JPH09201196A - Identification, purification and detection of white spot syndrome-related baculovirus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new isolated white spot syndrome-related baculovirus (WSBV), comprising the WSBV having a genome containing an SalI DNA fragment having a specific nucleotide sequence and useful for diagnosis, etc., of infection of penaeid shrimps, etc., with the WSBV.

SOLUTION: This new isolated white spot syndrome-related baculovirus (WSBV) is a substantially pure one of the WSBV having a genome containing an SalI DNA fragment having a nucleotide sequence represented by the formula and useful for diagnosis, etc., of infection of penaeid shrimps with the WSBV by identification, purification and detection, etc., of a new infectious viral substance in arthropods, especially lobsters or shrimps such as the penaeid shrimps. The isolated virus is obtained by extracting an outer shell and an epidermis backing the shell collected from Penaeus monodon which belongs to lobsters and shrimps with a cold extraction buffer, centrifuging and purifying the resultant extract at 24,000 r.p.m. for 60min using a sucrose linear gradient.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

FIELD OF THE INVENTION This invention relates to the identification, purification and detection of novel infectious viral agents in arthropods, especially shrimp. This virus is designated WSBV (white spot syndrome-associated baculovirus).

BACKGROUND OF THE INVENTION In recent years, due to the outbreak of disease in Asian countries, a large number of deaths have occurred during the cultivation of penaeid shrimps. Since 1992, in northern Taiwan,
A new, deadly disease outbreak is occurring in a population of farmed prawns (Penaeus japonicus). The disease is characterized by distinct vitiligo on the back, appendages and inner surface of the body, with a cumulative mortality rate of 10 to 10 within 2-7 days.
Reach 0%. In addition, the affected shrimp show signs of lethargy and the hepatopancreas stains red. In 1993, the giant tiger plow that was farmed [giant tiger
In the prawn] (P. monodon) and the redtail prawn (P. penicillatus) vitiligo syndrome (WS) was observed. W. in Taiwan S. S. Have been reported to cause serious damage to prawn production (Tung et al. Personal communica
tion).

[0003] The animal infectious disease survey of Japanese prawns in Japan also reported similar findings (Nakano et al., Fish
Pathology, 29 (2): 135-139, 1994). Evidence from electron microscopy and the results of challenge studies with filtrates from the lymphoid organs of the affected shrimp showed that the causative agent was a temporary shrimp (Penaeus japonicus), named RV-PJ. Rod-shaped virus [rod-shaped
nuclear virus] (Inouye et al., Fish Path
alogy, 29 (2): 149-158, 1994; Takahashi et al., Fish
Pathalogy, 29 (2): 121-125, 1994).

To date, many epidemics of baculovirus in farmed Kuruma prawn have been documented (Lightner et.
al., Aquaculture, 32: 209-233, 1983). Among these baculoviruses of prawns, monodon baculovirus (MBS), baculovirus-type midgut necrosis virus [baculoviral midgutne
crosis virus] (BMNV) and baulovirus penaei [BP] were considered to be the most important. This is because they often cause significant losses in infected shrimp populations (Couch, Nature 247 (5438): 22-231, 1974; J. I.
nvertebr.Pathol., 24: 311-331, 1974; Lightner & R
edman, J. Invertebr. Pathology, 38: 299-302, 1981;
Lightner et al. (1983), supra; Lightner et al., 1
987; Sano et al., Fish Pathology, 15: 185-191, 198.
1).

[0005] W. S. S. Baculovirus-like virions were observed in prawns naturally affected by
ng et al., personal communication). Possibly this virus is a W. cerevisiae that occurs in prawns in Taiwan.
S. S. Will be the main causative agent of. W. in shrimp
S. S. In order to prevent
Simple, accurate and W.I. S. S. There is a need to develop a diagnostic method that does not take much time to detect

SUMMARY OF THE INVENTION The present invention is based on the discovery of a novel causative agent responsible for the development of vitiligo syndrome in prawns. The causative agent has been isolated and purified and found to be non-occluded rod-shaped viral particles. This virus particle has an envelope, length 330 ± 20 nm, diameter 87.
± 7 nm. This virus is a baculovirus family Nudibaculovirinae subfamily NO
It has been identified as a member of the genus B (non-occluded baculovirus). In addition, this isolate was named PmNOBIII and was identified to be a PmNOBIII-related substance by WSBV (white spot syndrome-associated baculovirus).
Is also named. A WSBV genomic DNA library was constructed and, based on the sequence of one of the cloned WSBV DNA fragments, WSB in the prawn prawns
WSBV specific primer sets for PCR to detect V infection have been developed. PCR using this WSBV-specific primer set showed that causative agents of vitiligo syndrome in shrimp of different species are virtually closely related. The results of this invention provide a diagnostic tool for screening WSBV infections in host animals, especially in the epi. This tool seems to be very important in preventing the further spread of this viral disease. It is possible to develop a convenient, sensitive and specific ready-to-use diagnostic device for detecting the presence of WSBV and arresting the further spread of this viral disease. This diagnostic article comprises primers established based on the nucleotide sequence of a unique genomic DNA clone from WSBV.

The features and advantages of the present invention will become apparent in the following detailed description of the preferred embodiments, with reference to the accompanying drawings.

Detailed Description of the Invention In Taiwan, Penaeus monodo
n], prawns and P. Penicillus [P. penisil
The occurrence of diseases that cause severe economic loss among populations of farmed Kuruma prawns, including latus, is characterized by distinct vitiligo on the back, appendages and inner surface of the body. In order to identify the causative agent of vitiligo syndrome in prawns, electron microscopy of the affected shrimp was performed. Healthy prawn (P. japonicus) larvae were infected with P. japonicus and P. japonicus larvae showing marked vitiligo signs. It was exposed by immersing it in epidermal filtrate derived from monodon. In the challenge test, this filtrate was used against different size prawns.

Non-occlusive rod-shaped viral particles were found by electron microscopy in the epidermis of both naturally infected and experimentally infected Kuruma prawns. The virion has an envelope and is 330 ± 20 nm long.
And the diameter was 87 ± 7 nm. These experimentally infected shrimps resembled those naturally affected. Direct inoculation of this virus-containing filtrate into fish cell lines showed no cytotoxic effect. Cumulative mortality reached 100% within 5-7 days, but was significantly affected by capture and temperature stress.

The close similarity in external signs and viral morphology between naturally-affected shrimp and experimentally infected shrimp indicates that this rod-shaped virus is the major causative agent of the disease characterized by vitiligo syndrome in Taiwan. It is possible to be. Therefore, in this viral disease,
The name "white spot syndrome (WSS)" was presented. W. isolated from Penaeus monodon. S. S.
Further studies were conducted to determine the taxonomic position of the causative agent (viruses associated with vitiligo syndrome).

Shrimp suffering from vitiligo syndrome (Penaeus
The causative viral substance was purified from the monodone). Negative dyeing preparations show that this virus is polymorphic [pleiomorph
ic]. It is spindle-shaped or rod-shaped. In the negative dye preparations, the virions are 70-150 nm at their widest point and 250-380 nm in length. In some virions, a tail-like projection extends from one end. It is apparent that the capsid consists of a ring of subunits that form a continuous continuum. This ring is aligned perpendicular to the long axis of the capsid. The genome of this virus is a double-stranded DNA molecule that produces at least 22 HindIII fragments. The total length of this DNA is 150 kb
It has been confirmed to be longer. Based on the morphological characteristics and genomic structure of this virus, vitiligo syndrome-related virus (WSSV) has been confirmed to be a member of the baculoviridae genus Nudibaculovirus subfamily NOB (non-occluded baculovirus). . Furthermore, this isolate was named PmNOBIII and was also named WSBV (white spot syndrome associated baculovirus) to indicate that it is a PmNOBIII related substance.

WSBV is a subcutaneous tissue and hematopoietic necrotic baculovirus (HHNBV) (Cai et al, J. Fish. China, 19) reported as the pathogen (EEDS) of the epidemic epidemic of prone in China (1993-1994). : 11
2-117, 1995), and the systematic ectoderm and mesoderm baculovirus (SEMBV) of Black Tiger Prone and Penaeus monodon in Thailand (Wang e
t al, Dis. aquat. Org., printing, 1995; Wongteerasupa
Ya et al., Dis. aguat. Org., 21: 69-77, 1995).

The main clinical manifestation of this new viral disease is the presence on the exoskeleton and epidermis of the affected shrimp, vitiligo of varying size, from slightly visible to 3 mm in diameter. For histopathological studies, WS
BV is cuticular underlayer epidermal tissue [cuticular epidermis]
Has been shown to strike the most frequently. This is evidenced by the presence of altered cells in these tissues characterized by enlarged nuclei (Momoya
ma et al., Fish Pathol., 29: 141-148, 1994; Chou et
al., Dis. aquat. Org., printing, 1995; CH Wang et
al., 1995). Thus, it can be said that the vitiligo syndrome associated with non-occlusive baculovirus in Kuruma prawns is well defined, and in the study above, in order to develop a diagnostic tool for WSBV protection in prawns, P. The isolate from monodone was used as the starting material.

In order to develop a diagnostic tool to prevent the infection of WSBV and related substances in shrimp, WS
Black Tiger Shrimp, P.
The virion was purified from the monodon. Viral genomic DNA was extracted from purified virions by treating the virions with proteinase K and cetyltrimethylammonium bromide (CTAB), followed by phenol-chloroform extraction and ethanol precipitation. Shrimp D in the preparation of viral genomic DNA
Quality assessment was performed using polymerase chain reaction (PCR) against viral DNA and primers specific for shrimp genomic DNA to monitor NA contamination. A WSBV genomic DNA library was constructed, and a WSBV-specific primer set for PCR for detecting WSBV infection in prawns was designed based on the sequence of the cloned WSBV DNA fragment.

The sample containing WSBV DNA produced a clear amplification product which exhibited the expected mobility of the 1447 bp DNA fragment. In contrast, nucleic acids extracted from clinically healthy shrimp-derived tissue samples did not show such DNA fragments. This confirmed the specificity of the WSBV DNA-specific primer designed in this invention. PCR using this WSBV-specific primer set has shown that the causative agents of vitiligo syndrome in different species of shrimp are indeed closely related. Other host organisms including copepods [copecoda], crabs and insects were also tested for the presence of this novel causative agent and the experimental data currently collected are positive. The result of this invention is WSBV
Provide an effective diagnostic tool for screening shrimp for infections. It can be said that this tool is very important in preventing the viral disease from spreading further.

Materials and Methods Shrimp. The healthy prawns used in the antigen administration test are
Obtained from a hatchery and shrimp farm in southern Taiwan where no viral disease has been reported. All prawns were kept in an aerated aquarium at a temperature of 25-28 ° C and were fed with commercially available shrimp artificial feed 1
They were fed twice a day. Affected P. Japonics shrimp were collected from a farm in northern Taiwan in 1994, while
P. A sample of moribund prawns of the genus Monodon (average weight: 30 g) was collected from a shrimp farm located in southern Taiwan. These samples were examined by gross autopsy, light and electron microscopy using the methodology described below to confirm disease.

For normal light microscopy, both normal prawns and individuals exhibiting prominent vitiligo signs were stored in Davison's fixative (Bell & Lightner). Samples are stored in Davison fixative for 48 hours and then
Transfer to 0% ethanol, and then histologically follow the procedure to make a 5 μm paraffin wax section.
).

For transmission electron microscopy, a sample of epidermis covering the subdorsal gill cavity was removed from live, naturally and experimentally infected Kuruma prawns and immediately added to 0.1.
Prefix for 3 hours at 4 ° C. in a solution of 2.5% glutaraldehyde in M cold phosphate buffer solution (PBS, pH 7.4). Subsequently, the sample was washed several times in cold PBS and then 4% in 1% osmium tetroxide.
It was fixed after 3 hours at ° C. These samples were dehydrated and embedded in spool resin [Spurr's resin]. Richert-Jung Ultra Cut E Ultrotome [Ri
chert-jung Ultracut E Ultrotome] was prepared and stained with uranyl acetate and lead citrate. This section was observed with a Hitachi H-600 transmission electron microscope.

Antigen administration test. Infected P. The epidermis was removed from the monodon and homogenized in brackish water at 4 ° C. at a ratio of 1: 9. After centrifugation at 8510 × g (Sigma 2K15 rotor 12141) for 5 minutes, the supernatant was washed with 0.15 g.
Filtered through a 45 μm membrane. This filtrate is 14,549
Xg (Sigma 2K15 rotor 12139) 1.
After centrifugation for 5 hours, the resulting pellet was resuspended in sterile brackish water before being subjected to negative staining. For negative staining, 1 drop of the suspension was mixed with 0.1% bovine serum albumin and 2% phosphotungstic acid (1: 2,
Mixed with 4 drops of pH 7.0). The mixture was placed on a 300 mesh grid for 30-60 seconds and excess suspension was removed with filter paper. The preparation was dried before testing. The results were observed under a Hitachi H-600 transmission electron microscope.

Cytopathology assay. EPC (epithelioma papulitis carp [epithelioma carp
papulosum cyprini]), CHSE-214 (chinook salmon embryo), FHM (fathead minnow]) and SSE-5 (sockeye salmon embryo) cells were seeded. The filtrate was prepared from the epidermis of the affected shrimp and diluted 1/20 to 1/12500 with a 5-fold dilution. Inoculate the diluted solution into four fish cell lines,
These cells were observed at a seeding temperature of 20 ° C. for 2 weeks.

Live or frozen, naturally infected P. Japonix and P.P. An infection trial was performed using the epidermal filtrate derived from monodon. The filtrate was diluted 500-750 times in brackish water for use as a waterborne inoculum. Two groups of 35 1 year old Kuruma prawn larvae (average weight 0.08 g) were immersed in these diluted filtrates for 2 hours. Similarly, the other two groups were treated with normal P. Either the filtrate derived from the monodon epidermis or the Grace's insect medium [Grace's insect medium] was exposed. These were used as controls. After inoculation, the shrimp were bred in a glass aquarium while ventilating. Water temperature and salinity were 25-28 ° C and 25-30 ppt, respectively, during the experimental period. Mortality was observed daily and moribund shrimps were collected and examined by transmission electron microscopy.

Purification, genomic structure and taxonomic position of WSBV P. Purification of WSSV virions from shrimp of the genus Monodon was performed as follows. First, chill this shrimp 1 x T
E buffer (10 mM Tris-HCl, 1 mM ED
Rinse with TA, pH 7.6). Shells from 1 to 5 live or frozen shrimps and the epidermis lining them were placed in cold extraction buffer (20 mM
HEPES, 0.4N NaCl, 1 mM EDT
A, 1 mM EGTA, 1 mM DTT, 2.5 mM phenylmethylsulfonylfluoride, 1 μg / ml leupepsin, 1.6 μg / ml pepstatin, 2 μg / m
l aprotinin, 1 μg / bestatin) 20 ml, then centrifuge for 60 minutes at 74,700 × g (Hitachi SRP28SA rotor, 24,000 rpm) using a 35-65% (W / W) sucrose linear gradient. Purified by. Visible virus bands in the middle of this gradient were removed and pelleted by centrifugation at 74,700 xg for 30 minutes at 4 ° C. Cold this pellet 1
× Wash twice with TE buffer, 300-500ml
Resuspended in cold 1 × TE buffer (depending on pellet size) and used immediately for extraction of viral DNA. For ultrastructural studies of virions, a small amount of the purified virus suspension was added to 2% phosphotungstic acid (PTA) at pH 7.
It was negatively stained with.

Proteinase K and N-cetyl-N,
N, N-trimethylammonium bromide (CTA
B) treatment followed by phenol-chloroform extraction and ethanol precipitation (K. Wilson (1994), Preparation of gen.
omic DNA from bacteria.Miniprep of bacterial geno
mic DNA. in Ausubel, FM et al. (eds.) Current P
rotocols in Molecular Biology, Vol.1. Greene Pub.
Assoc. And Wiley-Interscience, NY, p. 2.4.1-2.4.5
) Performed extraction of viral genomic DNA from the gradient purified virions.

Estimation of the viral genome size was performed by restriction endonuclease analysis. Viral DNA
HindIII, SalI and XhoI restriction endonucleases (Boehringer Mannheim [Boehringer
Mannheim Company]) was used to digest viral DNA.
Tris-acetate buffer (0.04 M Tris-acetate, containing 0.5% μg / ml ethidium bromide,
0.8 with 0.1 mM EDTA, pH 8.0)
The restriction fragments were separated by electrophoresis in a% agarose gel (9 cm x 12 cm). 1 kilobase (k
b) DNA ladder and Lambda phage HindIII fragment marker (Life Technology [L ife Technolo
gies, Inc.]) was used as a DNA size standard on the gel.

Development of an Effective Diagnostic Tool To develop an effective diagnostic tool, a WSBV genomic library was constructed by cloning "ultra-pure" WSBV genomic DNA extracted from purified virions. In addition, amplification of selected DNA sequences by the polymerase chain reaction (PCR) promises to be a powerful diagnostic tool for identifying pathogens (Erlich et al., Nature 331: 461-462, 1988; Oste. ,
C., Biotechniques 6: 162-167, 1988). Clone W
A WSBV specific primer set for PCR is designed based on the sequence of the SBV DNA fragment. I. Construction of WSBV genomic DNA library A. Virus Purification and Extraction of Viral DNA The source of virus was the same WS used in the taxonomic study.
A batch of frozen BV-infected Black Tiger Shrimp and Penaeus Monodon, this strain of WSBV is F
It is named PmNOBIII (the third non-occluded baculovirus reported for P. monodon) according to the criteria set forth in rancki et al. (Arch. Virol., 2: 1-450, 1991). Purification of virions was performed as described in the above preparation. Extraction of viral genomic DNA from purified virions was performed by treating virions with proteinase K and N-cetyl N, N, N-trimethylammonium bromide (CTAB), followed by phenol-chloroform extraction and ethanol precipitation ( Wilson (1994), supra). Briefly, gradient purified virions were loaded with 100 mM KC
1, 1% SLS (N-lauryl sarcosine) and 0.
TE buffer containing 2 mg / ml proteinase K (10 mM Tris-HCl, 1 mM EDTA, pH
7.6) and incubated at 65 ° C. for 3 hours. After incubation, add 5M NaCl to add DNA
The NaCl concentration of the solution was adjusted to 0.7M. Then 1
/ 10 volume of CTAB / NaCl (0.7M NaCl
10% of CTAB) was added slowly, mixed thoroughly, and then incubated at 65 ° C. for 10 minutes.

Two extractions with approximately equal volumes of chloroform / isoamyl alcohol, and two extractions with an equal volume of phenol / chloroform / isoamyl alcohol, followed by DN with 2 volumes of absolute ethanol.
A was precipitated and washed with cold 70% ethanol. Dry D
NA pellets at 65 ° C for 30 minutes in an appropriate amount of 0.1 x T
After dissolving in E buffer, it was stored at 4 ° C until use. B. Preparation of shrimp DNA for PCR as a control
Used to monitor shrimp DNA contamination in SBV genomic DNA preparation. For this, the published sequence (Kim
& Abele, J. Crust. Biol., 10, 1-13, 1990), computerized data files (GeneBank [GenBank], National Institutes of Health, MD, USA) and PC / GENE program (Intelligenetics) Company [Intelligenetics,
Inc.] sequence alignment analysis [sequence alignment a
analysis, two primers were designed from the highly conserved region of the decapod 18S rRNA sequence. Forward primer 143F (5'-TGC CTT ATC AGC
TNT CGA TTG TAG-3 ', where N is G, A, T or C
Represents the reverse primer 145F (5'-TTC AGN TTT G
CA ACC ATA CTT CCC-3 ')
Shrimp DNA is 18S of Aztecs [P. aztecus]
It is expected to generate a PCR product of 848 bp corresponding to the nucleotide sequence 352 to 1200 of rRNA.

Healthy P. Monodon or P. Genomic DNA extracted from Japonica muscle was used as a positive control for PCR. Preparation of genomic DNA from mammalian tissue (Strauss, WM (1994) Preparation of genomic
DNA from mammalian tissue.In Ausubel FM, Brent R,
Kingston RE, Moore DD, Seidman JG, Smith JA, Struh
l K (Eds) Current Protocols in Molecular Biology,
Vol.1. Greene Publishing Associates, Inc. and John
Wiley and Sons, Inc., New York, p. 2.2.1-2.2.3)
Shrimp deproteinized genomic DNA was prepared according to Briefly, 200 muscle tissue excised from the shrimp abdomen
mg was frozen rapidly in liquid nitrogen and crushed to a fine powder. This treated tissue is digested with buffer (100 mM
NaCl, 10 mM Tris-HCl, pH 8, 25 mM
2.4 ml of EDTA, pH 8, 0.5% sodium dodecyl sulfate, 0.1 mg / ml proteinase K) was added and incubated at 65 ° C. for 12 to 18 hours. This digest is a continuous phenol / chloroform /
Deproteinize by isoamyl alcohol extraction, collect by ethanol precipitation, dry and resuspend in 0.1x TE buffer at 65 ° C for 30 minutes, then use for PCR 4
Stored at ° C. C. Construction of WSBV genomic DNA library Two kinds of WSBV genomic library, PmNOBIII
SalI (pms) and PmNOBIII Hind
III (pmh) was constructed as described below. DNA
To obtain the fragment, WSBV genomic DNA free of shrimp DNA contamination was digested with SalI or HindIII restriction endonucleases (BRL Life Technologies [BRL Li
fe Technologies Inc.]) and digested at 37 ° C. for 3 hours. This fragment was then cleaved with SalI or HindIII cleaved pU in the presence of T4 ligase at 16 ° C overnight.
It was ligated into the C19 plasmid vector. Escherichia coli DH5α cells were transformed with the obtained plasmid, and ampicillin / isopropyl-β-D-thiogalactopyranoside (IPTG) / 5-bromo-4-chloro-3-indolyl-β-D-galactopyrano was transformed. It was applied to Sid (X-Gal) agar plates. After using the miniprep method to screen for white transformants resistant to ampicillin due to the presence of a suitable recombinant plasmid,
Using both strands of the plasmid insert as a double-stranded DNA template, the Sequenase kit [Sequenase
Kit] (United States Biochemical Corp.) to M13 / p
UC Sequencing Primers [M13 / pUC Sequen
cing Primers] (GIBCO BRL Life Technologies), followed by sequencing with specific internal primers. Recombinant plasmids were isolated from transformants and screened for the presence of inserts by SalI or HindII digestion. The insert sizes are listed in Table 1. II. Amplification of WSBV DNA fragments derived from DNA extracted from purified WSBV virions Oligonucleotide primers (146F and 146
R) was used to amplify the WSBV DNA fragment. Primers 146F and 146R are recombinant plasmid pms
1461 bp S of WSBV cloned into 146
It is designed based on the DNA sequence of the alI DNA fragment, and 6 types of primer sets shown in FIG. 18 have been established. The 146R1 and 146F1 primer sets have the following nucleotide sequences: 146R1,
5'-TAA TGC GGG TGT AAT GTT CTTACG A-3 '146F
1, 5'-ACT ACT AAC TTC AGC CTA TCT AG-3 '. By using this primer set, WSBV genomic DN
It is expected that the A to 1447 bp fragment will be amplified. Internal primer set 146R2, 5'-TAC GGCAGC
TGC TGC ACC TTG T-3 ', and 146F2, 5'-GAT AC
T GCC CCT TCC ATCTCC A-3 'is used to confirm that the amplified fragment is indeed derived from the 941 bp SalI DNA fragment of WSBV.

PCR was performed on purified WSBV virions and deproteinized DNA samples extracted from healthy shrimp muscle.
DNA for evaluating the specificity of a primer
Used as a template. III. Shrimp naturally infected with WSBV and experimentally W
Amplification of WSBV DNA Fragments Derived from DNA Extracted from SBV-Infected Shrimp Tissue The diseased shrimp consisted of shrimp naturally infected with WSBV and experimentally shrimp infected with WSBV. As an experimental infection, healthy shrimp (average body weight: 0.5 g) was infected with WSBV using the method described in the preceding paragraph. Five days after infection, DNA was extracted from 3 experimentally infected shrimp and 3 healthy shrimp, WSBV specific primers (146F1 and 146R1) and shrimp DNA.
P using specific primers (143F and 145R)
Check by CR. IV. PCR amplification and analysis of products The total number of deproteinized DNA samples used for amplification was 1
0 mM Tris-HCl, pH 9 at 25 ° C, 50 mM K
Cl, 1.5 mM MgCl ₂ , 0.1% Triton X-
Reaction mix 100 containing 100, 200 μM of each dNTP, 100 pmol of each primer, 2.5 units of Taq DNA polymerase (Promega)
It was 0.1-0.3 μg in μl. Amplification is AG-
9600 Thermal Station [AG-9600 Thermal St
ation] (Biotronics Corp.)
At 94 ° C for 4 minutes, 55 ° C for 1 minute, 72 ° C for 3 minutes once; 94 ° C for 1 minute, 55 ° C for 1 minute
Min, 39 cycles of 72 ° C. for 3 min, and 40 min.
A final 5 minute extension at 72 ° C was made after the cycle. Control reactions without template DNA were run on all P
The CR reaction was performed. In some PCR reactions, controls also consisted of reaction mixtures with DNA extracted from healthy shrimp. PCR products were analyzed in a 1% agarose gel containing ethidium bromide at a concentration of 0.5 μg / ml and visualized under UV transillumination. V. WSBV-infected P. cerevisiae using the DIG-labeled 1447 bp PCR product. Monodon or healthy P.I. DNA dot hive extracted from monodon
Redission WSBV infection or healthy P. The DNA extracted from the monodon is subjected to a 96-well dot-blot vacuum filtration manifold device [96-well dot-blot vacuum filtration m
anifold apparatus] (Schleicher and Schuell, Inc.) was used to spot on Hybond-N paper [Hybond-N] (Amersham). The blots were air dried and denatured in 1.5M NaCl, 0.5N NaOH (10 minutes) before 1.5M NaCl, 1M.
Neutralized with Tris, pH 7.4 (10 minutes). These blots were subjected to DIG-labeled 1447 bp PCR.
The product is used to perform standard molecular cloning techniques (Sambrook
J, Fritsch EF, Maniatis T (1989), Molecular Clonin
g: A Laboratory Manual, 2nd. Cold Spring Harbor La
boratory Press, Cold Spring Harbor, New York). Dot blot and DIG-labeled probe were treated with 50% formamide, 5
X SSC, 1 mM EDTA, 50 mM Tris (pH
8) 5 × Denhardt's reagent (0.1% Ficoll-40
0, 0.1% polyvinylpyrrolidone, 0.1% BSA)
In the medium, prehybridization was carried out at 37 ° C. for 12 hours and then at 37 ° C. for 16 hours. 144
The 7 bp PCR product was used as a template for probe preparation using the random primer method (Boehringer Mannheim). After hybridization,
Detection of DIG-labeled nucleotides in the blot
Luminescent Detection Kit [DIG Lumine
scent Detetion Kit] (Boehringer Mannheim). This blot is 37 ℃
Exposed to Kodak XAR-5 film for 15-30 minutes at
The chemiluminescent signal was recorded. VI. Diseased P. cerevisiae using the DIG-labeled 1447 bp PCR product. Monodon or P. Southern hybridization of WSBV DNA from Japonix P. aeruginosa with vitiligo syndrome Monodon or P. Southern blot hybridization was performed to locate the 1447 bp PCR product in WSBV genomic DNA purified from Japonix.
To this end, genome D of WSBV isolated from diseased shrimp
200 ng NA was digested with SalI and then electrophoretically separated in a 0.8% agarose gel. Acid (0.25N HCl) depurination of DNA and alkali (1.
5M NaCl-0.5N NaOH) denaturation, then 1M
The gel was neutralized with Tris (pH 7.4) and 1.5 M NaCl, followed by a vacuum transfer unit (Hofer [Hofer
efer] TE80) was transferred to Hybond-N nylon membrane for 60 minutes. 20 x SSC (3MNa
Cl, 1.5 M sodium citrate) was used as the transfer buffer (Sambrook et al. (1989), supra). The blot was subjected to DIG-labeled 1447 bp PCR.
Used for hybridization with product. VII. Detection of WSBV in arthropods taken from areas where epizootic outbreaks PCR using 146R1 and 146F1 to detect WSBV in test organisms with DNA templates prepared from arthropods taken from areas where animal epidemics occur. Used for.

Results: A. Histopathological study: W. S.
S. The occurrence of is no longer clearly a limited regional problem. It is already pushing the aquaculture shrimp industry in Asia into a crisis. Further studies are underway on the physico-chemical characterization of this agent to better classify the causative virus and develop a rapid diagnostic method.

The main clinical sign of this disease in Penaeus monodon was vitiligo on the outer shell (FIG. 1). This vitiligo was particularly apparent on the back shell removed from the diseased shrimp, and was easily observed even on the back shell from animals with mild infection. Histopathological studies have revealed that the epidermis of affected shrimp has been attacked by viral agents. This is evidenced by the presence of degenerating cells in this tissue, characterized by enlarged nuclei with inclusion bodies (Fig. 2).

Ultrathin sections of the cuticular underlayer epidermis tissue from shrimp with vitiligo syndrome, observed under an electron microscope, showed numerous non-occluded baculo-like viral particles in the necrotic area. Virion-filled hypertrophic nuclei were also easily observed (Figure 3). This virus particle has a length of 330
It was ± 20 nm and the diameter was 87 ± 7 nm (n = 30).
The electron-dense central core of virus particles is about 220 in size.
× 70 nm nucleocapsid. It was confirmed that there was no difference in virion morphology between naturally-affected shrimp and experimentally infected shrimp (FIG. 4). B. Negative staining of filtrate and cytotoxicity assay for challenge test. The results of negative staining of pellets prepared from the monodone epidermal filtrate are shown in FIG. Viral particles in the form of rods can be seen. These are similar to the viral particles observed in ultrathin sections of naturally affected shrimp.

No cytopathic effect (CPE) was found in any of the four tested fish cell lines. The filtrate used as a waterborne inoculum was not cytotoxic. C. Antigen administration test: Healthy shrimp were infected with P. cerevisiae showing marked vitiligo signs. Japonix and P.P. It was exposed to the filtrate derived from monodon. These experimentally infected shrimps resembled those that were naturally adversely affected (FIG. 6), with a cumulative mortality rate of 5 compared to no dead shrimp in the control group.
It reached 100% within 7 days (Fig. 7).

Furthermore, this inoculum was highly pathogenic for the smallest shrimp tested (mean weight 0.08 g), all of which died within 5 days; 0.1
In the 6g size shrimp group, the mortality rate was 100 on the 12th.
%, But after 7 days the cumulative mortality rate was only 35
% Of the largest shrimp group (mean weight 0.26
In g), a mortality rate of 10% was observed within 2 weeks. No shrimp died in the control group. D. Purification, genomic structure and taxonomic location of WSBV: Purified virions were fusiform or rod-shaped with rounded ends. In the negative dye preparation, the virions were 70-150 nm at their widest point and 250-380 nm in length. This is often 10% greater than in ultrathin sections. In some virions, a tail-like protrusion extending from one end was observed (Fig. 8). The non-envelope nucleocapsid is usually 58 to 67 nm in diameter and 33 in length.
It was 0 to 350 nm. The capsid components formed parallel transverse grooves (Fig. 9). Therefore, the capsid appeared to consist of a ring of subunits in the form of a layered continuum. The thickness of this ring (20 nm) was quite constant and the ring was perpendicular to the long axis of the capsid. In terms of viral morphology, WSSV is similar to SEMBV (systematic ectoderm and mesoderm baculovirus), with BMN (baculovirus-type midgut necrosis virus) and PmSNPV (Penaeus monodon single nucleocapsid nuclear polyhedrosis). Virus [Penaeu
s monodon Single Nucleocapsid Nuclear Polyhedrosis
virus] = MBV) (Mari et al., D
is. aquat. Org. 16: 207-215, 1993; Sano et al., He
lgolander Meeresunters. 37: 255-264, 1984; Wongtee
rasupaya et al., Dis aquat. Org. 21: 69-77, 199
Five). However, the main clinical signs of vitiligo caused by WSSV have not been described in SEMBV infected shrimp. Until now, WSSV and SE
It is difficult to infer the association with MBV.

Single DNA from purified virions of WSBV
The molecules were extracted (Figure 10). WSB digested with HindIII, SalI and XhoI restriction endonucleases
The genomic DNA of V is shown in FIG. Genomic DNA of WSBV digested with HindIII restriction endonuclease
Each has an approximate size of 1 in the agarose gel.
9.4, 16.9, 14.9, 12.5, 10.0,
9.6, 8.4, 8.0, 7.3, 6.1, 5.5,
4.8, 4.3, 3.9, 3.6, 3.3, 3.0,
2.5, 2.0, 1.6, 1.4, and 1.1 kbp
At least 22 fragments were produced. Fragments smaller than 1 kbp will cross the gel, even if they are present. The length of WSBV DNA was estimated to be longer than 150 kbp. It settles within the size range 90-230 kbp found in insect baculovirus (Francki et al., Arch. Virol., Su.
ppl. 2: 1-450, 1991).

On the basis of morphological characteristics and genomic structure, WSSV is classified into the baculovirus family Nudibaculovirus subfamily non-occluded baculovirus (NOB) and its isolate is P. PmN because it is the third non-occlusive baculovirus reported for monodon
It was named OBIII (DV Lightner, Boca Raton,
p.393-486, 1993; Wongteerasupaya et al (1995), supra). This virus isolate, PmNOBIII, was identified by the Chinese Type Culture Collection Center [Ch.
ina Center for TypeCulture Collection] (CCT
CC) with the accession number CCTCC-V96001. WSBV (Baculovirus associated with vitiligo syndrome)
It is also proposed to be used to indicate mNOBIII related substances. E. FIG. Development of effective diagnostic tools for WSBV infection: I. Construction of WSBV genomic DNA library: a) Virus purification and extraction of viral DNA: WSB
The typical rod-shaped virions of V are readily observed after concentration and purification by sucrose gradient centrifugation. These virions were used to extract viral DNA.

Amplification of shrimp DNA using PCR and primers specific for 18S rRNA results in the reliably predicted 848 bp DNA fragment (FIG. 1).
2). This provided a convenient and sensitive method for detecting small amounts of shrimp DNA, which was subsequently used to monitor shrimp DNA contamination in WSBV genomic DNA preparation for library construction. The PCR analysis shown in Figure 13 shows that host DNA contamination was detected in most WSBV genomic DNA preparations. However, a small number of samples of WSBV genomic DNA extracted from the purified virion preparations contained virtually no host DN.
There was no A contamination. An example is shown in lane 3 of FIG. b) Genomic DNA library construction: SalI or H
indIII restriction endonuclease (BRL, life
Technologies) and pUC19 plasmid vector, two WSBV genomic libraries, P
mNOBIII SalI (pms) and PmNOBII
I HindIII (pmh) was constructed.

SalI digested WSBV DNA was checked, for example, by electrophoresing 5 μl aliquots in a 0.8% agarose gel containing ethidium bromide. WSBV genomic DNA was completely digested with SalI restriction endonuclease (FIG. 14). Digestion D
A 20 μl aliquot from the same batch of NA was used for library construction.

Recombinant plasmids isolated from the transformants were screened by SalI or HindIII digestion. Among them, 5 from the pms library
92 clones (pms1-pms592) and pm
410 clones from the h library (pmh1-p
mh245 and pmh419-pmh584) to S
Screened for the presence of inserts by digestion with alI or HindIII. Insert sizes range from 15 kbp to 100 bp, as shown in Table 1.
There were various miscellaneous items, even those that were less than. These libraries supply a large amount of WSBV DNA, which allows further study of the molecular biology of this virus and the development of nucleic acid and immunological diagnostic kits. II. Amplification of WSBV DNA fragment derived from deproteinized DNA extracted from purified virion: WSBV obtained
Designing several primer sets based on the DNA sequence of the SalI DNA fragment (data not shown),
Their ability to identify WSBV in infected tissues was evaluated by PCR.

FIG. 15 shows the SalI-1461bp DNA fragment cloned in the plasmid pms146, and FIG. 16 shows the position of the SalI-1461bp DNA fragment of the primers used for PCR amplification. 146F1 and 146R1 activate amplification of the 1447 bp fragment, while 146F2 and 146R2 have 9
Activates the amplification of the 41 bp fragment. SalI-1461
The positions of the two EcoRI sites in the bp DNA fragment are also indicated. FIG. 17 shows the detailed nucleotide sequence of the SalI-1461 bp fragment, which contains two primer sets 146F1 / 146R1 and 14
The 6F2 / 146R2, as well as the two EcoRI sites are also shown. FIG. 18 shows the nucleotide sequences of 6 primer sets developed from the SalI-1461 bp fragment. Among them, primer set 14
6F1 / 146R1 is WSBVD but not shrimp DNA
It provides stable and sufficient amplification of NA. Therefore, this primer set was selected for the next part of this study.

FIG. 19 shows the purified WSBV genomic DNA
The results of amplification used as a CR template and with a primer set specific to either WSBV DNA or shrimp DNA are shown. The reactions analyzed in lanes 2, 5 and 8 of Figure 16 represent amplifications using the WSBV DNA primer set 146F1-146R1 and three independent WSBV DNA preparations.
The results show that the intensity of 144 in these three lanes is
Relatively high amounts of WSBV genomic DNA in the three test samples as evidenced by the 7-bp PCR product.
Is present. In Lane 6 of FIG. 16, there is at least one WSBV, as evidenced by the absence of a detectable prawn DNA PCR product.
The DNA preparation is free of shrimp DNA contamination. The WSBV primer set 146 in the reaction mixture was used to approximate the proportion of WSBV DNA in the template DNA.
F1 / 146R1 and shrimp DNA primer set 1
43F / 145R were used simultaneously.

The data presented in FIG. 19 show that the WSBV-specific DNA fragment was detected as the major band in three independent WSBV preparations (lanes 4, 7 and 10), whereas shrimp DNA was detected in three WSBV DNA preparations. It was detected in two of them (lanes 4 and 10). Thus, the template DNA contains various proportions of shrimp DNA and WSBV DNA. It is also clear that the majority of the DNA extracted from WSBV virions purified by sucrose gradient centrifugation was WSBV DNA, despite contamination with shrimp DNA. On the other hand, total nucleic acid extracted from clinically healthy shrimp tissue and WSBV DNA-specific primer set 1
The reaction mixture with 46F1 / 146R1 was consistently negative (FIG. 19, lane 11), indicating the specificity of this primer set. III. WSBV DN derived from DNA extracted from shrimp tissue naturally or experimentally infected with WSBV
A fragment amplification Figure 17: Plasmid pms146 as well as naturally WSB
V. infected P. Monodon and P.M. 7 shows the results of amplification using DNA extracted from Japonix tissue as a DNA template. This DNA template is WS
BV specific primer set 146F1 / 146R1 or shrimp DNA specific primer set 143F / 145R
Was amplified by using either of The [comigrating] 1447-bp PCR product, which co-migrates with DNA amplified from the pure plasmid pms146 DNA, indicates that WSBV DNA is present in the total nucleic acid extracted from all naturally infected shrimp.
An example is shown in lanes 2, 5 and 8 of FIG.

Internal primer set 146F2 / 146
These products were reamplified by using R2 to generate a PCR product with the expected size of 941 bp (Figure 20, lanes 3, 6 and 9). These results confirm the identity of the amplification product with the template. As shown in lanes 7 and 10 of FIG. 20, shrimp DNA-specific primer set 143F / 14
Shrimp DNA could be amplified very efficiently by using 5R. The results shown in lanes 5 to 10 of FIG. 20 show that in the presence of a large excess of shrimp genomic DNA, WSBV DNA specific primer set 146F1 was used.
It shows that it was possible to detect WSBV DNA using / 146R1 and 146F2 / 146R2.

FIG. 21 shows P. coli which was experimentally infected with WSBV. The result of amplification which uses the DNA extracted from the tissue of a monodone as a template of PCR using primer set 146F1 / 146R1 is shown. Expected 1447 bp for all experimentally infected shrimp
It is clear that the fragment of A. is amplified. No 1447 bp amplification product was present in the control healthy shrimp. IV. WSBV infection or healthy P. cerevisiae using the DIG-labeled 1447-bp PCR product. Dot hybridization of DNA extracted from monodon: The result of dot hybridization is that the PCR product is WS
It shows that it hybridizes with DNA extracted from BV-infected shrimp, but does not hybridize with DNA extracted from healthy shrimp (FIG. 22). The result is
The specificity of the 1447-bp PCR product is shown. V. Diseased P. cerevisiae using the DIG-labeled 1447-bp PCR product. Monodon or P. Southern hybridization of WSBV DNA from Japonix 1447-bp PCR on WSBV genomic DNA
14 DIG-labeled to localize product
Using the 47-bp PCR product as a probe, W
Southern hybridization of the SBV genomic DNA SalI fragment was performed. The result is 1447-b
WSBV genomic DN with 1461 bp p PCR product
It shows that it hybridized specifically with the A SalI fragment (FIG. 23). P. Monodon and P.M. Both 1461 bp SalI fragments of WSBV genomic DNA, each prepared from Japonix, were found to be positive for this probe.

Detection of WSBV in arthropods collected from areas where epizootic diseases occur: Monodon, P.M. Japonix, crabs, copepods and insects (Family: Ephydridae [Ephydridae]) showed WSBV-positive results (FIG. 24).

Discussion: Affected shrimp have clear vitiligo on the back, appendages and inner surface of the body, and show signs of lethargy, with hepatopancreas staining red. Vibrio, viral infections, poor environmental control and nutritional imbalances have all been speculated to be responsible for these outbreaks.
However, based on electron microscopy, rod-shaped virus was considered to be the major causative agent. In this study, P. albicans suffering from vitiligo syndrome. Japonix and P.P.
The pathogenicity of the pathogenic virus from Monodon was investigated. The close resemblance of vitiligo signs and viral morphology between naturally-affected shrimp and experimentally infected shrimp showed that the virus was indeed the causative agent of disease development. The virus is highly pathogenic and threatens shrimp. Preliminary study of information on feeding affected shrimp to healthy species [inform
ation pilot studies] suggested that the virus could be transmitted orally in addition to water.

In addition to WSBV, various baculoviruses infect the decapod crust, first reported by Couch (Nature, 247 (5438): 229-231, 1974; J. Inv.
ertebr. Pathol., 24: 311-331, 1974) and some of them cause mortality in affected animals (Lightner & Redman, J. Invertebr. Pathol., 38:29).
9-302, 1981; Sano et al., Fish Pathol., 15: 185-19
1, 1981; Lester et al., Dis. Aquat. Org., 3: 217-21
9, 1987; Johnson PT, Dis. Aquat. Org., 5: 111-1
22, 1988; Johnson & Lightner, Dis. Aquat. Org., 5:
123-141, 1988; Bruce et al., J. Virol. Methods, 3
4: 245-254, 1991; Chang et al., Fish Pathol., 27
(3): 127-130, 1992; Chang et al., J. Invertebr. Pa
thol., 62: 116-120, 1993; Mari et al., Dis aquat. O
rg., 16: 207-215, 1993; Wongteerasupara et al., Di
s aquat. Org., 21: 69-77, 1995). These viruses are morphologically similar, and most researchers have found that the structure of the viral genome is a more necessary reference to determine the taxonomic position of crustacean baculoviruses. I agree that it should be. A rapid and reliable diagnostic tool using a molecular approach would be useful not only in the identification and comparison of viruses, but also in the screening of carriers in shrimp larvae and parental spawners. In view of these points, this work focuses on WSBV genomic structure and the development of rapid and sensitive diagnostic tools.

In experiments, shrimp DN was used in several evaluations.
A specific primer is used. The purpose of using the shrimp DNA specific primer set in this study is to
(I) evaluating the purity of the WSBV genomic DNA preparation,
(Ii) evaluating nucleic acid extraction methods for obtaining amplifiable DNA templates, and (iii) shrimp DNA and W in template DNA prepared from total nucleic acid of infected tissue.
It was to roughly estimate the ratio with SBV DNA.
At our laboratory, attempts have been made to purify WSBV virions from a variety of tissues including epidermis, muscle and gills. From these virions, different purity WSBV DNA was obtained as assessed by shrimp DNA specific primers. Examples of these evaluations are shown in Figures 13 and 20. Nucleic acid extracted from muscle tissue contains a large amount of WSBV
It produced DNA, but was heavily contaminated with shrimp DNA (Figure 19, lane 9). Infected epidermal cells under the outer shell are good starting materials for extracting "ultra-pure" WSBV genomic DNA (Figure 19, lanes 2 and 5).
By using shrimp DNA specific primers and PCR, this tool is
It can be used to evaluate the degree of shrimp DNA contamination in A preparations.

By using the WSBV DNA specific primers, all purified WSBV genomic DNA samples stably produce a clear amplification product showing the expected mobility of the 1447-bp DNA fragment. Nucleic acids extracted from tissues of shrimp naturally affected by vitiligo syndrome and experimentally infected with WSBV also stably produced PCR products of the same size. Nucleic acids extracted from clinically healthy shrimp tissue showed no positive results. These results show that the WS designed in this study
The specificity of the BV DNA specific primer is shown. In addition, the 1447-bp PCR product can be used to prepare WSBV-specific nucleic acid probes for detecting shrimp WSBV infection using dot blot hybridization, as shown in FIG. actually,
This study provides three effective diagnostic tools for screening WSBV infections of prawns, as shown in FIGS. 20, 22 and 23.

Using PCR (FIG. 20) and Southern blot hybridization (FIG. 23), we show that the causative agents of vitiligo syndrome in shrimp of different species are indeed closely related. WSBV in shrimp
Screening for infection should be undertaken immediately to prevent the disease from spreading further. on the other hand,
The PCR diagnostic technique for WSBV developed in this study is based on Japanese RV-PJ (Inouye et al (1994),
(Above), Chinese HHNBV (Cai et al., J. Fish. Chi
na, 19: 112-117, 1995), SEMBV (Wong, Thailand)
teerasupaya et al. (1995), supra), WS of this invention
It provides an effective tool for comparative studies on shrimp non-occluded baculoviruses such as BV isolate PmNOBIII and other crustacean non-occluded baculoviruses.

From the above technology, it is apparent that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, it should be understood that the invention may be practiced other than as specifically described herein.

[0051]

[Table 1]

[0052]

[Table 2]

[0053]

[Table 3]

[0054]

[Table 4]

[0055]

[Table 5]

[0056]

[Table 6]

[0057]

[Table 7]

[0058]

[Table 8]

[0059]

[Table 9]

[0060]

[Table 10]

[0061]

[Table 11]

[0062]

[Table 12]

[0063]

[Table 13]

[0064]

[Table 14]

[0065]

[Table 15]

[0066]

[Table 16]

[0067]

[Table 17]

[0068]

[Table 18]

[0069]

[Table 19]

[0070]

[Table 20]

[0071]

[Sequence list]

(1) General information (i) Applicant: Guo Mitsuo Wang Shigeo Ratakeyoshi (ii) Title of invention: Identification, purification and detection of WSBV (Baculovirus associated with vitiligo syndrome) (iii) Number of sequences: 13 (iv) Addressee : (A) Address: (B) Street: (C) City: (D) State: (E) Country: (v) Computer readable form: (A) Media type: Floppy disk-3.
5 inches, capacity 1.44M (B) Computer: IBM PC / 386 compatible (C) Operating system: MS-DOS5.0 (D) Software: PE2 (vi) Data of this application: (A) Application number: None (B) Filing date: New application (C) Classification: None (vii) Prior application data: (A) Application number: None (B) Application date: None (viii) Agent information: (A) Name: (B) Registration number: (C) Reference / Docket number: (ix) Telecommunications information (A) Telephone: (B) Facsimile: (C) Telex: (2) Sequence number 1 information: (i) Array Properties: (A) Length: 1461 base pairs (B) Type: Nucleic acid (C) Number of strands: Double strand (only one strand is shown) (D) Topology: Linear (ii) Sequence type : Genomic DNA (vi) Origin: (A) Organism name: WSBV (Vitiligo) Group-related baculovirus) (B) Strain name: PmNOBIII (vii) Direct origin: (A) Library name: (B) Clone name: (ix) Sequence characteristics: (A) Characteristic symbols: (B) Location: (ix) Sequence feature: (A) Feature symbol: (B) Location: (ix) Sequence feature: (A) Feature symbol: (B) Location: (xi) Sequence: GTCGACAGAC TACTAACTTC AGCCTATCTA GTAAAACAAG CTAAAAGATT CGACGGAGTT 60 GACCCAGCCT TCCCTGCCGC CCTCACCTGC GCTTCTCACC TCATGCTTTC TTCCATGGAT 120 TCCCATACAA AGTCATCTTT CATGGACAAC ATCAAATTGC ACATGACTGA TACTCAATGC 180 TTCTTCAAGA ACATTGAACG ATTTGAGAAA TTCTTGGGAA GATATGGGGA CGAATACGCC 240 ATGTCCCACA AGCAAAATTG TAACTGCCCC TTCCATCTCC ACCACACTTT TACTCCCTCA 300 GATAACGAGC ATCTGGTATC CTCTTTCGCA TTCGCCCGCC CAGAAGTCTC CATGGAAGAA 360 ATTAGAGCCA CACCCTATCA GGCCAACAAG CTTATTAGTG ACAAACATTA CGTGATGAAC 420 ATGTCCAAGA TCGATTCTAG AGTAACAGGA TCTTCCCTCC TTAAGAAGGT TAGCGAATGG 480 ACTGAAATGA GAATGAACTC CAACTTTAAT GGAACATTTG AACCATCAAG ACTCGCCCTC 540 TCCAACTCTG GCATGACAAC GGCAGGAGTC AACCTCGACG TTATTGTCAA ACCAAATAAT 600 GCAAGAAGTG TACTAGGAAT ATTGGAATGT CATCGCCAGC ACGTGTGCAC CGCCGACGCC 660 AAGGGAACTG TCGCTTCAGC CATGCCAGCC GTCTTCCAGG CAACCGATGG AAACGGTAAC 720 GAATCTGAAC TGATCCAGAA TGCTCTGCCA AGGAACAGAT ACATCCAAAA GAGCACAATG 780 AACGCTCAAA CTGTCGTGTT TGCTAATGTT TTGGAACAAC TTATCGCCGA TCTTGGAAAG 840 GTTATCGTGA ACGAACTGGC CGGCACCATC GCTGAATCTG TACCAGAAAG CGTATATGAA 900 AACACCAAGG AAATGATTGA TAGACTAGGC TCTGACGACC TCTTCAAATC TAATAATAAT 960 GGAGGAGTAG AATCAATGGA TTATGAAGAT AGCGAAACAA CATCCAACAA TGGTCCCGTC 1020 CTCATCTCAG AAGCCATGAA GAATGCCGTC TATCACACAC TAATTTCCGG CAAGGCAGCT 1080 CGCCCGGAAA ATGTACCATT CGCCTCATGC GCCAGCGGCC CTCTCGCCTT TGATTTCCTT 1140 CTGTCAAAGG GAGATACATT CGAAGAAAAG AACGCCGAAC AAGGTGCAGC AGCTGCCGTA 1200 TCCTCTACCT ATTCTTCCTC TTCTAACACT ACTCTTCGTA AGCATTTGGC TCGAGTTTTC 1260 GAAGCCATCT CTAAGCAAGT AACTGATGCT GAATTCAAGG ATATCCTCAA CGATATCGAA 1320 CGTAATATTT CT TCTGACTA TACTAACTGT CCACCAAATA CTAACCAAAA TGCCTTTGCT 1380 CTAGCTATCA AGAGAGAATT CAGCAGAATT GTTTCCTTCT TAACCATTCT TCGTAAGAAC 1440 ATTACACCCG CATTAGTCGA C (2) Sequence No. 2 information (i) Sequence characteristics: (A) Length: 1447 base pairs (C) type Number: double-stranded (only one strand is shown) (D) topology: linear (ii) sequence type: Genomic DNA (vi) origin: (A) organism name: WSBV (white spot syndrome-associated baculovirus) ) (B) Strain name: PmNOBIII (vii) Direct origin: (A) Library name: (B) Clone name: (ix) Sequence features: (A) Feature symbol: (B) Location: (A) ix) Sequence feature: (A) Feature symbol: (B) Location: (ix) Sequence feature: (A) Feature symbol: (B) Location: (xi) Sequence: ACTACTAACT TCAGCCTATC TAGTAAAACA AGCTAAAAGA TTCGACGGAG TTGACCCAGC 60 CTTCCCTGCC GCCCT CACCT GCGCTTCTCA CCTCATGCTT TCTTCCATGG ATTCCCATAC 120 AAAGTCATCT TTCATGGACA ACATCAAATT GCACATGACT GATACTCAAT GCTTCTTCAA 180 GAACATTGAA CGATTTGAGA AATTCTTGGG AAGATATGGG GACGAATACG CCATGTCCCA 240 CAAGCAAAAT TGTAACTGCC CCTTCCATCT CCACCACACT TTTACTCCCT CAGATAACGA 300 GCATCTGGTA TCCTCTTTCG CATTCGCCCG CCCAGAAGTC TCCATGGAAG AAATTAGAGC 360 CACACCCTAT CAGGCCAACA AGCTTATTAG TGACAAACAT TACGTGATGA ACATGTCCAA 420 GATCGATTCT AGAGTAACAG GATCTTCCCT CCTTAAGAAG GTTAGCGAAT GGACTGAAAT 480 GAGAATGAAC TCCAACTTTA ATGGAACATT TGAACCATCA AGACTCGCCC TCTCCAACTC 540 TGGCATGACA ACGGCAGGAG TCAACCTCGA CGTTATTGTC AAACCAAATA ATGCAAGAAG 600 TGTACTAGGA ATATTGGAAT GTCATCGCCA GCACGTGTGC ACCGCCGACG CCAAGGGAAC 660 TGTCGCTTCA GCCATGCCAG CCGTCTTCCA GGCAACCGAT GGAAACGGTA ACGAATCTGA 720 ACTGATCCAG AATGCTCTGC CAAGGAACAG ATACATCCAA AAGAGCACAA TGAACGCTCA 780 AACTGTCGTG TTTGCTAATG TTTTGGAACA ACTTATCGCC GATCTTGGAA AGGTTATCGT 840 GAACGAACTG GCCGGCACCA TCGCTGAATC TGTACCAGAA AGCGTATATG AAAACACCAA 900 GGAAATGATT GATAGACTAG GCTCTGACGA CCT CTTCAAA TCTAATAATA ATGGAGGAGT 960 AGAATCAATG GATTATGAAG ATAGCGAAAC AACATCCAAC AATGGTCCCG TCCTCATCTC 1020 AGAAGCCATG AAGAATGCCG TCTATCACAC ACTAATTTCC GGCAAGGCAG CTCGCCCGGA 1080 AAATGTACCA TTCGCCTCAT GCGCCAGCGG CCCTCTCGCC TTTGATTTCC TTCTGTCAAA 1140 GGGAGATACA TTCGAAGAAA AGAACGCCGA ACAAGGTGCA GCAGCTGCCG TATCCTCTAC 1200 CTATTCTTCC TCTTCTAACA CTACTCTTCG TAAGCATTTG GCTCGAGTTT TCGAAGCCAT 1260 CTCTAAGCAA GTAACTGATG CTGAATTCAA GGATATCCTC AACGATATCG AACGTAATAT 1320 TTCTTCTGAC TATACTAACT GTCCACCAAA TACTAACCAA AATGCCTTTG CTCTAGCTAT 1380 CAAGAGAGAA TTCAGCAGAA TTGTTTCCTT CTTAACCATT CTTCGTAAGA ACATTACACC 1440 CGCATTA (2) Sequence No. 3 information: (i) Sequence characteristics (A) Length: 23 base pairs (B) Type: Nucleic acid (C) Number of chains: 1 Strand (5 ′ to 3 ′) (D) Topology: Linear (ii) Sequence type: Oligonucleotide probe (iv) Antisense: (xi) Sequence: ACTACTAACT TCAGCCTATC TAG (2) Information of SEQ ID NO: 4 (i ) Distribution Properties: (A) Length: 25 base pairs (B) Type: Nucleic acid (C) Number of strands: Single-stranded (5 ′ to 3 ′) (D) Topology: Linear (ii) Sequence type: Oligonucleotide probe (iv) Antisense: (xi) Sequence: TAATGCGGGT GTAATGTTCT TACGA (2) Information of SEQ ID NO: 5: (i) Sequence characteristics: (A) Length: 22 base pairs (B) Type: nucleic acid (C ) Number of strands: Single strand (5 ′ to 3 ′) (D) Topology: Linear (ii) Sequence type: Oligonucleotide probe (iv) Antisense: (xi) Sequence: GTAACTGCCC CTTCCATCTC CA (2) Information of SEQ ID NO: 6: (i) Sequence characteristics (A) Length: 22 base pairs (B) Type: nucleic acid (C) Number of strands: single-stranded (5 ′ to 3 ′) (D) Topology: direct Chain (ii) Type of sequence: oligonucleotide probe (iv) Antisense: (xi) Sequence: TACGGCAGCT GCTGCACCTT GT (2) Sequence No. 7 information: (i) Sequence characteristics: (A) Length: 21 base pairs (B) Type: Nucleic acid (C) Number of strands: Single-stranded (5 'to 3') (D) Topology: Direct Chain type (ii) Sequence type: oligonucleotide probe (iv) Antisense: (xi) Sequence: TGGGAAGATA TGGGGACGAA T (2) Information of SEQ ID NO: 8: (i) Sequence characteristics: (A) Length: 23 bases Pair (B) type: nucleic acid (C) Number of strands: single-stranded (5 ′ to 3 ′) (D) Topology: linear (ii) Sequence type: oligonucleotide probe (iv) Antisense: (xi ) Sequence: CGAAGAGTAG TGTTAGAAGA GGA (2) Information of SEQ ID NO: 9: (i) Sequence characteristics: (A) Length: 21 base pairs (B) Type: Nucleic acid (C) Number of strands: Single strand (5 ' From 3 ') (D) Topology: Linear (ii) Sequence type: Oligonucleotide probe (iv) Antisense: (xi) Sequence: AGAAGGTTAG CGAATGGACT G (2) Information of SEQ ID NO: 10: (i) Sequence characteristics: (A) Length: 21 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (5 ′ to 3 ′) (D) Topology: Linear (ii) Sequence type: Oligonucleotide probe (iv) Antisense: (xi) Sequence: TTGAAGAGGT CGTCAGAGCC T (2) Information of SEQ ID NO: 11: (i) Sequence characteristics: (A) ) Length: 23 base pairs (B) Type: nucleic acid (C) Number of strands: single-stranded (5 ′ to 3 ′) (D) Topology: linear (ii) Sequence type: oligonucleotide probe (iv ) Antisense: (xi) Sequence: GAAACGGTAA CGAATCTGAA CTG (2) Information of SEQ ID NO: 12: (i) Sequence characteristics: (A) Length: 18 base pairs (B) Type: Nucleic acid (C) Number of strands: Single-stranded (5 ′ to 3 ′) (D) Topology: Linear (ii) Sequence type: Oligonucleotide probe (iv) Anti Sequence: (xi) Sequence: CAGTCCATTC GCTAACCT (2) Information of SEQ ID NO: 13: (i) Sequence characteristics: (A) Length: 18 base pairs (B) Type: Nucleic acid (C) Number of strands: single strand (5 ′ to 3 ′) (D) Topology: Linear (ii) Sequence type: Oligonucleotide probe (iv) Antisense: (xi) Sequence: CGTCCCCATA TCTTCCCA

[Brief description of drawings]

FIG. 1 is a photograph of Penaeus monodon with vitiligo syndrome showing vitiligo that is slightly visible to a diameter of 3 mm. Bar: 1 cm.

FIG. 2 shows basophilic inclusions in hypertrophic nuclei of degenerated cells,
Optical micrograph of cuticle underlayer epidermis under the craniothoracic shell (C) from Penaeus monodon suffering from vitiligo syndrome.
Bar: 10 μm.

FIG. 3 is a transmission electron micrograph of a thin section of infected tissue under the dorsal shell cuticle (C) from Penaeus monodon with vitiligo syndrome, showing viral particles in necrotic areas and hypertrophic nuclei (arrows). Bar: 0.5 μm.

FIG. 4. Diseased P. The filtrate obtained from monodon was used to experimentally infect P. A photograph showing rod-shaped virus particles in the epidermis of Japonix. Scale bar: 200 nm.

FIG. 5. Diseased P. Negative staining micrograph of pellets prepared from monodon epidermis filtrate. Viral particles in the form of rods (arrows) were observed. Scale bar: 500n
m.

FIG. 6: Experimentally infected P. A photograph showing the white spots (arrows) on the back of the Japonyx removed.

FIG. 7. Diseased P. Japonix and P.P. Experimentally infected P. cerevisiae by immersion in a filtrate prepared from monodon. The figure which shows the cumulative mortality rate of Japonix (mean weight 0.08g) compared with a healthy shrimp control group.

FIG. 8: Tail-like protrusion (P) extending from one end of the virus
Is a transmission electron micrograph of the negative-stained purified virion. Bar: 0.1 μm.

FIG. 9 is a transmission electron micrograph of a negative-stained envelope-free nucleocapsid showing the trough on the capsid formed by the ring-shaped subunit. These rings line up at right angles to the long axis of the capsid. Bar: 0.1 μm.

FIG. 10: PmNOBIII extracted from purified virions
A photograph showing an ethidium bromide-stained agarose gel of DNA. A single molecule of DNA is observed in the gel. Lane 1: Lambda phage DNA HindIII fragment marker; Lanes 2-4: PmNOBIII DNA extracted from each of the three preparations.

FIG. 11 is a photograph showing an ethidium bromide-stained agarose gel of PmNOBIII DNA digested with three types of restriction endonucleases. At least 22 DNA fragments (arrows) can be identified in this gel. Lane 1: Lambda phage DNA HindIII fragment marker; Lane 2: PmNOBIII DNA Hi.
ndIII fragment; lane 3: PmNOBIII DNA S
alI fragment; Lane 4: PmNOBIII DNA Xh
oI fragment; and lane 5: 1 Kb DNA ladder.

FIG. 12: PCR amplified 18Sr derived from shrimp genomic DNA
A photograph showing an agarose gel of a DNA fragment stained with ethidium bromide. Primers to the highly conserved region of the decapod 18S rRNA sequence, 143F and 145R, were used in the reaction to activate amplification of the 848-bp fragment from a DNA template prepared from healthy Penaeus monodon (lane 2). . Lane 1 is pGEM
It is a DNA size marker. The size of the DNA marker is shown in base pairs (bp).

FIG. 13: Shrimp DN in WSBV genomic DNA preparation
A. Photographs showing quality assessment using PCR and shrimp DNA specific primer sets 143F and 145R to monitor A contamination. PCR products were analyzed on a 1% agarose gel. Shrimp DNA contamination is evidenced by the presence of the 848 bp PCR product. Lane 1: pGEN DNA size marker; Lanes 2-6: WSBV genomic DNA preparation as DNA template; Lanes 7-8: healthy Penaeus monodon (lane 7) and P. Shrimp genomic DNA prepared from Japonix (lane 8); lane 9: no DNA template. The size of the DNA marker is given in base pairs (bp).

FIG. 14 is a photograph showing a WSBV DNA fragment digested with SalI. WSBV genomic DNA was digested with SalI restriction endonuclease for 3 hours at 37 ° C. A 5 μl aliquot was analyzed on a 0.8% agarose gel containing ethidium bromide and showing fragments of size 15 kbp to less than 1 kbp (lane 2). From the same batch of digested DNA, aliquot 20 μl W
Used for construction of SBV DNA library. Lane 1, lambda phage DNA HindIII fragment marker. The size of the DNA marker is shown in base pairs (bp).

FIG. 15 shows a SalI-1461bp DNA fragment cloned in plasmid pms146.

FIG. 16 is a diagram showing the positions of primers used for PCR amplification in a SalI-1461 bp DNA fragment. 146F1 and 146R1 activate amplification of the 1447-bp fragment. In contrast, 146F2 and 146R2 activate amplification of the 941-bp fragment. Also shown are the positions of the two EcoRI sites in the SalI 1461 bp DNA fragment.

FIG. 17 shows a detailed nucleotide sequence of the SalI-1461 bp fragment. There are two primer sets 146F1 / 146R1 and 146F2 / 146R
Two as well as two EcoRI sites are also shown.

FIG. 18 shows the nucleotide sequences of 6 types of primer sets developed from the SalI-1461 bp fragment.

FIG. 19: WSBV purified by sucrose gradient centrifugation
Using a DNA template prepared from virions,
Photographs showing PCR amplification of WSBV and shrimp DNA specific fragments. WS producing a 1447-bp PCR product
BV specific primers 146F1 and 146R1 were used for the reactions in lanes 2, 5, 8 and 11. 848
Shrimp DNA specific primers 143F and 145R, which produce a -bp PCR product, were used in the reactions in lanes 3, 6, 9 and 12. In lanes 4, 7, 10 and 13, primer 143F,
145R, 146F1 and 146R1 were added together. PCR products were analyzed on a 1% agarose gel. Lane 1, pGEN DNA size marker; Lane 2-
4, PCR product showing shrimp DNA and WSBV DNA bands using DNA template extracted from virions purified from the epidermis of diseased shrimp # 1; lane 5
-7, PCR product showing only WSBV DNA band using DNA template extracted from virions purified from diseased shrimp # 2; lanes 8-10: extracted from virions purified from muscle of diseased shrimp # 2 DN
Strong Shrimp DNA and WSBV using A template
PCR product showing DNA band; lane 11-1
3. PCR product showing only shrimp DNA band using DNA template extracted from healthy shrimp. DNA
Marker size is shown in base pairs (bp).

FIG. 20. Plasmid pms146 and D extracted from Penaeus monodon naturally infected with WSBV.
WS using NA as PCR DNA template
Photographs showing PCR amplification of BV and shrimp DNA specific fragments. WSBV specific primers 146F1 and 146R
1 was used for the reactions in lanes 2, 5, 8 and 11. The PCR product is the 1447-bp fragment. 1
Internal primer specific for the 447-kbp fragment, 146
F2 and 146R2 were used for reactions in lanes 3, 6, 9 and 12; these activate amplification of the 941-bp fragment. Shrimp DNA specific primers 143F and 145R were used for the reactions in lanes 4, 7 and 10. These activate the amplification of the 848-bp fragment.
Amplification products were analyzed on a 1% agarose gel. Lane 1, pGEN DNA size marker; Lanes 2-4,
Plasmid pms146; lanes 5-7, naturally infected P. DNA extracted from monodon; Lane 8-1
0, naturally infected P. D extracted from Japonix
NA; Lanes 11 and 12, control reaction without template. The size of the DNA marker is given in base pairs (bp).

FIG. 21: Penaeus experimentally infected with WSBV
Using a DNA template prepared from monodon, W
Photographs showing PCR amplification of SBV and shrimp DNA specific fragments. Primers 146F1 and 146R1 which generate a 1447-bp PCR product were used in the reaction. PC
R products were analyzed on a 1% agarose gel. Lane 1,
pGEN DNA size marker; lanes 2-4, 3 experimentally infected P. DNA extract derived from monodon; lanes 5-7, control healthy P. D from monodon
NA extract. The size of the DNA marker is base pair (bp)
Indicated by

FIG. 22 is a photograph showing dot hybridization of DNA extracted from WSBV-infected Penaeus monodon or healthy Penaeus monodon using a DIG-labeled 1447-bp PCR product. 2 WS
BV-infected shrimp (1 and 2) and 2 healthy shrimp (3
DNA from 4 and 4) was blotted onto two sites of Hybond-N. Paper (A and B) and examined with the DIG-labeled 1447-bp PCR product. This probe hybridized with the DNA from the infected shrimp, but did not hybridize with the DNA from the healthy shrimp.

FIG. 23: Diseased P. cerevisiae using DIG labeled 1447-bp PCR product. Monodon and P.M. The photograph which shows the Southern hybridization of WSBV DNA derived from Japonix. P. Monodon and P.M. SalI digested WSBV DNA from Japonics was blotted to Hybond-N. Paper and labeled 144 with DIG.
Checked with 7-bp PCR product. This probe is a SalI digested WSBV DNA from any source of shrimp.
And the 1461 bp fragment of H. This shows these close relationships.
A: 0.8% agarose gel stained with ethidium bromide; B: Autoradiograph of Southern blot of gel (A). Lane 1, pGEN DNA size marker; Lane 2, P. WSBV genomic DNA SalI fragment purified from monodon; lane 3, P.I. WSBV genomic DNA SalI purified from Japonix
fragment.

FIG. 24 is a photograph showing PCR amplification of WSBV DNA-specific fragments using the primer set 146F1 / 146F2 and a DNA template prepared from arthropods collected from an endemic area of animal diseases. Lane 1: pGEN marker; Lane 2: P.I. Monodon; Lane 3: P.M. Japonyx; Lane 4: Crab; Lane 5: Copepod; Lane 6: Insect (family: Effidridae); Lane 7: Positive control; Lane 8: Negative control, sample without template.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12Q 1/70 7823-4B C12Q 1/70 (72) Inventor Guo Mitsuo 4 Lusafuku Road, Taipei City, Taiwan Dan No. 1 National Taiwan University Animal System (72) Inventor Wang Shigeo Lusufuku Road, Taipei City, Taiwan Dan Dan No. 1 National Taiwan University Plant Disease Diseases and Diseases (72) Inventor Luzhu Fang Taiwan, Taipei City Lusafuku Road 4 Dan No. 1 National Taiwan University Animal System

Claims (27)

[Claims] 1. A vitiligo syndrome-related baculovirus (WS)
BV) a substantially pure viral isolate. 2. The WSBV of claim 1, wherein the virus comprises a genome having the restriction enzyme fragment profile shown in FIG. 3. The viral genome comprises a SalI DNA fragment having the following nucleotide sequence:
Or WSBV according to any one of 2 above. 1 GTCGA CAGAC TACTA ACTTC AGCCT ATCTA GTAAA ACAAG CTAAA AGATT 51 CGACG GAGTT GACCC AGCCT TCCCT GCCGC CCTCA CCTGC GCTTC TCACC 101 GACATA TGATCGA TGACATCGAGATCA TGA GGGGA CGAAT ACGCC ATGTC CCACA 251 AGCAA AATTG TAACT GCCCC TTCCA TCTCC ACCAC ACTTT TACTC CCTCA 301 GATAA CGAGC ATCTG GTATAT CTCGACACAGACCACAGACCAAGACCAACCCA CCACAAGCACACCACACAACCCA CATCA ACA TCTTC CCTCC TTAAG AAGGT TAGCG AATGG ACTGA AATGA GAATG AACTC 501 CAACT TTAAT GGAAC ATTTG AACCA TCAAG ACTCG CCCTC TCCAA CTCTG 551 GCATG ACAAC GGCAG CAGTCAG ATC ATCATGATGC AGA ATAAT 601 AGA CATGC CAGCC GTCTT CCAGG 701 CAACC GATGG AAACG GTAAC GAATC TGAAC TGATC CAGAA TGCTC TGCCA 751 AGGAA CAGAT ACATC CAAAA GAGCA CAATG AAC GC TCAAA CTGTC GTGTT 801 TGCTA ATGTT TTGGA ACAAC TTATC GCCGA TCTTG GAAAG GTTAT CGTGA 851 ACGAA CTGGC CGGCA CCATC GCTGA ATC TACCA GAAAG CGTAT ATGAAGA 901 AACACCAATAG CATGATGAAAG 901 AACAC CAAGA AACAA TGGTC CCGTC CTCAT CTCAG AAGCC ATGAA GAATG CCGTC 1051 TATCA CACAC TAATT TCCGG CAAGG CAGCT CGCCC GGAAA ATGTA CCATT 110TCCTC CTCTCGCTCGCTCTCTCTCTCTCTCTCTCGCTCGATCATCTAGCTC TCGTA AGCAT TTGGC EcoRI 1251 TCGAG TTTTC GAAGC CATCT CTAAG CAAGT AACTG ATGCT GAATT CAAGG 1301 ATATC CTCAA CGATA TCGAA CGTAA TATTT CTTCT GACTA TACTA ACTGT EcoRI 1351 CCACCACT GACACTAGACT GACAC CAGA TGACTGACAGAT CAGA TGACTGACAGAGA CATTA GTCGA C 4. The viral genome comprises nucleotides 9 to 1 of the nucleotide sequence shown in claim 1.
The WS according to claim 1, comprising a SalI DNA fragment having a partial nucleotide sequence consisting of 455 nucleotides.
BV. 5. The WS according to claim 1, wherein the virus is PmNOBIII or a PmNOBIII-related factor.
BV. 6. A method for obtaining large amounts of substantially pure non-occluded baculovirus (NOB) from a host organism infected with them, comprising the steps of: a) obtaining a sample from a host organism infected with NOB virus; b. A.) Treating the sample with a protease inhibitor in an amount sufficient to prevent degradation of the NOB virus, and c) purifying the virus. 7. The method according to claim 6, wherein the purification step c) is performed by centrifugation. 8. The method according to claim 7, wherein the purification step c) is performed by sucrose gradient centrifugation. 9. The method of claim 7, wherein the protease inhibitor is removed after centrifugation. 10. The method of claim 6, wherein the virus is WSBV. 11. The method according to claim 6, wherein the virus is PmNOBIII or a PmNOBIII-related factor. (I) The following nucleotide sequence: 1 GTCGA CAGAC TACTA ACTTC AGCCT ATCTA GTAAA ACAAG CTAAA AGATT 51 CGACG GAGTT GACCC AGCCT TCCCT GCCGC CCTCA CCTGC GCTTC TCACC 101 TCATG CTTTC TTCCA TGGAT TCCCA TACAA ATCCA ACATCA ACATCA ATCCA ATCCA ACATCA ACA TACTC AATGC TTCTT CAAGA ACATT GAACG 201 ATTTG AGAAA TTCTT GGGAA GATAT GGGGA CGAAT ACGCC ATGTC CCACA 251 AGCAA AATTG TAACT GCCCACACACATCA TACCATCCATCCA ATCCATCCA ATCATCATCCATCATCCATCATCCATCATCCATCATCCATCATCGACCATCCATCCATCCATCATCAG 401 ACAAA CATTA CGTGA TGAAC ATGTC CAAGA TCGAT TCTAG AGTAA CAGGA 451 TCTTC CCTCC TTAAG AAGGT TAGCG AATGG ACTGA AATGA GAATG AGAGA CATGA AGA CATGA CGA CGA ATCGAC CATCA AGA AATGT CATCG CCAGC ACGTG TGCAC 651 CGCCG ACGCC AAGGG AACTG TCGCT TCAGC CATGC CAGCC GTCTT CCAGG 701 CAACC GATGG AAACG GTAAC GAATC TGAAC TGA TC CAGAA TGCTC TGCCA 751 AGGAA CAGAT ACATC CAAAA GAGCA CAATG AACGC TCAAA CTGTC GTGTT 801 TGCTA ATGTT TTGGA ACAAC TTATC GCCGA TCTTG GAAAG GTTAT CGTGA TAC ACGAC ACCTGA ACT ACGAAAG ATAAT GGAGG AGTAG AATCA ATGGA TTATG AAGAT AGCGA AACAA 1001 CATCC AACAA TGGTC CCGTC CTCAT CTCAG AAGCC ATGAA GAATG CCGTC 105AG CTC GACGACAG GAG CTC GCCAG CTC CTC CATGC GCC GCAGC AGCTG CCGTA 1201 TCCTC TACCT ATTCT TCCTC TTCTA ACACT ACTCT TCGTA AGCAT TTGGC EcoRI 1251 TCGAG TTTTC GAAGC CATCT CTAAG CAAGT AACTG ATGCT GAATT CAAGG CATACA CACACACACACACAA TATTA TACTTATATATATACTACATATATATACTACATATATACTACATATATACTACATAA CAGCA GAATT GTTTC CTTCT TAACC ATTCT TCGTA AGAAC ATTAC ACCCG 1451 CATTA GTCGA C (ii) (i) nucleotide sequence A nucleotide sequence complementary to; or (iii) (i) or a nucleotide sequence capable of hybridising to the nucleotide sequence of (ii),
A polynucleotide comprising: 13. A nucleotide fragment derived from the polynucleotide defined in claim 12, which is capable of hybridizing with WSBV genomic DNA. 14. The nucleotide fragment according to claim 13, wherein the nucleotide fragment is labeled with a reporter digoxigenin (DIG). 15. A nucleotide fragment derived from the polynucleotide defined in claim 12, which is useful as a primer in PCR amplification of WSBV genomic DNA. 16. The nucleotide fragment according to claim 15, which is labeled with a reporter digoxigenin (DIG). 17. (146F1, 146R1, 146F
2 or 146R2). 18. A method for detecting WSBV virus infection in shrimp, comprising the steps of: a) collecting a sample from the leg or blood lymph of a live shrimp; b) i) the nucleotide fragment of claim 13. Detect the presence of WSBV in the sample by Southern hybridization used, ii) dot blotting with the nucleotide fragment of claim 13 or iii) PCR amplification reaction with the pair of primers defined in claim 15. A method comprising the steps of: 19. The method of claim 16, wherein the nucleotides are labeled with the reporter digoxigenin (DIG) for dot blotting. (I) A nucleotide sequence consisting of nucleotides 9 to 1455 of the following sequence: 1 GTCGA CAGAC TACTA ACTTC AGCCT ATCTA GTAAA ACAAG CTAAA AGATT 51 CGACG GAGTT GACCC AGCCT TCCCT GCCGC CCTCA CCTGC GCTTC TCACC 101 TCATG CTTTC TTCCA TGGAT TCCCA TACAA AGTCA TCTTT CATGG ACAAC 151 ATCAA ATTGC ACATG ACTGA TACTC AATGC TTCTT CAAGA ACATT GAACG 201 ATTTG AGAAA TTCTT GGGAA GATAT GGGGA CGAAT ACGCC CTC CTC CTC ATCTCCCTCCTC ATCTCCCTCATCTCCCTCATCTCTCATCTCCCTCATCTCATCTCCCTCATCTCTCATCTCCCTCATCTCTCATCTCCCTCATCTCTCATCTCCCTCTCCATCATCTCCCTCATCTCTCATCTCCCCA 351 CATGG AAGAA ATTAG AGCCA CACCC TATCA GGCCA ACAAG CTTAT TAGTG 401 ACAAA CATTA CGTGA TGAAC ATGTC CAAGA TCGAT TCTAG AGTAAG CTCTATACAT AAGTC TAGCG ATCGATCAG ATCGATCATGCATG ATCGAATCAG ATCGA AGA CGACG TTATT GTCAA ACCAA ATAAT 601 GCAAG AAGTG TACTA GGAAT ATTGG AATGT CATCG CCAGC ACGTG TGCAC 651 CGCCG ACGCC AAGG G AACTG TCGCT TCAGC CATGC CAGCC GTCTT CCAGG 701 CAACC GATGG AAACG GTAAC GAATC TGAAC TGATC CAGAA TGCTC TGCCA 751 AGGAA CAGC AGA CGA CGA CGA CGA CGA CGA CGA TGA CGTAT ATGAA 901 AACAC CAAGG AAATG ATTGA TAGAC TAGGC TCTGA CGACC TCTTC AAATC 951 TAATA ATAAT GGAGG AGTAG AATCA ATGGA TTATG AAGAT AGCGA AACAA 1001 CATCC AACAA TGGTC CAGCT CACTA CCAGAC CACTA CACTA CCAGAC CAATC CACATCACAGAA TCCTCGAC CGGCC CTCTC GCCTT TGATT TCCTT CTGTC AAAGG 1151 GAGAT ACATT CGAAG AAAAG AACGC CGAAC AAGGT GCAGC AGCTG CCGTA 1201 TCCTC TACCT TACCT TACCT GACTATGATCAGAC CAATCCAGAC CAATCCAGAC CAATCCAGAC TACTA ACTGT EcoRI 1351 CCACC AAATA CTAAC CAAAA TGCCT TTGCT CTAGC TATCA AGAGA GAATT 1401 CAGCA GAATT GTTTC CTTCT TAACC ATTCT TCGTA AGAAC ATTAC ACCCG 1451 CATTA GTCGA C (ii) a nucleotide sequence complementary to the nucleotide sequence of (i); or (iii) a nucleotide sequence capable of hybridizing with the nucleotide sequence of (i) or (ii),
A polynucleotide comprising: 21. A nucleotide fragment derived from the polynucleotide defined in claim 20, which is hybridizable with WSBV genomic DNA. 22. The nucleotide fragment according to claim 21, wherein the nucleotide fragment is labeled with a reporter digoxigenin (DIG). 23. A nucleotide fragment derived from the polynucleotide defined in claim 22, which is useful as a primer in PCR amplification of WSBV genomic DNA. 24. The nucleotide fragment according to claim 23, wherein the nucleotide fragment is labeled with a reporter digoxigenin (DIG). 25. (146F1, 146R1, 146F
2 or 146R2). 26. A method for detecting WSBV virus infection in shrimp, comprising the steps of: a) collecting a sample from the leg or blood lymph of a live shrimp, b) i) the nucleotide fragment of claim 21. The presence of WSBV in the sample is detected by Southern hybridization used, ii) dot blotting with the nucleotide fragment of claim 21 or iii) PCR amplification reaction with the pair of primers defined in claim 23. A method comprising the steps of: 27. The method of claim 26, wherein the nucleotides are labeled with the reporter digoxigenin (DIG) for dot blotting.