Papers by Jiann-Horng Leu
Life
The shrimp aquaculture industry has encountered many diseases that have caused significant losses... more The shrimp aquaculture industry has encountered many diseases that have caused significant losses, with the most serious being white spot syndrome (WSS). Until now, no cures, vaccines, or drugs have been found to counteract the WSS virus (WSSV). The purpose of this study was to develop an oral delivery system to transport recombinant proteinaceous antigens into shrimp. To evaluate the feasibility of the oral delivery system, we used white shrimp as the test species and maggots as protein carriers. The results indicated that the target protein was successfully preserved in the maggot, and the protein was detected in the gastrointestinal tract of the shrimp, showing that this oral delivery system could deliver the target protein to the shrimp intestine, where it was absorbed. In addition, the maggots were found to increase the total haemocyte count and phenoloxidase activity of the shrimp, and feeding shrimp rVP24-fed maggots significantly induced the expression of penaeidins 2. In th...
Journal of Marine Science and Technology, 2013
Piscine iridoviruses infect a wide variety of fish and are classified into three genera: Lymphocy... more Piscine iridoviruses infect a wide variety of fish and are classified into three genera: Lymphocystivirus, Ranavirus and Megalocytivirus. Lymphocystiviruses cause non-fatal, dermal infections, while ranaviruses and megalocytiviruses produce devastating, systemic infections with mortality reaching up to 100%. Although both ranaviruses and megalocytiviruses cause fatal systemic infections, they induce different pathology. In Taiwan, both ranaviruses and megalocytiviruses have caused serious epidemics in several mariculture fish species, including groupers. In this study, we infected the orange-spotted grouper (Epinephelus coioides) with either a megalocytivirus (TGIV, grouper iridovirus of Taiwan) or a ranavirus (GIV, grouper iridovirus), and then the two iridoviruses were investigated and compared in respect of their target organs, virulence, and effects on the expression of several immune-related genes in the spleen and head kidney. By measuring cumulative mortality rate, GIV was shown to have higher virulence than TGIV. By PCR, we found that TGIV mainly infected the spleen, head kidney, kidney, heart and gill, while GIV mainly infected the spleen and intestine. The assayed immune genes were hemoglobin subunit-β-2 (Hb), CC chemokine 19, Toll-like receptor 9 isoform A and B (TLR9-A and B) and Mx (myxovirus resistance). By real-time RT-PCR, we found that of the assayed genes, the expression of CC chemokine 19 was strongly induced in spleen by both viruses, whereas the expression of Mx were strongly induced by both viruses in both organs and the expression of Hb gene was induced only by TGIV in head kidney.
BMC genomics, Jan 25, 2014
Penaeus monodon nudivirus (PmNV) is the causative agent of spherical baculovirosis in shrimp (Pen... more Penaeus monodon nudivirus (PmNV) is the causative agent of spherical baculovirosis in shrimp (Penaeus monodon). This disease causes significant mortalities at the larval stage and early postlarval (PL) stage and may suppress growth and reduce survival and production in aquaculture. The nomenclature and classification status of PmNV has been changed several times due to morphological observation and phylogenetic analysis of its partial genome sequence. In this study, we therefore completed the genome sequence and constructed phylogenetic trees to clarify PmNV's taxonomic position. To better understand the characteristics of the occlusion bodies formed by this marine occluded virus, we also compared the chemical properties of the polyhedrin produced by PmNV and the baculovirus AcMNPV (Autographa californica nucleopolyhedrovirus). We used next generation sequencing and traditional PCR methods to obtain the complete PmNV genome sequence of 119,638 bp encoding 115 putative ORFs. Phyl...
Virology, 2002
To identify the protein encoded by a 687-bp open reading frame (ORF) of a salI genomic DNA fragme... more To identify the protein encoded by a 687-bp open reading frame (ORF) of a salI genomic DNA fragment of shrimp white spot syndrome virus (WSSV), we expressed the ORF in a baculovirus/insect cell expression system. The apparent molecular mass of the recombinant protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 35 kDa in insect cells. Antibody raised against bacterially synthesized protein of the ORF identified a nucleocapsid protein (VP35) in the extracts of both the purified WSSV virions and the nucleocapsids which comigrated with the 35-kDa baculovirus-expressed recombinant protein on SDS-PAGE. We also show by transient expression in insect cells (Sf9) that VP35 targets the nucleus. Two potential nuclear localization signals (NLSs) were characterized, but only one of them was important for targeting VP35 to the nuclei of transfected insect cells. Replacement of a cluster of four positively charged residues (24 KRKR 27) at the N terminus of the protein with AAAA resulted in mutant proteins that were distributed only in the cytoplasm, thus confirming that this sequence is a critical part of the functionally active NLS of VP35.
Virology, 2000
From previously constructed genomic libraries of a Taiwan WSSV isolate, a putative WSSV tk-tmk ge... more From previously constructed genomic libraries of a Taiwan WSSV isolate, a putative WSSV tk-tmk gene was identified. Uniquely, the open reading frame (ORF) of this gene was predicted to encode a novel chimeric protein of 388 amino acids with significant homology to two proteins: thymidine kinase (TK) and thymidylate kinase (TMK). Northern blot analysis with a WSSV tk-tmk-specific riboprobe detected a major transcript of 1.6 kb. When healthy adult Penaeus monodon shrimp were inoculated with WSSV, the tk-tmk gene transcript was first detected by RT-PCR analysis at 4 h postinfection and transcription levels continued to increase over the first 18 h. The gene's major in vitro transcription and translation product, equivalent to the predicted size (43 kDa), is a single chimeric protein that includes both the TK and TMK functional motifs. Evidence for phylogenetic analysis and sequence alignment suggested that the gene may have resulted from the fusion of a cellular-type TK gene and a cellular-type TMK gene. Its unique arrangement may also provide a valuable gene marker for WSSV.
Virology, 2009
The genome of the white spot syndrome virus (WSSV) Taiwan isolate has many structural and non-str... more The genome of the white spot syndrome virus (WSSV) Taiwan isolate has many structural and non-structural genes that are arranged in clusters. Screening with Northern blots showed that at least four of these clusters produce polycistronic mRNA, and one of these (vp31/vp39b/vp11) was studied in detail. The vp31/vp39b/ vp11 cluster produces two transcripts, including a large 3.4-kb polycistronic transcript of all three genes. No monocistronic vp39b mRNA was detected. TNT and in vitro translation assays showed that vp39b translation was independent of vp31 translation, and that ribosomal reinitiation was not a possible mechanism for vp39b. An unusually located IRES (internal ribosome entry site) element was identified in the vp31/vp39b coding region, and this region was able to promote the expression of a downstream firefly luciferase reporter. We show that vp31/vp39b/vp11 is representative of many other WSSV structural/non-structural gene clusters, and argue that these are also likely to produce polycistronic mRNAs and that use an IRES mechanism to regulate their translation.
European Journal of Biochemistry, 2003
The STAT5 (signal transducer and activator of transcription 5) gene was isolated and characterize... more The STAT5 (signal transducer and activator of transcription 5) gene was isolated and characterized from a round‐spotted pufferfish genomic library. This gene is composed of 19 exons spanning 11 kb. The full‐length cDNA of Tetraodon fluviatilis STAT5 (TfSTAT5) contains 2461 bp and encodes a protein of 785 amino acid residues. From the amino acid sequence comparison, TfSTAT5 is most similar to mouse STAT5a and STAT5b with an overall identity of 76% and 78%, respectively, and has < 35% identity with other mammalian STATs. The exon/intron junctions of the TfSTAT5 gene were almost identical to those of mouse STAT5a and STAT5b genes, indicating that these genes are highly conserved at the levels of amino acid sequence and genomic structure. To understand better the biochemical properties of TfSTAT5, a chimeric STAT5 was generated by fusion of the kinase‐catalytic domain of carp Janus kinase 1 (JAK1) to the C‐terminal end of TfSTAT5. The fusion protein was expressed and tyrosine‐phospho...
Proceedings of the National Academy of Sciences, 2008
White spot syndrome virus (WSSV) is a large (≈300 kbp), double-stranded DNA eukaryotic virus that... more White spot syndrome virus (WSSV) is a large (≈300 kbp), double-stranded DNA eukaryotic virus that has caused serious disease in crustaceans worldwide. ICP11 is the most highly expressed WSSV nonstructural gene/protein, which strongly suggests its importance in WSSV infection; but until now, its function has remained obscure. We show here that ICP11 acts as a DNA mimic. In crystal, ICP11 formed a polymer of dimers with 2 rows of negatively charged spots that approximated the duplex arrangement of the phosphate groups in DNA. Functionally, ICP11 prevented DNA from binding to histone proteins H2A, H2B, H3, and H2A.x, and in hemocytes from WSSV-infected shrimp, ICP11 colocalized with histone H3 and activated-H2A.x. These observations together suggest that ICP11 might interfere with nucleosome assembly and prevent H2A.x from fulfilling its critical function of repairing DNA double strand breaks. Therefore, ICP11 possesses a functionality that is unique among the handful of presently know...
Marine Biotechnology, 2010
Journal of Virology, 2004
White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infec... more 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 ...
Journal of Virology, 2008
Immediate-early proteins from many viruses function as transcriptional regulators and exhibit tra... more Immediate-early proteins from many viruses function as transcriptional regulators and exhibit transactivation activity, DNA binding activity, and dimerization. In this study, we investigated these characteristics in white spot syndrome virus (WSSV) immediate-early protein 1 (IE1) and attempted to map the corresponding functional domains. Transactivation was investigated by transiently expressing a protein consisting of the DNA binding domain of the yeast transactivator GAL4 fused to full-length IE1. This GAL4-IE1 fusion protein successfully activated the Autographa californica multicapsid nucleopolyhedrovirus p35 basal promoter when five copies of the GAL4 DNA binding site were inserted upstream of the TATA box. A deletion series of GAL4-IE1 fusion proteins suggested that the transactivation domain of WSSV IE1 was carried within its first 80 amino acids. A point mutation assay further showed that all 12 of the acidic residues in this highly acidic domain were important for IE1's...
Journal of Virology, 2004
One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a gia... more 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,...
Journal of Virology, 2006
The protein components of the white spot syndrome virus (WSSV) virion have been well established ... more 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 res...
Journal of Virology, 2011
High temperature (32 to 33°C) has been shown to reduce mortality in white spot syndrome virus (WS... more High temperature (32 to 33°C) has been shown to reduce mortality in white spot syndrome virus (WSSV)-infected shrimps, but the mechanism still remains unclear. Here we show that in WSSV-infected shrimps cultured at 32°C, transcriptional levels of representative immediate-early, early, and late genes were initially higher than those at 25°C. However, neither the IE1 nor VP28 protein was detected at 32°C, suggesting that high temperature might inhibit WSSV protein synthesis. Two-dimensional gel electrophoresis analysis revealed two proteins, NAD-dependent aldehyde dehydrogenase (ALDH) and the proteasome alpha 4 subunit (proteasome α4), that were markedly upregulated in WSSV-infected shrimps at 32°C. Reverse transcription-PCR (RT-PCR) analysis of members of the heat shock protein family also showed that hsp70 was upregulated at 32°C. When aldh , proteasome α4 , and hsp70 were knocked down by double-stranded RNA interference and shrimps were challenged with WSSV, the aldh and hsp70 knoc...
PloS one, 2012
White spot syndrome virus (WSSV), a large enveloped DNA virus, can cause the most serious viral d... more White spot syndrome virus (WSSV), a large enveloped DNA virus, can cause the most serious viral disease in shrimp and has a wide host range among crustaceans. In this study, we identified a surface protein, named glucose transporter 1 (Glut1), which could also interact with WSSV envelope protein, VP53A. Sequence analysis revealed that Glut1 is a member of a large superfamily of transporters and that it is most closely related to evolutionary branches of this superfamily, branches that function to transport this sugar. Tissue tropism analysis showed that Glut1 was constitutive and highly expressed in almost all organs. Glut1's localization in shrimp cells was further verified and so was its interaction with Penaeus monodon chitin-binding protein (PmCBP), which was itself identified to interact with an envelope protein complex formed by 11 WSSV envelope proteins. In vitro and in vivo neutralization experiments using synthetic peptide contained WSSV binding domain (WBD) showed tha...
DNA and Cell Biology, 2000
We have previously reported the isolation of the JAK1 gene from the round-spotted pufferfish. In ... more We have previously reported the isolation of the JAK1 gene from the round-spotted pufferfish. In the present study, we cloned and characterized genomic sequences encoding pufferfish JAK2, JAK3, and TYK2, which are other members of JAK family. To our knowledge, this is the first report to demonstrate the existence of four JAK genes in fish. All pufferfish JAK genes except JAK1 are composed of 24 exons; JAK1 has an additional exon. A comparison of the exon-intron organization of these genes revealed that the splice sites of JAK genes are nearly identical. In addition, all pufferfish JAK genes have one intron in the 59 untranslated region. Taken together, these data suggest that the pufferfish JAK genes may have evolved from a common ancestor. By 59 rapid amplification of cDNA ends and sequence analysis, we deduced the promoter regions for all JAK genes and found they do not contain typical TATA or CCAAT boxes but rather numerous other potential binding sites for transcription factors. Interestingly, the TYK2 gene is linked to CDC37 in a head-to-tail manner with a small intergenic region of 292 bp. Within this region, there are two potential binding sites for transcriptional factors such as c-Myb and NF-IL6. The putative promoter regions of all JAK genes were tested either in a carp CF cell line or in zebrafish embryos using CAT or lacZ as reporter genes. Both assays confirmed the transcriptional activities of these promoters in vitro and in vivo.
DNA and Cell Biology, 1996
A 3.7-kb cDNA encodes the carp JAK1 kinase of 1,156 amino acid residues. The overall amino acid s... more A 3.7-kb cDNA encodes the carp JAK1 kinase of 1,156 amino acid residues. The overall amino acid sequence identity between carp JAK1 and murine JAK1, JAK2, JAK3, and human TYK2 is 57%, 35.5%, 31.3%, and 42.4%, respectively. In addition, carp JAK1 shows higher sequence homology to mammalian JAK1 in both the kinase-like (JH2) and kinase (JH1) domains (approximately 70% identity). Therefore, carp JAK1 is a homolog of mammalian JAK1. To investigate the possible function of JH2 domain, full-length, and various truncated forms of carp JAK1 were produced in the baculovirus system. Our results demonstrate that c-JHl and C-JH2 associate with each other and C-JH2 can be tyrosine-phosphorylated by c-JAKl and by c-JH(l + 2). The JAK I gene was also isolated from a carp genomic library and characterized. This gene is divided into 24 exons spanning at least 31 kb of genomic DNA. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. The 2.5-kb DNA region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including NF-IL6, HNF-5, API, GHF-5, and E2A. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 16 times more efficiently than the promoterless pCAT-Basic. Deletion analysis defined a positive regulatory region between-1,023 and-528. A smaller region (-181 to +59) without any typical TATA-box sequences, G + Crich sequences, or other binding sequences for known transcription factors still had promoter activity. Constructs without this region did not have detectable promoter activity. This suggests that this region of DNA may play an important role in the expression of carp JAK I gene.
Diseases of Aquatic Organisms, 1996
White spot syndrome associated baculovlrus (WSBV) is the causative agent of a dlsease which has r... more 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 62 Inter-Research 1996 Resale of full artlcle not permitted
Diseases of Aquatic Organisms, 1995
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Papers by Jiann-Horng Leu