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1.
Mol Immunol ; 124: 172-179, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32585511

RESUMO

The family of TRIM proteins with E3 ubiquitin ligase activity plays important roles in virus infection in vertebrates and invertebrates. In this study, a novel Trim gene shows high similarity to Trim23 (designated as MnTrim23) was identified from Macrobrachium nipponense. The MnTrim23 protein contains three conserved domains (one RING finger domain, two B-box, and one Coiled-coil region) at its N-terminal and one ARF domain at its C-terminal. The ARF domain characterizes the members of the Trim23 family. MnTrim23 belongs to C-IX family. Phylogenetic analysis shows that MnTrim23 has a closer genetic distance with other Trim23 proteins from invertebrates than that from vertebrates. MnTrim23 has higher expression level in the intestine and hepatopancreas than in the other immune tissues. The expression levels of MnTrim23 in the gills, stomach, and intestines are significantly up-regulated after white spot syndrome virus (WSSV) infection. Moreover, knockdown of MnTrim23 inhibits WSSV replication and VP28 expression, suggesting that MnTrim23 plays a positive role in WSSV infection. Further studies revealed that MnTrim23 negatively regulates the Relish transcription factor-mediated expression of antimicrobial peptides (AMPs). Synthetic AMPs inhibit VP28 expression and WSSV replication. These findings indicate that Trim23 promotes WSSV replication by inhibiting the expression of AMPs that are positively regulated by the host NF-κB signal pathway.


Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Interações entre Hospedeiro e Microrganismos/fisiologia , Penaeidae/virologia , Proteínas com Motivo Tripartido/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Regulação da Expressão Gênica/fisiologia , Penaeidae/imunologia , Penaeidae/metabolismo , Proteínas com Motivo Tripartido/imunologia , Replicação Viral/fisiologia
2.
Arch Virol ; 165(6): 1433-1440, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32318832

RESUMO

So far, there have been no studies on the distribution of viral white spot syndrome in wild Indian white shrimp (Fenneropenaeus indicus) brooders at Iranian capture sites. This study was conducted to investigate the presence of white spot syndrome virus (WSSV) in wild Indian white shrimps in Iran, using PCR, histopathologic, and electron microscopic surveys. The samples were collected within two seasons (autumn and spring) and from two provinces (six capture sites), from the major hatcheries providing spawners. Eight hundred thirty-three samples were collected and analyzed first by PCR, after which the positive samples were examined using histological tests, and if inclusion bodies were observed, electron microscopy was also used. White spot syndrome virus was detected only at the capture sites in Sistan and Baluchistan Province, where the mean infection rate was significantly higher in the spring (8.7%) than in the autumn (2.03%). At the Chabahar, Pasabandar, and Govater capture sites, the mean infection rate was significantly higher (4.9%, 2.1%, and 9.2%, respectively), than in Hormozgan Province. The results showed that there was no significant difference in infection rate between the two different sizes and sexes of shrimps (P < 0.05). Phylogeny analysis revealed a close relationship between the viruses from this study and those in other Asian countries, including China, India, Bangladesh, Thailand, Taiwan, and South Korea. It is possible that the virus has spread across the Indian Ocean to other countries. Therefore, the spawners in this study, particularly those collected during the spring and those from capture sites in Sistan and Baluchistan Province, were found to be more susceptible to WSSV infection, and the virus might have been transmitted vertically from WSSV-infected brooders to post-larvae.


Assuntos
Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Feminino , Brânquias/patologia , Irã (Geográfico) , Masculino , Microscopia Eletrônica de Transmissão , Filogenia , Reação em Cadeia da Polimerase , Estações do Ano , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/ultraestrutura
3.
Artigo em Inglês | MEDLINE | ID: mdl-32048310

RESUMO

White spot syndrome virus has been a threat to the global shrimp industry since it was discovered in Taiwan in 1992. Thus, shrimp-producing countries have launched regulations to prevent import of WSSV-infected commodity shrimp from endemic areas. Recently, cooked shrimp that is infected with WSSV tested positive by PCR. However, there is no study to determine the infectivity of WSSV in cooked shrimp that tested positive by PCR. In the present study, WSSV-infected shrimp were cooked at boiling temperature for different times including 0, 1, 3, 5, 10 and 30 min. Upon exposure to boiling temperature, WSSV-infected shrimp were fed to SPF shrimp (Litopenaeus vannamei). The result showed experimentally challenged shrimp from 0-min treatment (positive control) indeed got infected with WSSV. However, experimentally challenged shrimp that were fed tissues boiled at 1, 3, 5, 10 and 30 min were not infected with WSSV. Mortality data showed that only the positive control (0-min) treatment displayed high mortality, whereas no mortality was observed in any other treatment category. These findings suggest that cooking shrimp at boiling temperature for at least 1 min might prevent any potential spread of WSSV from endemic countries to other geographical areas where WSSV has not yet been reported.


Assuntos
Culinária , Infecções por Vírus de DNA/transmissão , Contaminação de Alimentos/prevenção & controle , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/prevenção & controle , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Doenças Transmitidas por Alimentos/virologia , Longevidade , Penaeidae , Organismos Livres de Patógenos Específicos , Fatores de Tempo
4.
Fish Shellfish Immunol ; 98: 522-533, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31911290

RESUMO

Troponin C (TnC) is one member of the EF-hand superfamily. In many species, this gene had been identified and related functions had been elucidated. The TnC gene was still blank in the Scylla paramamosain. We obtained the TnC gene for the first time in the S. paramamosain. And we systematically analyzed the possible role of this gene in the innate immunity of S. paramamosain while infected with white spot syndrome virus (WSSV) or Vibrio alginolyticus. The full-length 1427 bp sequence of TnC contains a 453 bp open reading frame (ORF) for encoding a 151 amino acid protein. Detection of tissue specificity of gene expression showed that the TnC was primarily expressed in muscle tissue. The expression of TnC was successfully inhibited by RNA interference technology, and several immune genes were affected. The activity of phenoloxidase and superoxide dismutase increased, and the total hemocytes counts increased after RNAi of TnC. It was found that after infection with V. alginolyticus and WSSV, the expression of TnC in hemocytes decreased. Infected with V. alginolyticus and WSSV, the cumulative mortality and apoptotic rate of hemocytes increased after silencing the TnC gene. Our results indicate that TnC takes participate in the innate immunity of S. paramamosain and may plays a different role in the antiviral and antibacterial immune response.


Assuntos
Braquiúros/microbiologia , Troponina C/metabolismo , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Braquiúros/metabolismo , Braquiúros/virologia , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , Troponina C/genética
5.
Fish Shellfish Immunol ; 98: 354-363, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31945483

RESUMO

L-type lectins (LTLs) belong to the lectin family and are characterized by a conserved structural motif in their carbohydrate recognition domain. LTLs are homologous to leguminous lectins. In this study, we identified and functionally characterized an LTL from kuruma shrimp Marsupenaeus japonicus. We designated this LTL as MjLTL2. MjLTL2 contains a signal peptide, a Lectin_leg domain, a coiled coil, and transmembrane domain. MjLTL2 is distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestine; higher expression levels are seen in hemocytes and the hepatopancreas than in other tissues. MjLTL2 was upregulated following challenge of shrimp with Vibrio anguillarum and white spot syndrome virus (WSSV). MjLTL2 can agglutinate several bacteria without Ca2+. In addition, MjLTL2 could bind to several Gram-positive and -negative bacteria by binding to their lipopolysaccharide and peptidoglycan. However, MjLTL2 could not enhance the clearance of V. anguillarum in vivo. In the presence of WSSV infection, MjLTL2 knockdown by RNA interference resulted in a 7-day lower cumulative mortality of M. japonicus. Moreover, less VP19, VP24, VP26, and VP28 mRNAs were extracted from the hemocytes of MjLTL2 knockdown shrimp than from the control. These results suggest that MjLTL2 is involved in immune responses in shrimp.


Assuntos
Proteínas de Artrópodes/metabolismo , Lectinas/metabolismo , Penaeidae/imunologia , Testes de Aglutinação , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Resistência à Doença/genética , Regulação da Expressão Gênica , Imunidade Inata , Lectinas/química , Lectinas/genética , Penaeidae/classificação , Penaeidae/genética , Filogenia , Polissacarídeos Bacterianos/metabolismo , Alinhamento de Sequência , Taxa de Sobrevida , Distribuição Tecidual , Vibrio/fisiologia , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia
6.
Fish Shellfish Immunol ; 98: 245-254, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31945484

RESUMO

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F1F0-ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation.


Assuntos
Proteínas de Artrópodes/metabolismo , Penaeidae/virologia , Proteínas/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Regulação da Expressão Gênica , Hemócitos/metabolismo , Mitocôndrias/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Penaeidae/classificação , Penaeidae/genética , Filogenia , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Superóxidos/metabolismo , Taxa de Sobrevida , Distribuição Tecidual , Replicação Viral/efeitos dos fármacos
7.
Fish Shellfish Immunol ; 98: 255-261, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31945486

RESUMO

Previous studies have indicated that white spot syndrome virus (WSSV) infection induces apoptosis in many shrimp organs. However, the mechanism by which WSSV causes host apoptosis remains largely unknown. In this study, we demonstrated the function of wsv152, the first mitochondrial protein identified as encoded by WSSV. Glutathione S-transferase pulldown and co-immunoprecipitation analysis revealed that wsv152 interacts with the shrimp mitochondrial protein cytochrome c oxidase 5a (COX5a), a subunit of the COX complex. We also found that wsv152 expression significantly increased the rate of apoptosis, suggesting a role of wsv152 in WSSV-induced apoptosis in shrimp. Knockdown of wsv152 in vivo led to downregulation of several apoptosis-related shrimp genes, including cytochrome c, apoptosis-inducing factor and caspase-3. Suppression of wsv152 also resulted in significant reductions in the number of WSSV genome copies in tissues and in the mortality of WSSV-infected shrimp. Together, these results suggest that wsv152 targets host COX5a and is associated with the expression profiles of apoptosis-related shrimp genes. Wsv152 is likely also involved in WSSV-induced apoptosis, thereby facilitating virus infection and playing a complex role in WSSV pathogenesis.


Assuntos
Apoptose/genética , Penaeidae/virologia , Proteínas Virais/metabolismo , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hemócitos/metabolismo , Hemócitos/patologia , Interações Hospedeiro-Patógeno , Mitocôndrias/metabolismo , Penaeidae/metabolismo , Ligação Proteica , Taxa de Sobrevida , Carga Viral , Proteínas Virais/genética , Vírus da Síndrome da Mancha Branca 1/patogenicidade
8.
Fish Shellfish Immunol ; 98: 236-244, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31953197

RESUMO

Astakine is a crucial factor in the proliferation and differentiation of hematopoietic stem cells and is directly involved in hematopoiesis in crustaceans. To assess the role of Astakine in the innate immune system of Scylla paramamosain, the immune responses in healthy and Astakine-inhibited S. paramamosain were investigated in the present study. The RNA transcripts of Astakine were widely distributed in all examined tissues, with significantly higher levels of expression in hemocytes of both healthy and challenged S. paramamosain with Vibrio alginolyticus and WSSV. When Astakine was knocked down by RNA interference technology, immune-related genes, including Janus kinase, prophenoloxidase, hemocyanin, ß-actin, myosin II essential light chain-like protein, signal transducer and activator of transcription, Relish, and C-type-lectin, were significantly down-regulated in hemocytes. The levels of phenoloxidaseactivity (PO), total hemocyte counts (THC) and hemocyte proliferation decreased significantly in hemocytes of Astakine-dsRNA treated S. paramamosain. After being challenged with V. alginolyticus and WSSV, the THC decreased significantly and the levels of hemocyte apoptosis increased significantly in Astakine-dsRNA treated S. paramamosain in comparison with those in infected groups without Astakine-dsRNA treatment. After being challenged with WSSV, the WSSV copies were significantly lower in Astakine-dsRNA treated groups than those in the WSSV infection group, which suggested that knockdown of Astakine was not conductive to WSSV replication and this might be associated with the decreasing THC. The results of survival analysis showed that the survival rate of V. alginolyticus or WSSV infected S. paramamosain decreased significantly following Astakine knockdown. These results suggested that RNA interference of Astakine might weaken the resistance of S. paramamosain to V. alginolyticus or WSSV infection. The weaken resistivity after knockdown Astakine might be related to the changes of important immune-related gene expression, THC, PO activity, proliferation and apoptosis of hemocytes.


Assuntos
Proteínas de Artrópodes/metabolismo , Braquiúros/microbiologia , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Apoptose , Proteínas de Artrópodes/genética , Braquiúros/imunologia , Braquiúros/virologia , Proliferação de Células , Resistência à Doença/genética , Regulação da Expressão Gênica/imunologia , Hemócitos/metabolismo , Hemócitos/patologia , Imunidade Humoral , Taxa de Sobrevida , Distribuição Tecidual , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Replicação Viral
9.
Fish Shellfish Immunol ; 98: 271-284, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31968265

RESUMO

The histone deacetylase, sirtuin 6 (SIRT6), plays an essential role in the regulation of oxidative stress, mitochondrial function and inflammation in mammals. However, the specific role of SIRT6 in invertebrate immunity has not been reported. Here, we characterized for the first time, a sirtuin 6 homolog in Litopenaeus vannamei (LvSIRT6), with full-length cDNA of 2919 bp and 1536 bp open reading frame (ORF) encoding a putative protein of 511 amino acids, which contains a typical SIR2 domain. Sequence and phylogenetic analysis revealed that LvSIRT6 shares a close evolutionary relationship with SIRT6 from invertebrates. Real-time quantitative PCR analysis of LvSIRT6 transcripts revealed that they were ubiquitously expressed in shrimp and induced in hepatopancreas and hemocytes upon challenge with Vibrio parahaemolyticus, Streptococcus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting the involvement of LvSIRT6 in shrimp immune response. Moreover, knockdown of LvSIRT6 decreased mitochondrial membrane potential and increased total ROS level in hemocytes, especially upon V. parahaemolyticus challenge. Depletion of LvSIRT6 also increased hemocytes apoptosis in terms of decreased expression of pro-survival LvBcl-2, but increased expression of pro-apoptotic LvBax and LvCytochrome C, coupled with high LvCaspase3/7 activity. Shrimp were rendered more susceptible to V. parahaemolyticus infection upon LvSIRT6 knockdown. Taken together, our present data suggest that LvSIRT6 plays an important role in shrimp immune response by modulating hemocytes ROS production and apoptosis during pathogen challenge.


Assuntos
Proteínas de Artrópodes/metabolismo , Hemócitos/metabolismo , Hemócitos/patologia , Penaeidae/imunologia , Sirtuínas/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Sequência de Bases , Clonagem Molecular , Resistência à Doença/genética , Regulação da Expressão Gênica , Fases de Leitura Aberta , Penaeidae/classificação , Penaeidae/microbiologia , Penaeidae/virologia , Filogenia , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência , Sirtuínas/química , Sirtuínas/genética , Streptococcus iniae/fisiologia , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
10.
Fish Shellfish Immunol ; 96: 319-329, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31805414

RESUMO

Viral immediate early (IE) genes encode regulatory proteins that are critical for viral replication. WSV056 is an IE protein of white spot syndrome virus (WSSV), an important pathogen of farmed shrimp. It targets the host Rb protein(s) and, according to a previous study, may enhance the replication of the viral genome. However, the ectopic expression of WSV056 in transgenic Drosophila melanogaster exerted an inhibitory effect on the replication of Drosophila C virus (DCV). Transcriptome study using Affymetrix GeneChip suggested that the enrichment of serine proteases (SPs) likely accounts for DCV inhibition in WSV056-overexpressing Drosophila. Injection of recombinant WSV056 to the WSSV natural host Litopenaeus vannamei enhanced the expression of the SP family member prophenoloxidase-activating enzyme 2 (LvPPAE2) and conferred shrimp with more resistance to WSSV infection. LvPPAE2 knockdown contributed to decreased expression of antimicrobial peptides LvAlf1 and LvLyz1, reduced hemolymph phenoloxidase activity, and increased virus load, suggesting that LvPPAE2 is involved in the host defense against WSSV infection. Taken together, these results suggest that wsv056 plays a role in restricting viral replication by inducing the SP-mediated immune responses in the host.


Assuntos
Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/imunologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Análise Serial de Proteínas
11.
Arch Virol ; 165(2): 407-412, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31811441

RESUMO

In this study, two aspects of the ultrastructure of white spot syndrome virus (WSSV) were identified: (i) The virus nucleocapsids were disassembled, and transmission electron microscopy (TEM) image analysis confirmed that the nucleocapsids were composed of stacked ring segments rather than the usual helix system, with each ring segment consisting of three rows of subunits linked by filaments. (ii) In addition, the morphological characteristics of virus self-assembly at different stages were observed, and two different enveloping morphologies were found, implying that the virion matures through two distinct envelopments. Thus, we propose a viral membrane assembly process for WSSV virion.


Assuntos
Nucleocapsídeo/ultraestrutura , Montagem de Vírus , Vírus da Síndrome da Mancha Branca 1/fisiologia , Vírus da Síndrome da Mancha Branca 1/ultraestrutura , Microscopia Eletrônica de Transmissão
12.
Dev Comp Immunol ; 102: 103476, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445053

RESUMO

White spot syndrome (WSS) caused by white spot syndrome virus (WSSV) is a severe infectious disease in shrimp aquaculture. To find effective therapeutics to control WSSV, it is indispensable to understand the innate immune responses of shrimp to WSSV infection. Previous report demonstrated that the Litopenaeus vannamei heat shock protein 70 (LvHSP70) could induce shrimp innate immunity against bacterial infection. Herein, we further investigate the role of LvHSP70 in anti-WSSV infection. The temporal expression of LvHSP70 was significantly upregulated 2.5- and 1.5-fold at 6 and 24 h post systemic WSSV infection suggesting that the LvHSP70 was a WSSV responsive gene. The recombinant protein of LvHSP70 (rLvHSP70) was produced in an Escherichia coli system and its effect in protection against WSSV infection was investigated. Intramuscularly injection of juvenile shrimp with 1 nmol of rLvHSP70 could significantly prolong 50% mortality of WSSV-infected shrimp from 3 days to 5 days as compared to the control group injected with bovine serum albumin (BSA). Consistently, the injection of rLvHSP70 resulted in 24-fold, 20-fold and 100-fold decrease in the viral copy number after 6, 12 and 24 h post injection, respectively, compared to the control shrimp injected with BSA. Interestingly, it was found that the rLvHSP70 enhanced the expression of the key gene in the prophenoloxidase (proPO) activating system, LvproPO, but reduced the expression of Lvcaspase2 and LvIAP in WSSV-infected shrimp. These results suggested that the LvHSP70 is an important molecule involved in antiviral defense in shrimp presumably via modulating the proPO system and apoptosis.


Assuntos
Proteínas de Artrópodes/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Penaeidae/imunologia , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Apoptose , Proteínas de Artrópodes/administração & dosagem , Proteínas de Artrópodes/genética , Catecol Oxidase/genética , Resistência à Doença/genética , Precursores Enzimáticos/genética , Regulação da Expressão Gênica/imunologia , Proteínas de Choque Térmico HSP70/administração & dosagem , Proteínas de Choque Térmico HSP70/genética , Hemócitos/imunologia , Hemócitos/virologia , Imunidade Inata/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/genética , Taxa de Sobrevida , Regulação para Cima/genética
13.
Dev Comp Immunol ; 102: 103491, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494218

RESUMO

As the most productive crustacean species in aquaculture, Litopenaeus vannamei is seriously threatened by white spot syndrome virus (WSSV), which has caused huge economic damage in the past decades. Shrimp cuticle proteins are the important components in the frontier target tissues, including cuticle and the chitinous lining of the digestive tract. In present study, a novel cuticle protein gene, named LvCPAP1, was isolated and demonstrated to play an important role in WSSV infection. The deduced amino acid sequence of LvCPAP1 contained a signal peptide and a conserved chitin-binding domain type 2 (ChBD2). Tissue distribution analysis revealed that LvCPAP1 was predominantly expressed in epidermis and stomach. The transcription levels of LvCPAP1 in epidermis and stomach were significantly regulated upon WSSV challenge. DsRNA silencing of LvCPAP1 decreased the in vivo WSSV copy numbers and the death rate of shrimp after WSSV infection, indicating that LvCPAP1 might facilitate WSSV invasion. In addition, the interaction between LvCPAP1 and the major envelop protein VP24 of WSSV was revealed by yeast two-hybrid system and further confirmed by dot blot and pull-down assays. The present study implied that cuticle protein LvCPAP1 might favor the entry process of WSSV, which provided new clues for understanding the role of cuticle proteins during virus infection.


Assuntos
Proteínas de Artrópodes/metabolismo , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Quitina/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Fases de Leitura Aberta , Penaeidae/metabolismo , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sinais Direcionadores de Proteínas , Frutos do Mar/virologia , Taxa de Sobrevida , Distribuição Tecidual , Proteínas do Envelope Viral/metabolismo , Carga Viral
14.
Fish Shellfish Immunol ; 95: 227-235, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31654766

RESUMO

Myeloid differentiation factor 88 (MyD88) is a universal and essential adaptor protein required for the Toll-like receptors (TLRs) pathway activation in invertebrates as well as in vertebrates. Herein, we characterized a MyD88 (Pt-MyD88) cDNA sequence in the swimming crab (Portunus trituberculatus). The Pt-MyD88 ORF is predicted to encode 469 peptides with an N-terminal death domain and a typical C-terminal TIR domain. Real-Time quantitative PCR analysis showed that the Pt-MyD88 transcriptions were constitutively expressed in hemocytes, gill, intestine, heart and muscle in normal crab. The expressions of Pt-MyD88 would be down-regulated by V. alginolyticus or LPS challenge, and be up-regulated by WSSV infection in hemocytes. Intracellular localization showed Pt-MyD88 was distributed mainly in the cytoplasm when it was over-expressed in human cell HEK293T or in Drosophila Schneider 2 (S2). Functionally, over-expression of Pt-MyD88 could either activate the NF-κB in HEK293T cells or activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. In primary cultured hemocytes of swimming crab, after Pt-MyD88 was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), hyastatin3, crustin1 and crustin3 have been significantly inhibited, while the expression of other AMPs is normal compared to non-specific dsRNA treated cells.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Linhagem Celular , Regulação para Baixo/imunologia , Drosophila , Feminino , Células HEK293 , Hemócitos/imunologia , Humanos , Lipopolissacarídeos/fisiologia , Masculino , Modelos Animais , Fator 88 de Diferenciação Mieloide/química , Filogenia , Regulação para Cima/imunologia , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
15.
J Fish Dis ; 42(11): 1471-1491, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31637760

RESUMO

Samples from multiple animals may be pooled and tested to reduce costs of surveillance for infectious agents in aquatic animal populations. The primary advantage of pooling is increased population-level coverage when prevalence is low (<10%) and the number of tests is fixed, because of increased likelihood of including target analyte from at least one infected animal in a tested pool. Important questions and a priori design considerations need to be addressed. Unfortunately, pooling recommendations in disease-specific chapters of the 2018 OIE Aquatic Manual are incomplete and, except for amphibian chytrid fungus, are not supported by peer-reviewed research. A systematic review identified only 12 peer-reviewed aquatic diagnostic accuracy and surveillance studies using pooled samples. No clear patterns for pooling methods and characteristics were evident across reviewed studies, although most authors agreed there is a negative effect on detection. Therefore, our purpose was to review pooling procedures used in published aquatic infectious disease research, present evidence-based guidelines, and provide simulated data examples for white spot syndrome virus in shrimp. A decision tree of pooling guidelines was developed for use by peer-reviewed journals and research institutions for the design, statistical analysis and reporting of comparative accuracy studies of individual and pooled tests for surveillance purposes.


Assuntos
Crustáceos/virologia , Testes Diagnósticos de Rotina/normas , Monitoramento Epidemiológico/veterinária , Doenças dos Peixes/epidemiologia , Guias como Assunto , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Doenças Transmissíveis/epidemiologia , Doenças Transmissíveis/veterinária , Vigilância da População/métodos , Prevalência
16.
Fish Shellfish Immunol ; 94: 907-915, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31604147

RESUMO

Previous studies have demonstrated that white spot syndrome virus (WSSV) could induce hemocytes apoptosis in shrimps, however the inter-relationship between apoptotic process and the WSSV infection status is still currently underexplored. In the present work, the apoptosis and the viral proliferation in hemocytes of Litopenaeus vannamei were simultaneously investigated post WSSV infection by two-color immunofluorescence flow cytometry and real-time quantitative PCR. The apoptotic hemocytes of WSSV-infected shrimp was significantly increased at 12 h post infection (hpi), whereas underwent a slight decline at 24 hpi subsequently. Since 24 hpi, the apoptotic rate of hemocytes in the WSSV-infected shrimp exhibited a rapid and significant increase, and reached the peak level at 48 hpi with the ratio of 18.1 ±â€¯2.0%. Meanwhile, the percentage of WSSV-infected hemocytes and WSSV copies in hemocytes significantly increased at 24 hpi and maintained at a high level afterwards. With the rapid increase of hemocytes apoptosis, hemocyte density in hemolymph decreased dramatically to less than 20% of the mean value of control. Co-localization assay showed that the apoptotic WSSV-infected hemocytes occupied the dominant proportion of total apoptotic hemocytes, which reached the peak at 48 hpi with 12.6 ±â€¯1.5%. The expression profiles of seven pro-apoptotic genes and two apoptosis-inhibiting genes showed significant differential responses at different stages of WSSV infection, reflecting the interplay between the virus and the host immune response. Our results demonstrated that the apoptotic response of shrimp hemocytes could be significantly influenced by the WSSV infection process, which might provide an insight into deeper relationships between viral infection and apoptosis.


Assuntos
Apoptose , Proteínas de Artrópodes/genética , Hemócitos/imunologia , Penaeidae/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/imunologia
17.
Fish Shellfish Immunol ; 94: 852-860, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31600594

RESUMO

Bcl-2 associated athanogene-1 (BAG1) is involved in various signalling pathways including apoptosis, cell proliferation, gene transcriptional regulation and signal transduction in animals. However the functions of BAG1 during the antiviral response of mud crab Scylla paramamosain is still unclear. In this study, the mud crab BAG1 (SpBAG1) was characterized to consist of 1761 nucleotides, containing an opening frame of 630bp encoding 209 amino acids with an ubiquitin domain and a BAG1 domain. SpBAG1 was found to be significantly up-regulated at 6 h-24 h, but down-regulated from 48 h-72 h in the hemocytes of mud crab after challenge with white spot syndrome virus (WSSV). RNAi knock-down of SpBAG1 significantly reduced the copies of WSSV and increased the apoptotic rate in mud crabs. The finding from this study suggested that SpBAG1 could promote the WSSV infection by inhibiting apoptosis in mud crab. Therefore, to the best of our knowledge, this is the first study demonstrating the role of SpBAG1 as a novel apoptosis inhibitor to promote virus infection in mud crab.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/imunologia , Sequência de Aminoácidos , Animais , Apoptose , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Proteína de Morte Celular Associada a bcl/química
18.
Fish Shellfish Immunol ; 94: 434-446, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31536767

RESUMO

Carboxypeptidase plays an important physiological role in the tissues and organs of animals. In this study, we cloned an entire 2316 bp carboxypeptidase B-like (CPB) sequence with a 1302 bp open reading frame encoding a 434 amino acid peptide from Scylla paramamosain. The CPB gene was expressed highly in hepatopancreas and decreased in crab hemocytes after challenges with white spot syndrome virus (WSSV) or Vibrio alginolyticus. After CPB gene knockdown using double-stranded RNA (CPB-dsRNA), the expression of JAK, STAT, C-type lectin, crustin antimicrobial peptide, Toll-like receptors, prophenoloxidase, and myosin II essential light chain-like protein were down-regulated in hemocytes at 24 h post dsRNA treatment. CPB knockdown decreases total hemocyte count in crabs indicated that CPB may negatively regulate crab hemocyte proliferation in crabs. CPB showed an inhibitory effect on hemocyte apoptosis in crabs infected with WSSV or V. alginolyticus. The phagocytosis rate of WSSV by hemocytes was increased after CPB-dsRNA treatment. After WSSV challenge, the mortality and WSSV copy number were both decreased but the rate of hemocyte apoptosis was increased in CPB-dsRNA-treated crabs. The results indicate that the antiviral activity of the crabs was enhanced when CPB was knocked down, indicating WSSV may take advantage of CPB to benefit its replication. In contrast, the absence of CPB in crabs increased mortality following the V. alginolyticus challenge. The phagocytosis rate of V. alginolyticus by hemocytes was increased after CPB-dsRNA treatment. It was revealed that CPB may play a positive role in the immune response to V. alginolyticus through increasing the phagocytosis rate of V. alginolyticus. This research further adds to our understanding of the CPB and identifies its potential role in the innate immunity of crabs.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Carboxipeptidase B/genética , Carboxipeptidase B/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Carboxipeptidase B/química , Perfilação da Expressão Gênica , Hemócitos/imunologia , Fagocitose , Filogenia , Distribuição Aleatória , Alinhamento de Sequência , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
19.
Ecotoxicol Environ Saf ; 184: 109626, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31536848

RESUMO

Of late, Pacific white shrimp Penaeus vannamei culture has intensified globally and is a major contributor to the cultured shrimp produced worldwide. Intensification of its culture has led to elevated ammonia concentration during grow-out. Ammonia toxicity is a function of water pH, temperature, salinity and beyond the optimum range, creates stress to cultured aquatic species which can reduce growth, increase susceptibility to diseases and eventually mortality. The present study was aimed at quantifying the toxic effect of total ammonia nitrogen (TAN) (1, 3, 6 & 9 mg/l) and pH levels (6, 8 & 10) individually and in combination on median survival (50% lethal time) of shrimp (8 g) after exposure for 14 days followed by post-stress challenge with white spot syndrome virus (WSSV) for 9 days. Mortality risk factor and the toxicity effect on the immune variables were evaluated. Individual stressors showed a risk factor of 1-13 times, whereas combined treatments considerably increased the risk of dying compared to control. Low survival (15%) was observed in pH6TAN9 and pH10TAN3 treatments and was substantiated by prominent histological obliteration in gills of shrimp. The cumulative mortality in post-stress WSSV challenged trials was 1-5 times and 1-35 times in individual and combination treatments, respectively compared to control. The study revealed that variations in ammonia and pH beyond the optimal range significantly influence the non-specific immune mechanisms in P.vannamei and increases the susceptibility to WSSV especially in combination treatments.


Assuntos
Amônia/toxicidade , Penaeidae/efeitos dos fármacos , Penaeidae/imunologia , Estresse Salino , Poluentes Químicos da Água/toxicidade , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Concentração de Íons de Hidrogênio , Compostos de Nitrogênio/toxicidade , Penaeidae/virologia , Estresse Salino/imunologia , Análise de Sobrevida
20.
Fish Shellfish Immunol ; 94: 417-426, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31491531

RESUMO

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway as a negative regulator in mammals and Drosophila, but the function of PIAS in crustaceans is still unclear. In this study, a PIAS termed LvPIAS was cloned and characterized from Litopenaeus vannamei. The full length of LvPIAS was 3065 bp, including a 2361 bp open reading frame (ORF) coding for a protein of 786 aa. LvPIAS expression was most abundant in muscle and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly I: C and white spot syndrome virus (WSSV). LvPIAS could be induced by the transcription factor LvSTAT, but LvPIAS could inhibit the transcriptional activity of LvSTAT to the LvPIAS promoter conversely, which indicated that there was a negative feedback loop between LvSTAT and LvPIAS. Furthermore, RNAi-mediated knockdown of LvPIAS shrimps showed higher survival rate to WSSV infection than those in the control group (dsGFP injection), suggesting that LvPIAS may play a negatively role against WSSV infection.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Proteínas Inibidoras de STAT Ativados/química , Alinhamento de Sequência , Staphylococcus aureus/fisiologia , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
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