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
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ABSTRACT. White spot syndrome (WSS) has been found in many species of shrimp and crabs, not just In Asia but globally. The causative agent is known as white spot syndrome virus (WSSV). In order to clarify the relatedness of WSSV from various geographic regions, we compared the viral DNA of a number of clinical samples of WSSV. (1) China96-116A from Penaeus chlnens~s, (2) India95-314 from Penaeus monodon, (3) grocery store95-204 and grocery store96-l15 from P. rnonodon possibly originating from Thailand, (4) crayfish97-25 from Orconectespunctimanuscollected from the U.S. National Zoo, (5) Thailand95-46 from experimentally infected Penaeus vannamei. (6) South Carolina97-64 from P. vannamei, and (7) Texas95-242 and Texas96-7 from P. vannarnei. These specimens were first examined by dot hybridization analysis with nucleic acid probes derived from a WSSV Taiwan isolate.Although the intensity of the hybridization signals varied, and although some specimens of India95-314, crayfish97-25, Texas95-242 and Texas96-7 failed to give a detectable hybriduation signal with certain probes, the broad consistency of dot hybridization data suggests that these WSSV clinical samples from different geographical locations are closely related. Following this analysis, all the specimens were examined using 10 virus-specific polymerase chain reactions (PCR). The amplification products were subsequently digested with Cfo I, Hae 111, Hpa I1 and Rsa I restriction endonucleases to determine if there were any DNA fragment polymorphisms in the WSSV clinical samples. The results highlighted the genetic relatedness of all the WSSV clinical samples with the possible exception of a series of Texas viral samples which could be distinguished from the other geographic samples in some of the PCR-based tests.
The white spot syndrome virus DNA polymerase (DNA pol) gene (WSSV dnapol) has already been tentatively identified based on the presence of highly conserved motifs, but it shows low overall homology with other DNA pols and is also much larger (2351 amino acid residues vs 913-1244 aa). In the present study we perform a transcriptional analysis of the WSSV dnapol gene using the total RNA isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with a WSSV dnapol-specific riboprobe found a major transcript of 7.5 kb. 5'-RACE revealed that the major transcription start point is located 27 nucleotides downstream of the TATA box, at the nucleotide residue A within a CAGT motif, one of the initiator (Inr) motifs of arthropods. In a temporal expression analysis using differential RT-PCR, WSSV dnapol transcripts were detected at low levels at 2-4 h.p.i., increased at 6 h.p.i., and remained fairly constant thereafter. This is similar to the previously reported transcription patterns for genes encoding the key enzyme of nucleotide metabolism, ribonucleotide reductase. Phylogenetic analysis showed that the DNA pols from three different WSSV isolates form an extremely tight cluster. In addition, similar to an earlier phylogenetic analysis of WSSV protein kinase, the phylogenetic tree of viral DNA pols further supports the suggestion that WSSV is a distinct virus (likely at the family level) that does not belong to any of the virus families that are currently recognized.
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