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1.
Fish Shellfish Immunol ; 90: 126-133, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059814

RESUMO

To investigate the role of the Rab7 effector RILP (Rab-interacting lysosomal protein) in white spot syndrome virus (WSSV) infection, the full-length cDNA of RILP (LvRILP) was cloned in Litopenaeus vannamei, which consists of 1595 bp and encodes a polypeptide of 411 amino acids. Sequence analysis and multiple sequence alignment displayed that LvRILP contained a conserved RILP region from 277 amino acid to 325 amino acid. Both the LvRILP and Rab7 mRNA were most highly expressed in stomach and most lowly expressed in hemocyte, which were significantly up-regulated and exhibited similar kinetics post WSSV infection. The interaction of Rab7 with LvRILP was verified by both GST Pull-down and ELISA. Meanwhile, the results of Pull-down assays showed that the GST-tagged VP28 (GST-VP28), His-tagged Rab7 (His-Rab7) and His-RILP formed a tripartite complex. After silencing by specific LvRILP dsRNA, the LvRILP mRNA level exhibited a significant reduction, and the expression levels of three WSSV genes ie1, wsv477 and vp28 all exhibited decreases at 24, 36 and 48 h post WSSV infection. These results suggested that the Rab7 effector RILP was involved in WSSV infection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Vírus da Síndrome da Mancha Branca 1/fisiologia
2.
Fish Shellfish Immunol ; 89: 458-467, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30954523

RESUMO

White spot disease (WSD) is a highly virulent viral disease in shrimps. Clinical signs and high mortality of WSD is generally observed after a few days of infection by White Spot Syndrome virus (WSSV). Mud crabs are the major carrier and persistent host for the WSSV. However, an elucidation of viral interaction and persistent mode of WSSV infection in mud crab is still limited. We investigated the defensive role of mud crab (Scylla olivacea) hemocytes against WSSV infection by using comparative proteomic analysis coupled with electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS). The proteomic maps of expressed proteins obtained from WSSV infected hemocytes revealed differential proteins related to various biological functions, including immune response, anti-apoptosis, endocytosis, phosphorylation signaling, stress response, oxygen transport, molting, metabolism, and biosynthesis. Four distinctive cell types of crab hemocytes: hyaline cells (HC), small granular cells (SGC), large granular cells (LGC) and mixed granular cells (MGC) were found susceptible to WSSV. However, immunohistochemistry analysis demonstrated a complete replication of WSSV only in SGC and LGC. WSSV induced apoptosis was also observed in HC, SGC and MGC except for LGC. These results suggested that HC and MGC may undergo apoptosis prior to a complete assembly of virion, while SGC is more susceptible showing higher amplification and releasing of virion. In contrast, WSSV may inhibit apoptosis in infected LGC to stay in latency. This present finding provides an insight for the responsive roles of crustacean hemocyte cells involved in molecular interaction and defense mechanism against WSSV.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/genética , Hemócitos/imunologia , Proteoma/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Braquiúros/imunologia , Cromatografia Líquida , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
3.
Fish Shellfish Immunol ; 89: 555-563, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999041

RESUMO

In shrimp, the JAK-STAT pathway is essentially implicated in both antiviral and antibacterial responses. However, few regulatory target genes of the JAK-STAT pathway in shrimp have been reported so far. In this study, a novel single WAP domain-containing peptide (LvSWD4) was identified from Pacific white shrimp Litopenaeus vannamei. The promoter of LvSWD4 was predicted to harbor multiple STAT-binding DNA motifs. Over-expression of the JAK-STAT pathway components STAT, JAK and Domeless in vitro significantly enhanced the transcriptional activity of the LvSWD4 promoter, and in vivo silencing of STAT and the the JAK-STAT pathway upstream regulator IRF down-regulated the expression of LvSWD4, suggesting that LvSWD4 could be a target gene of the JAK-STAT pathway. The expression of LvSWD4 was significantly increased after infection with Gram-negative and positive bacteria, fungi and virus, and silencing of LvSWD4 increased the susceptibility of shrimp to V. parahaemolyticus and WSSV infections. In vitro experiments also demonstrated that the recombinant LvSWD4 protein had significant inhibitory activities against Gram negative bacteria V. parahaemolyticus and E. coli and Gram positive bacteria S. aureus and B. subtilis. Furthermore, silencing of LvSWD4 in vivo significantly affected expression of various immune functional genes and attenuated the phagocytic activity of hemocytes. These suggested that as a target gene of STAT, LvSWD4 was essentially implicated in shrimp immunity, which could constitute part of the mechanism underlying the immune function of the shrimp JAK-STAT pathway.


Assuntos
Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Transdução de Sinais , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
4.
Fish Shellfish Immunol ; 88: 1-8, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30826412

RESUMO

In this study, GST Pull-down and mass spectrometry was applied to the precipitation and identification of the small GTP-binding protein (Rab7) interacting protein in hemocyte of Litopenaeus vannamei. According to the search in GenBank with the peptide mass fingerprint, the 45 kDa protein which was pulled down with the GST-tagged Rab7 (GST-Rab7, GTP bound form) was identified to be ß-actin with 28% coverage of amino acid sequences. The interaction of Rab7 with ß-actin was verified by both GST Pull-down and ELISA in vitro. Meanwhile, confocal microscopic observation showed that Rab7 could be co-localized with ß-actin in hemocytes at 12 h post white spot syndrome virus (WSSV) infection (hpi). GST Pull-down and western blotting were used to analyze the cross-interaction between WSSV VP28, Rab7 and ß-actin. The results showed that the GST-VP28, His-tagged Rab7 (His-Rab7) and His-ß-actin formed a tripartite complex. At 12 hpi, confocal microscopic observation showed that WSSV could be co-localized with Rab7 and ß-actin in hemocytes respectively. Furthermore, based on the in vivo neutralization assay, recombinant His-ß-actin accelerated the infection of WSSV, conversely, recombinant His-Rab7 delayed WSSV infection in shrimp. These results suggested the interaction of Rab7 with ß-actin and this interaction was involved in WSSV infection.


Assuntos
Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Actinas , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/metabolismo , Hemócitos/virologia , Microscopia Confocal , Penaeidae/metabolismo
5.
Fish Shellfish Immunol ; 88: 47-52, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30831245

RESUMO

Herein, we evaluated the immunomodulatory and the antiviral protective properties of a cyanobacteria-enriched diet on the immune responses of the Pacific white shrimp Litopenaeus vannamei challenged with the White spot syndrome virus (WSSV). Shrimp were fed with an Arthrospira platensis supplemented feed during 20 days, and its effects were examined by evaluating well-known standardized shrimp immune parameters (total hemocyte counts, total protein concentration, phenoloxidase activity, and serum agglutination titer). Additionally, we assessed the expression of crucial genes involved in both hemolymph- and gut-based immunities related to the shrimp capacity to circumvent viral and microbial infections. Dietary supplementation improved shrimp survival rates after challenge with a median lethal dose of WSSV. From all immune parameters tested, only the serum agglutination titer was higher in treated animals. On the other hand, the expression of some representative marker genes from different immune response pathways was only modulated in the midgut and not in the circulating hemocytes, suggesting that this feed supplementation can be used as an attractive strategy to enhance immunity in shrimp gut. Altogether, our results evidence the immunomodulatory properties of A. platensis supplemented feed in shrimp humoral and intestinal defenses and highlight the potential use of cyanobacteria-based immunostimulants in shrimp farming for protection against infectious diseases.


Assuntos
Ração Animal/análise , Penaeidae/imunologia , Spirulina , Adjuvantes Imunológicos , Animais , Aquicultura/métodos , Dieta/veterinária , Expressão Gênica , Hemolinfa/imunologia , Intestinos/imunologia , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
6.
Fish Shellfish Immunol ; 87: 755-764, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30790658

RESUMO

Vibrio parahaemolyticus and white spot syndrome virus (WSSV) are pathogens that cause epidemics in kuruma shrimp (Marsupenaeus japonicus) during aquaculture, resulting in severe economic losses to local farmers. To characterise the mechanisms of the molecular responses to V. parahaemolyticus and WSSV infection in M. japonicus, the transcriptome of hepatopancreas was sequenced using next-generation sequencing after infection. A total of 29,180 unigenes were assembled, with an average length of 1,151 bp (N50 = 1,951 bp). After BLASTX searching against the Nr database (E-value cut-off = 10-5), 15,176 assembled unigenes remained, with 3,039 and 1,803 differentially expressed transcripts identified in the V. parahaemolyticus- and WSSV-infected groups, respectively. Of these, 1466 transcripts were up-regulated and 1573 were down-regulated in V. parahaemolyticus-infected shrimps, and 970 transcripts were up-regulated and 833 were down-regulated in the WSSV-infected shrimps. Additionally, 761 transcripts were differentially expressed in both V. parahaemolyticus- and WSSV-infected shrimps. Several known immune-related genes including caspase 4, integrin, crustin, ubiquitin-conjugating enzyme E2, C-type lectin, and α2-macroglobulin were among the differentially expressed transcripts. These results provide valuable information for characterising the immune mechanisms of the shrimp responses of to V. parahaemolyticus andWSSV infection.


Assuntos
Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Hepatopâncreas/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Transcriptoma/imunologia , Animais , Perfilação da Expressão Gênica , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
7.
Fish Shellfish Immunol ; 88: 161-169, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30802628

RESUMO

Lysozyme is an important defense molecule of the innate immune system and possess high antimicrobial activities. In this study, a full-length c-type lysozyme cDNA (Fplysc) was cloned and characterized from Fenneropenaeus penicillatus. The cDNA contains an open reading frame of 477 bp encoding 158 amino acids, with 53-94% identity with those of other crustaceans. The recombinant Fplysc had antibacterial activities against Gram-positive bacteria (Streptococcus agalactiae and Micrococcus luteus) and Gram-negative bacteria (Vibrio alginolyticus and Escherichia coli), and showed antiviral activity against WSSV and IHHNV. The qRT-PCR analysis showed that Fplysc expression levels were most abundant in hemocytes and less in eyestalk. The expression levels of Fplysc were significantly upregulated in gill, intestine and hemocytes when challenged with WSSV and V. alginolyticus. Fplysc-silencling suppressed Fplysc expression in cephalothoraxes and increased mortality caused by WSSV and V. alginolyticus, and exogenous rFplysc led to a significant decrease of shrimp mortality by injecting rFplysc into Fplysc silenced shrimp, suggesting Fplysc is the important molecule in shrimp antimicrobial and antiviral response. In conclusion, the results provide some insights into the function of Fplysc in shrimp against bacterial and viral infection.


Assuntos
Proteínas de Artrópodes/imunologia , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Clonagem Molecular , Densovirinae/fisiologia , Escherichia coli/fisiologia , Hemócitos , Imunidade Inata , Micrococcus luteus/fisiologia , Muramidase/química , Muramidase/genética , Muramidase/metabolismo , Penaeidae/genética , Penaeidae/microbiologia , Penaeidae/virologia , Streptococcus agalactiae/fisiologia , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
8.
J Fish Dis ; 42(4): 497-510, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30742312

RESUMO

The replication profile of white spot syndrome virus (WSSV) in crayfish, Procambarus clarkii, at different water temperature was investigated in this study. The WSSV detections were negative at 15 ± 1°C, and the natural infection ratio increased at 19 ± 1°C (24.2% ± 2.25%), reached 100% at 25 ± 1°C and decreased at 30 ± 1°C (93.2% ± 3.37%). The WSSV genome copies number was much higher at 25 ± 1°C (≥5 × 106.45 ± 0.35 /mg) than at 15 ± 1°C (≤5 × 101.13 ± 0.12 /mg), 19 ± 1°C (≤5 × 102.74 ± 0.48 /mg) and 32 ± 1°C (≤5 × 103.18 ± 0.27 /mg). Meanwhile, the significant transcription signals of immediate early gene ie1 and late gene vp28 and a large number of virus particles were detected in epitheliums of stomach, gut and gill, hepatopancreas, heart and muscle cells at 25 ± 1°C by using in situ hybridization (ISH) and transmission electron microscopy. The experimental infection of P. clarkii with WSSV infection showed reduced mortality and lower virus copies number at 19 ± 1°C (23.51% ± 0.84%, ≤5 × 103.41 ± 0.11 /mg) and 32 ± 1°C (38.42% ±  1.21%, ≤5 × 103.72 ± 0.13 /mg) compared to 25 ± 1°C (100%, ≥5 × 104.99 ± 0.24 /mg). The water temperature regulated the transcription of immune-related genes (crustin2, prophenoloxidase (proPO) and heat shock protein70 (Hsp70)), with some differences between WSSV treatments and control treatments. These results demonstrate that water temperature has effect on WSSV proliferation, which may due to transcriptional response of immune-related genes to temperature.


Assuntos
Astacoidea/virologia , Infecções por Vírus de DNA/veterinária , Temperatura Ambiente , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Infecções por Vírus de DNA/virologia , Alimentos Marinhos/virologia , Ativação Transcricional
9.
Fish Shellfish Immunol ; 87: 297-306, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30682407

RESUMO

Relish is a transcription factor and forms an important part of the immune deficiency signaling pathway. In the current study, a Relish homolog was cloned from the hemolymph of Scylla paramamosain using RT-PCR and RACE. The full length cDNA of Relish consists of 4263 base pairs (bp), including a 3552 bp open reading frame encoding a 1184 amino acid protein. The data showed that Relish was highly expressed in the gonad and digestive organs of S. paramamosain. Furthermore, the expression of Relish was up-regulated by infection with white spot syndrome virus (WSSV) or Vibrio alginolyticus. When Relish was knocked down, immune genes such as Janus Kinase, signal transducer and activator of transcription, crustin antimicrobial peptide, prophenoloxidase, C-type-lectin and myosin-II-essential-light-chain-like-protein were significantly down-regulated (P < 0.01), and Toll-like receptor was significantly up-regulated (P < 0.01) in hemocytes. The mortality of WSSV-infected or V. alginolyticus-infected crabs was enhanced following Relish knockdown. Thus, Relish is very important in the progression of WSSV and V. alginolyticus infection. It was found that Relish knockdown caused the highest level of apoptosis in the disease-free group, and higher levels of apoptosis in the WSSV group and V. alginolyticus group compared with that in the control group. Knockdown of Relish influenced the activity of phenoloxidase (PO) and superoxide dismutase (SOD), and total hemocyte count (THC) following WSSV or V. alginolyticus infection, indicating that Relish plays a regulatory role in the immune response to WSSV or V. alginolyticus infection in crabs. Thus, we conclude that Relish may anticipate host defense mechanisms against pathogen infection by affecting apoptosis, THC, PO activity and SOD activity.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Fatores de Transcrição/química , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
10.
Fish Shellfish Immunol ; 86: 882-891, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30553892

RESUMO

Ferritin is a protein related to the storage of iron and widely distributed in animals. It participates in many biological process, including antioxidation, cell activation, angiogenesis, regulation of iron metabolic balance and immune defense. In the present study, a novel ferritin gene was identified from red swamp crayfish Procambarus clarkii, with a cDNA sequence encoding a predicted 221 amino-acid residues. The ferritin protein contains a 19-residue signal peptide and 145-residue classic ferritin domain. The NJ phylogenetic analysis showed PcFer clustered with other crustacean peptides. The recombinant PcFer protein was produced and purified in E. coli, and the anti-rabbit polyclonal antibody was obtained. The rPcFer exhibited iron binding activity at a dose-dependent effect. The qPCR and western blot analysis revealed that PcFer was highly expressed in hemocytes, hepatopancreas, and gills. After challenged with WSSV and Aeromonas hydrophila, the mRNA and protein expression patterns of PcFer were significantly up-regulated in hemocytes and hepatopancreas. dsRNA interfering technique was utilized to silence the expression of PcFer gene. The WSSV copy number in PcFer silenced shrimp was much higher than that in the control group. The present study indicated that PcFer was involved in the immune defense against WSSV and Aeromonas hydrophila, and might inhibit WSSV replication in P. clarkii. These results will provide important data support for further study of the functional role of the ferritin gene.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Ferritinas/genética , Ferritinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Ferritinas/química , Perfilação da Expressão Gênica , Brânquias/metabolismo , Hemócitos/metabolismo , Hepatopâncreas/metabolismo , Filogenia , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia
11.
Dev Comp Immunol ; 90: 1-9, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30031870

RESUMO

Lectin is a protein with multiple functions. In this study, the full-length cDNA of the Agrocybe aegerita lectin (AAL) gene was cloned, recombinant AAL (AAL-His) was expressed, and the activities of AAL-His were analyzed. Northern blot analysis showed that the major AAL transcript is approximately 900 bp. Sequence analysis showed that the coding region of AAL is 489 bp with a transcription start site located 39 nucleotides upstream of the translation initiation codon. In an agglutination test, AAL-His agglutinated rabbit erythrocytes at 12.5 µg/ml. AAL-His also showed antiviral activity in protecting shrimp from white spot syndrome virus (WSSV) infection. This anti-WSSV effect might be due to the binding of AAL-His on WSSV virions via the direct interactions with four WSSV structural proteins, VP39B, VP41B, VP53A and VP216. AAL demonstrates the potential for development as an anti-WSSV agent for shrimp culture. It also implies that these four AAL interaction WSSV proteins may play important roles in virus infection.


Assuntos
Agrocybe/genética , Antígenos de Fungos/genética , Infecções por Vírus de DNA/imunologia , Lectinas/genética , Penaeidae/imunologia , Transgenes/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Antivirais/metabolismo , Clonagem Molecular , Agregação Eritrocítica , Imunidade Inata , Lectinas/metabolismo , Penaeidae/virologia , Ligação Proteica , Proteínas Virais/metabolismo
12.
Dev Comp Immunol ; 90: 130-137, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30227218

RESUMO

Argonaute (Ago) proteins, the catalytic component of an RNA-induced silencing complex (RISC) in RNA interference pathway, function in diverse processes, especially in antiviral defense and transposon regulation. So far, cDNAs encoding four members of Argonaute were found in Penaeus monodon (PmAgo1-4). Two PmAgo proteins, PmAgo1 and PmAgo3 shared high percentage of amino acid identity to Ago1 and Ago2, respectively in other Penaeid shrimps. Therefore, the possible roles of PmAgo1 and PmAgo3 upon viral infection in shrimp were characterized in this study. The level of PmAgo1 mRNA expression in shrimp hemolymph was stimulated upon YHV challenge, but not with dsRNA administration. Interestingly, silencing of either PmAgo1 or PmAgo3 using sequence-specific dsRNAs impaired the efficiency of PmRab7-dsRNA to knockdown shrimp endogenous PmRab7 expression. Inhibition of yellow head virus (YHV) replication and delayed mortality rate were also observed in both PmAgo1-and PmAgo3-knockdown shrimp. In addition, silencing of PmAgo3 transcript, but not PmAgo1, revealed partial inhibition of white spot syndrome virus (WSSV) infection and delayed mortality rate. Therefore, our study provides insights into PmAgo1and PmAgo3 functions that are involved in a dsRNA-mediated gene silencing pathway and play roles in YHV and WSSV replication in the shrimp.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Artrópodes/metabolismo , Hemolinfa/metabolismo , Infecções por Nidovirales/imunologia , Penaeidae/imunologia , Roniviridae/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Antivirais/metabolismo , Proteínas Argonauta/genética , Proteínas de Artrópodes/genética , Clonagem Molecular , Elementos de DNA Transponíveis/genética , Imunidade Inata , Interferência de RNA , RNA Interferente Pequeno/genética , Replicação Viral
13.
Dev Comp Immunol ; 91: 50-61, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30339874

RESUMO

Recent studies have shown that hemocyanin plays immune-related functions apart from its canonical respiratory function. While shrimp hemocyanin is found to generate antimicrobial peptides, antiviral related peptides have not been reported. In the present study, the serum of white spot syndrome virus (WSSV) infected Litopenaeus vannamei analyzed by two-dimensional gel electrophoresis, revealed 45 consistently down-regulated protein spots and 10 up-regulated protein spots. Five of the significantly up-regulated spots were identified as hemocyanin derived peptides. One of the five peptides, designated LvHcL48, was further characterized by analyzing its primary sequence via Edman N-terminal sequencing, C-terminal sequencing and amino acid sequence alignment. LvHcL48 was found to be a 79 amino acid fragment (aa584-662) from the C-terminal domain of L. vannamei hemocyanin protein (ADZ15149). Both in vivo and in vitro functional studies revealed that LvHcL48 has immunological activities, as recombinant LvHcL48 protein (rLvHcL48) significantly inhibited the transcription of the WSSV genes wsv069 and wsv421 coupled with a significant reduction in WSSV copy numbers. Further analysis showed that LvHcL48 could interact with the WSSV envelope protein 28 (VP28). Our present data therefore reveals the generation of an antiviral hemocyanin derived peptide LvHcL48 from WSSV infected shrimp, which binds to the envelope protein VP28 of WSSV.


Assuntos
Antivirais/imunologia , Proteínas de Artrópodes/imunologia , Infecções por Vírus de DNA/imunologia , Hemocianinas/imunologia , Penaeidae/imunologia , Peptídeos/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Clonagem Molecular , Imunidade Inata , Penaeidae/virologia , Ligação Proteica , Ativação Transcricional , Proteínas do Envelope Viral/metabolismo , Replicação Viral
14.
Dev Comp Immunol ; 91: 101-107, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385317

RESUMO

Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Caspases/genética , Infecções por Vírus de DNA/imunologia , Hemócitos/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Astacoidea/virologia , Caspases/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Imunidade Inata/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral
15.
Artigo em Inglês | MEDLINE | ID: mdl-30041062

RESUMO

Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor that induces genes involved in glucose metabolism. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive ß-subunit (HIF-1ß). The white spot syndrome virus (WSSV) induces a shift in glucose metabolism and oxidative stress. HIF-1α is associated with the induction of metabolic changes in tissues of WSSV-infected shrimp. However, the contributions of HIF-1 to viral load and antioxidant responses in WSSV-infected shrimp have been not examined. In this study, the effect of HIF-1 silencing on viral load and the expression and activity of antioxidant enzymes (superoxide dismutase-SOD, glutathione S-transferase-GST, and catalase) along with oxidative damage (lipid peroxidation and protein carbonyl) in tissues of white shrimp infected with the WSSV were studied. The viral load increased in hepatopancreas and muscle after WSSV infection, and the accumulative mortality was of 100% at 72 h post-infection. The expression and activity of SOD, catalase, and GST decreased in each tissue evaluated after WSSV infection. Protein carbonyl concentrations increased in each tissue after WSSV infection, while lipid peroxidation increased in hepatopancreas, but not in muscle. Silencing of HIF-1α decreased the WSSV viral load in hepatopancreas and muscle of infected shrimp along with shrimp mortality. Silencing of HIF-1α ameliorated the antioxidant response in a tissue-specific manner, which translated to a decrease in oxidative damage. These results suggest that HIF-1 is essential for restoring the antioxidant response, which counters the oxidative injury associated with WSSV infection.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Animais , Aquicultura , DNA Viral/isolamento & purificação , Inativação Gênica , Hepatopâncreas/crescimento & desenvolvimento , Hepatopâncreas/metabolismo , Hepatopâncreas/virologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Injeções Intramusculares , Peroxidação de Lipídeos , México , Músculos/metabolismo , Músculos/virologia , Especificidade de Órgãos , Estresse Oxidativo , Oxirredutases/genética , Oxirredutases/metabolismo , Penaeidae/crescimento & desenvolvimento , Penaeidae/metabolismo , Carbonilação Proteica , Interferência de RNA , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/metabolismo , Carga Viral , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Vírus da Síndrome da Mancha Branca 1/fisiologia
16.
J Fish Dis ; 41(7): 1129-1146, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29745450

RESUMO

White spot syndrome virus (WSSV)-infected shrimp samples collected from grow-out ponds located at Nellore, Andhra Pradesh, India, showed WSSV negative and positive by PCR using primer sets specific to ORF119 and VP28 gene of WSSV, respectively. This indicated the deletion of genetic fragments in the genome of WSSV. The WSSV isolate along with lab strain of WSSV was subjected to next-generation sequencing. The sequence analysis revealed a deletion of 13,170 bp at five positions in the genome of WSSV-NS (new strain) relative to WSSV-TH and WSSV-LS (lab strain). The PCR analysis using the ORF's specific primer sets revealed the complete deletion of 10 ORFs in the genome of WSSV-NS strain. The primer set was designed based on sequence covering ORF161/162/163 to amplify a product of 2,748 bp for WSSV-LS and 402 bp for WSSV-NS. Our surveillance programme carried out since 2002 revealed the replacement of WSSV-LS by WSSV-NS in Indian shrimp culture system.


Assuntos
DNA Viral/análise , Genoma Viral , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Deleção de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Índia , Vírus da Síndrome da Mancha Branca 1/genética
17.
Fish Shellfish Immunol ; 79: 130-139, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29738871

RESUMO

White spot syndrome virus (WSSV) is a dangerous threat to shrimp farming that also attacks a wide range of crustaceans. Knowledge of the surface protein-protein interactions between the pathogen and host is very crucial to unraveling the molecular pathogenesis mechanisms of WSSV. In this study, LvBiP (Litopenaeus vannamei immunoglobulin heavy-chain-binding protein) was identified as a novel WSSV binding protein of L. vannamei by a biotinylation based affinity chromatography method. By using pull-down and ELISA assays, the binding of recombinant LvBiP to WSSV was proved to be specific and ATP- dependent. The interaction was also confirmed by the result of co-immunoprecipitation assay. Immunofluorescence studies revealed the co-localization of LvBiP with WSSV on the cell surface of shrimp haemocytes. Additionally, LvBiP is likely to play an important role in WSSV infection. Treatment of gill cellular membrane proteins (CMPs) with purified rLvBiP and antibody that specifically recognizes LvBiP, led to a significant reduction in the binding of WSSV to gill CMPs. In the in vivo neutralization assay, rLvBiP and anti-LvBiP polyclonal antibody partially blocked the infection of WSSV. Taken together, the results indicate that LvBiP, a molecular chaperon of the HSP70 family, is a novel host factor involved at the step of attachment of the WSSV to the host cells and a potential candidate of therapeutic target.


Assuntos
Cromatografia de Afinidade/métodos , Regulação da Expressão Gênica/imunologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Biotinilação , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/química , Interações Hospedeiro-Patógeno , Ligação Proteica , Vírus da Síndrome da Mancha Branca 1/fisiologia
18.
Fish Shellfish Immunol ; 79: 274-283, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29775740

RESUMO

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression at the posttranscriptional level. In this study, the function of microRNA-7 (miR-7) in host-virus interaction was investigated. Replication of White spot syndrome virus (WSSV) was enhanced with the overexpression of miR-7 and inhibited with the downregulation of miR-7 by using anti-miRNA oligonucleotide AMO-miR-7. The target gene of miR-7 was predicted using bioinformatics methods. Results showed that crab myeloid differentiation factor 88 (Myd88) could be targeted by miR-7. When the expression of Myd88 was knocked down by sequence-specific siRNA, WSSV copies in crabs were significantly increased. Further findings revealed that knockdown of Myd88, Tube, or Pelle inhibited the expressions of interleukin enhancer-binding factor 2 homolog (ILF2) and interleukin-16-like gene (IL-16L). While ILF2 was silenced, IL-16L expression was inhibited. The overexpression of miR-7 inhibited the expressions of ILF2 and IL-16L. Moreover, when ILF2 or IL-16L was silenced, WSSV copies in crabs were increased. Thus, the upregulated expression of miR-7 during WSSV challenge suppressed the host Myd88-ILF2-(IL-16L) signaling pathway in crabs and enhanced WSSV replication. Our study indicated that WSSV utilized crab miR-7 to enhance virus replication during infection.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/genética , MicroRNAs/genética , Fator 88 de Diferenciação Mieloide/genética , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Braquiúros/imunologia , MicroRNAs/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais
19.
Fish Shellfish Immunol ; 78: 299-309, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29709591

RESUMO

Glutaminyl-peptide cyclotransferase (QPCT) catalyzes the posttranslational modification of an N-terminal glutamate of proteins to pyroglutamate. This renders the protein more resistant to protease degradation, more susceptible to hydrophobic interactions, aggregation, and neurotoxic. In this study, we evaluated the influence of QPCT in the crab Scylla paramamosain infected with white spot syndrome virus (WSSV) or with Vibrio alginolyticus. A cDNA clone, encompassing the entire 2445 bp of the S. paramamosain QPCT gene, containing a 1113 bp open reading frame (ORF) encoding a 370 amino acid protein was cloned from S. paramamosain. Real-time PCR indicated that QPCT was primarily expressed in the digestive tract of S. paramamosain, was up-regulated in hemocytes after infection with V. alginolyticus, and down-regulated in hemocytes after infection with WSSV. Knockdown of QPCT expression by double-stranded RNA (QPCT-dsRNA) resulted in down-regulation of prophenoloxidase (proPO) and crustin antimicrobial peptide, whereas myosin-II-essential-light-chain-like-protein was significantly up-regulated in hemocytes at 24 h post QPCT-dsRNA treatment. WSSV challenge in crabs treated with QPCT-dsRNA resulted in a reduction in viral burden and in the apoptotic rate of crab hemocytes, while the phagocytic activity of crab hemocytes and overall mortality rate were increased. This suggests that WSSV might take advantage of QPCT to benefit its replication. In contrast, V. alginolyticus infection in crabs treated with QPCT-dsRNA indicated that the apoptotic rate and phagocytic activity of hemocytes, and overall incidence of mortality, were increased compared to mock-treated animals, indicating that QPCT might be a resistance factor in bacterial infection. These results increase our understanding of the function of QPCT and its role in the innate immunity of S. paramamosain.


Assuntos
Aminoaciltransferases/genética , Aminoaciltransferases/imunologia , Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Aminoaciltransferases/química , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Masculino , Filogenia , Distribuição Aleatória , Alinhamento de Sequência , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
20.
Fish Shellfish Immunol ; 78: 1-9, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29656126

RESUMO

Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea and exhibits potential antibacterial and anticancer activities. In this study, EGCG was used in pathogen-challenge experiments in shrimp to discover its effect on the innate immune system of an invertebrate. Kuruma shrimp Marsupeneaus japonicus was used as an experimental model and challenged with white spot syndrome virus (WSSV) and the Gram-negative bacterium Vibrio alginolyticus. Pathogen-challenge experiments showed that EGCG pretreatment significantly delayed and reduced mortality upon WSSV and V. alginolyticus infection, with VP-28 copies of WSSV also reduced. Quantitative reverse transcription polymerase chain reaction revealed the positive influence of EGCG on several innate immune-related genes, including IMD, proPO, QM, myosin, Rho, Rab7, p53, TNF-alpha, MAPK, and NOS, and we observed positive influences on three immune parameters, including total hemocyte count and phenoloxidase and superoxide dismutase activities, by EGCG treatment. Additionally, results showed that EGCG treatment significantly reduced apoptosis upon V. alginolyticus challenge. These results indicated the positive role of EGCG in the shrimp innate immune system as an enhancer of immune parameters and an inhibitor of apoptosis, thereby delaying and reducing mortality upon pathogen challenge. Our findings provide insight into potential therapeutic or preventive functions associated with EGCG to enhance shrimp immunity and protect shrimp from pathogen infection.


Assuntos
Catequina/análogos & derivados , Imunidade Inata/efeitos dos fármacos , Penaeidae/efeitos dos fármacos , Penaeidae/imunologia , Substâncias Protetoras/farmacologia , Vibrio alginolyticus/efeitos dos fármacos , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Antivirais/farmacologia , Catequina/farmacologia , Penaeidae/enzimologia , Penaeidae/genética , Distribuição Aleatória , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
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