Chu Fang Lo

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.

PmRab7 Is a VP28-Binding Protein Involved in White Spot Syndrome Virus Infection in Shrimp

ABSTRACT Our aim was to isolate and characterize white spot syndrome virus (WSSV)-binding proteins from shrimp. After a blot of shrimp hemocyte membrane proteins was overlaid with a recombinant WSSV envelope protein (rVP28), the reactive bands on the blot were detected using anti-VP28 antibody. Among three membrane-associated molecules identified by liquid chromatography-tandem mass spectrometry, there was a 25-kDa protein that bound to both rVP28 and WSSV. Since it had a primary structure with high homology to the small GTP-binding protein Rab7, we named it Penaeus monodon Rab7 (PmRab7). The full-length PmRab7 cDNA was obtained, and results from a glutathione S-transferase pull-down assay confirmed specific binding to rVP28. Reverse transcriptase PCR analysis revealed PmRab7 expression in many tissues, and real-time PCR analysis revealed that expression was constitutive. Binding of PmRab7 to rVP28 or WSSV occurred in a dose-dependent manner and was inhibited by anti-Rab7 antibody. In an in vivo neutralization assay, the number of dead shrimp after challenge with WSSV plus PmRab7 (15%) or WSSV plus anti-Rab7 antibody (5%) was significantly lower than after challenge with WSSV alone (95%). In contrast to the WSSV-injected group, shrimp injected with WSSV plus PmRab7 or WSSV plus anti-Rab7 showed no WSSV-type histopathology. We conclude that PmRab7 is involved in WSSV infection in shrimp. This is the first study to identify a shrimp protein that binds directly to a major viral envelope protein of WSSV.

Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion.

ABSTRACT 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 Annexes a Shrimp STAT To Enhance Expression of the Immediate-Early Gene ie1

ABSTRACT Although the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is part of the antiviral response in arthropods such as Drosophila, here we show that white spot syndrome virus (WSSV) uses a shrimp STAT as a transcription factor to enhance viral gene expression in host cells. In a series of deletion and mutation assays using the WSSV immediate-early gene ie1 promoter, which is active in shrimp cells and also in insect Sf9 cells, an element containing a STAT binding motif was shown to be important for the overall level of WSSV ie1 promoter activity. In the Sf9 insect cell line, a specific protein-DNA complex was detected by using electrophoresis mobility shift assays (EMSA) with the 32P-labeled STAT binding motif of the WSSV ie1 promoter as the probe. When recombinant Penaeus monodon STAT (rPmSTAT) was overexpressed in Sf9 cells, EMSA with specific antibodies confirmed that the STAT was responsible for the formation of the specific protein-DNA complex. Another EMSA showed that in WSSV-infected P. monodon, levels of activated PmSTAT were higher than in WSSV-free P. monodon. A transactivation assay of the WSSV ie1 promoter demonstrated that increasing the level of rPmSTAT led to dose-dependent increases in ie1 promoter activity. These results show that STAT directly transactivates WSSV ie1 gene expression and contributes to its high promoter activity. We conclude that WSSV successfully annexes a putative shrimp defense mechanism, which it uses to enhance the expression of viral immediate-early genes.

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Significance Since 2009, an emergent shrimp disease, acute hepatopancreatic necrosis disease (AHPND), has been causing global losses to the shrimp farming industry. The causative agent of AHPND is a specific strain of Vibrio parahaemolyticus. We present evidence here that the opportunistic V. parahaemolyticus becomes highly virulent by acquiring a unique AHPND-associated plasmid. This virulence plasmid, which encodes a binary toxin [V. parahaemolyticus Photorhabdus insect-related toxins (PirAvp and PirBvp)] that induces cell death, is stably inherited via a postsegregational killing system and disseminated by conjugative transfer. The cytotoxicity of the PirAvp/PirBvp system is analogous to the structurally similar insecticidal pore-forming Cry toxin. These findings will significantly increase our understanding of this emerging disease, which is essential for developing anti-AHPND measures. Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirAvp and PirBvp) proteins and found that the overall structural topology of PirAvp/PirBvp is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirABvp heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirABvp may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected Penaeus monodon

BackgroundWhite spot syndrome (WSS) is a viral disease that affects most of the commercially important shrimps and causes serious economic losses to the shrimp farming industry worldwide. However, little information is available in terms of the molecular mechanisms of the host-virus interaction. In this study, we used an expressed sequence tag (EST) approach to observe global gene expression changes in white spot syndrome virus (WSSV)-infected postlarvae of Penaeus monodon.ResultsSequencing of the complementary DNA clones of two libraries constructed from normal and WSSV-infected postlarvae produced a total of 15,981 high-quality ESTs. Of these ESTs, 46% were successfully matched against annotated genes in National Center of Biotechnology Information (NCBI) non-redundant (nr) database and 44% were functionally classified using the Gene Ontology (GO) scheme. Comparative EST analyses suggested that, in postlarval shrimp, WSSV infection strongly modulates the gene expression patterns in several organs or tissues, including the hepatopancreas, muscle, eyestalk and cuticle. Our data suggest that several basic cellular metabolic processes are likely to be affected, including oxidative phosphorylation, protein synthesis, the glycolytic pathway, and calcium ion balance. A group of immune-related chitin-binding protein genes is also likely to be strongly up regulated after WSSV infection. A database containing all the sequence data and analysis results is accessible at http://xbio.lifescience.ntu.edu.tw/pm/.ConclusionThis study suggests that WSSV infection modulates expression of various kinds of genes. The predicted gene expression pattern changes not only reflect the possible responses of shrimp to the virus infection but also suggest how WSSV subverts cellular functions for virus multiplication. In addition, the ESTs reported in this study provide a rich source for identification of novel genes in shrimp.

WSSV infection activates STAT in shrimp

Although the JAK/STAT signaling pathway is usually involved in antiviral defense, a recent study suggested that STAT might be annexed by WSSV (white spot syndrome virus) to enhance the expression of a viral immediate early gene in infected shrimps. In the present study, we clone and report the first full-length cDNA sequence for a crustacean STAT from Penaeus monodon. Alignment and comparison with the deduced amino acid sequences of other STATs identified several important conserved residues and functional domains, including the DNA binding domain, SH2 domain and C-terminal transactivation domain. Based on these conserved sequences, a phylogenetic analysis suggested that shrimp STAT belongs to the ancient STAT family, while the presence of the functional domains suggested that shrimp STAT might share similar functions and regulating mechanisms with the well-known STATs isolated from model organisms. Real-time PCR showed a decreased transcription level of shrimp STAT after WSSV infection, but a Western blot analysis using anti-phosphorylated STAT antibody showed an increased level of phosphorylated (activated) STAT in the lymphoid organ of shrimp after WSSV infection. We further show that a primary culture of lymphoid organ cells from WSSV-infected shrimp resulted in activated STAT being translocated from the cytoplasm to the nucleus. This report provides experimental evidence that shrimp STAT is activated in response to WSSV infection. Our results support an earlier finding that WSSV does not disrupt JAK/STAT pathway, but on the contrary benefits from STAT activation in the shrimp host.

Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12.3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family.

Studies on transmission of white spot syndrome associated baculovirus (WSBV) in Penaeus monodon and P. japonicus via waterborne contact and oral ingestion

WSBV (white spot syndrome associated baculovirus) is considered to be the main causative agent of a recently reported disease which has resulted in serious mortality among cultured penaeid shrimp in Taiwan and is characterized by obvious white spots on the body. Shrimp infectivity tests of WSBV were carried out by means of waterborne contact and oral ingestion. Healthy juvenile P. monodon and P. japonicus and healthy P. penicillatus postlarvae were immersed in filtrates prepared from either diseased P. japonicus or diseased P. monodon, both of which exhibited marked white spot signs. Cumulative mortalities of the three tested shrimp species reached 100% within 4–6 days. Using PCR with a specific primer set, WSBV was first detected in the previously healthy P. monodon immersed in filtrate from diseased P. monodon 6 h postinoculation (h p.i.). At 24 h p.i. detection rates reached 90%, and even though the tested shrimp failed to show visible evidence of disease, they nonetheless suffered 33% mortality. The appearance of WSBV in experimentally infected P. penicillatus postlarvae was detected at 24 h p.i. and reached 100% by 72 h p.i. Healthy P. monodon fed with diseased P. japonicus as well as those fed with diseased P. monodon became 80–90% WSBV-positive 24 h p.i. by PCR and all of the tested shrimp died within 5 d. Obvious white spots appeared on the exoskeleton of shrimp whether they were infected by waterborne contact or orally. WSBV was found highly pathogenic to the three tested shrimp species and was readily transmitted across different penaeid shrimp.