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1.
Artigo em Inglês | MEDLINE | ID: mdl-31760077

RESUMO

Glutathione peroxidases (GPx) are parts of the enzymatic antioxidant system that can eliminate the peroxides produced as effect of reactions of molecules with reactive oxygen species (ROS). In this study, a selenium-dependent glutathione peroxidase 3 cDNAs (designated as SpGPx3) was obtained from the mud crab Scylla paramamosain. The open reading frame (ORF) of SpGPx3 was 639 bp, which encoded a putative protein of 212 amino acids. SpGPx3 protein contained a characteristic GPx signature motif, and an active site motif. Mud crabs were exposed to 20 mg L-1 nitrite for 72 h. Quantitative real-time PCR analysis revealed that the SpGPx3 mRNA was distributed abundantly in mud crab. The transcript levels of antioxidant enzyme genes (SpGPx3, SpSOD and SpCAT) were obviously induced after acute nitrite exposure. After knockdown of the SpGPx3 level, the mortality of mud crabs and malondialdehyde (MDA) content significantly increased under nitrite stress. These results suggested that SpGPx3 played an important role in protecting organisms against oxidative stress.

2.
Artigo em Inglês | MEDLINE | ID: mdl-31683012

RESUMO

The mud crab, Scylla paramamosain, is an economically-important crab in China. Air exposure is an important environmental stressor during mud crab culture and transportation. Adaptive mechanisms responding to air exposure in mud crabs are still poorly understood. In this study, mud crabs were exposed to air for 120 h. Air exposure decreased total hemocyte counts, led to cytological damage, and caused high mortality. Transcriptomic analysis was conducted at 0, 6 and 96 h after air exposure. A total of 3530 differentially expressed genes (DEGs) were identified. DEGs were mainly involved in the oxidative stress response, metabolism, cellular processes, signal transduction, and immune functions. Transcriptomic analysis also revealed that genes of glycolysis and of the tricarboxylic acid cycle were key factors in regulating the mud crab adaptation to air exposure.

3.
Chemosphere ; 239: 124668, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31494325

RESUMO

Nitrite is one of major environmental pollutants that can impact immunological parameters in aquatic organisms. In the present study, we investigated the effects of nitrite exposure on oxidative stress, DNA damage and apoptosis in mud crab (Scylla paramamosain). Mud crab were exposed to 0, 5, 10 and 15 mg L-1 nitrite for 72 h. These data showed that acid phosphatase (ACP) and alkaline phosphatase (ALP) activity significantly decreased in treatments with various concentrations of nitrite (5, 10 and 15 mg L-1) after 24 and 48 h, while the levels of nitric oxide (NO) significantly increased in these treatments. Nitrite exposure could suppress superoxide dismutase (SOD) and catalase (CAT) activity, and increase the formation of malondialdehyde (MDA) after 48 and 72 h of exposure. In addition, nitrite exposure decreased total haemocyte counts after 48 and 72 h of exposure. Cytological damage, DNA damage and apoptosis was observed obviously at 72 h after nitrite exposure. Moreover, nitrite exposure significantly induced the mRNA levels of phosphorylated Jun N-terminal kinases (JNK), and eventually activated p53 signaling and caspase-3. These results indicated that nitrite exposure could induce oxidative stress, which further caused DNA damage and apoptosis in mud crab. Our results will be helpful to understand the mechanism of nitrite toxicity on crustaceans.


Assuntos
Apoptose/efeitos dos fármacos , Braquiúros/efeitos dos fármacos , Dano ao DNA/genética , Nitritos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Braquiúros/genética , Catalase/metabolismo , Hemócitos , Malondialdeído/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais , Superóxido Dismutase/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-31654827

RESUMO

Glutathione S-transferase (GST) plays important roles in cellular detoxification and antioxidant defense. A Mu-type glutathione S-transferase (designated as SpMu-GST) was obtained from the mud crab Scylla paramamosain. The open reading frame of SpMu-GST was comprised a 690 bp, which encoded a putative protein of 229 amino acids. Quantitative real-time PCR (qRT-PCR) revealed that the SpMu-GST mRNA was expressed in all examined tissues, with highest expression in hepatopancreas. During ammonia exposure, the SpMu-GST transcriptions in hepatopancreas and gill were significantly up-regulated at early exposure time. Moreover, RNA interference (RNAi) experiment was designed to understand the roles of SpMu-GST under ammonia exposure. Ammonia exposure reduced the levels of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and total antioxidative capacity (T-AOC), and increased the formation of malondialdehyde (MDA). After knockdown of the SpMu-GST level, GST activity and T-AOC were significantly decreased at some exposure time after ammonia exposure. However, the mortality of mud crabs and malondialdehyde (MDA) contents significantly increased under ammonia exposure. These results further suggested that SpMu-GST played a critical role in mud crab antioxidant defenses in response to environmental stress.

5.
Dev Comp Immunol ; 98: 54-64, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30986432

RESUMO

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection.

6.
Ecotoxicol Environ Saf ; 179: 9-16, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31022654

RESUMO

Ammonia is a major aquatic environmental pollutants. However, the underlying molecular mechanism of ammonia-induced toxicity is not fully understood. In this study, we investigated the physiological response and molecular mechanism in mud crab (Scylla paramamosain) exposed to the acute total ammonia (30 mg L-1) for 48 h. The results shown that ammonia exposure induced oxidative stress, and subsequently led to cytological damage and DNA damage. Transcriptome analysis was applied to investigate the key genes and pathways involved in the responses to ammonia exposure. A total of 722 differentially expressed genes (DEGs) (526 up-regulated and 196 down-regulated) were identified. DEGs mainly involved in pathways including metabolism, cellular processes, signal transduction and immune functions. Additionally, transcriptome analysis revealed that ATM/p53-Caspase3 pathway involved in apoptosis induced by ammonia stress. These results provided a new insight into the mechanism of the potential toxic effects of ammonia on crustaceans.


Assuntos
Amônia/toxicidade , Braquiúros/efeitos dos fármacos , Dano ao DNA , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Braquiúros/genética , Braquiúros/fisiologia , Perfilação da Expressão Gênica , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Transdução de Sinais
7.
Fish Shellfish Immunol ; 77: 457-464, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29656127

RESUMO

The present study was aimed to investigate the low temperature toxicity and its protection by taurine in pufferfish. The experimental basal diets supplemented with taurine at the rates of 250 (control), 550, 850, 1140, 1430, 1740 mg kg-1 were fed to fish for 8 weeks. The results showed that fish fed diet with taurine had significantly improved weight gain and specific growth rate. After the feeding trial, the fish were then exposed to low temperature stress. The results showed that low temperature stress could induce reactive oxygen species (ROS) generation, disturb the cytoplasm Ca2+ homeostasis, and lead to oxidative stress and apoptosis. Compared with the control group, dietary taurine supplementation groups increased antioxidant enzyme genes such as manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT), heat shock proteins (HSP70) and complement C3 (C3) mRNA levels under low temperature stress. Meanwhile, dietary taurine supplementation groups reduced ROS generation, and stabilized the cytoplasm Ca2+ under low temperature stress. Furthermore, dietary taurine supplementation groups reduced apoptosis via decreasing caspase-3 activity. This is the first report to demonstrate the mechanisms of taurine against low temperature stress in fish.


Assuntos
Apoptose/efeitos dos fármacos , Expressão Gênica/imunologia , Substâncias Protetoras/farmacologia , Takifugu/imunologia , Taurina/farmacologia , Ração Animal/análise , Animais , Cálcio/metabolismo , Temperatura Baixa , Citoplasma/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Estresse Oxidativo/efeitos dos fármacos , Distribuição Aleatória , Takifugu/genética , Takifugu/crescimento & desenvolvimento , Takifugu/metabolismo , Ganho de Peso/efeitos dos fármacos
8.
Fish Physiol Biochem ; 44(2): 735-745, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29349632

RESUMO

This study was conducted to determine the effects of vitamin E on growth performance, biochemical parameters, and antioxidant capacity of pufferfish (Takifugu obscurus) exposed to ammonia stress. The experimental basal diets supplemented with vitamin E at the rates of 2.31 (control), 21.84, 40.23, 83.64, 158.93, and 311.64 mg kg-1 dry weight were fed to fish for 60 days. After the feeding trial, the fish were exposed to 100 mg L-1 ammonia-nitrogen for 48 h. The results shown that the vitamin E group significantly improved weight gain, specific growth rate, and the expression levels of growth hormone receptors and insulin-like growth factor. Fish fed with the vitamin E-supplemented diets could increase plasma alkaline phosphatase activities and decrease plasma glutamicoxalacetic transaminase and glutamic-pyruvic transaminase activities. The relative expression levels of heat shock proteins (40.23-311.64 mg kg-1 vitamin E diet group), manganese superoxide dismutase (83.64-158.93 mg kg-1 vitamin E diet group), catalase (40.23-311.64 mg kg-1 vitamin E diet group), and glutathione reductase (40.23-311.64 mg kg-1 vitamin E diet group) were upregulated. On the other hand, the decreased level of reactive oxygen species (ROS) was observed in the 83.64-311.64 mg kg-1 vitamin E additive group. These results showed that vitamin E might have a potentially useful role as an effective antioxidant to improve resistance in pufferfish.


Assuntos
Amônia/toxicidade , Dieta/veterinária , Estresse Oxidativo/efeitos dos fármacos , Takifugu/crescimento & desenvolvimento , Vitamina E/farmacologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Antioxidantes/farmacologia , Suplementos Nutricionais , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Takifugu/fisiologia
9.
Fish Physiol Biochem ; 44(1): 209-218, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28936571

RESUMO

The present study was conducted to investigate the effects of astaxanthin on growth performance, biochemical parameters, ROS production, and immune-related gene expressions of the pufferfish (Takifugu obscurus) under high temperature stress. The experimental basal diets supplemented with astaxanthin at the rates of 0 (control), 20, 40, 80, 160, and 320 mg kg-1 were fed to fish for 8 weeks. The results showed that the fish fed diet with 80, 160, and 320 mg kg-1 astaxanthin significantly improved weight gain and specific growth rate. Furthermore, fish fed the moderate dietary astaxanthin increased plasma alkaline phosphatase activities, and decrease plasma aspartate aminotransferase and alanine aminotransferase activities. After the feeding trial, the fish were exposed to high temperature stress for 48 h. The results shown that astaxanthin could suppress ROS production induced by high temperature stress. Meanwhile, compared with the control group, the astaxanthin groups increased SOD, CAT, and HSP70 mRNA levels under high temperature stress. These results showed that the basal diet supplemented with 80-320 mg kg-1 astaxanthin could enhance growth, nonspecific immune responses, and antioxidant defense system and improve resistance against high temperature stress in pufferfish.


Assuntos
Dieta/veterinária , Suplementos Nutricionais , Takifugu/metabolismo , Ração Animal , Fenômenos Fisiológicos da Nutrição Animal , Animais , Antioxidantes/metabolismo , Catalase/genética , Catalase/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Temperatura Alta , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Takifugu/imunologia , Temperatura Ambiente , Xantofilas/administração & dosagem , Xantofilas/farmacologia
10.
Ecotoxicol Environ Saf ; 150: 190-198, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29276954

RESUMO

Water temperature is an important environmental factor that affects physiology and biochemical activities of fish. In this study, we investigated of high temperature on biochemical parameters, oxidative stress, DNA damage and apoptosis of pufferfish. Thermal stress could significantly increase the levels of AST, ALT, LDH, GLU and TG, whereas the levels of ALP and TP decrease significantly. In addition, thermal stress also decreased total blood cell count, inhibited cell viability, and subsequently lead to DNA damage and apoptosis. The mRNA levels of p53, caspase-9 and caspase-3 were up-regulated under thermal stress. These results suggested that caspase-dependent and p53 signaling pathways could play important roles in thermal stress-induced apoptosis in fish. Furthermore, the gene expression of SOD, CAT, HSP90 and C3 were induced by thermal stress. This study provides new insights into the mechanism whereby thermal stress affects physiological responses and apoptosis in pufferfish.


Assuntos
Apoptose , Dano ao DNA , Temperatura Alta/efeitos adversos , Estresse Oxidativo , Takifugu/metabolismo , Poluição da Água/efeitos adversos , Animais , Apoptose/fisiologia , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , China , Pesqueiros , Expressão Gênica , Estresse Oxidativo/fisiologia , Transdução de Sinais , Takifugu/sangue , Takifugu/genética
11.
Fish Physiol Biochem ; 43(4): 965-975, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28553691

RESUMO

Apoptosis plays a crucial role in many biological processes, including development, cellular homeostasis, and immune responses. Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that protects cells from endoplasmic reticulum stress-induced apoptosis. In this study, a BI-1 gene from the pufferfish Takifugu obscurus (Pf-BI-1) was identified and characterized. The full length of Pf-BI-1 cDNA was 1387 bp, including a 5'-UTR of 82 bp, a 3'-UTR of 591 bp containing a poly-(A) tail, and an open reading frame (ORF) of 714 bp that encodes a polypeptide of 237 amino acids. Pf-BI-1 was ubiquitously expressed in various tissues, with the highest expression levels in the blood, brain, and gill. The expression of Pf-BI-1 was up-regulated in a time-dependent manner after heat shock stress, ammonia stress, and bacterial challenge. Intracellular localization revealed that Pf-BI-1 was primarily localized in the cell cytoplasm. Furthermore, over-expression of Pf-BI-1 could active NF-кB reporter genes in HeLa cells. These results indicated that Pf-BI-1 may be involved in the apoptosis and immunity process against ambient stressors in pufferfish.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Regulação da Expressão Gênica/fisiologia , Takifugu/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Sequência de Bases , Clonagem Molecular , DNA Complementar , Células HeLa , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
12.
Fish Shellfish Immunol ; 64: 137-145, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28263881

RESUMO

Low temperature is an important environmental factor in aquaculture farming that affects the survival and growth of organisms. In the present study, we investigated the effects of low temperature on biochemical parameters, oxidative stress and apoptosis in pufferfish. In the stress group, water temperature decreased from 25 °C to 13 °C at a rate of 1 °C/1 h. Fish blood and liver were collected to assay biochemical parameters, oxidative stress and expression of genes at 25 °C, 21 °C, 17 °C, 13 °C and 13 °C for 24 h. The results showed that low temperature could decrease total blood cell count, inhibit cell viability, and subsequently lead to DNA damage. Biochemical parameters such as plasma protein and ALP significantly declined in fish under low temperature, while a significant increase in AST, ALT, LDH and glucose was observed. The gene expression of antioxidant enzymes (SOD and CAT), HSP90 and C3 were induced by low temperature stress. Furthermore, the gene expression of apoptotic related genes including P53, caspase-9 and caspase-3 were up-regulated, suggesting that caspase-dependent pathway could play important roles in low temperature-induced apoptosis in fish. This study may provide baseline information about how cold stress affects the physiological responses and apoptosis in fish.


Assuntos
Temperatura Baixa , Regulação da Expressão Gênica , Estresse Oxidativo , Takifugu/fisiologia , Animais , Antioxidantes/metabolismo , Apoptose , Dano ao DNA , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Distribuição Aleatória , Takifugu/sangue , Takifugu/genética , Takifugu/imunologia
13.
Fish Shellfish Immunol ; 59: 312-322, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27815196

RESUMO

The tumor suppressor protein p53 plays a critical role in cell cycle, apoptosis and DNA repair. In this study, the full-length pufferfish p53 (Pf-p53) was obtained, containing an open reading frame of 1095 bp, a 5'UTR of 157 bp and a 3'UTR of 285 bp with a poly (A) tail. The Pf-p53 encoded a polypeptide of 364 amino acids with a theoretical isoelectric point of 8.03 and predicted molecular weight of 40.6 kDa. Pf-p53 was ubiquitously expressed in various tissues with a high-level expression in kidney, liver and gill. Vibrio alginolyticus challenge could induce ROS production and disrupt Ca2+ homeostasis, subsequently leading to the induction of DNA damage and apoptosis, while the Vibrio alginolyticus-induced oxidative stress can also increase the non-specific immunity. The pufferfish challenged with Vibrio alginolyticus showed a sharp increase of Pf-p53 transcript in liver. Subcellular localization analysis revealed that Pf-p53 was primarily localized in nucleus. Furthermore, overexpression of Pf-p53 in Hela cells could inhibit cell proliferation and the transcriptional activities of the NF-ĸB promoter. Taken together, our results indicated that Pf-p53 may play an important role in the immune response to Vibrio alginolyticus challenge.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Takifugu , Proteína Supressora de Tumor p53/genética , Vibrioses/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Alinhamento de Sequência/veterinária , Distribuição Tecidual , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio alginolyticus/fisiologia
14.
Fish Shellfish Immunol ; 40(1): 190-6, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25010674

RESUMO

Guava (Psidium guajava L.) leaf extracts have antiviral and antibacterial activity against shrimp pathogens such as yellow-head virus (YHV), white spot syndrome virus (WSSV), and Vibrio harveyi, which make it a potential water disinfectant for use in shrimp culture. In this study, the safety of guava leaf supplementation in shrimp was evaluated by studying its influence on growth and the non-specific immune response of Penaeus monodon. Six diets containing different levels of guava leaves (0% [basal diet], 0.025% [G1], 0.05% [G2], 0.1% [G3], 0.2% [G4], and 0.4% [G5]) were fed to groups of shrimp (1.576 ± 0.011 g body weight) in triplicate for 56 days. Growth performance (final body weight, WG, PWG, SGR) of shrimp fed guava leaf diets was significantly higher (P < 0.05) than that of shrimp fed on the basal diet. The G1 diet resulted in the highest body weight gain (308.44%), followed by the G2 (295.45%), G3 (283.05%), G5 (281.29%), G4 (276.11%), and finally the basal diet (214.58%). Survival of shrimp in the G1 diet group was higher than that of shrimp in the control and the other experimental groups; however, no statistical differences (P > 0.05) were found. Dietary supplementation with guava leaf improved the activities of prophenoloxidase (PO) and nitric oxide synthase (NOS) in serum, and of superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LSZ) both in serum and hepatopancreas of shrimp. In the experimental groups, the activities of these enzymes followed a similar pattern of change; they increased initially at low levels of dietary supplementation and then decreased with increasing concentrations of dietary guava leaf. Serum PO and SOD activities in shrimp fed the G1 diet reached 7.50 U ml(-1) and 178.33 U ml(-1), respectively, with PO activity being significantly higher than in controls. In shrimp fed the G1 diet, SOD, ACP, and AKP activities in hepatopancreas were significantly higher than in the controls, reaching 57.32 U g(-1), 23.28 U g(-1), and 19.35 U g(-1) protein, respectively. The highest activities of serum ACP, AKP, LSZ, and of hepatopancreas LSZ, were observed in the G3 diet group. Total nitric oxide synthase (TNOS) activity was highest (64.80 U ml(-1)) in the G4 diet group, which was significantly higher than that observed in the control group. These results suggest that dietary guava leaf supplementation could enhance the growth performance and non-specific immune response of P. monodon. Therefore, guava leaf is considered safe for use as a water disinfectant in shrimp culture.


Assuntos
Imunidade Inata/efeitos dos fármacos , Penaeidae/efeitos dos fármacos , Extratos Vegetais/efeitos adversos , Psidium/efeitos adversos , Ração Animal/efeitos adversos , Ração Animal/análise , Animais , Aquicultura , Dieta/efeitos adversos , Suplementos Nutricionais/efeitos adversos , Suplementos Nutricionais/análise , Penaeidae/enzimologia , Penaeidae/crescimento & desenvolvimento , Penaeidae/imunologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Folhas de Planta/efeitos adversos , Folhas de Planta/química , Psidium/química
16.
Virus Res ; 171(1): 8-14, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23073178

RESUMO

A virus with a particle diameter of approximately 30 nm and no envelope was purified from diseased mud crab, Scylla paramamosain and it was demonstrated to be pathogenic to mud crab. The complete nucleotide sequence analysis indicated that its genome was a single molecule of linear positive-sense ssRNA with a length of 10,415 nucleotides, excluding the 3'poly (A) tail. It consisted of two open reading frames (ORF) separated by an intergenic region (IGR) and flanked by a 5'untranslated region (5'-UTR) and a 3'untranslated region (3'-UTR). The 5'-ORF encode five putative non-structural proteins, including BIR (Baculovirus Inhibitor of Apoptosis Protein Repeat), helicase, VPg (the genome-linked viral protein), 3C-like protease and RdRP (RNA-dependent RNA polymerase), while the 3'-ORF encode the structural protein precursors. This genome organization was consistent with the typical organization of dicistrovirus and the virus was designated as mud crab dicistrovirus-1 (MCDV-1). The results of the phylogenetic analysis of the putative structural protein precursor suggest that MCDV-1 has a closer genetic relationship with Taura syndrome virus (TSV) than do other dicistroviruses and that MCDV-1 is a new member of the family Dicistroviridae and assigned into the genus Aparavirus.


Assuntos
Braquiúros/virologia , Dicistroviridae/genética , Genoma Viral , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas do Capsídeo/genética , Dicistroviridae/classificação , Dicistroviridae/patogenicidade , Dicistroviridae/ultraestrutura , Ordem dos Genes , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Vírion/isolamento & purificação , Vírion/ultraestrutura
17.
J Virol Methods ; 184(1-2): 15-20, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22609257

RESUMO

Haliotis diversicolor (small abalone) is an important seafood found along the southern coast of China. Since 1999, the yields of cultured abalone in China have been severely affected by an epidemic of continuous outbreaks of a fatal disease. A novel double-stranded DNA virus, abalone shriveling syndrome-associated virus (AbSV), was proven to be one of the main causative agent. Although the pathogenicity and genome of AbSV has been ascertained, the epidemiology of AbSV remains to be investigated. In this study, four pairs of AbSV-specific primers were designed on the basis of the AbSV genome, and were tested for their specificities and sensitivities in quantitative real-time PCRs (qPCRs) after optimization of the annealing temperature. The 3F3/3B3 primer pair was finally chosen with a good specificity and high efficiency of amplification, with a detection limit of about 10 copies of recombinant plasmid containing AbSV genes in a 20-µL reaction mixture. In the detection of AbSV in abalone samples along the southern coast of China, most of the diseased samples had more than 80 virus copies in 1ng host genome DNA. AbSV was also demonstrated in mature hybrid (LY) and juvenile (JH) abalones from assays of healthy animals collected in recent years.


Assuntos
Vírus de DNA/isolamento & purificação , Gastrópodes/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Virologia/métodos , Animais , China , Primers do DNA/genética , Vírus de DNA/genética , Sensibilidade e Especificidade
18.
J Virol Methods ; 184(1-2): 21-6, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22633927

RESUMO

Haliotis diversicolor (small abalone) is an economic seafood found off the Southern coast of China. Since 1999, the cultured abalone yields in China have been affected severely by continual outbreaks of a fatal epidemic disease caused by abalone shriveling syndrome associated virus (AbSV), a double-stranded DNA virus. Although the pathogenicity and genome of AbSV have been ascertained, the epidemiology of AbSV infection remains to be investigated. In the present study, four pairs of AbSV-specific primers were designed on the basis of open reading frame (ORF)24 and ORF25 sequences in the AbSV genome. Two nested PCR detection methods were established by optimization of the annealing temperatures of primers. The results showed that the specificity of primers for AbSV detection could not be interfered with by the host genome and other aquaculture species or viruses. The detection limits of the two methods were about 10 copies of recombinant plasmid containing AbSV genes in 20µL reaction mixture. The results of detection of the AbSV epidemic showed that AbSV was still present in juvenile abalones in some farms along the Southern coast of China (Fujian and Guangdong).


Assuntos
Vírus de DNA/isolamento & purificação , Gastrópodes/virologia , Reação em Cadeia da Polimerase/métodos , Animais , China , Primers do DNA/genética , Sensibilidade e Especificidade
19.
Virology ; 422(2): 185-94, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22088215

RESUMO

Mud crab reovirus (MCRV) is the causative agent of a serious disease with high mortality in cultured mud crab (Scylla serrata). This study sequenced and analyzed 12 genome segments of MCRV. The 12 genome segments had a total length of 24.464 kb, showing a total G+C content of 41.29% and predicted 15 ORFs. Sequence analysis showed that the majority of MCRV genes shared low homology with the counterpart genes of other reoviruses, e.g., the amino acid identity of RNA-dependent RNA polymerase (RdRp) was lower than 13.0% compared to the RdRp sequences of other reoviruses. Nucleotide and amino acid sequences of RdRp and capping enzyme suggested MCRV as a single group. Further genome-based phylogenetical analysis of conserved termini and reovirus polymerase motif indicates that this MCRV belongs to a new genus of the Reoviridae family, tentatively named as Crabreovirus.


Assuntos
Genoma Viral , Reoviridae/genética , Sequência de Aminoácidos , Animais , Braquiúros , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Regulação Viral da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Viral/química , RNA Viral/genética
20.
Mar Genomics ; 4(3): 189-96, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21867971

RESUMO

The small abalone, Haliotis diversicolor, is a widely distributed and cultured species in the subtropical coastal area of China. To identify and classify functional genes of this important species, a normalized expressed sequence tag (EST) library, including 7069 high quality ESTs from the total body of H. diversicolor, was analyzed. A total of 4781 unigenes were assembled and 2991 novel abalone genes were identified. The GC content, codon and amino acid usage of the transcriptome were analyzed. For the accurate annotation of the abalone library, different influencing factors were evaluated. The gene ontology (GO) database provided a higher annotation rate (69.6%), and sequences longer than 800bp were easily subjected to a BLAST search. The taxonomy of the BLAST results showed that lancelet and invertebrates are most closely related to abalone. Sixty-seven identified plant-like genes were further examined by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, only seven of these were real transcripts in abalone. Phylogenic trees were also constructed to illustrate the positions of two Cystatin sequences and one Calmodulin protein sequence identified in abalone. To perform functional classification, three different databases (GO, KEGG and COG) were used and 60 immune or disease-related unigenes were determined. This work has greatly enlarged the known gene pool of H. diversicolor and will have important implications for future molecular and genetic analyses in this organism.


Assuntos
Etiquetas de Sequências Expressas , Biblioteca Gênica , Moluscos/genética , Animais , Sequência de Bases , Evolução Biológica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica
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