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.

Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.

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