Guang-Hsiung Kou

Experimental infection of white spot baculovirus in some cultured and wild decapods in Taiwan

Techniques for the detection of white spot baculovirus virus (WSBV) by polymerase chain reaction are well established. In this study, two primer sets designed from an isolate of WSBV from Penaeus monodon, PmNOB III, were used to detect WSBV infection in cultured and wild decapods in Taiwan. WSBV positive cases were found in all of four major marine cultured shrimp, P. monodon, P. japonicus, P. penicillatus and Metapenaeus ensis. Wild P. semisulcatus was also found to be naturally infected by WSBV. On the other hand, no cases of naturally occurring WSBV infection have yet been found in the wild shrimp Exopalaemen orientalis (from a milkfish culture farm), Trachypenaeus curvirostris, M. ensis (from the coast of Taiwan), Macrobrachium sp. and Procambarus clarkii (from rivers in Taiwan). Furthermore, neither the wild crabs, Calappa lophos, Portunus sanguinolentus, Charybdis granulata and C. feriata, nor the wild lobsters Panulirus ornatus, P. versicolor, P. longipes and P. penicillatus, collected from the coast of Taiwan showed any evidence of being naturally infected by WSBV. When captured specimens of these decapods were artificially infected by feeding them with tissues from severely PmNOB III infected P. monodon, wild shrimp mortality reached moderate to high levels at 18 days post infection. Using PCR analysis, WSBV DNA could be detected in the moribund specimens during the experimental period and in the survivors on the final day of the experiment. The mortalities in wild crabs and lobsters, however, were not significantly different from control groups. Nevertheless, WSBV DNA was also detectable in these specimens. WSBV was thus shown to have a wide host range and to exhibit different infectivity in the various decapods investigated in the present study.

Genomic and Proteomic Analysis of Thirty-Nine Structural Proteins of Shrimp White Spot Syndrome Virus

ABSTRACT 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 sulfate-polyacrylamide 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 Unique Stacked Rings in the Nucleocapsid of the White Spot Syndrome Virus Virion Are Formed by the Major Structural Protein VP664, the Largest Viral Structural Protein Ever Found

ABSTRACT One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.

A 3D Model of the Membrane Protein Complex Formed by the White Spot Syndrome Virus Structural Proteins

Background Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus), is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process. Principal Findings In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument) proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers. Conclusions From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented.

Major Viral Diseases of Penaeus Monodon in Taiwan

Preliminary results are reported on an automatic manipulation system for the masspropagation and chromosome manipulation of the small abalone,Haliotis diversicolor.The system was designed based on the optimum conditions obtained from tests simulatingthe possible conditions during automatic manipulation.Small abalone eggs couldwithstand seawater and air exposure for up to 210 min and 80 min,respectively,beforefertilization.Fertilized eggs could accumulate up to 0.8 cm thick for 60 min withoutdamage.Transfer of the fertilized eggs through outlet pipes up to 4 m in length did notresult in visible damage or decline in hatching rate.Fertilized eggs were not resistant tocontinuous rotation at 150,200,or 250 rpm for 20 min.The rotating drum speed of theautomatic manipulation system within 40 rpm and the pump flow at 30±3 liter/min wererecommended.Effect of aeration and stirring of the rotating drum on the fertilized eggswithin the concentration tank improved hatching rates.The final design of the automaticmanipulation system incorporated these features.The automatic control box of thesystem ensures that specific treatments,such as those for chromosome manipulation,canbe precisely carried out.As a whole,the system has been found highly applicable forregular mass propagation and chromosome manipulation procedures of the small abaloneand is the first one of its kind.

Sequencing and Amplified Restriction Fragment Length Polymorphism Analysis of Ribonucleotide Reductase Large Subunit Gene of the White Spot Syndrome Virus in Blue Crab (Callinectes sapidus) from American Coastal Waters

Abstract: In the present study, the existence of white spot syndrome virus (WSSV) in blue crab (Callinectes sapidus) collected from 3 different American coastal waters (New York, New Jersey, and Texas) was confirmed by 2-step diagnostic polymerase chain reaction and in situ hybridization analysis. When geographic isolates were also compared using a gene that encodes the WSSV ribonucleotide reductase large subunit RR1 (WSSV rr1), a C1661-to-T point mutation was found in the New Jersey WSSV isolated. This point mutation, which resulted in the creation of an additional RsaI endonuclease recognition site, was not found in the WSSV from the New York and Texas blue crab samples, or in the WSSV Taiwan isolate, or in any of the other WSSV geographical isolates for which data are available. WSSV rr1-specific RsaI amplified restriction fragment length polymorphism of an amplified 1156-bp fragment thus distinguished the New Jersey blue crab samples from the other WSSV isolates.

Molecular mechanism of the interactions between white spot syndrome virus anti-apoptosis protein AAP-1 (WSSV449) and shrimp effector caspase.

AAP-1 (WSSV449), an anti-apoptosis protein encoded by white spot syndrome virus (WSSV), blocked apoptosis in insect cells (SF9) induced by Penaeus monodon effector caspase (Pm caspase). Here, to characterize in detail the anti-Pm caspase activity of AAP-1, both proteins were expressed and purified from Escherichia coli and their interactions were assayed in vitro. We found that although AAP-1 could inhibit Pm caspase activity, the inhibition was not as efficient as that of baculovirus anti-apoptosis protein P35. We further confirmed the binding and cleavage of AAP-1 by Pm caspase, and detected three AAP-1 cleavage products. Mutational analysis and protein N-terminal sequencing revealed that whereas both Asp233 and Asp272 residues of AAP-1 are involved in binding and cleavage by Pm caspase, only the Asp272 is involved in Pm caspase inhibition. Asp233, on the other hand, negatively regulates AAP-1s anti-Pm caspase activity. Lastly, AAP-1 homotypically interacts with each other both in vitro and in insect cells.

Identification and characterization of a hyperglycemic hormone from freshwater giant prawn, Macrobrachium rosenbergii.

Crustacean hyperglycemic hormone (CHH), a physiologically important neurohormone stored in the sinus gland of eyestalks, primarily regulates carbohydrate metabolism and also plays significant roles in reproduction, molting and other physiological processes. In the freshwater giant prawn, Macrobrachium rosenbergii, an injection of X-organ sinus gland (XOSG) extract evoked a hyperglycemic response, peaked in 1 h. The hyperglycemic effect of the eyestalk extract was maximal at the dose of 0.5 eyestalk equivalent. CHH fractionated by RP-HPLC, in M. rosenbergii was identified by its hyperglycemic activity and partial amino acid sequence, and the molecular weight of 8534 was determined by matrix-assisted laser desorption ionization mass spectrometry--time of flight analysis (MALDI-TOF). The amino acid sequence of the first 25 residues of CHH showed 72% homology with the first 25 residues of CHH A and CHH B of the American lobster Homarus americanus.

Penaeus monodon TATA Box-Binding Protein Interacts with the White Spot Syndrome Virus Transactivator IE1 and Promotes Its Transcriptional Activity

ABSTRACT We show here that the white spot syndrome virus (WSSV) immediate-early protein IE1 interacts with the Penaeus monodon TATA box-binding protein (PmTBP) and that this protein-protein interaction occurs in the absence of any other viral or cellular proteins or nucleic acids, both in vitro and in vivo. Mapping studies using enhanced green fluorescent protein (EGFP) fusion proteins containing truncations of IE1 and PmTBP delimited the interacting regions to amino acids (aa) 81 to 180 in IE1 and, except for aa 171 to 230, to aa 111 to 300 in PmTBP. A WSSV IE1 transactivation assay showed that large quantities (>800 ng) of the GAL4-IE1 plasmid caused “squelching” of the GAL4-IE1 activity and that this squelching effect was alleviated by the overexpression of PmTBP. Gene silencing of WSSV ie1 and PmTBP by pretreatment with double-stranded RNAs (dsRNAs) prior to WSSV challenge showed that the expression of these two target genes was specifically inhibited by their corresponding dsRNAs 72 and 96 h after dsRNA treatment. dsRNA silencing of ie1 and PmTBP expression also significantly reduced WSSV replication and the expression of the viral early gene dnapol (DNA polymerase gene). These results suggest that WSSV IE1 and PmTBP work cooperatively with each other during transcription initiation and, furthermore, that PmTBP is an important target for WSSV IE1s transactivation activity that can enhance viral gene expression and help in virus replication.

Hepatopancreas and ovary are sites of vitellogenin synthesis as determined from partial cDNA encoding of vitellogenin in the marine shrimp, Penaeus vannamei

Summary The site of yolk protein synthesis in crustaceans has long been a subject of controversy. A portion of the vitellogenin gene structure was reported recently in a freshwater giant prawn (Macrobrachium rosenbergii) and black tiger shrimp (Penaeus monodori), in which the hepatopancreas was confirmed to be the extraovarian site of vitellogenin synthesis. The ovary is also frequently reported to be the site of yolk protein synthesis in penaeid shrimp. The same PCR product was obtained using cDNA from the hepatopancreas or the ovary as a template. The deduced amino acid sequence of Vg in P. vannamei showed high identities of 57% and 78% with those from M. rosenbergii and P. monodon, respectively. The same location of the intron in the sequenced region of genomic DNA was also found between these three species. We therefore concluded that the hepatopancreas and ovary are sites of vitellogenin synthesis in P. vannamei. The partial structure of the vitellogenin gene is further presented.