the immunogenic protein VP28, but it can not cause infection due to lack of PAP.

It is however important to be able to discriminate between the presence, in a shrimp farming environment, of such an attenuated WSSV or a wild-type virulent WSSV. A diagnostic test that e.g. comprises separate wells coated with either antibodies against VP28 or antibodies against PAP can easily do this job. In such a test, wild-type virus would attach to the wells comprising VP28-antibodies and to the wells comprising PAP antibodies, whereas the PAP -minus mutant virus would attach only to the wells comprising VP28. A standard color reaction with conjugated anti-WSSV-antibodies can be used for the screening of the presence/absence of the virus in the respective wells.

Therefore, another embodiment of the present invention relates to diagnostic tests for the detection of antigenic material of White Spot Syndrome virus, where such tests comprise antibodies against the White Spot Syndrome virus protein PAP according to the invention or against a fragment of said protein PAP capable of inhibiting the binding between WSSV and crustacean host cells.

EXAMPLES

Example 1 Cloning of the PAP protein

For the expression of WSSV PAP or fragments thereof, an expression construct of PAP fused to a (HΙS)ό-tag using the pET28a vector (Novagen) is used. The complete PAP protein can be cloned as a BamHl / Pstl fragment into the pF.T28a vector after PCR from the WSSV genome using the 5'- and 3'-terminal 20-mers with additional respective BamHl / Pstl sites.

Expression is done in expression strain E coii BL21. An empty pET28a vector can be used as a control. Both the (HiS)ό-PAP and pF.^'T28a control vector are ovcrexpressed according to the manufacturer^'s instructions.

Example 2

Expression of the PAP protein The His6-PAP and control proteins are overexpressed according to the manufacturer's instructions and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and Western blot with a WSSV polyclonal antiserum. The bacterial concentration after inactivation is determined with a Beckman DU-7500 photo spectrometer, where an optical density at 600 nm of 1 equals 10⁹ bacteria per ml. The bacteria are inactivated in 0.5% formalin, incubated for 15 min at 20⁰C, checked for inactivation levels, and stored at 4°C until further use.

Legend to the figure:

Figure 1 : a tree indicating the evolutionary distance between proteins involved in virus to host attachment of large DNA-viruses.

AcMNPV = Autographa californica nucleopolyhedrovirus; CpGV = Cydia pomonella granulovirus; NeIeNPV = Neodiprion lecontii nucleopolyhedrovirus; CuniNPV = Culex nigripalpus nucleopolyhedrovirus; GbNV = Gryllus bimaculatus nudivirus; HzNV = Heliothis zea nudivirus; GpSGHV = Glossina pallipides salivary gland hypertrophy virus; MdSGHV =

Musca domestica salivary gland hypertrophy virus; WS SV = white spot syndrome virus

Claims

1) White Spot Syndrome virus (WSSV) nucleic acid molecule having a nucleotide sequence according to SEQ ID NO.: 1 or a nucleic acid molecule capable of hybridizing under stringent conditions to said nucleic acid molecule having a nucleotide sequence according to SEQ ID NO.: 1.

2) White Spot Syndrome virus protein or a fragment of said protein capable of inhibiting the binding between WSSV and crustacean host cells, characterized in that said protein or fragment thereof is encoded by a nucleic acid molecule according to claim 1. 3) White Spot Syndrome virus protein according to claim 2, characterized in that it has an amino acid sequence according to SEQ ID NO. : 2 or a fragment of said protein capable of inhibiting the binding between WSSV and crustacean host cells.

4) Pharmaceutical composition comprising a White Spot Syndrome virus protein or a fragment of said protein according to claim 2 or 3, and a pharmaceutically acceptable carrier.

5) White Spot Syndrome virus protein or a fragment thereof according to claim 2 or 3, for use in a vaccine.

6) Use of a White Spot Syndrome virus protein or a fragment thereof according to claim 2 or 3 for the manufacturing of a vaccine for combating White Spot Syndrome virus infection.

7) Vaccine for combating White Spot Syndrome virus infection characterized in that said vaccine comprises a White Spot Syndrome virus protein or a fragment thereof according to claim 2 or 3 and a pharmaceutically acceptable carrier.

8) Combination vaccine for combating White Spot Syndrome virus infection according to claim 7, characterized in that it additionally comprises White Spot Syndrome virus protein VP28.

9) Combination vaccine for combating White Spot Syndrome virus infection according to claim 7, characterized in that it additionally comprises White Spot Syndrome virus protein VPl 10. 10) Combination vaccine for combating White Spot Syndrome virus infection according to claim 7, characterized in that it additionally comprises White Spot Syndrome virus protein VP 187. 11) Diagnostic test for the detection of White Spot Syndrome virus specific DNA characterized in that the test comprises a nucleic acid molecule according to claim 1 or PCR-primers based upon said nucleic acid molecule.

12) Diagnostic test for the detection of antigenic material of White Spot Syndrome virus, characterized in that said test comprises antibodies against a White Spot Syndrome virus protein or a fragment thereof according to claim 2 or 3.

13) Antibodies capable of binding to a White Spot Syndrome virus protein or a fragment of said protein, characterized in that said protein or fragment thereof is encoded by a nucleic acid molecule according to claim 1. 14) Antibodies capable of binding to a White Spot Syndrome virus protein or a fragment thereof according to claim 2 or 3, said antibodies being at least capable of binding to a fragment of said protein involved in inhibiting the binding between WSSV and crustacean host cells.