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1.
PLoS Pathog ; 19(5): e1011330, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37141203

RESUMO

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.


Assuntos
Toxinas Bacterianas , Penaeidae , Photorhabdus , Vibrio parahaemolyticus , Animais , Photorhabdus/metabolismo , Penaeidae/microbiologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Insetos/metabolismo , Vibrio parahaemolyticus/metabolismo
2.
Life (Basel) ; 11(8)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34440562

RESUMO

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 the WSSV challenge, the survival rate of rVP24-fed maggots was approximately 43%. This study showed that maggots can be used as effective oral delivery systems for aquatic products and may provide a new method for aquatic vaccine delivery systems.

3.
Dev Comp Immunol ; 120: 104058, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33657430

RESUMO

Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.


Assuntos
Bass/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , L-Aminoácido Oxidase/metabolismo , Sequência de Aminoácidos , Animais , Bass/genética , Bass/microbiologia , Clonagem Molecular , Proteínas de Peixes/genética , Proteínas de Peixes/isolamento & purificação , L-Aminoácido Oxidase/genética , L-Aminoácido Oxidase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Células Sf9 , Spodoptera , Especificidade por Substrato/imunologia , Vibrio parahaemolyticus/imunologia
4.
Fish Shellfish Immunol ; 106: 910-919, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32841684

RESUMO

Since the mechanisms by which cellular factors modulate replication of the shrimp viral pathogen white spot syndrome virus (WSSV) are still largely unknown, here we consider the sirtuins, a family of NAD+-dependent protein deacetylases that are known to function as regulatory factors that activate or suppress viral transcription and replication in mammals. In particular, we focus on SIRT1 by isolating and characterizing LvSIRT1 from white shrimp (Litopenaeus vannamei) and investigating its involvement in WSSV infection. DsRNA-mediated gene silencing led to the expression of WSSV genes and the replication of genomic DNAs being significantly decreased in LvSIRT1-silenced shrimp. The deacetylase activity of LvSIRT1 was significantly induced at the early stage (2 hpi) and the genome replication stage (12 hpi) of WSSV replication, but decreased at the late stage of WSSV replication (24 hpi). Treatment with the SIRT1 activator resveratrol further suggested that LvSIRT1 activation increased the expression of several WSSV genes (IE1, VP28 and ICP11). Lastly, we used transfection and dual luciferase assays in Sf9 insect cells to show that while the overexpression of LvSIRT1 facilitates the promoter activity of WSSV IE1, this enhancement of WSSV IE1 expression is achieved by a transactivation pathway that is NF-κB-independent.


Assuntos
Proteínas de Artrópodes/genética , Infecções por Vírus de DNA/genética , Penaeidae/genética , Sirtuína 1/genética , Proteínas Virais/genética , Vírus da Síndrome da Mancha Branca 1/genética , Animais , Sítios de Ligação , Linhagem Celular , Infecções por Vírus de DNA/veterinária , Inativação Gênica , Insetos , Mutação , NF-kappa B , Penaeidae/virologia , Regiões Promotoras Genéticas
5.
Dev Comp Immunol ; 97: 1-10, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30904428

RESUMO

In this study, we describe 19 different CC chemokine genes from the orange-spotted grouper, Epinephelus coioides, identified by the analysis of the spleen transcriptome. Multiple sequence alignment of the 19 CC chemokines showed that although two genes, EcSCYA115 and EcSCYA117, shared 80% amino acid similarity (72% identity), the majority exhibited low similarity to each other. Phylogenetic analysis divided the 19 CC chemokines into six major groups. Tissue distribution analysis by RT-PCR showed that most of these chemokines were ubiquitously expressed in the 9 examined tissues, whereas some exhibited tissue-preferential expression patterns. For example, EcSCYA103 was preferentially expressed in fin and gill; EcSCYA109 in head kidney and spleen; EcSCYA114 in fin, gill, and liver; and EcSCYA119 in fin and stomach. Quantitative RT-PCR showed that after challenge with grouper iridovirus (GIV), four of the 19 CC chemokine genes, EcSYCA102, EcSYCA103, EcSYCA116, and EcSYCA118, were highly induced in the spleen. The expression of these four genes could also be upregulated by LPS and poly (I:C) challenges, suggesting that these four genes might be involved in immune response against invading pathogens.


Assuntos
Bass/genética , Quimiocinas CC/genética , Proteínas de Peixes/genética , Família Multigênica , Baço/metabolismo , Transcriptoma/genética , Sequência de Aminoácidos , Nadadeiras de Animais/metabolismo , Animais , Bass/virologia , Quimiocinas CC/classificação , Perfilação da Expressão Gênica/métodos , Brânquias/metabolismo , Interações Hospedeiro-Patógeno , Iridovirus/fisiologia , Especificidade de Órgãos/genética , Homologia de Sequência de Aminoácidos
6.
Dev Comp Immunol ; 49(1): 7-18, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25445906

RESUMO

A series of deletion and mutation assays of the white spot syndrome virus (WSSV) immediate-early gene WSSV108 promoter showed that a Krüppel-like factor (KLF) binding site located from -504 to -495 (relative to the transcription start site) is important for the overall level of WSSV108 promoter activity. Electrophoretic mobility shift assays further showed that overexpressed recombinant Penaeus monodon KLF (rPmKLF) formed a specific protein-DNA complex with the (32)P-labeled KLF binding site of the WSSV108 promoter, and that higher levels of Litopenaeus vannamei KLF (LvKLF) were expressed in WSSV-infected shrimp. A transactivation assay indicated that the WSSV108 promoter was strongly activated by rPmKLF in a dose-dependent manner. Lastly, we found that specific silencing of LvKLF expression in vivo by dsRNA injection dramatically reduced both WSSV108 expression and WSSV replication. We conclude that shrimp KLF is important for WSSV genome replication and gene expression, and that it binds to the WSSV108 promoter to enhance the expression of this immediate-early gene.


Assuntos
Proteínas de Artrópodes/metabolismo , Genes Precoces/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Virais/genética , Vírus da Síndrome da Mancha Branca 1/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Proteínas Imediatamente Precoces , Fatores de Transcrição Kruppel-Like/genética , Dados de Sequência Molecular , Penaeidae/genética , Penaeidae/metabolismo , Penaeidae/virologia , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Proteínas Virais/metabolismo , Replicação Viral/genética , Vírus da Síndrome da Mancha Branca 1/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia
7.
Dev Comp Immunol ; 49(2): 239-48, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25499032

RESUMO

By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54-59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp.


Assuntos
Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Ditiotreitol/farmacologia , Chaperona BiP do Retículo Endoplasmático , Técnicas de Silenciamento de Genes , Brânquias/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Penaeidae/genética , Penaeidae/metabolismo , Análise de Sequência de DNA , Células Sf9 , Tunicamicina/farmacologia
8.
BMC Genomics ; 15: 628, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-25063321

RESUMO

BACKGROUND: 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). RESULTS: We used next generation sequencing and traditional PCR methods to obtain the complete PmNV genome sequence of 119,638 bp encoding 115 putative ORFs. Phylogenetic tree analysis showed that several PmNV genes and sequences clustered with the non-occluded nudiviruses and not with the baculoviruses. We also investigated the characteristics of PmNV polyhedrin, which is a functionally important protein and the major component of the viral OBs (occlusion bodies). We found that both recombinant PmNV polyhedrin and wild-type PmNV OBs were sensitive to acid conditions, but unlike the baculoviral OBs, they were not susceptible to alkali treatment. CONCLUSIONS: From the viral genome features and phylogenetic analysis we conclude that PmNV is not a baculovirus, and that it should be assigned to the proposed Nudiviridae family with the other nudiviruses, but into a distinct new genus (Gammanudivirus).


Assuntos
Organismos Aquáticos/virologia , Baculoviridae/genética , Baculoviridae/fisiologia , Genômica , Penaeidae/virologia , Animais , Baculoviridae/classificação , Baculoviridae/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Boca/virologia , Fases de Leitura Aberta/genética , Filogenia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo , Montagem de Vírus/genética
9.
J Gen Virol ; 95(Pt 8): 1799-1808, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24836670

RESUMO

White spot syndrome virus (WSSV) is a large enveloped virus which has caused severe mortality and huge economic losses in the shrimp farming industry. The enveloped virus must be combined with the receptors of the host cell membrane by the virus envelope proteins. In the case of WSSV, binding of envelope proteins with receptors of the host cell membrane was discovered in a number of previous studies, such as VP53A and 10 other proteins with chitin-binding protein (CBP), VP28 with Penaeus monodon Rab7, VP187 with ß-integrin, and so on. WSSV envelope proteins were also considered capable of forming a protein complex dubbed an 'infectome'. In this study, the research was focused on the role of CBP in the WSSV infection process, and the relationship between CBP and the envelope proteins VP24, VP28, VP31, VP32 VP39B, VP53A and VP56. The results of the reverse transcription-PCR analyses showed that CBP existed in a variety of shrimp. The speed of WSSV infection could be slowed down by inhibiting CBP gene expression. Far-Western blot analysis and His pull-down assays were conducted, and a protein complex was found that appeared to be composed of a 'linker' protein consisting of VP31, VP32 and VP39B together with four envelope proteins, including VP24, VP28, VP53A and VP56. This protein complex was possibly another part of the infectome and the possible binding region with CBP. The findings of this study may have identified certain points for further WSSV research.


Assuntos
Penaeidae/virologia , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Far-Western Blotting , Proteínas de Transporte/metabolismo , Centrifugação , Perfilação da Expressão Gênica , Complexos Multiproteicos/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
PLoS One ; 9(1): e85779, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24465701

RESUMO

White spot syndrome virus (WSSV) is a large enveloped virus. The WSSV viral particle consists of three structural layers that surround its core DNA: an outer envelope, a tegument and a nucleocapsid. Here we characterize the WSSV structural protein VP11 (WSSV394, GenBank accession number AF440570), and use an interactome approach to analyze the possible associations between this protein and an array of other WSSV and host proteins. Temporal transcription analysis showed that vp11 is an early gene. Western blot hybridization of the intact viral particles and fractionation of the viral components, and immunoelectron microscopy showed that VP11 is an envelope protein. Membrane topology software predicted VP11 to be a type of transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal. Based on an immunofluorescence assay performed on VP11-transfected Sf9 cells and a trypsin digestion analysis of the virion, we conclude that, contrary to topology software prediction, the C-terminal of this protein is in fact inside the virion. Yeast two-hybrid screening combined with co-immunoprecipitation assays found that VP11 directly interacted with at least 12 other WSSV structural proteins as well as itself. An oligomerization assay further showed that VP11 could form dimers. VP11 is also the first reported WSSV structural protein to interact with the major nucleocapsid protein VP664.


Assuntos
Proteínas do Envelope Viral/metabolismo , Vírus da Síndrome da Mancha Branca 1/metabolismo , Regulação Viral da Expressão Gênica , Imunoprecipitação , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Reprodutibilidade dos Testes , Fatores de Tempo , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/ultraestrutura , Vírion/metabolismo , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/ultraestrutura
11.
Fish Shellfish Immunol ; 35(3): 707-15, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23747417

RESUMO

The white spot syndrome virus (WSSV) has had a serious economic impact on the global shrimp aquaculture industry in the past two decades. Although research has clarified a lot about its genome and structure, the mechanism of how WSSV enters a cell is still unclear. In this study to determine this mechanism, primary cultured hemocytes were used as an experimental model to observe the process of WSSV entry because the stable shrimp cell lines for WSSV infection are lacking. After labeling virions and endosomes with fluorescent dyes followed by observation with a confocal microscope, the results show that the WSSV colocalizes with early endosomes. Hemocytes are further treated with different endocytic inhibitors, methyl-ß-cyclodextrin (MßCD) and chlorpromazine (CPZ). WSSV still can be detected in the hemocytes treated with CPZ, but not in the hemocytes treated with MßCD. Thus, we conclude that WSSV adopts the caveolae-mediated endocytosis to enter the shrimp cell.


Assuntos
Endocitose , Hemócitos/virologia , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Antieméticos/farmacologia , Células Cultivadas , Clorpromazina/administração & dosagem , Clorpromazina/farmacologia , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Hemócitos/fisiologia , Rodaminas/metabolismo , Coloração e Rotulagem , beta-Ciclodextrinas/administração & dosagem , beta-Ciclodextrinas/farmacologia
12.
Fish Shellfish Immunol ; 34(4): 1011-7, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22683516

RESUMO

White spot syndrome virus (WSSV) is an enveloped, large dsDNA virus that mainly infects penaeid shrimp, causing serious damage to the shrimp aquaculture industry. Like other animal viruses, WSSV infection induces apoptosis. Although this occurs even in by-stander cells that are free of WSSV virions, apoptosis is generally regarded as a kind of antiviral immune response. To counter this response, WSSV has evolved several different strategies. From the presently available literature, we construct a model of how the host and virus both attempt to regulate apoptosis to their respective advantage. The basic sequence of events is as follows: first, when a WSSV infection occurs, cellular sensors detect the invading virus, and activate signaling pathways that lead to (1) the expression of pro-apoptosis proteins, including PmCasp (an effecter caspase), MjCaspase (an initiator caspase) and voltage-dependent anion channel (VDAC); and (2) mitochondrial changes, including the induction of mitochondrial membrane permeabilization and increased oxidative stress. These events initiate the apoptosis program. Meanwhile, WSSV begins to express its genes, including two anti-apoptosis proteins: AAP-1, which is a direct caspase inhibitor, and WSV222, which is an E3 ubiquitin ligase that blocks apoptosis through the ubiquitin-mediated degradation of shrimp TSL protein (an apoptosis inducer). WSSV also induces the expression of a shrimp anti-apoptosis protein, Pm-fortilin, which can act on Bax to inhibit mitochondria-triggered apoptosis. This is a life and death struggle because the virus needs to prevent apoptosis in order to replicate. If WSSV succeeds in replicating in sufficient numbers, this will result in the death of the infected penaeid shrimp host.


Assuntos
Apoptose/imunologia , Infecções por Vírus de DNA/imunologia , Penaeidae/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Infecções por Vírus de DNA/patologia , Infecções por Vírus de DNA/virologia , Penaeidae/virologia
13.
Dev Comp Immunol ; 38(1): 78-87, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22564858

RESUMO

The members of the inhibitor of apoptosis protein (IAP) family are involved in the regulation of diverse cellular processes, including apoptosis, signal transduction and mitosis. Here, we report the cloning and characterization of three IAP genes from Pacific white shrimp Litopenaeus vannamei: LvIAP1, LvIAP2 and LvSurvivin. LvIAP1, the orthologue of Penaeus monodon IAP (PmIAP), consists of three BIR domains and one RING domain; LvIAP2 consists of two BIR domains and LvSurvivin has only one BIR domain. Expression profiling by absolute quantitative real-time RT-PCR revealed that of the three IAP genes, LvIAP1 had the highest expression levels in almost all examined tissues and LvSurvivin had the lowest expression levels. Furthermore, among the examined tissues, the lymphoid organs most strongly expressed all three genes. When LvIAP1 expression was silenced by injection of its corresponding dsRNA, the shrimp died within 48h after injection, whereas injection of the other two dsRNAs did not cause shrimp death. In LvIAP1-silenced shrimp, the number of circulating haemocytes decreased dramatically because of extensive apoptosis. This suggested that LvIAP1 is central to the regulation of shrimp haemocyte apoptosis.


Assuntos
Proteínas de Artrópodes/fisiologia , Proteínas Inibidoras de Apoptose/fisiologia , Penaeidae/fisiologia , Animais , Apoptose , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Clonagem Molecular , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/metabolismo , Análise de Sequência de DNA
14.
PLoS One ; 7(3): e33216, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22427993

RESUMO

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 that the WBD peptide could inhibit WSSV infection in primary cultured hemocytes and delay the mortality in shrimps challenged with WSSV. These findings have important implications for our understanding of WSSV entry.


Assuntos
Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Penaeidae/genética , Filogenia , Proteínas do Envelope Viral/metabolismo , Vírus da Síndrome da Mancha Branca 1/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Biologia Computacional , Biblioteca Gênica , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Fases de Leitura Aberta/genética , Penaeidae/virologia , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-Híbrido , Internalização do Vírus
15.
Antioxid Redox Signal ; 17(6): 914-26, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22332765

RESUMO

AIMS: In this study we identified viral gene targets of the important redox regulator thioredoxin (Trx), and explored in depth how Trx interacts with the immediate early gene #1 (IE1) of the white spot syndrome virus (WSSV). RESULTS: In a pull-down assay, we found that recombinant Trx bound to IE1 under oxidizing conditions, and a coimmunoprecipitation assay showed that Trx bound to WSSV IE1 when the transfected cells were subjected to oxidative stress. A pull-down assay with Trx mutants showed that no IE1 binding occurred when cysteine 62 was replaced by serine. Electrophoretic mobility shift assay (EMSA) showed that the DNA binding activity of WSSV IE1 was downregulated under oxidative conditions, and that Penaeus monodon Trx (PmTrx) restored the DNA binding activity of the inactivated, oxidized WSSV IE1. Another EMSA experiment showed that IE1's Cys-X-X-Cys motif and cysteine residue 55 were necessary for DNA binding. Measurement of the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in WSSV-infected shrimp showed that oxidative stress was significantly increased at 48 h postinfection. The biological significance of Trx was also demonstrated in a double-strand RNA Trx knockdown experiment where suppression of shrimp Trx led to significant decreases in mortality and viral copy numbers. INNOVATION AND CONCLUSION: WSSV's pathogenicity is enhanced by the virus' use of host Trx to rescue the DNA binding activity of WSSV IE1 under oxidizing conditions.


Assuntos
DNA Viral/metabolismo , Tiorredoxinas/metabolismo , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Animais , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Imunoprecipitação , Penaeidae/metabolismo , Penaeidae/virologia , Ligação Proteica
16.
J Virol ; 85(7): 3517-25, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21228234

RESUMO

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 knockdown shrimps became severely infected at 32°C, while the proteasome α4 knockdown shrimps remained uninfected. Our results therefore suggest that ALDH and Hsp70 both play an important role in the inhibition of WSSV replication at high temperature.


Assuntos
Aldeído Desidrogenase/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Penaeidae/virologia , Temperatura , Replicação Viral/efeitos da radiação , Vírus da Síndrome da Mancha Branca 1/fisiologia , Vírus da Síndrome da Mancha Branca 1/efeitos da radiação , Animais , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/metabolismo
17.
Mar Biotechnol (NY) ; 13(4): 608-21, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20401624

RESUMO

By economic value, shrimp is currently the most important seafood commodity worldwide, and these animals are often the subject of scientific research in shrimp farming countries. High throughput methods, such as expressed sequence tags (ESTs), were originally developed to study human genomics, but they are now available for studying other important organisms, including shrimp. ESTs are short sequences generated by sequencing randomly selected cDNA clones from a cDNA library. This is currently the most efficient and powerful method for providing transcriptomic data for organisms with an uncharacterized genome. This review will summarize the sixteen major shrimp EST studies that have been conducted to date. In addition, we analyzed the EST data downloaded from NCBI dbEST for the four major penaeid shrimp species and constructed a database to host all of these EST data as well as our own analysis results. This database provides the shrimp aquaculture research community with an outline of the shrimp transcriptome as well as a tool for shrimp gene identification.


Assuntos
Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica/genética , Imunidade Inata/genética , Penaeidae/genética , Transcriptoma/genética , Animais , Reprodução/genética , Processos de Determinação Sexual/genética , Especificidade da Espécie
18.
Dev Comp Immunol ; 35(4): 469-75, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21130805

RESUMO

Fortilin plays an important role in anti-apoptotic mechanisms and cell proliferation in many eukaryotic organisms. This work confirmed previous reports that Sf9 can support the replication of white spot syndrome virus (WSSV) genomic material by using immunohistochemistry with a specific antibody to detect the immediate early gene 1 (ie1) and by amplification of WSSV DNA and mRNA products. Using this insect-cell model system, we show that overexpression of Pm-fortilin in Sf9 cells inhibited the expression of WSSV early genes and late genes (WSSV-DNA polymerase, VP15 and VP28) but not an immediate early gene ie1. This is the first time that an insect cell line has been used to demonstrate interaction between a shrimp gene and genes of a shrimp virus.


Assuntos
Penaeidae/imunologia , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1 , Animais , Genes Precoces , Insetos/citologia , Proteínas/genética , Proteínas/imunologia
19.
Fish Shellfish Immunol ; 30(2): 576-82, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21182965

RESUMO

Previous studies showed that heat-shock protein 60 (HSP60) was known to function as a molecular chaperone and is an important factor in the innate immune system in mammals. However, little was known about the physiological relevance of HSP60 in marine invertebrates. This study focuses on long-term monitoring of the differential expression of LvHSP60 in shrimp Litopenaeus vannamei in response to environmental stress. The thermal aggregation assay elucidated that LvHSP60 was an effective chaperone. It also suggested that LvHSP60 may employ the cell's intrinsic mechanism to start the immunizing process. Using quantitative real-time PCR to monitor gene expression showed that LvHSP60 was variable under different stresses including environmental stress and pathogenic infection. LvHSP60 was speculated to regulate the adaptive responses to overcome environmental stresses. In conclusion, our study proved that LvHSP60 plays an important role in the intrinsic immune system and stress responses of shrimp.


Assuntos
Chaperonina 60/imunologia , Meio Ambiente , Regulação da Expressão Gênica , Penaeidae/imunologia , Estresse Fisiológico/imunologia , Animais , Western Blotting , Chaperonina 60/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Chaperonas Moleculares , Penaeidae/microbiologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Vibrio alginolyticus/imunologia
20.
Biosens Bioelectron ; 26(3): 964-9, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20863681

RESUMO

We have investigated rapid, label free detection of white spot syndrome virus (WSSV) using the first longitudinal extension resonance peak of five lead-magnesium niobate-lead titanate (PMN-PT) piezoelectric microcantilever sensors (PEMS) 1050-700 µm long and 850-485 µm wide constructed from 8 µm thick PMN-PT freestanding films. The PMN-PT PEMS were encapsulated with a 3-mercaptopropyltrimethoxysilane (MPS) insulation layer and further coated with anti-VP28 and anti-VP664 antibodies to target the WSSV virions and nucleocapsids, respectively. By inserting the antibody coated PEMS in a flowing virion or nucleocapsid suspension, label free detection of the virions and nucleocapsids were respectively achieved by monitoring the PEMS resonance frequency shift. We showed that positive label free detection of both the virion and the nucleocapsid could be achieved at a concentration of 100virions(nucleocapsids)/ml or 10 virions(nucleocapsids)/100 µl, comparable to the detection sensitivity of polymerase chain reaction (PCR). However, in contrast to PCR, PEMS detection was label free, in situ and rapid (less than 30 min), potentially requiring minimal or no sample preparation.


Assuntos
Técnicas Biossensoriais/métodos , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Animais , Anticorpos Antivirais , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Chumbo , Nióbio , Nucleocapsídeo/imunologia , Nucleocapsídeo/isolamento & purificação , Óxidos , Reação em Cadeia da Polimerase , Titânio , Vírion/imunologia , Vírion/isolamento & purificação , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/imunologia
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