ABSTRACT-White spot syndrome associated baculovlrus (WSBV) is the causative agent of a dlsease which has recently caused high shrimp mortahties and severe damage to shrimp cultures. In thls study, a strain of WSBV from black tiger shrimp Penaeus monodon was used to develop a diagnostlc tool for the detection of WSBV and related agent lnfect~ons in shnmp The vlnons were punfied from P monodon Infected with LVSBV V~ral genomlc DNA was extracted from purlfled vinons by treatlng the vlnons \ n t h proteinase K dnd cetyltnmethylammonium bromlde (CTAB) followed by phenol-chloroform extraction and ethanol precipitation A qualitative assessment Ivas performed using polymerase chain reaction (PCR) analys~s on the viral DNA and primers specif~c to shrimp genomic DNA in order to mon~t o r shrimp DNA contamination In the viral genomic DNA preparations A WSBV genomlc DNA llbrary was constructed and based upon the sequence of the cloned WSBV DNA fragment, we deslgned a LVSBV-specific prlmer set for PCR to detect WSBV Infection in penaeld shrimp Samples which contained WSBV DNA yielded a n evident ampl~f~catlon product showing the expected moblllty of a 1447-bp DNA fragment whereas n u c l e~c aclds extracted from tissue samples of clln~cally healthy shnmp showed no such DNA fragment, thereby confirming the speclficity of our pnmers By PCR with thls prlmer set, ~t was demonstrated that the causative agents of white spot syndrome in different shnmp specles are closely related An effective diagnostlc tool is thus provided for screening shnmp for \.VSBV infections, and may be important In preventing the further spread of this d~s e a s e KEY WORDS: WSBV . W h~t e s p o t . PmNOBIII . Detection . Penaeid shnmp baculovirus . PCR

In cultured shrimp, w h~t e spot syndrome 'baculovirus' (WSBV) Infection IS characterized by a wide range of target tissues, rapid disease onset and high mortality Dunng the viremic phase of infection, the virus is present in many organs However, the s~tuation in the natural environment remalns unclear To identify the pattern of the t~s s u e t r o p~s m of WSBV infection in adult Penaeus monodon (black tiger shnmp) of wild ongin, w e conducted a c o m b~n e d study using currently available nucleic acid diagnostic tools and conventional histological observat~ons uslng light (LM) and transnxssion electron (TEM) mlcroscopy to examine the sites for virus mult~plication S~x t e e n parts excised from shnmp specimens were examined pleopods, gills, stomach, abdominal muscle, hemolymph. m~d g u t , heart, pereiopods lymphoid organs, integument, nervous tissue, hepatopancreas, testes, ovaries, spermatophores, and eye stalks All these tissues/organs were found to support WSBV replic a t~o n For the f~r s t t~m e in s~t u hybridization and TEM showed evidence of WSBV in reproduct~ve organs of black tiger shrlmp In testes, WSBV-positive cells were located in the connective Ussue layer surrounding the seminiferous tubules and no germ cells were found to b e infected In the spermatophore only muscle and connective t~s s u e cells were WSBV posltive In the ovary, foll~cle cells oogonla oocytes and connective tissue cells were WSBV positlve However, the fact that w e were unable to find any Infected mature eggs suggested that infected e g g cells were killed by the virus before maturation

ABSTRACT. The causative viral agent was purified from diseased shrimp Penaeus monodon with white spot syndrome. Negatively stained preparations show that the virus is pleiomorphic. It is fusiform or rod-shaped. In negatively stained preparations, the virion measures 70 to 150 nm at its broadest point and is 250 to 380 nm long. In some virions, a tail-like projection extends from one end. The capsid is apparently composed of rings of subunits in a stacked series. The rings are aligned perpendicular to the longitudinal axis of the capsid. The genome of the virus is a double-stranded DNA molecule which produces at least 22 Hind 111 fragments. The full length of the DNA is estimated to be longer than 150 kbp. Based on the morphological characteristics and genomic structures of the virus, we confirm that white spot syndrome associated virus (MJSSV) is a member of genus NOB (Non-Occluded Baculovirus) of the subfamily Nudibaculovirinae of Baculoviridae, name the present isolate PmNOBIII, and propose the use of WSBV (Baculovirus associated with White Spot syndrome) to indicate PmNOBIII related agents.

White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands were then excised from the gel, and following trypsin digestion of the reduced and alkylated WSSV proteins in the bands, the peptide sequence of each fragment was determined by liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) using a quadrupole/time-of-flight mass spectrometer. Comparison of the resulting peptide sequence data against the nonredundant database at the National Center for Biotechnology Information identified 33 WSSV structural genes, 20 of which are reported here for the first time. Since there were six other known WSSV structural proteins that could not be identified from the SDS-PAGE bands, there must therefore be a total of at least 39 (33 ؉ 6) WSSV structural protein genes. Only 61.5% of the WSSV structural genes have a polyadenylation signal, and preliminary analysis by 3 rapid amplification of cDNA ends suggested that some structural protein genes produced mRNA without a poly(A) tail. Microarray analysis showed that gene expression started at 2, 6, 8, 12, 18, 24, and 36 hpi for 7, 1, 4, 12, 9, 5, and 1 of the genes, respectively. Based on similarities in their time course expression patterns, a clustering algorithm was used to group the WSSV structural genes into four clusters. Genes that putatively had common or similar roles in the viral infection cycle tended to appear in the same cluster.

The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.White spot syndrome virus (WSSV) is a widespread and disastrous viral pathogen of cultured shrimp that also attacks crabs and crayfish as well as many other crustaceans (3,10,16,22). The WSSV virion is an enveloped particle of approximately 275 by 120 nm with an olivaceous-to-bacilliform shape, and it has a nucleocapsid (300 by 70 nm) with periodic striations (22,25). The most obvious feature of WSSV is the presence of a thread-like extension at one end of the virion (2, 25), which gives this virus the family name Nimaviridae (13).A virion is a complex assembly of macromolecules exquisitely suited for the protection and delivery of viral genomes. Its structural proteins are particularly important, since these proteins are the first molecules to interact with the host, and they therefore play critical roles in cell targeting as well as in the triggering of host defenses. Recently, thanks to the introduction of proteomic methods, the total number of known WSSV structural proteins increased to 39 (5, 17).Immunogold electron microscopy (IEM) has been used to identify VP28, VP26, VP31, VP51C, VP36B, VP41A, VP12B, and VP180 as envelope proteins (4,5,8,9,(28)(29)(30) and VP664 as a nucleocapsid protein (7). Other studies (1,6,(18)(19)(20) that combined detergent treatment and Western blotting confirmed and expanded most of these results (VP28, VP19, and VP73 as envelope proteins; VP24, VP15, and VP35 as nucleocapsid proteins) but also identified VP26 as a nucleocapsid protein.Here for the first time we embark on a systematic study of the structural proteins of WSSV that not only allows us to resolve the question of VP26's location but also reveals the existence of a previously unreported component of the WSSV virion. This co...