Indian Journal of Experimental Biology Vol. 43. July 2005, pp. 654-661

White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and

electron microscopy

S S Mishra' & M S Shekhar

Central Institute of Brackishwater Aquaculture, 75, Santhome High Road. R.A.Puram, Chennai 600 028, India

Received 23 December 2003. revised 17 February 2005

Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus. Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation . Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of llOx320 ± 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.

Keywords: Electron microscopy, PCR, Shrimp, White spot disease, WSSV

White spot disease (WSD) caused by White spot syndrome virus (WSSV) is one of the most dreaded diseases of shrimp. This has caused mass mortalities in cultured shrimp and inflicted severe economic damage in India as in other Asian countries. The disease affects shrimps of all sizes, ages and culture conditions and is characterized by formation of white spots or patches on carapace over head, body cuticle and appendages with reddish body discoloration. The cumulative mortalities may reach up to 100% within 2-7 days '-4. The disease was first recognized in Taiwan5 in 1992 and China6

•7 in 1993. Since then

several outbreaks of WSD in shrimp culture farms and hatcheries have been reported in Japan8

,

Thailand9 and Taiwan 1o. Various researchers have isolated and identified different pathogenic non-

* Present address : Biotechnology Unit, Central Inland Fisheries Research Institute, Barrackpore, Kolkata 700 120, India, Phone : (033)- 25921190/91 Fax : 033-25920388 E-Mail: ss_mishra60@yahoo.co.uk

occluded bacilliform viruses associated with white spot syndrome (WSS). At least 5 different viruses have been implicated in WSS in shrimp like rod shaped virus of Penaeus japonicus (RV-PJ) in Japan7

,1J, which was later re-designated as Penaeid rod shaped DNA virus (PRDV)12.13, Hypodermal and hematopoietic necrosis baculovirus (HHNBV) in China, also called as China virus6

, Systemic ectodermal and mesodermal baculovirus (SEMBV) of tiger shrimp in Thailand9 and White spot baculovirus (WSBV) in TaiwanID. WSD causing high mortality in cultured shrimps and hatcheries have been reported in India since J 994 and the causative agent was identified as SEMBV2,14,15.

Although the clinical signs and histopathology of WSSV have been studied and virus detected using PCR technique by different researchers I6

-2o, there still

exists conflicting reports of variable virulence, pathology, morphology of viruses involved in white

d . I d ' 21-23 d I h 12.1624 spot syn rome ill n Ja an e sew ere '. Information on detailed comparative analysis of WSSV/SEMBV isolates prevalent in India and those in other countries is lacking. Keeping this in view, the

MISHRA & SHEKHAR : WHITE SPOT SYNDROME VIRUS OF TIGER SHRIMPS 655

present investigation was carried out to study the pathogenecity and PCR detection of SEMBVIWSSV isolates collected from different shrimp farms located along the East-Coast of India, using different primers as used for detection of exotic isolatesI6.20.24.

Materials and Methods Collection of shrimp samples - Shrimps suffering

from WSD were collected from 12 different shrimp culture farms located along East-Coast of India, viz. in Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa). A total 200 samples were screened and a representative random sample of 20 infected shrimps, was kept for microbiological analysis and PCR screening. At farm site, gills, hepatopancreas, muscle and gut samples from dying and moribund shrimp were kept in Davidson's fixative25 for histopathological analysis. For electron microscopy, gills and eyes talk samples (3-4 mm size) were fixed in cold 3% glutaraldehyde (Electron microscopy grade, Sigma, USA) in O.lM phosphate buffer (PB, sodium dihydrogen phosphate, 3.2g; di­sodium hydrogen phosphate, Na2HP04.12H20, 27.6g; distilled water up to 1000 ml), kept in small glass vials, brought to the laboratory and stored at 4°C for further analysis. Besides, some moribund shrimp specimen were packed, transported to the laboratory in iced condition for microbiological analysis and some samples were stored at -80°C, for virological studies. Healthy tiger shrimp Penaeus monodon for use in infectivity trials were obtained from a commercial farm located near Mutukadu, Mahabalipuram Road, Chennai. The animals were acclimated for one week in 500 I fiberglass tanks containing ".erated seawater (25 ppm salinity) at ambient temperature and fed with CIBA shrimp feed 26.

Analysis of samples - Tissue samples kept in Davidson's fixative for 48-72hr were transferred to 70% ethanol and processed for histopathological analysis25. Microtome sections (4-5 ttm thick) were prepared, stained with hematoxylin and eosin and observed under microscope. For microbiological analysis, tissue samples of hepatopancreas, gills and hemolymph from moribund shrimp were directly streaked onto Zobell marine agar 2216 (ZMA), Aeromonas isolation agar (AlA) and TCBS agar (HiMedia, Mumbai). A total 20 samples from each location were processed for microbiological analysis. After incubation at 35°C for 72 hr, the pure cultures of bacteria were obtained on ZMA, AlA and TCBS. The

pure cultures were subjected to biochemical characterization and bacteria were identified using the standard microbiological methods27.29. The bacterial isolates were tested for their pathogenecity by experimental infection in shrimp. Briefly, bacteria were grown overnight in Zobell marine broth (ZMB) at 35°C until the middle exponential phase and the growth was determined by measuring optical density at 550 nm (OD550 = 0.5). Different to-fold dilutions (10.3

- 10.5) of bacteria were prepared in normal saline (0.85% NaCI in distilled water). Apparently healthy Penaeus mOllodon (5g size) were inoculated (0.05 ml) at 3rd abdominal segment and the control group animals were inoculated with similar dose of normal saline. The animals were maintained in aerated sea water in glass fibre tanks and observed daily for mortality and development of clinical symptoms; up to 14 days.

Infectivity trials - WSD infected shrimp samples collected from different shrimp farms and preserved in deep freeze . (-80°C) were used for disease induction in healthy P. monodon. The cephalothorax of shrimps were cut and homogenized in PBS (NaCI, 8.0g; KCI, 0.20g; Na2HP04. 2H20, 1.44g; KH2P04,

0.20g, distilled water up to lOOOml, pH 7.2) to prepare 10% (w/v) suspension. After centrifugation at 2000g for 10 min at 4°C the supernatants were collected and again centrifuged at 8000 g for 10 min at 4°C to clear most tissue debris. The supernatants were collected filtered through membrane filters (0.45 tt pore size, Milipore) and stored at -20°C for infectivity trials. Infectivity studied were carried out both by water borne exposure and injection method. Groups of healthy P. monodon (3-5g size) were immersed in glass fibre tanks containing 100 I sea water mixed with 1ml of diluted (10.2 and 10-4) virus extract prepared in PBS. After 1 hr, the tanks were filled up to 500 I of filtered sea water. In second set of experiment, healthy P. monodom (lO-12g size) were injected, im at 3rd abdominal segment at upper side with 0.05 ml of diluted (10.2 and 10-4) virus extracts of shrimp and kept in 500 I glass fibre tanks. The control groups were kept in filtered seawater or injected with 0.05ml of PBS, pH 7.2 and maintained in glass fibre tanks. The animals were fed with CIBA shrimp feed26

and monitored daily for mortality up to 14 days. The. dead or affected shrimp during this period were collected . and preserved for pathological and virological studies . For bioassay test, cephalothorax portion of dying or moribund shrimp from infectivity

656 INDIAN J EXP BIOL, JULY 2005

trials was taken and homogenized in PBS to prepare 10% suspension . From this, virus suspension was prepared and inoculated to groups of healthy shrimps for infectivity trials as described above and observed up to 14 days for mortality or clinical symptoms.

Extraction of DNA - Viral DNA was extracted20

from the eye stalk tissue. Briefly, tissue homogenates were mixed with TE buffer (l OmM Tris, I mM EDT A, pH 8.0) containing 0.5% (w/v) sodium dodecyl sulphate (SDS), 100mM NaCI and 1 mg/ml Proteinase K (Bangalore Genei Pvt. Ltd, Bangalore) and incubated in water bath at 55°C for 30 min. After phenol-chloroform (1:1) extraction, the DNA was precipitated in 2 volume of chilled absolute ethanol and solubilized in distilled water. Genomic DNA extracted from infected shrimp samples obtained from different farms was used as template in PCR.

Polymerase chain reaction - Three sets of primers previously used for detection of viruses involved in White spot syndrome in different countries 16.20,24, were selected for screening the samples (Table 1). The primers were synthesized commercially (Bangalore Genei Pvt. Ltd, Bangalore) and 20 shrimp samples from each location were screened using PCR. The reaction conditions were 100 ng of extracted DNA, 1.0 p,M of each primer, 200 p,M deoxynucleotide triphosphate, 1 U of Taq DNA polymerase in lOx buffer supplied with the commercially available PCR kit (Bangalore Genei Pvt. Ltd, Bangalore). The total volume was made up to 50 p,l with sterile distilled water. The mixtures were incubated at 95°C for 5 min followed by 30 cycles of amplification in an automated thermal cycler (Perkin­Elmer 2400, USA) programmed for 0.5 min at 95°C, 1

min at 58°C, and 1 min at noc. Final extension for 5min at n oc was given after which the 20p,1 PCR products were mixed with 5p,1 of DNA sample buffer (Bangalore Genei Pvt Ltd, Bangalore) and electrophoresed in 1 % agarose gel. The gels were stained with ethidium bromide and viewed under UV­transilluminator (UVP-INC, USA).

Transmission Electron microscopy - Organ samples of infected shrimp kept in 3% glutaraldehyde were given several rinses with PB. Small tissue sections 0-3mm2) were selected and post fixed in 1 % osmium tetroxide (OS04, Johnson Metty Chemicals, London) prepared in PB (2% OS04 in distilled water mixed with equal volume of 0.2M PB) for 1 hr. The tissues were dehydrated in a series of ethanol (50,70,95,100%) with 15 min washing in each, followed by 15 min washing in two changes of propylene oxide (PO). In the mean time Epon-Araldite embedding medium was prepared (Epon resin 812, 25ml; Araldite resin 502, 20ml; Dodecenyl succinic anhydride, DDSA, 60 ml ; 2,4,6-Tri-Dimethyl aminomethyl phenol, DMP, 3rnl, Sigma). The tissues were then treated with PO: Epon-Araldite (2:1) mix for 1 hr, followed by PO:Epon-Araldite 0:2) mix for overnight. Blocks in Epon-Araldite embedding medium was prepared in beam capsules by heating at 60°C for 24-48 hr in a hot air oven. Ultrathin sections were cut using Ultramicrotome (LKB Broma,8800) and glass block knife. The sections were stained with 2% uranyl acetate (BDH, England) and 0.5% lead citrate (Sigma). Presence of viral bodies were viewed under transmission electron microscope (Jeol Gem lOOx, Japan). Result and Discussion

Aquaculture has grown significantly in most Asian countries including China, Taiwan, Thailand,

Table 1 - Different primer sets selected for use in the present investigation for screening of shrimp samples using PCR

SI.No Sequence of primers Size (bp) Size of Reference Remarks

2

3

Forward FI: 5'-GAC AGA GAT ATG CAC GCC AA -3'

20

Reverse R I :5' - ACC AGT GTT TCG 20 TCA TGG AG-3

Forward: 146FI-5'- ACT ACT AAC TTC AGC CTA TCT AG -3 '

Reverse: 146RI-5'- TAA TGC GGG TGT AAT GTT CTT ACG -3'

23

24

Forward: 102FI 5'- TCA CATCGA 20 GAC CTC TGT AC -3'

Reverse : 102RI 5'- TCT AGG ACG 20 GAC TAT GGC AA -3'

amplified product (bp)

643

1447

520

Takahashi et Used for PCR detection of R V -PJ al. 20 and SEMBV in P.1Il0nOdOIl in Japan

Lo et al. 16

Kasorncha­ndra el al. 24

Used for detection of WSBV in P.mollodoll in Taiwan

Reactive with WSSV infected shrimp samples from six different Asian countries

MISHRA & SHEKHAR : WHITE SPOT SYNDROME VIRUS OF TIGER SHRIMPS 657

Philippines, Japan, Indonesia and Asia now stands in the forefront both in terms of volume and value, having China, India and Japan accounting for over 65% of world production3o. Although shrimp aquaculture has been an age old practice in India, modern methods of improved culture practices has only taken place since late 1980s and aquaculture has grown significantly in the maritime states of India, particularly in Andhra Pradesh (AP), Tamil Nadu (TN), Orissa and West Bengal (WB), which has shown tremendous potential of growth. In particular, one of the most significant constraints for aquaculture development in India is realised to be the threat of viral disease in shrimp farming, ·3.'5.21. Considering the extent of shrimp farming in TN, AP and Orissa, the three regions representing the epicenter of shrimp farming, viz. Chidambram, Nellore and Balasore, respectively, were selected in the present investigation. Shrimp showing symptoms of WSO in different shrimp farms, were collected and out of 200 shrimp samples screened, a total 60 representative samples were analyzed using microbiological and PCR techniques. The clinical signs of affected shrimp observed were reddish coloration of body and appendages, presence of white spots which were prominent on carapace over head and body cuticle, as reported by others as characteristic feature of WS05.9,2IJI. Microbiological analysis revealed presence of Vibrio spp, in hepatopancreas, gills, hemolymph and in most organ samples and Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas spp. were isolated. The experimental

Fig.l- Histopathological analysis of gills tissues from WSD infected shrimp from Nellore (AP), showing characteristic intranuclear eosino-basophilic bodies (arrow)

infection trials in p, monodon using these isolates did not induce any acute mortality or formation 'of white spots on carapace or cuticle, indicating these bacterial isolates to be secondary invaders, Vibrio spp, have been isolated from WSSV infected shrimp and

d b d ' 831 reporte to e secon ary 111 nature ' , Histopathological analysis demonstrated

degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ, Cellular degeneration, with hypertrophyoid nuclei, marginated chromatin were evident with presence of intranuclear basophilic (Fig, 1) or eosino-basophilic bodies in tubular cells and inter cellular spaces (Fig.2). In the infectivity trials, P.monodon given oral mode of infection with WSSV, showed mortality as early as 24 hr post inoculation (hpi) in Nellore sample and after 2 days post inoculation (dpi) in other samples (Table 2). Infected animals became lethargic with reduced feeding and preening activities. As the infection progressed beyond 2 dpi, most shrimp stopped feeding, surfaced frequently and exhibited a loss of balance. Cannibalistic behavior of some shrimp feeding on parts of infected shrimp were also noted. Cumulative mortality of 100% was noted by the end of 7dpi. Characteristic white spots on carapace, reddening of appendages and empty gut were other features noticed in the infected shrimp. However, the control groups remained normal without any mortality till the end of 14th day of experiment period. In groups injected with virus suspension, the

Fig. 2 - Showing cellular degeneration, tissue disorganization with hypertrophied nuclei , marginated chromatin and presence of intranuclear eosino-basophilic bodies (thin arrow) in tubular cells and inter-cellular spaces of hepatopancreas in infected shrimp samples collected from Nellore (AP),

658 INDIAN J EXP BIOL. JULY 2005

mortality rate was higher and some animals died even without showing any gross signs of disease. The rate of mortality with Nellore samples was much higher and all shrimps died by 2 dpi. whereas shrimps inoculated with Balasore and Chidambram samples died within 4 dpi. Characteristic white spots on carapace and cuticle were not detected in most animals died within 2 dpi but minute spots were visible in few shrimps which died at 4dpi. The groups infected through water exhibited gradual mortality spreading over 7 days. by which time 100% mortality could be recorded in all groups except in the control group (Table 2). In dead and moribund shrimp, clinical signs of the disease with presence of white spots on carapace and cuticle were revealed. Presence of virus in shrimp samples in both groups could be detected using bioassay/PCR test, as described earlier.

PCR test has been shown to be useful for detection of WSSV in different shrimp species and screening samples for presence of virusI 7.18.32,33. In the present investigation, PCR test was used for screening

eyestalk samples from shrimps, for presence of WSSV using primers of three distinct sequences (Table 1) and the results indicated amplification of DNA (Figs.3A, Band 4). In our earlier laboratory

. I . h . h . . b PCR 191415 tna s wIt vanous s nmp tissues y .... , amplified DNA could be produced with ti ssue samples such as pleopods, lymphoid organ, muscle, cuticular epidermis and eye stalk, but no amplification could be achieved when whole eye tissues containing eye lens was used, indicating the presence of inhibitory substances in the compound eyes. Hence eyestalk devoid of eye lens was used in the present study for PCR amplification. In the present trials, a total number of 60 samples were screened using PCR. Significantly, all the three primers could amplify the specific regions of WSSV/SEMBV in samples collected from all the three locations in India and amplified product of approximately 643bp (Fig. 3A), 1447bp (Fig. 3B) and 520 bp (Fig. 4) could be generated. However, one sample collected from Balasore, did not get amplified with 146F and 146R

Table 2 - Details of experimental infection trials in P.monodo/! with WSSV extracts from infected shrimp collected from different shrimp culture farms

Animals Dilutions Mode of Day wise mortality (%) **Tcsted for WSSV by used of inoculation

extracts*

0 2 3 4 5 6 7 8 9 10 Gross Bioassay PCR

P.JIlol1odon Chi IO~' Oral 0 0 20 40 50 80 100 + ND ND 3-5g size

Chi 1O~4 0 0 10 30 50 70 80 100 + ND ND

Nel IO~' 0 20 50 70 100 + ND +

NellO"' 0 0 30 40 70 100 + ND ND

Bal 1O~1 0 0 40 50 80 90 100 ~ + + + Bal IO~· 0 0 20 30 50 50 80 100 + ND ND

Control 0 0 0 0 0 0 0 0 0 0 0 ND

P~mollodoll Chi 1O~2 im 0 40 80 100 + + 1O-12g size

Chi 10-4 0 20 50 80 100 + ND ND

Nel 1O~2 0 60 100 + ND

NellO-4 40 80 100 + +

BallO ' 0 20 50 100 + ND + BallO-4 0 10 40 60 100 + ND ND

Control 0 0 0 0 0 0 0 0 0 0 0 ND

* Chi = Samples from Chidambram (TN). Nel = Samples from Nellore (AP). Bal = Samples from Balasore (Orissa) ** P.monodon from experimental trials were tested for presence of WSSV in tissues Gross = Presence of characteristics white spots on carapace and cuticle Bioassay = Re-infection trials in healthy P.11lonodon. PCR = Tested for presence of WSSV using PCR + Posilive for WSSV. - Negative for WSSV. ND = Not done

MISHRA & SHEKHAR : WHITE SPOT SYNDROME VIRUS OF TIGER SHRIMPS 659

primers but specific amplified products could be obtained with other two sets of primers (Fig.3A). This indicated genomic closeness of SEMBVIWSSV prevalent in India with RV-PJ, SEMBV prevalent in Japan , WSBV in Taiwan and WSSV in Thailand, China and Japan.

TEM examination of eyestalk samples revealed numerous rod shaped, non-occluded, enveloped

. virions accumulat~d in hypertrophied nuclei and some in the cytoplasm (Figs.5 and 6). There was much variation in the size of virions and the mean size of the virion was in the range of 110 x 320 ± 20 nm. Some empty capsids and circular envelopes were also observed in the hypertrophied nuclei (Figs 5B and 6A). Similar TEM observations of WSSV have also been made7

,9.J6 and the observation of immature viruses with empty capsids, capsid originators, nucleocapsids and circular envelops suggests that virogenesis was taking place in the nucleus4.' 8. The observation of few virus particles in the cytoplasm indicated these to be released from infected nuclei after degeneration and rupture. Occurrence of small sized virions in samples collected from WSD outbreaks in Japan8,24 with the size of complete virion 83x275 nm and the size of nucleocapsid 54x216 nm, was reported8

, whereas comparatively larger viruses with size of 298 ± 21 x 107 ± 8 nm in giant tiger shrimp, P. monodon and 248 ± 12 x 104 ± 8 nm in

LANE

21226 4268

1904

2 3 4 567

Kuruma shrimp, P. japonicus, was reported in Taiwan4, indicating size variability in different species. Presence of paracrystalline arrays of the virions in the nuclei of infected cells was reported by others 18,23. Presence of tube like projections or appendages at one or both ends of the virus have been reported4

,22 , but similar features were not observed in the present investigation as also reported by others 16.20,24 .

It has been observed that WSD virus is morphologically similar to SEMBV and RV-PJ isolated in Thailand and Japan4, respectively. Lo et al. '6 reported WSBV to be closely related to HHNBV,

LANE 1 2 3 4 5 6 7 8 9 : 0 1 I 12

51 7 344 221

163 1

721

Fig.4 - Screening of shrimp samples using 102 FI and 102 RI primers in PCR. See amplification of approximately 520bp DNA band (arrow) with all shrimp samples. Lane I and 12 indicate position of pBR 322 DNA-Hinfl digest DNA marker; Lane 2, 3. 4 = shrimp samples from Chidambram (TN); Lane 5,6.7 = shrimp samples from Nellore (AP); Lane 8,9,10 = shrimp samples from Balasore (Orissa); Lane 11= known positive control sample

B 9 10 11 12 13 .W(bp)

21226 4188 1904

947

LANE

1631

6 7 8 9 10 11 12 13

1631

517

396

Fig.3 - Screening of shrimp samples collected from different shrimp farms using PCR. DNA band of 1447 bp (arrow) was amplified (Fig.3A) in all samples except one (Lanell) using 146FI and 146Rl primers. Lane 1 and 13 indicate position of DNA molecular weight marker (Lamda DNA EcoRJ-HindIlI double digest). DNA band of 643 bp '(arrow) was visualized with all samples when FI and R I primers were used in PCR (Fig.3B). Lane 1 and 13 indicate position of pBR 322 DNA-Hinfl digest DNA marker. In both A and B, Lane 3, 4,5 = shrimp samples from Chidambram (TN); Lane 6,7,8 = shrimp samples from Nellore (AP); Lane 9,10,11 = shrimp samples from Balasore (Orissa); Lane 12 = known positive control DNA and Lane2 = known negative control DNA •

660 INDIAN J EXP BIOL, JULY 2005

Fig.5- Transmission electron micrograph of eyestalk tissue from WSD infected P.lI1onodon, from Chidambram (TN) showing WSSV virus at different stages of development. A- Show presence of viral bodies (arrow) in the hypertrophied nucleus (N) with degenerated nuclear membrane (Nm). (Cy = cytoplasm, Bar = 100 nm). Some complete virions, immature viral particles and empty capsids are seen in the nucleus (Bar = 100 nm). Enlarged view of viral particles (C}-- Bar = 20 nm and (DHBar = 50 nm)

. Fig.o -Transmission electron micrograph of eyestalk tissue from infected P.mollodoll. collected from Balasore, (Orissa) showing WSSV in various stages of development (Fig.6A) with mature virions, immature particles (double head arrow and deep arrow head) and empty capsids (thin arrow) in the nucleus (Bar = 20 11111). Enlarged view of viral particles of different sizes have been shown in Fig.6 (B - G).

responsible for shrimp explosive epidermis disease in China and SEMBV in P. monodon in Thailand. Using pathological and PCR amplification, it was also observed that viral isolates collected from six Asian countries were similar in nature24

. Based on the symptoms, clinical signs, pathology, viral morphology and PCR amplification using three sets of primers and correlating the previous work1.l 6

.18

.2 1.22,24,35 it can be concluded that the viral

isolates collected from East-Coast of India in the present investigation, are probably the same type of virus. However, further studies on their polypeptide pattern, antigenicity and genomic DNA restriction profile would provide further information in this regard.

Acknowledgement The authors are thankful to the Dean, Madras

Vetelinary College, TAN U V AS, Chennai , and Prof. P. Ramdass, Head, Animal Biotechnology Department, for providing necessary facilities in PCR analysis and to Dr. Andrew Chandra Mohan, for assistance in electron microscopical analysis of samples.

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MlSHRA & SHEKHAR : WHITE SPOT SYNDROME VIRUS OF TIGER SHRIMPS 661

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