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Current Trends in Biotechnology and PharmacyVol. 5 (1) 1011-1020 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)Protein Expression and Microscopic Evidence of White SpotSyndrome Virus (WSSV) in Tiger prawns Penaeus monodonBhatt, S.S. a* , Chavda, D. V. a and Sreepada, R. A. baB.R. Doshi School of Biosciences, Zoology Laboratory, Bakrol Vadtal Road,Satellite Campus, Sardar Patel University, Vallabh Vidhyanagar 388120, IndiabNational Institute of Oceanography, Dona Paula, Goa 403 004, India*For Correspondence - bhatt.sujata@gmail.com1011AbstractShrimp culture is being practiced in largeareas of coastal farms of South Gujarat region inWestern India. The disease caused due to whitespot syndrome virus (WSSV) has been detectedin the cultured Penaeus monodon in this region.White spots were clearly visible in the carapaceof infected shrimps. The scanning electronmicrographs of the upper and lower surfaces ofcarapace revealed the presence of white spotsmeasuring about 78µm to 135µm. In the infectedshrimps, rod shaped viral particles have also beenidentified from the epidermis associated withcarapace by TEM using negative staining. Thehisto-pathological analysis of gills,hepatopancreas, gut and muscles of P. monodoninfected with WSSV indicated hypertrophiednuclei, presence of intranuclear inclusion bodiesand chromatin margination in gill epithelial cellsand necrosis of gill lamellae. In the infectedmuscles, necrosis, haemocytic infiltration anddegeneration of muscle fibers were clearlyevident. Damage to the mucosal epithelial cellsof gut was accompanied by haemocyticinflammatory response. Inflammatory responseand necrotic changes in hepatopancreas ofinfected shrimps have been observed.Hypertrophied and piknotic nuclei as well asinfiltration of viral particles into connective tissuesassociated with vacuolization were observed inall the analyzed infected tissues. Changes in theexpression of proteins have been detected bySDS-PAGE in the infected muscles andhepatopancreas. Four new proteins of 79kDa,58kDa, 36kDa and 17kDa were found to bedifferentially expressed in muscles. Inhepatopancreas, three proteins viz. 46kDa, 20kDaand 17kDa were differentially expressed ininfected shrimps; whereas 62kDa protein, whichwas expressed in healthy shrimps, was notexpressed in infected shrimps. No change in theprotein expression pattern was detected in gills.The current study has shown that WSSV, besidescausing damage to the tissues, has also drasticallyaltered the protein expression patterns of theinfected tissues of P. monodon.Key words: WSSV, protein expression, Penaeusmonodon, SDS-PAGEIntroductionIntensive shrimp aquaculture is one ofthe most important aquaculture practices in theworld. Intensive culture practice has also resultedin the development of diseases including viraldiseases in shrimp culture units (1). White spotsyndrome virus (WSSV) is one of the highlypathogenic shrimp viruses affecting the shrimpculture worldwide with severe economic losses;and its characterization is critical for developingeffective control methods (2,3). Though, prawnsinfected with WSSV or YHV have been reportedto be safe for human consumption (4); theProtein expression in WSSV
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1011-1020 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1012pathogenic effects of WSSV infected shrimps inhuman cannot be completely ruled out. The viruswas first reported in India during 1994 (5) and ithas been isolated and characterized from Indianshrimp, Penaeus indicus and Penaeusmonodon (6,7). The WSSV has also been foundto be pathogenic to other aquatic hosts like crabs,copepods and also to fresh water prawns (6,7).White spot syndrome virus is characterizedby the development of white spots in exoskeleton(8,9) and is reported to infect mainly tissues ofmesodermal and ectodermal in origin (10). Variousdiagnostic methods like histology, immunologicalmethods, polymerase chain reaction, in-situhybridization and several other methods are beingused for the identification of WSSV infection(1,11-13). The WSSV infection to shrimp is alsoknown to show changes in the expression ofproteins in several tissues (14).Shrimp culture is being extensivelypracticed in the coastal farms of South Gujarat(India). During the monitoring of hygienicconditions of these aquaculture units, WSSV hasbeen observed in cultured P. Monodon in growoutponds by morphological observations. The presentstudy was undertaken to determine the effect ofWSSV on the cultured P. monodon andidentification of pathogen. During theinvestigations, the morphology and chemicalcomposition of white spots of inside and outsidesurfaces of carapace were examined by scanningelectron microscopy (SEM) along with energydispersiveX-ray analysis (EDX). The WSSVvirus has been identified in the cuticular epitheliumby transmission electron microscopy (TEM) withnegative staining. Damage caused by viruses todifferent tissues have been analysed byhistopathological techniques. Along withhistopathological studies, changes in theexpression of proteins in several tissues of WSSVinfected shrimps have also been studied.Materials and Methods1. Sample collection and tissue processing :Penaeus monodon with white spots on carapaceand other body parts as well as without whitespots (healthy shrimps) were collected from thecostal farms of South Gujarat (Olpad, Suratdistrict, 21° 19' 60N, 72° 45' 0E) region of WesternIndia during June-2008 and brought to thelaboratory at 4 ºC. Carapace from the shrimpswith and without infection were collected andstored at -20ºC for morphological analysis of whitespot and for the study of change in the chemicalcomposition of cuticle of white spot region.Hepatopancreas and muscles from infected andhealthy shrimps were removed and stored at -20ºC to study the changes in the expression ofproteins during infection. Epidermis from thecarapace of infected shrimps is removed andstored at -20ºC for identification of pathogen.Hepatopancreas, muscles, gills and gut from bothinfected and healthy shrimps were fixed to studyhistopathological changes in these tissues duringinfection.2. Scanning electron microscopy and EDX :For the morphological analysis of white spots, thecarapaces of diseased shrimps were air dried andthe morphology and size of white spots wereexamined under SEM (Philips ESEM series). TheEDX quantification of calcium (Ca), carbon (C),oxygen (O) and phosphorus (P) from upper andlower surface of the carapace for both infected(from white spot region) and healthy shrimps hasbeen performed with SEM.3. Negative Staining : Epidermis from thecarapace region of infected Penaeus monodonwas removed and homogenized in sterile brackishwater at 4ºC (1:9 V/W), centrifuged at 8510×g at-4ºC for 5 min and the supernatant was filteredthrough a 0.45ì m membrane. The collectedfiltrate was again centrifuged at 14549×g at -4ºCfor 1.5 h and the resulting pellet was resuspendedBhatt et al
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