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1.
J Fish Dis ; 45(1): 59-68, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34536027

RESUMO

White spot syndrome virus (WSSV) is a pathogenic and threatening virus in shrimp culture for which there is no effective control strategy. Finding antiviral lead compounds for the development of anti-WSSV drugs is urgent and necessary; in this study, esculin from 12 monomeric compounds exhibited an excellent anti-WSSV activity. The results showed that esculin increased the survival rate of WSSV-infected shrimps by 59% and reduced the virus copy number in vivo over 90% at 100 µM. In the pre-treatment and post-treatment experiments, esculin could prevent and treat WSSV infection. Compared with the control group, the virus copy number decreased by 30% after 6 h of esculin pre-incubation with WSSV particles and inhibited horizontal transmission of WSSV to a certain extent. Considering that the antiviral activity of esculin was stable in the aquacultural water for 2 days, we evaluated the dosing pattern of continuous medication changes. Obviously, the survival rate of WSSV-infected shrimps was 0% at 108 h when no esculin exchange was made, while at 120 h the survival rate was over 40% at continuous medicine changes. In addition, esculin significantly increased the expression of antimicrobial peptides and thus improved the ability of shrimp to resist WSSV. Overall, our findings suggest that esculin has the potential to be developed into an anti-WSSV medicine.


Assuntos
Antivirais/farmacologia , Esculina/farmacologia , Doenças dos Peixes , Penaeidae , Vírus da Síndrome da Mancha Branca 1 , Animais , Aquicultura , Surtos de Doenças , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/virologia , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos
2.
PLoS One ; 16(10): e0258655, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34653229

RESUMO

Diseases have remained the major issue for shrimp aquaculture industry for decades by which different shrimp species demonstrated alternative disease resistance or tolerance. However, there had been insufficient studies on the underlying host mechanisms of such phenomenon. Hence, in this study, the main objective involves gaining a deeper understanding into the functional importance of shrimp STAT gene from the aspects of expression, sequence, structure, and associated genes. STAT gene was selected primarily because of its vital signalling roles in stress, endocrine, and immune response. The differential gene expressions of Macrobrachium rosenbergii STAT (MrST) and Penaeus monodon STAT (PmST) under White Spot Syndrome Virus (WSSV) and Vibrio parahaemolyticus/VpAHPND infections were identified through qPCR analysis. Notably, during both pathogenic infections, MrST demonstrated significant gene expression down-regulations (during either early or later post-infection time points) whereas PmST showed only significant gene expression up-regulations. Important sequence conservation or divergence was highlighted through STAT sequence comparison especially amino acid alterations at 614 aa [K (Lysine) to E (Glutamic Acid)] and 629 aa [F (Phenylalanine) to V (Valine)] from PmST (AY327491.1) to PmST (disease tolerant strain). There were significant differences observed between in silico characterized structures of MrST and PmST proteins. Important functional differentially expressed genes (DEGs) in the aspects of stress, endocrine, immune, signalling, and structural were uncovered through comparative transcriptomic analysis. The DEGs associated with STAT functioning were identified including inositol 1,4,5-trisphosphate receptor, hsp90, caspase, ATP binding cassette transmembrane transporter, C-type Lectin, HMGB, ALF1, ALF3, superoxide dismutase, glutathione peroxidase, catalase, and TBK1. The main findings of this study are STAT differential gene expression patterns, sequence divergence, structural differences, and associated functional DEGs. These findings can be further utilized for shrimp health or host response diagnostic studies. STAT gene can also be proposed as a suitable candidate for future studies of shrimp innate immune enhancement.


Assuntos
Palaemonidae/genética , Penaeidae/genética , Fatores de Transcrição STAT/genética , Vibrio parahaemolyticus/patogenicidade , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Substituição de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Simulação por Computador , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Palaemonidae/virologia , Penaeidae/virologia , Conformação Proteica , Fatores de Transcrição STAT/química , Transdução de Sinais
3.
PLoS One ; 16(9): e0257792, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34559852

RESUMO

Spray-dried animal plasma (SDP) in feed for several animal species provides health benefits, but research about use of SDP in shrimp feed is very limited. The objectives of the present study were to investigate the effects of dietary SDP on growth performance, feed utilization, immune responses, and prevention of Vibrio parahaemolyticus infection in Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the post-larvae were divided into five groups (four tank/group and 80 shrimp/tank) and fed four times daily diets with porcine SDP at 0, 1.5, 3, 4.5, and 6% of the diet for 45 days. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups: four SDP groups as in Experiment 1 plus the positive and negative controls (four tank/group and 30 shrimp/tank). They were then challenged with V. parahaemolyticus by immersion at 105 colony-forming units (CFU)/mL and were fed with the same diets for another 4 days. In Experiment 1, shrimp fed 4.5% or 6% SDP diets had significantly higher body weight, survival rate, and improved feed conversion ratio. The immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) of the shrimp fed 3-6% SDP diets also showed significant enhancement compared to the control. In Experiment 2, the survival rates of the 3-6% SDP groups were significantly higher than the positive control at day 4 after the immersion challenge. Likewise, the histopathological study revealed milder signs of bacterial infection in the hepatopancreas of the 3-6% SDP groups compared to the challenged positive control and 1.5% SDP groups. In conclusion, shrimp fed diets with SDP, especially at 4.5-6% of the diet, showed significant improvement in overall health conditions and better resistance to V. parahaemolyticus infection.


Assuntos
Suplementos Nutricionais/análise , Resistência à Doença , Penaeidae/crescimento & desenvolvimento , Plasma/química , Vibrio parahaemolyticus/imunologia , Ração Animal/análise , Animais , Peso Corporal , Hemócitos/metabolismo , Imunidade Inata , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/virologia , Penaeidae/imunologia , Penaeidae/virologia , Fagócitos/metabolismo , Secagem por Atomização , Suínos
4.
Arch Virol ; 166(10): 2763-2778, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34342747

RESUMO

White spot syndrome virus (WSSV) is a significant threat to the aquaculture sector, causing mortality among crabs and shrimps. Currently available diagnostic tests for WSSV are not rapid or cost-effective, and a new detection method is therefore needed. This study demonstrates the development of a biosensor by functionalization of magnetosomes with VP28-specific antibodies to detect WSSV in seafood. The magnetosomes (1 and 2 mg/ml) were conjugated with VP28 antibody (0.025-10 ng/µl), as confirmed by spectroscopy. The magnetosome-antibody conjugate was used to detect the VP28 antigen. The binding of antigen to the magnetosome-antibody complex resulted in a change in absorbance. The magnetosome-antibody-antigen complex was then concentrated and brought near a screen-printed carbon electrode by applying an external magnetic field, and the antigen concentration was determined using impedance measurements. The VP28 antigen (0.025 ng/µl) bound more efficiently to the magnetosome-VP28 antibody complex (0.025 ng/µl) than to the VP28 antibody (0.1 ng/µl) alone. The same assay was repeated to detect the VP28 antigen (0.01 ng/µl) in WSSV-infected seafood samples using the magnetosome-VP28 antibody complex (0.025 ng/µl). The WSSV in the seafood sample was also drawn toward the electrode due to the action of magnetosomes controlled by the external magnetic field and detected using impedance measurement. The presence of WSSV in seafood samples was verified by Western blot and RT-PCR. Cross-reactivity assays with other viruses confirmed the specificity of the magnetosome-based biosensor. The results indicate that the use of the magnetosome-based biosensor is a sensitive, specific, and rapid way to detect WSSV in seafood samples.


Assuntos
Técnicas Biossensoriais/veterinária , Magnetossomos , Alimentos Marinhos/virologia , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Aquicultura , Reações Cruzadas , Espectroscopia Dielétrica , Ensaio de Imunoadsorção Enzimática , Microbiologia de Alimentos , Magnetossomos/química , Magnetossomos/imunologia , Penaeidae/virologia , Reprodutibilidade dos Testes , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia
5.
Fish Shellfish Immunol ; 117: 240-247, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34418555

RESUMO

The cytosolic DNA-sensing immune response is essential for recognizing and establishing an effective host immune response to pathogens. However, the importance of the cytosolic signalling molecules responsible for facilitating an appropriate immune response following infection with a DNA virus in shrimps remains unknown. Here, we report the discovery of the Penaeus monodon stimulator of interferon gene (PmSTING) and interferon regulatory factor (PmIRF) genes and their important roles in the host defense against viral infection. High expression levels of PmSTING transcripts were detected in the midgut, hepatopancreas, and hindgut, with lower levels in foregut, while PmIRF was highly expressed in the hindgut, foregut, and hepatopancreas of P. monodon. The mRNA expression level of both PmSTING and PmIRF was up-regulated in the foregut in response to white spot syndrome virus (WSSV; dsDNA virus) infection. RNA-interference-mediated gene silencing of PmSTING and PmIRF rendered shrimps to be more susceptible to WSSV infection; suppression of PmIRF decreased the mRNA transcript level of PmSTING; and silencing of the cytosolic sensor PmDDX41 suppressed both PmSTING and PmIRF gene transcript levels. Thus, PmSTING and PmIRF are likely to be important for the antiviral innate response against the dsDNA WSSV pathogen and may mediate the antiviral immune defenses via PmDDX41/PmSTING/PmIRF signaling cascade in P. monodon.


Assuntos
Proteínas de Artrópodes/imunologia , Infecções por Vírus de DNA/imunologia , Fatores Reguladores de Interferon/imunologia , Proteínas de Membrana/imunologia , Penaeidae , Vírus da Síndrome da Mancha Branca 1 , Animais , Proteínas de Artrópodes/genética , Infecções por Vírus de DNA/veterinária , Fatores Reguladores de Interferon/genética , Proteínas de Membrana/genética , Penaeidae/genética , Penaeidae/imunologia , Penaeidae/virologia
6.
Fish Shellfish Immunol ; 117: 104-112, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34333126

RESUMO

Cell survival is based on the stability of intracellular state. It was well known that biochemical reactions in cells require specific intracellular environments, such as pH and calcium concentration. While the mechanism of stabilizing the intracellular environment is complex and far from clear. In this study, a Sma and Mad related protein 5 gene (LvSmad5) of Litopenaeus vannamei was cloned. LvSmad5 was located to both cytoplasm and nucleus. And subcellular localization of LvSmad5 was responsed to the changing of cells internal and external environment. Besides, it was found that subcellular localization of LvSmad5 was also regulated by unfolded protein response. Moreover, it was proved that nucleic localization of LvSmad5 could significantly increase the white spot syndrome virus (WSSV) infection in shrimp, and knockdown expression of LvSmad5 decreased the cumulative mortality of WSSV infection shrimp. Further investigation revealed that cytoplasm LvSmad5 could interplay with shrimp hexokinase 1, and contribute to glycolysis. These results indicated that LvSmad5 played a role in L. vannamei environmental stress response, and was used by WSSV for its replication.


Assuntos
Infecções por Vírus de DNA/genética , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Penaeidae/genética , Proteína Smad5/genética , Estresse Fisiológico/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Núcleo Celular , Clonagem Molecular , Citoplasma , Infecções por Vírus de DNA/mortalidade , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/mortalidade , Doenças dos Peixes/virologia , Penaeidae/virologia , Resposta a Proteínas não Dobradas/genética , Replicação Viral
7.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372583

RESUMO

The present study was intended to screen the wild crustaceans for co-infection with Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Andaman and Nicobar Archipelago, India. We screened a total of 607 shrimp and 110 crab samples using a specific polymerase chain reaction, and out of them, 82 shrimps (13.5%) and 5 (4.5%) crabs were found positive for co-infection of IHHNV and WSSV. A higher rate of co-infection was observed in Penaeus monodon and Scylla serrata than other shrimp and crab species. The nucleotide sequences of IHHNV and WSSV obtained from crab in this present study exhibited very high sequence identity with their counterparts retrieved from various countries. Histopathological analysis of the infected shrimp gill sections further confirmed the eosinophilic intra-nuclear cowdry type A inclusion bodies and basophilic intra-nuclear inclusion bodies characteristics of IHHNV and WSSV infections, respectively. The present study serves as the first report on co-infection of WSSV and IHHNV in Andaman and Nicobar Archipelago, India and accentuates the critical need for continuous monitoring of wild crustaceans and appropriate biosecurity measures for brackishwater aquaculture.


Assuntos
Braquiúros/virologia , Coinfecção/epidemiologia , Penaeidae/virologia , Animais , Animais Selvagens/virologia , Aquicultura/métodos , Densovirinae/genética , Densovirinae/patogenicidade , Índia , Reação em Cadeia da Polimerase/métodos , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/patogenicidade
8.
Fish Shellfish Immunol ; 117: 140-147, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34314788

RESUMO

Invertebrates are considered completely dependent on their innate immunity to defend themselves against pathogens as they lack an adaptive immunity. However, a growing body of evidence has indicated a specific acquired immunity called 'immune priming' may exist. The Pacific white shrimp, Penaeus vannamei is one of the most economically important shrimp species in the world. In the previous research, we investigated the hepatopancreas immune response of shrimp immunized with trans -vp28 gene Synechocystis sp. PCC6803 at the protein level. In this study, on the basis of the previous research, the shrimp were then challenged with WSSV, and hepatopancreas analyzed using isobaric tags for relative and absolute quantification (i TRAQ) labeling. In total, 308 differentially expressed proteins (DEPs) were identified including 84 upregulated and 224 downregulated. Upregulated proteins such as calmodulin B and calreticulin, and downregulated proteins such as calnexin, and signaling pathways like Ras, mTOR were differentially expressed in both studies. Data from this study are more significant than previous work and indicate increased sensitivity to WSSV after immunization with trans-vp28 gene Synechocystis sp. PCC6803. In addition, selected DEPs (upregulated: A0A3R7QHH6 and downregulated: A0A3R7PEF6, A0A3R7MGX8, A0A423TPJ4, and A0A3R7QCC2) were randomly analyzed using parallel reaction monitoring (PRM). These data preliminarily confirm immune priming in P. vannamei, and show that the initial stimulation with trans -vp28 gene Synechocystis sp. PCC6803 regulate P. vannamei immune responses and they provide shrimp with enhanced immune protection against secondary stimulation.


Assuntos
Infecções por Vírus de DNA/imunologia , Genes Bacterianos/imunologia , Hepatopâncreas/imunologia , Penaeidae/imunologia , Synechocystis/genética , Vírus da Síndrome da Mancha Branca 1 , Imunidade Adaptativa , Animais , Infecções por Vírus de DNA/veterinária , Penaeidae/virologia , Proteômica
9.
Viruses ; 13(6)2021 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199268

RESUMO

White Spot Disease (WSD) presents a major barrier to penaeid shrimp production. Mechanisms underlying White Spot Syndrome Virus (WSSV) susceptibility in penaeids are poorly understood due to limited information related to early infection. We investigated mRNA and miRNA transcription in Penaeus vannamei over 36 h following infection. Over this time course, 6192 transcripts and 27 miRNAs were differentially expressed-with limited differential expression from 3-12 h post injection (hpi) and a more significant transcriptional response associated with the onset of disease symptoms (24 hpi). During early infection, regulated processes included cytoskeletal remodelling and alterations in phagocytic activity that may assist WSSV entry and translocation, novel miRNA-induced metabolic shifts, and the downregulation of ATP-dependent proton transporter subunits that may impair cellular recycling. During later infection, uncoupling of the electron transport chain may drive cellular dysfunction and lead to high mortalities in infected penaeids. We propose that post-transcriptional silencing of the immune priming gene Dscam (downregulated following infections) by a novel shrimp miRNA (Pva-pmiR-78; upregulated) as a potential mechanism preventing future recognition of WSSV that may be suppressed in surviving shrimp. Our findings improve our understanding of WSD pathogenesis in P. vannamei and provide potential avenues for future development of prophylactics and treatments.


Assuntos
Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , Penaeidae/genética , Penaeidae/virologia , RNA Mensageiro/genética , Vírus da Síndrome da Mancha Branca 1 , Doenças dos Animais/genética , Doenças dos Animais/patologia , Doenças dos Animais/virologia , Animais , Biologia Computacional , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/química , Modelos Biológicos , RNA Mensageiro/química , Transcriptoma , Carga Viral
10.
Sci Rep ; 11(1): 12766, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140570

RESUMO

White spot syndrome virus (WSSV) is one of the most devastating pathogens in penaeid shrimp and can cause massive damage in shrimp aquaculture industries. Previously, the WSSV structural protein VP15 was identified as an antigenic reagent against WSSV infections. In this study, we truncated this protein into VP15(1-25), VP15(26-57), VP15(58-80), and VP15(1-25,58-80). The purified proteins from the E. coli expression system were assayed as potential protective agents in Kuruma shrimp (Marsupenaeus japonicus) using the prime-and-boost strategy. Among the four truncated constructs, VP15(26-57) provided a significant improvement in the shrimp survival rate after 20 days of viral infection. Subsequently, four peptides (KR11, SR11, SK10, and KK13) from VP15(26-57) were synthesized and applied in an in vivo assay. Our results showed that SR11 could significantly enhance the shrimp survival rate, as determined from the accumulated survival rate. Moreover, a multiligand binding protein with a role in the host immune response and a possible VP15-binding partner, MjgC1qR, from the host M. japonicus were employed to test its binding with the VP15 protein. GST pull-down assays revealed that MjgC1qR binds with VP15, VP15(26-57), and SR11. Taken together, we conclude that SR11 is a determinant antigenic peptide of VP15 conferring antiviral activity against WSSV.


Assuntos
Antígenos Virais/química , Antivirais/farmacologia , Proteínas do Nucleocapsídeo/química , Penaeidae/virologia , Peptídeos/química , Sequência de Aminoácidos , Animais , Bombyx , Proteínas do Nucleocapsídeo/metabolismo , Filogenia , Domínios Proteicos , Vacinação , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos , Vírus da Síndrome da Mancha Branca 1/fisiologia
11.
J Gen Virol ; 102(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34106826

RESUMO

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded ß-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.


Assuntos
Heparina/metabolismo , Sulfatos/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Vírus da Síndrome da Mancha Branca 1/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Penaeidae/virologia , Ligação Proteica , Conformação Proteica , Conformação Proteica em Folha beta , Domínios Proteicos , Estrutura Quaternária de Proteína , Ressonância de Plasmônio de Superfície , Proteínas do Envelope Viral/genética , Vírus da Síndrome da Mancha Branca 1/genética
12.
Front Immunol ; 12: 682562, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34046043

RESUMO

Most tripartite motif (TRIM) family proteins are critical components of the autophagy machinery and play important roles in host defense against viral pathogens in mammals. However, the roles of TRIM proteins in autophagy and viral infection have not been studied in lower invertebrates, especially crustaceans. In this study, we first identified a TRIM50-like gene from Penaeus monodon (designated PmTRIM50-like), which, after a white spot syndrome virus (WSSV) challenge, was significantly upregulated at the mRNA and protein levels in the intestine and hemocytes. Knockdown of PmTRIM50-like led to an increase in the WSSV quantity in shrimp, while its overexpression led to a decrease compared with the controls. Autophagy can be induced by WSSV or rapamycin challenge and has been shown to play a positive role in restricting WSSV replication in P. monodon. The mRNA and protein expression levels of PmTRIM50-like significantly increased with the enhancement of rapamycin-induced autophagy. The autophagy activity induced by WSSV or rapamycin challenge could be inhibited by silencing PmTRIM50-like in shrimp. Further studies showed that rapamycin failed to induce autophagy or inhibit WSSV replication after knockdown of PmTRIM50-like. Moreover, pull-down and in vitro ubiquitination assays demonstrated that PmTRIM50-like could interact with WSSV envelope proteins and target them for ubiquitination in vitro. Collectively, this study demonstrated that PmTRIM50-like is required for autophagy and is involved in restricting the proliferation of WSSV through its ubiquitination. This is the first study to report the role of a TRIM family protein in virus infection and host autophagy in crustaceans.


Assuntos
Doenças dos Animais/etiologia , Autofagia/genética , Penaeidae/genética , Penaeidae/virologia , Ubiquitina-Proteína Ligases/genética , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia , Doenças dos Animais/metabolismo , Animais , Interações Hospedeiro-Patógeno/imunologia , Penaeidae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
13.
Sci Rep ; 11(1): 10534, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006863

RESUMO

Yellow head virus (YHV) is a pathogen which causes high mortality in penaeid shrimp. Previous studies suggested that YHV enters shrimp cells via clathrin-mediated endocytosis. This research investigated the roles of clathrin adaptor protein 2 subunit ß (AP-2ß) from Penaeus monodon during YHV infection. PmAP2-ß was continuously up-regulated more than twofold during 6-36 hpi. Suppression of PmAP2-ß significantly reduced YHV copy numbers and delayed shrimp mortality. Quantitative RT-PCR revealed that knockdown of PmAP2-ß significantly enhanced the expression level of PmSpätzle, a signaling ligand in the Toll pathway, by 30-fold at 6 and 12 hpi. Moreover, the expression levels of gene components in the Imd and JAK/STAT signaling pathways under the suppression of PmAP2-ß during YHV infection were also investigated. Interestingly, anti-lipopolysaccharide factor isoform 3 (ALFPm3) was up-regulated by 40-fold in PmAP2-ß knockdown shrimp upon YHV infection. In addition, silencing of PmAP2-ß dramatically enhanced crustinPm1 expression in YHV-infected shrimp. Knockdown of ALFPm3 and crustinPm1 significantly reduced shrimp survival rate. Taken together, this work suggested that PmAP2-ß-deficiency promoted the Toll pathway signalings, resulting in elevated levels of ALFPm3 and crustinPm1, the crucial antimicrobial peptides in defence against YHV.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Penaeidae/virologia , Roniviridae/isolamento & purificação , Transdução de Sinais , Receptores Toll-Like/metabolismo , Animais , Inativação Gênica , Penaeidae/genética
14.
Fish Shellfish Immunol ; 113: 89-95, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33823247

RESUMO

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.


Assuntos
Densovirinae/fisiologia , Penaeidae/imunologia , RNA de Cadeia Dupla/administração & dosagem , RNA Viral/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Proteínas do Envelope Viral/química , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Penaeidae/virologia , Interferência de RNA
15.
Fish Shellfish Immunol ; 114: 36-48, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33864947

RESUMO

By using immunohistochemistry detection, yellow head virus (YHV) was found to replicate in granule-containing hemocytes including semi-granular hemocytes (SGC) and granular hemocytes (GC) during the early phase (24 h post injection) of YHV-infected shrimp. Higher signal of YHV infection was found in GC more than in SGC. Comparative phosphoproteomic profiles between YHV-infected and non-infected GC reveal a number of phosphoproteins with different expression levels. The phosphoprotein spot with later on identified as caspase-3 in YHV-infected GC is most interesting. Blocking caspase-3 function using a specific inhibitor (Ac-DEVD-CMK) demonstrated high replication of YHV and consequently, high shrimp mortality. The immunohistochemistry results confirmed the high viral load in shrimp that caspase-3 activity was blocked. Caspase-3 is regulated through a variety of posttranslational modifications, including phosphorylation. Analysis of phosphorylation sites of shrimp caspase-3 revealed phosphorylation sites at serine residue. Taken together, caspase-3 is a hemocytic protein isolated from shrimp granular hemocytes with a role in anti-YHV response and regulated through the phosphorylation process.


Assuntos
Caspase 3/metabolismo , Hemócitos/enzimologia , Penaeidae/virologia , Roniviridae , Animais , Caspase 3/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia
16.
J Fish Dis ; 44(8): 1131-1145, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33835515

RESUMO

Whiteleg shrimp is a widely cultured crustacean, but frequent disease outbreaks have decreased production and caused significant losses. Toll-like receptors (TLRs) comprise a large innate immune family that is involved in the innate immune response. However, understanding of their regulatory mechanism is limited. In this study, PacBio sequencing and Illumina sequencing were applied to the gill and hepatopancreas tissues of whiteleg shrimp and an integrated transcript gene set was established. The upregulation of Toll1, Toll2 and Toll3 transcripts in the hepatopancreas tissue of whiteleg shrimp after iridescent virus infection implies that these proteins are involved in the immune response to the virus; simultaneously, the TRAF6 and relish transcripts in the Toll pathway were also upregulated, implying that the Toll pathway was activated. We predicted the three-dimensional structure of the five Toll proteins in whiteleg shrimp and humans and constructed a phylogenetic tree of the Toll protein family. In addition, there was a large discrepancy of Toll1 between invertebrates and vertebrates, presumably because of the loss of Toll1 protein sequence during the evolution process from invertebrates to vertebrates. Our research will improve the cognition of Toll protein family in invertebrates in terms of evolution, structure and function and provide theoretical guidance for researchers in this field.


Assuntos
Proteínas de Artrópodes/genética , Evolução Molecular , Iridoviridae/fisiologia , Penaeidae/genética , Receptores Toll-Like/genética , Animais , Proteínas de Artrópodes/metabolismo , Penaeidae/virologia , Análise de Sequência de DNA , Análise de Sequência de Proteína , Receptores Toll-Like/metabolismo , Transcrição Genética
17.
J Invertebr Pathol ; 182: 107567, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33711317

RESUMO

Decapod iridescent virus 1 (DIV1) is a new virus discovered in recent years that infects farmed shrimp. DIV1 is highly infectious and causes substantial economic loss to the aquaculture industry of China. To prevent and control the spread and outbreak of DIV1 in a timely manner, it is necessary to establish an efficient method for DIV1 diagnosis. In this study, quantitative real-time polymerase chain reaction (qPCR) and quantitative real-time loop-mediated isothermal amplification (qLAMP) detection methods were established based on the specific sequence of the viral ATPase gene. The results indicated that the minimum detection limits of qPCR and qLAMP were 1.9 × 101 copies/µL and 1.9 × 102 copies/µL, respectively; the designed primer had good specificity for DIV1 and did not react with 13 other viruses, including white spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreatic necrosis disease (AHPND), infectious hypodermal and haematopoietic necrosis virus (IHHNV), etc. A total of 43 clinical samples suspected of DIV1 infection were diagnosed by qPCR and qLAMP. Our qPCR demonstrated results consistent with a qPCR assay published previously, and the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of qLAMP were 85.71% and 100%, respectively. This result indicates that qPCR and qLAMP have good accuracy in the detection of DIVI in clinical samples. As established in this study, qPCR and qLAMP combined with a comprehensive comparative analysis can provide effective new solutions for the detection of DIV1.


Assuntos
Iridoviridae/isolamento & purificação , Penaeidae/virologia , Animais , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo Real
18.
Fish Shellfish Immunol ; 112: 8-22, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33600947

RESUMO

In the present study, a hot water crude extract from Ulva intestinalis (Ui-HWCE) was used as a dietary supplement, and the effects on growth, immune responses, and resistance against white spot syndrome virus (WSSV) and yellowhead virus (YHV) infection in Pacific white shrimp (Litopenaeus vannamei) were investigated. Chemical analyses of Ui-HWCE revealed 13.75 ± 0.41% sulfate, 37.86 ± 5.96% uronic acid, and 46.63 ± 5.16% carbohydrate contents. The monosaccharide content of Ui-HWCE contained glucose (6.81 ± 0.94%), xylose (4.15 ± 0.11%), and rhamnose (25.84 ± 0.80%). Functional group analysis of Ui-HWCE by Fourier transform infrared (FTIR) spectroscopy revealed a typical infrared spectrum of ulvan similar to the infrared spectrum of commercially purified ulvan from Ulva armoricana (77.86 ± 2.19% similarity). Ui-HWCE was added to shrimp diets via top-dressing at 0, 1, 5, and 10 g/kg diet. After 28 days, Ui-HWCE supplementation at 5 g/kg diet efficiently improved shrimp growth performance, as indicated by weight gain, average daily growth, specific growth rates, and villus height determined by observing gut morphology. Additionally, Ui-HWCE feed supplementation at 5 g/kg diet significantly increased immune responses against a pathogenic bacterium (Vibrio parahaemolyticus AHPND stain), including phagocytic activity and clearance efficiency. Furthermore, Ui-HWCE feed supplementation upregulated the expression of several immune-related genes in the hemocytes and gills. Ui-HWCE supplementation at 1 and 5 g/kg resulted in effective anti-YHV but not anti-WSSV activity, which significantly decreased the mortality rate and YHV burden in surviving shrimp. It was concluded that Ui-HWCE supplied at 5 g/kg diet exhibits growth-promoting, immune-stimulatory, and antiviral activity that could protect L. vannamei against YHV infection.


Assuntos
Penaeidae/imunologia , Extratos Vegetais/metabolismo , Roniviridae/fisiologia , Ulva/química , Vírus da Síndrome da Mancha Branca 1/fisiologia , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Penaeidae/crescimento & desenvolvimento , Penaeidae/virologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Distribuição Aleatória
19.
J Invertebr Pathol ; 180: 107545, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33571511

RESUMO

Outbreaks of white spot syndrome virus (WSSV) have caused serious damage to penaeid shrimp aquaculture worldwide. Despite great efforts to characterize the virus, the conditions that lead to infection and the infection mechanisms, there is still a lack of understanding regarding these complex virus-host interactions, which is needed to develop consistent and effective treatment methods for WSSV. In this study, we used a gas chromatography - mass spectrometry (GC-MS)-based metabolomics approach to compare the metabolite profiles of gills, haemolymph and hepatopancreas from whiteleg shrimp (Penaeus vannamei) exposed to WSSV and corresponding controls. The results revealed clear discriminations between metabolite profiles of WSSV-challenged shrimp and controlled shrimp in each tissue. The responses of shrimp gills to WSSV infection were characterized by increases of many fatty acids and amino acids in WSSV-challenged shrimp compared to the controls. Changes in haemolymph metabolite profiles include the increased levels of itaconic acid, energy-related metabolites, metabolites in glutathione cycle and decrease of amino acids. The WSSV challenge led to the decreases of several fatty acids and amino acids and increases of other amino acids, lactic acid and other organic compounds (levulinic acid, malonic acid and putrescine) in hepatopancreas. These alterations of shrimp metabolites suggest several immune responses of shrimp to WSSV in a tissue-specific manner, including upregulation of osmoregulation, antimicrobial activity, metabolic rate, gluconeogenesis, glutathione pathway in control of oxidative stress and shift from aerobic to anaerobic metabolism in shrimp which indicates the Warburg effect. The findings from this study provide a better understanding of molecular process of shrimp response against WSSV invasion which may be useful for development of disease management strategies.


Assuntos
Penaeidae/metabolismo , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Aquicultura , Cromatografia Gasosa-Espectrometria de Massas , Brânquias/virologia , Hemolinfa/virologia , Hepatopâncreas/virologia
20.
J Invertebr Pathol ; 179: 107536, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33472086

RESUMO

We surveyed 130 shrimp farms located on the eastern coast of India to determine the prevalence of emerging diseases in Litopenaeus vannamei and Penaeus monodon. Live shrimps were collected from the farms based on external symptoms. The biochemical, molecular, and histopathology results confirmed infection with Enterocytozoon hepatopenaei (32.4%), Vibrio parahaemolyticus (27.7%), White Spot Syndrome Virus (25.4%), Vibrio alginolyticus (16.1%), Vibrio harveyi (13.1%), Monodon-type baculovirus (4.61%), and infectious Hematopoietic Necrosis Virus (2.3%) in the collected shrimps. Enterocytozoon hepatopenaei (EHP) occurred more frequently in L. vannamei than P. monodon, with the microsporidian spores in the hepatopancreas. In P. monodon, Monodon-type Baculovirus infection (33.3%) was dominant and small percentages of WSSV, IHHNV, V. alginolyticus, and V. harveyi were observed. A few ponds were observed with co-infection of EHP and WSSV (7.6%), V. parahaemolyticus and WSSV (4.6%) and also V. parahaemolyticus and EHP (6.1%). Among the Vibrio spp, V. parahaemolyticus showed the highest percentage of infection in L. vannamei. Overall, we found that shrimp were chiefly infected with EHP and V. parahaemolyticus. The impact of water quality parameters on shrimp diseases was not addressed in this study. In an antibiotic susceptibility study, V. parahaemolyticus isolated from L. vannamei ponds was susceptible to nitrofurantoin, chloramphenicol, oxytetracycline and tetracycline, but resistant to erythromycin and nalidixic acid. In a preliminary in vitro antibacterial activity assay, probiotics against V. parahaemolyticus showed high inhibitory activity and the results encourage further in-depth studies on the efficacy of probiotics for disease control and prevention in shrimp farms.


Assuntos
Antibacterianos/farmacologia , Penaeidae/microbiologia , Probióticos/farmacologia , Vibrio parahaemolyticus/efeitos dos fármacos , Animais , Aquicultura , Índia , Penaeidae/virologia , Organismos Livres de Patógenos Específicos
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