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1.
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
2.
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
3.
Fish Shellfish Immunol ; 76: 279-286, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29496475

RESUMO

The PI3K/AKT signaling pathway is commonly exploited to regulate viral replication and affect the fate of infected cells. In the present study, a PI3K-specific inhibitor (LY294002) was employed to pretreat crayfish to evaluate the effects of PI3K/AKT signaling pathway in WSSV replication. The results showed that the WSSV copy numbers in crayfish pretreated with LY294002 were significantly lower than those in Tris-HCl pretreatment crayfish on the sixth and tenth day after WSSV infection. In semigranular cells, the apoptosis rates were up-regulated on the third day post-WSSV infection, and a significantly lower proportion of apoptosis cells were observed in LY294002-pretreatment group. The expression level of Bax, Bax inhibitor-1 and lectin mRNA in haemocytes of crayfish were increased after WSSV infection. After the secondary stimulation with Tris-HCl, the Bax expression level in LY294002-pretreatment crayfish was significantly higher than that of crayfish pretreated with Tris-HCl on the third or sixth day, but the Toll and lectin mRNA expression decreased significantly on the third, sixth and tenth day. The Bax mRNA expression levels in LY294002-WSSV group were significantly higher than those in Tris-HCl-WSSV group on the third and tenth day. The Bax inhibitor-1 mRNA expression levels in LY294002-WSSV group were significantly lower than those in Tris-HCl-WSSV crayfish on the third day. These results together indicated that the hosts PI3K/AKT signaling pathway play positive roles in WSSV replication through the balance between host cell apoptois and innate immune responses. This information is helpful to further understand the role of PI3K/AKT signaling pathway on WSSV replication in Decapoda crustaceans.


Assuntos
Proteínas de Artrópodes/antagonistas & inibidores , Astacoidea/imunologia , Cromonas/farmacologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Astacoidea/genética , Astacoidea/virologia , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos
4.
Dev Comp Immunol ; 84: 109-116, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29428488

RESUMO

The ß-thymosins are a group of structurally related, highly conserved intracellular small peptides in vertebrates with various biological functions, including cytoskeletal remodeling, neuronal development, cell migration, cell survival, tissue repair and inhibition of inflammation. In contrast to vertebrates, the function of ß-thymosin is not fully understood in crustaceans. Previously, we found that a thymosin-repeated protein1 (CqTRP1) gene was up-regulated after white spot syndrome virus (WSSV) challenge in hematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus. To further identify the effect of CqTRP1 on WSSV infection, a full length cDNA sequence of ß-thymosin homologue was cloned and analyzed from red claw crayfish followed by functional study. The CqTRP1 cDNA contains an open reading frame of 387 nucleotides encoding a protein of 129 amino acids with a putative molecular mass of 14.3 kDa. The amino acid sequence showed high identity with other ß-thymosins and contained three characteristic thymosin ß actin-binding motifs, suggesting that CqTRP1 was a member of the ß-thymosin family. Tissue distribution analysis revealed a ubiquitous presence of CqTRP1 in all the examined tissues with the highest expression in hemocytes, Hpt and gonad at the transcriptional level. Interestingly, the gene silencing of endogenous CqTRP1 by RNAi enhanced the WSSV replication in Hpt cells. Meanwhile, the WSSV replication was significantly reduced in the Hpt cell cultures if overloaded with a recombinant CqTRP1. Taken together, these data clearly indicated that CqTRP1 was likely to be associated with the anti-WSSV response in a crustacean C. quadricarinatus, which provides new strategy against white spot disease in crustacean aquaculture.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Infecções por Vírus de DNA/imunologia , Gônadas/metabolismo , Hemócitos/metabolismo , Timosina/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Aquicultura , Proteínas de Artrópodes/metabolismo , Astacoidea/virologia , Clonagem Molecular , Gônadas/imunologia , Gônadas/virologia , Hemócitos/imunologia , Hemócitos/virologia , Proteínas dos Microfilamentos/genética , RNA Interferente Pequeno/genética , Frutos do Mar , Timosina/metabolismo , Replicação Viral
5.
Dev Comp Immunol ; 82: 104-112, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29341872

RESUMO

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.


Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/imunologia , Infecções por Vírus de DNA/imunologia , Ferritinas/metabolismo , Sistema Hematopoético/metabolismo , Ferro/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/genética , Astacoidea/virologia , Células Cultivadas , Clonagem Molecular , DNA Complementar/genética , Ferritinas/genética , Imunidade Inata , Transporte de Íons , Miocárdio/metabolismo , Replicação Viral
6.
J Virol Methods ; 251: 139-144, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29050792

RESUMO

Chequa iflavirus (+ve sense ssRNA virus) infects redclaw crayfish (Cherax quadricarinatus) and it may cause mortality reaching 20-40% after about three weeks following stress. The sequence of the RNA-dependent RNA polymerase at nucleotide position 8383-9873 was used for developing and comparing PCR-based detection protocols. The reverse transcription, quantitative, polymerase chain reaction (RT-qPCR) was specific against nine Picornavirales and crustacean viruses and its' measurement of uncertainty (0.07-1.37) was similar to PCRs for other crustacean viruses. In vitro, the reverse transcription loop-mediated isothermal amplification (RT-LAMP) read at 60min had poor repeatability for a linearized plasmid with an iflavirus insert when compared with RT-PCR visualised on an electrophoretic gel and RT-qPCR; both sensitive to 102 copies. In a limited, comparative sample of clinical crayfish haemolymph, the lowest, non-zero copies were 2.88×104 for RT-PCR and 4.60×101 for the RT-qPCR. In 68 further clinical crayfish haemolymph samples tested by RT-qPCR only, copy numbers ranged from 0 to 1.14×106. For RT-qPCR, the amplification plots, melt curves and the CT values indicated that the CT above 34.0 is a potential negative result but examination of the melt curve is necessary for an accurate interpretation. A suggested program of testing for crayfish farmers would consist of non-destructive bleeding, labelling of crayfish and screening with RT-qPCR. Only those crayfish nominally negative (below detectable limits) would be used for broodstock or selective breeding.


Assuntos
Astacoidea/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Vírus de RNA/veterinária , Vírus de RNA/isolamento & purificação , Animais , Água Doce , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética
7.
J Virol Methods ; 251: 133-138, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29089143

RESUMO

Developing a rapid, accurate and quantitative method for detecting white spot syndrome virus (WSSV) is extremely urgent and critical for reducing the risk of white spot disease outbreaks. In the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for quantitative detection of WSSV. The method employed rabbit polyclonal antibodies against WSSV as the capture antibody and previously produced anti-WSSV monoclonal antibodies as the detector antibody. A standard curve of the log concentration of WSSV versus OD value was established, which was linear in the concentration range of 120-7680ng/mL, and the linear regression equation was y=0.166x-0.151. Viral proteins in different tissues of crayfish (Procambarus clarkia) post artificial infection with WSSV were quantitatively measured using the DAS-ELISA. WSSV proliferated quickly within 60h post infection and gradually slowed down afterwards. According to the linear regression relationship, the viral proteins in hemolymph, gut and gonad were firstly able to be quantified at 24h post infection with the concentrations of 186, 158 and 128ng/mL, respectively. These three tissues also contained higher viral proteins than the gill, heart, hepatopancreas and muscle during the entire infection period. The viral protein concentration in gut reached the highest level of 6220ng/mL at 72h post infection. Real time quantitative PCR was also used to detect the dynamic change of viral copies in crayfish hemolymph post WSSV infection, with similar results for both assays. The developed DAS-ELISA could detect WSSV propagation from initial to moribund stage in infected crayfish and demonstrated potential application for diagnosis of WSSV.


Assuntos
Anticorpos Antivirais/imunologia , Astacoidea/virologia , Infecções por Vírus de DNA/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Carga Viral/métodos , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Estruturas Animais/virologia , Animais , Infecções por Vírus de DNA/virologia , Coelhos , Fatores de Tempo , Vírus da Síndrome da Mancha Branca 1/imunologia
8.
PLoS One ; 12(11): e0187760, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29121070

RESUMO

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in the innate immunity system of invertebrates, especially in the aspect of antivirus. In the present study, high-throughput small RNA Illumina sequencing systems were used to identify differentially expressed miRNAs (DEMs) from the intestines of Procambarus clarkii that were infected with white spot syndrome virus (WSSV). As a result, 39 known and 12 novel miRNAs were identified in both NG and WG small RNA libraries. Seven DEMs were determined to be involved in the antiviral innate immunity in the intestines of P. clarkii. The results of the target gene predictions of the DEMs showed that the putative target genes of these 7 DEMs are related to tight junctions, vascular smooth muscle contraction regulation of the actin cytoskeleton, focal adhesion, RNA transport, mRNA surveillance, viral carcinogenesis, and Salmonella infection. These results provide theoretical insights for future studies on the antiviral immunity of crustaceans.


Assuntos
Astacoidea/genética , Astacoidea/virologia , Mucosa Intestinal/metabolismo , MicroRNAs/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Astacoidea/imunologia , Sequência de Bases , Imunidade Inata/genética , Intestinos/imunologia , Análise de Sequência de RNA
9.
J Gen Virol ; 98(10): 2589-2595, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28874231

RESUMO

A novel iridovirus, Cherax quadricarinatus iridovirus (CQIV), was identified from diseased C. quadricarinatus in 2014. This virus is considered as a new threat to crustacean aquaculture because it is lethal to both peneaid shrimp and crayfish. Here, we determined the complete genome sequence of CQIV. The double-stranded DNA genome is 165 695 bp in length with a G+C content of 34.6 %. A total of 178 open reading frames (ORFs) have been predicted, encoding hypothetical proteins ranging from 50 to 1327 amino acids. Forty-seven of these exhibit similarities to proteins of known functions. Phylogenetic analysis based on multiple alignments of conserved proteins shows that CQIV clusters with the members of the family Iridoviridae, but is placed in a distinct clade from all the five known genera. It indicates that CQIV may represent a new genus in the family Iridoviridae, for which we propose the name Cheraxvirus based on the host organism.


Assuntos
Astacoidea/virologia , DNA Viral/genética , Genoma Viral/genética , Iridoviridae , Animais , Composição de Bases , Sequência de Bases , Iridoviridae/classificação , Iridoviridae/genética , Iridoviridae/isolamento & purificação , Fases de Leitura Aberta/genética , Análise de Sequência de DNA , Proteínas Virais/genética
10.
J Invertebr Pathol ; 150: 41-44, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28888768

RESUMO

Chequa iflavirus and a bunya-like virus infect redclaw crayfish (Cherax quadricarinatus) and they may cause mortality reaching 20-40% after about three weeks after a stress event. To complete River's postulates for viruses, virus-free animals are needed. Due to a lack of chequa iflavirus and bunya-like virus-free crayfish (testing shows>85% infection rate) coupled with the facts that iflavirus and bunyaviruses are found in insects and that crickets had been successful alternate hosts for crustacean viruses before, Acheta domesticus was trialled asa bioassay animal. There was no significant difference (P>0.05) in mortality rates between uninfected control crickets and infected crickets. Reverse transcriptase polymerase chain reaction for both viruses failed to find any trace of the RNA viruses in fed or inoculated crickets after 30days. The search for an alternative bioassay host will have to be widened.


Assuntos
Astacoidea/virologia , Gryllidae/virologia , Músculo Esquelético/virologia , Vírus de RNA , Animais , Bioensaio
11.
Fish Shellfish Immunol ; 69: 78-84, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28803958

RESUMO

MicroRNAs (miRNAs) were important post-transcriptional regulators and played vital roles in innate immunity system of invertebrates, especially in the aspect of antivirus. In this study, using high-throughput small RNAs Illumina sequencing system, differentially expressed miRNAs (DEMs) from lymph organs in red swamp crayfish, Procambarus clarkii, infected with white spot syndrome virus, were identified. As a result, 32 known miRNAs and 7 novel miRNAs were identified in crayfish lymph organ small RNAs library of NG and WG. Among them, 7 differentially expressed miRNAs (DEMs) were predicted to be involved in the lymph organ antiviral innate immunity of P. clarkii. Besides, the results showed that putative target genes of these DEMs were related with tight junction, RNA transport, regulation of actin cytoskeleton, focal adhesion, vascular smooth muscle contraction, mRNA surveillance pathway, NOD-like receptor signaling pathway, leukocyte transendothelial migration, and protein processing in endoplasmic reticulum. These results might provide the guiding theoretical foundation for future studies about crustaceans' antiviral innate immunity.


Assuntos
Astacoidea/imunologia , Astacoidea/virologia , Imunidade Inata , MicroRNAs/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Astacoidea/genética , Sequenciamento de Nucleotídeos em Larga Escala , Tecido Linfoide/metabolismo
12.
Virus Res ; 238: 148-155, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28676258

RESUMO

In 2014, northern Queensland crayfish from farms affected by particularly transportation and translocation stress, started to die with mortality reaching 20-40% after about three weeks and then mortalities subsided. Crayfish from 1 farm had 65% mortalities within 11 weeks. With histological examination of broodstock and juveniles, the muscle fibres were fractured with haemocytic infiltration reminiscent of viral infection or vitamin E/selenium deficiencies. Sequence dependent and independent PCRs failed to identify a viral aetiology. However, the whole transcriptomes of a case crayfish and an unaffected crayfish from a different population were assembled producing over 500,000 contigs. The complete sequence of a positive sense, single stranded RNA virus (+ve ssRNA virus; 9933bp) and the large and medium segments of a bunya-like virus were detected. Transcript back-mapping and newly developed PCRs indicated that the viruses were in the case crayfish but not the control crayfish. The +ve ssRNA virus is clearly in the order Picornavirales, marginally in the genus Iflavirus in a clade of Chinese and Northern American terrestrial arthropod viruses. The internal Picornavirales motifs; RNA-dependent RNA polymerase, helicase (P-loop) and 2 viral capsids genes were easily identified. This is the first iflavirus identified from crustacea and is named Chequa iflavirus. Whether these viruses are responsible for the stress-related mortalities is unproven but the Chequa virus' role seems limited as it appears it has been present in crayfish from at least the early 1990s; unless low-grade, chronic mortalities have been largely unnoticed.


Assuntos
Astacoidea/virologia , Infecções por Picornaviridae/veterinária , Picornaviridae/classificação , Picornaviridae/isolamento & purificação , Animais , Aquicultura , Histocitoquímica , Filogenia , Picornaviridae/genética , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/virologia , Queensland , Homologia de Sequência , Proteínas Virais/genética
13.
Fish Shellfish Immunol ; 68: 211-219, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28705723

RESUMO

Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PlcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S) motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PlcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.


Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/enzimologia , Astacoidea/virologia , Imunidade Inata , Serina Proteases/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Astacoidea/genética , Astacoidea/imunologia , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Hemócitos/enzimologia , Hemócitos/virologia , Alinhamento de Sequência , Serina Proteases/química , Serina Proteases/genética , Proteínas do Envelope Viral/metabolismo
14.
Gene ; 610: 140-147, 2017 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-28213041

RESUMO

Tolls or Toll-like receptors (TLRs) have an essential role in initiating innate immune responses against pathogens. In this study, a novel Toll gene, PcToll4, was first identified from the intestinal transcriptome of the freshwater crayfish, Procambarus clarkii. The PcToll4 cDNA is 4849bp long with a 3036bp open reading frame that encodes a 1011-amino acid protein. PcToll4 contains a signal peptide, 13 LRR domains, 3 LRR TYP domains, 2 LRR CT domains, an LRR NT domain, a transmembrane region, and a TIR domain. Quantitative RT-PCR analysis revealed that PcToll4 mRNA was detected in all tested tissues, and the expression of PcToll4 in the intestine was significantly upregulated after white spot syndrome virus (WSSV) challenge. Overexpression of PcToll4 in Drosophila Schneider 2 (S2) cells activates the antimicrobial peptides (AMPs) of Drosophila, including metchnikowin, drosomycin, attacin A, and shrimp Penaeidin-4. Results of RNA interference by siRNA also showed that PcToll4 regulates the expressions of 5 anti-lipopolysaccharide factors (ALFs) in the intestine of crayfish. Our findings suggest that PcToll4 is important for the innate immune responses of P. clarkii because this gene regulates the expressions of AMPs against WSSV.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Astacoidea/genética , Astacoidea/imunologia , Receptores Toll-Like/imunologia , Animais , Astacoidea/virologia , Linhagem Celular , Drosophila melanogaster , Imunidade Inata , Filogenia , Receptores Toll-Like/metabolismo , Transcrição Genética , Vírus da Síndrome da Mancha Branca 1/fisiologia
15.
Fish Shellfish Immunol ; 59: 469-483, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27825947

RESUMO

White spot syndrome virus (WSSV) is one of the most prevalent and widespread viruses in both shrimp and crayfish aquaculture. MicroRNAs (miRNAs) are crucial post-transcriptional regulators and play critical roles in cell differentiation and proliferation, apoptosis, signal transduction and immunity. In this study, miRNA expression profiles were identified via deep sequencing in red claw crayfish Cherax quadricarinatus haematopoietic tissue (Hpt) cell cultures infected with WSSV at both early (i.e., 1 hpi) and late (i.e., 12 hpi) infection stages. The results showed that 2 known miRNAs, namely, miR-7 and miR-184 play key roles in immunity. Meanwhile, 106 novel miRNA candidates were predicted by software in these combined miRNA transcriptomes. Compared with two control groups, 36 miRNAs showed significantly different expression levels after WSSV challenge. Furthermore, 10 differentially expressed miRNAs in WSSV-exposed Hpt cells were randomly selected for expression analysis by quantitative real-time RT-PCR. Consistent with the expression profiles identified by deep sequencing, RT-PCR showed a significant increase or decrease in miRNA expression in Hpt cells after WSSV infection. Prediction of targets of miRNAs such as miR-7, cqu-miR-52, cqu-miR-126 and cqu-miR-141 revealed that their target genes have diverse biological roles, including not only immunity but also transcriptional regulation, energy metabolism, cell communication, cell differentiation, cell death, autophagy, endocytosis and apoptosis. These results provide insight into the molecular mechanism of WSSV infection and highlight the function of miRNAs in the regulation of the immune response against WSSV infection in crustaceans.


Assuntos
Astacoidea/genética , Astacoidea/virologia , Imunidade Inata , MicroRNAs/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Astacoidea/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
16.
Fish Shellfish Immunol ; 57: 213-221, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27544268

RESUMO

It is well-known that anti-lipopolysacchride factors (ALFs) are involved in the recognition and elimination of invading pathogens. In this study, the full-length ALF cDNA sequence of the red claw crayfish Cherax quadricarinatus (termed CqALF) was cloned from a suppression subtractive hybridization library constructed using red claw crayfish hematopoietic tissue cell (Hpt cell) cultures following challenge with white spot syndrome virus (WSSV). The full-length cDNA sequence of CqALF was 863 bp, and the open reading frame encoded 123 amino acids with a signal peptide in the N-terminus and a conserved LPS-binding domain. Unlike most ALFs, which are highly expressed in haemocytes, high expression levels of CqALF were detected in epithelium, the stomach and eyestalks, while lower expression was detected in Hpt, nerves, the heart, muscle tissue, gonads, haemocytes, intestines, gills and the hepatopancreas. To further explore the biological activities of CqALF, mature recombinant CqALF protein (rCqALF) was expressed and purified using a eukaryotic expression system, and an antimicrobial activity test was carried out. rCqALF clearly exerted antiviral activity, as evidenced by the severe disruption of the envelope of intact WSSV virions following co-incubation of virions with rCqALF. Additionally, pre-incubation of WSSV with rCqALF resulted in both a significant reduction in WSSV replication in red claw crayfish Hpt cell cultures and an increased survival rate among animals. Furthermore, rCqALF was effective against both Gram-negative bacteria and Gram-positive bacteria, particularly Shigella flexneri and Staphylococcus aureus. A membrane integrity assay suggested that rCqALF was unlikely to disrupt bacterial membrane integrity compared to cecropin P1. Taken together, these data suggest that CqALF may play an important role in immune defence in the crustacean C. quadricarinatus.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Artrópodes/genética , Astacoidea/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Astacoidea/microbiologia , Astacoidea/virologia , Sequência de Bases , Candida albicans/fisiologia , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Vírus da Síndrome da Mancha Branca 1/fisiologia
17.
Dis Aquat Organ ; 120(2): 143-50, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27409237

RESUMO

The red swamp crayfish Procambarus clarkii represents an important aquaculture species responsible for over half of all commercial aquaculture profits in Louisiana, USA. White spot syndrome virus (WSSV) is highly pathogenic in crustacean species and induces mass mortality in aquaculture operations worldwide. Natural outbreaks of WSSV occur yearly in cultured populations of crayfish in Louisiana. The goal of this study was to better understand the infectivity of WSSV in P. clarkii, by determining the minimum lethal dose necessary to initiate infection and to measure the resulting cumulative mortality following infection with different doses. A real time quantitative PCR (qPCR) method was used to detect WSSV in DNA extracted from gill tissue to ensure P. clarkii study populations were WSSV-free before the start of trials. Viable viral particles were isolated from naturally infected P. clarkii gill tissue and quantified using a novel digital PCR approach. Three infectivity trials were performed, and WSSV inocula were created by serial dilution, generating 5 treatments per trial. Five crayfish (weighing ~25 g) per dilution per trial received viral inoculations. Mortality was monitored daily for the duration of the trial in order to construct a median lethal dose (LD50) curve, and probit regression analysis was used to determine LD50 concentrations of viral particles. Knowledge of the infectivity of WSSV in native crayfish populations is of critical importance to the management of the commercial crayfish aquaculture industry in Louisiana. This is the first study to investigate the infectivity and to determine the LD50 of the Louisiana strain of WSSV in native crayfish.


Assuntos
Astacoidea/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Interações Hospedeiro-Patógeno
18.
Sci Rep ; 6: 28694, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27385304

RESUMO

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-ß-tubulin and Cq-ß-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm.


Assuntos
Proteínas de Artrópodes/fisiologia , Família da Proteína 8 Relacionada à Autofagia/fisiologia , Endocitose , Internalização do Vírus , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Astacoidea/citologia , Astacoidea/virologia , Autofagia , Células Cultivadas , Invaginações Revestidas da Membrana Celular/ultraestrutura , Invaginações Revestidas da Membrana Celular/virologia , Ligação Proteica , Proteínas do Envelope Viral/metabolismo , Replicação Viral
19.
Sci Rep ; 6: 29924, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27411341

RESUMO

C-type lectins are important immune molecules that participate in host defense response. The present work reports a novel C-type lectin (PcLec3) from the red swamp crayfish Procambarus clarkii. Sequence analysis found that PcLec3 encodes a polypeptide with252 amino acid residues, which contains an immunoglobulin-like domain (IG) and a C-type lectin domain (CTLD) arranged in tandem. Tissue distribution analysis indicated that PcLec3 is enriched expressed in hemocytes and hepatopancreas cells, in which PcLec3 was up-regulated following bacterial challenge by Vibrio anguillarum. Function analysis using recombinant full-length PcLec3, IG, and CTLD proteins revealed that these recombinant proteins had the capacity to bind carbohydrates and bacteria, while IG determined the cell binding activity. However, only full-length PcLec3 promotes the phagocytic activity of hemocytes and subsequent clearance of invasive bacteria. Taken together, these results manifest that PcLec3 acts as a hemocyte adhesion molecule to promote hemocyte phagocytosis against invasive V. anguillarum.


Assuntos
Astacoidea/citologia , Hemócitos/citologia , Domínios de Imunoglobulina , Lectinas Tipo C/química , Fagocitose , Sequência de Aminoácidos , Animais , Astacoidea/virologia , Sequência de Bases , Adesão Celular/efeitos dos fármacos , DNA Complementar/genética , Perfilação da Expressão Gênica , Hemócitos/efeitos dos fármacos , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Fagocitose/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Açúcares/metabolismo , Regulação para Cima/efeitos dos fármacos , Vibrio/fisiologia
20.
Dis Aquat Organ ; 120(1): 17-26, 2016 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-27304867

RESUMO

We report the preliminary characterization of a new iridovirus detected in diseased Cherax quadricarinatus collected from a farm in Fujian, China. Transmission electron microscopy identified numerous icosahedral particles (~150 nm in diameter) in the cytoplasm and budding from the plasma membrane of hematopoietic tissue cells. SDS-PAGE of virions semi-purified from the hemolymph of moribund C. quadricarinatus identified 24 proteins including a 50 kDa major capsid protein (MCP). By summing the sizes of DNA restriction endonuclease fragments, the viral genome was estimated to be ~150 kb in length. A 34 amino acid sequence deduced from a 103 bp MCP gene region amplified by PCR using degenerate primers targeted to MCP gene regions conserved among iridoviruses and chloriridoviruses was most similar (55% identity) to Sergestid iridovirus. Based on virion morphology, protein composition, DNA genome length, and MCP sequence relatedness, the virus identified has tentatively been named Cherax quadricarinatus iridovirus (CQIV). In addition, experimental infection of healthy C. quadricarinatus, Procambarus clarkii, and Litopenaeus vannamei with CQIV caused the same disease and high mortality, suggesting that CQIV poses a potential threat to cultured and wild crayfish and shrimp.


Assuntos
Astacoidea/virologia , Iridoviridae/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Viral/genética , Regulação Viral da Expressão Gênica , Genoma Viral , Interações Hospedeiro-Patógeno , Iridoviridae/classificação , Iridoviridae/genética , Iridoviridae/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Proteínas Virais/genética , Proteínas Virais/metabolismo
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