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1.
BMC Bioinformatics ; 20(Suppl 7): 192, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31074372

RESUMO

BACKGROUND: The Iridoviridae family is categorized into five genera and clustered into two subfamilies: Alphairidovirinae includes Lymphocystivirus, Ranavirus (GIV), and Megalocystivirus (TGIV), which infect vertebrate hosts and Betairidovirinae includes Iridovirus and Chloriridovirus, which infect invertebrate hosts. Clustered Iridoviridae subfamilies possess host-specific characteristics, which can be considered as exclusive features for in-silico prediction of effective epitopes for vaccine development. A voting mechanism-based linear epitope (LE) prediction system was applied to identify and endorse LE candidates with a minimum length requirement for each clustered subfamily RESULTS: The experimental results showed that four conserved epitopes among the Iridovirideae family, one exclusive epitope for invertebrate subfamily and two exclusive epitopes for vertebrate family were predicted. These predicted LE candidates were further validated by ELISA assays for evaluating the strength of antigenicity and cross antigenicity. The conserved LEs for Iridoviridae family reflected high antigenicity responses for the two subfamilies, while exclusive LEs reflected high antigenicity responses only for the host-specific subfamily CONCLUSIONS: Host-specific characteristics are important features and constraints for effective epitope prediction. Our proposed voting mechanism based system provides a novel approach for in silico LE prediction prior to vaccine development, and it is especially powerful for analyzing antigen sequences with exclusive features between two clustered groups.


Assuntos
Infecções por Vírus de DNA/imunologia , Epitopos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Invertebrados/imunologia , Iridoviridae/imunologia , Vertebrados/imunologia , Proteínas Virais/imunologia , Animais , Infecções por Vírus de DNA/virologia , Invertebrados/virologia , Iridoviridae/classificação , Iridoviridae/genética , Vertebrados/virologia
2.
Fish Shellfish Immunol ; 89: 468-476, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30940578

RESUMO

Interferon regulatory factor (IRF) 3 and IRF7 are key regulators of type I interferon (IFN) gene expression for the antiviral immune response. In the present study, interferon regulatory factor 3 and 7 from Asian seabass, namely AsIRF3 and AsIRF7 were cloned and characterized. The full-length cDNA sequence of IRF3 and IRF7 consisted of 2965 and 2343 bp respectively. AsIRF3 and AsIRF7 were true orthologes of vertebrate IRF3/7 and showed similar domain organization, with an N-terminal DBD which consisted five tryptophan residues in IRF3 and four in IRF7, a C-terminal IRF3 domain and a serine rich region. Both IRF3 and 7 constitutively expressed during the ontogenesis and in all tissues of healthy fish. The expression of both genes was up-regulated following NNV challenge with obvious transcript abundance in brain heart and kidney. Ectopic expression of AsIRF3 and AsIRF7 displayed activation of ISRE/NF-κB promoters and modulation of interferon, ISGs and pro-inflammatory cytokine gene expression. These observations indicated that IRF3 and IRF7 play an important role in Asian seabass's antiviral defense and the RIG-IRF-IFN axis is conserved in the species.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Vírus de DNA/imunologia , Proteínas de Peixes/química , Perfilação da Expressão Gênica/veterinária , Fator Regulador 3 de Interferon/química , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 7 de Interferon/química , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Nodaviridae/fisiologia , Filogenia , Alinhamento de Sequência/veterinária
3.
Fish Shellfish Immunol ; 89: 710-718, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999043

RESUMO

The horizontal transmission of lymphocystis disease virus (LCDV) through contaminated water and feed (using artemia as vehicle) and the associated immune gene expression profiles in Senegalese sole post-larvae were investigated. All specimens analyzed were positive for LCDV DNA detection at 1-day post-challenge (1 dpc) with the highest viral levels in specimens infected through the immersion route. However, the percentage of LCDV-positive animals and number of viral DNA copies dropped progressively at 2 and 7 dpc. The histological analysis identified structural changes in the skin, muscle and gills of sole post-larvae LCDV-challenged by immersion. In situ hybridization confirmed a wide distribution of LCDV in the skin, gut, surrounding vessels in trunk muscle and head kidney in the immersion route, while the signals were restricted to the liver and lamina propria in the feeding treatment. Expression analysis using a set of 22 genes related to innate immune defense system demonstrated clear differences in the time-course response to LCDV as function of the infection route. Most antiviral defense genes, the proinflammatory cytokines, the complement c3, g-type lysozyme and T-cell markers cd4 and cd8a were rapidly induced in the feeding-infected post-larvae, and they were remained activated at 2 dpc. In contrast, in the immersion-infected post-larvae the induction of most defensive genes was delayed, with a low intensity at 2 dpc. All these data demonstrate that LCDV can horizontally infect Senegalese sole post-larvae through the water or feed although with different patterns of histopathological disorders, virus distribution and route-specific expression profiles.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Linguados , Iridoviridae/fisiologia , Transcriptoma/imunologia , Carga Viral , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/metabolismo , Distribuição Tecidual
4.
New Microbiol ; 42(2): 118-120, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31034081

RESUMO

Gemycircularviruses (GemyCV) are a vast array of viruses belonging to the Genomoviridae family. Prevalence and pathogenesis in humans are still poorly understood. Different GemyCV species were investigated in 661 Italian subjects by species-specific PCRs. Only the GemyCV-C1c species was detected, with low prevalence and the highest rate in HIV immunosuppressed patients.


Assuntos
Vírus de DNA , DNA Viral , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/virologia , Vírus de DNA/genética , DNA Viral/isolamento & purificação , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Hospedeiro Imunocomprometido , Itália , Prevalência
5.
Fish Shellfish Immunol ; 89: 677-686, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30905839

RESUMO

Krϋppel-like factor 9 (KLF9) is a member of the SP/KL family, which are transcription factors implicated in several biological processes, including cell proliferation, differentiation, development and apoptosis. Studies have focused on the function of KLF9 in mammalian disease and the immune system, such as its regulatory role in the growth of tumors and its impact on interferon-related genes and inflammatory cytokines. In fish, little is known about the role of KLF9, especially its regulatory function in the innate antiviral immune response. In this study, we characterized the grouper KLF9 gene (EcKLF9) and investigated its role in viral infection. Amino acid alignment analysis showed that EcKLF9 was approximately 228 amino acids long and contained a typical three-tandem Krϋppel-like zinc fingers. Phylogenetic tree analysis revealed that EcKLF9 clustered with three fish species: Amphiprion ocellaris, Acanthochromis pollyacanthus and Stegastes partitus. Comparison analyses showed that the three Kruppel-like zinc finger domains of KLF9 were highly conserved in different fish species. Tissue expression analysis showed that EcKLF9 was constitutively expressed in all 12 tissues tested, in the healthy grouper, the highest expression being detected in the gonads. The relative expression levels of EcKLF9 in the head kidney, spleen and brain was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infections. Using fluorescence microscopy, EcKLF9 was primarily localized to the nucleus and cytoplasm. The in vitro ectopic expression of EcKLF9 significantly increased the severity of vacuoles induced by RGNNV and the cytopathic effect progression evoked by SGIV infection. Real-time PCR results showed that the transcription levels of viral genes, such as the Singapore grouper iridovirus infection genes, MCP (major capsid protein), LITAF (lipopolysaccharide induced TNF-α factor), VP19 (envelop protein) ICP-18 (infected cell protein-18) and the red-spotted grouper nervous necrosis virus genes, CP (coat protein), RdRp (RNA-dependent RNA polymerase), were all significantly increased in EcKLF9 overexpressing cells, when compared to control cells. Furthermore, western blotting analyses showed that protein levels of the RGNNV gene, CP and the SGIV gene, MCP were also increased in EcKLF9 overexpressing cells, suggesting EcKLF9 may promote viral activity against iridovirus and nodavirus, in vitro. Moreover, the overexpression of EcKLF9 significantly inhibited the expression of several interferon related cytokines and several inflammatory cytokines. Accordingly, we speculate that EcKLF9 may exert stimulatory effects on RGNNV and SGIV replication, through the negative regulation of host immune and inflammation responses.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Fatores de Transcrição Kruppel-Like/química , Nodaviridae/fisiologia , Filogenia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária , Especificidade da Espécie
6.
Fish Shellfish Immunol ; 89: 52-60, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30904683

RESUMO

Siniperca chuatsi is an economically important fish in China, but infectious spleen and kidney necrosis virus (ISKNV) causes high mortality and significant economic losses. Currently, vaccination is the most promising strategy to prevent infectious diseases, while adjuvant can effectively enhance immune responses. In this study, inactivated ISKNV vaccine was prepared, then poly (I:C), chitosan, anisodamine and ims1312 were used as adjuvants to evaluate the effect on the immune responses and ISKNV replication. Chitosan could strongly boost the protection of liver and spleen tissues by pathological sections. In serum, poly (I:C) and chitosan group had protective effect on catalase, acid phosphatase, blood urea nitrogen. mRNA expressions showed these adjuvants induced the cytokines of early immune responses (TNF-α, Viperin) in both spleen and mesonephron by real time quantitative RT-PCR assays. Meanwhile, poly (I:C), chitosan and anisodamine were significantly improved the antiviral function and inhibited ISKNV replication. Chitosan and anisodamine played a significantly protective role in the immune protective rate test. The results indicated that all the four adjuvants are valid in the inactivated ISKNV vaccine, and chitosan is recommended preferentially. The present study provides reference for other animal vaccine adjuvants.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Quitosana/imunologia , Iridoviridae/imunologia , Perciformes/imunologia , Alcaloides de Solanáceas/imunologia , Vacinas Virais/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Enzimas/sangue , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Perfilação da Expressão Gênica/veterinária , Imunidade Inata/efeitos dos fármacos , Perciformes/genética , Poli I-C/imunologia , Replicação Viral/efeitos dos fármacos
7.
Fish Shellfish Immunol ; 89: 27-34, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30910614

RESUMO

Granulocyte colony stimulating factor (GCSF) is a key regulator of neutrophil production, and plays a vital role in immune response of mammals and teleost against pathogen. Sequences of GCSF were identified in several teleost species, however, the function and activity of GCSF in teleost remain largely unknown. In this study, we examined the biological activity and the immunomodulatory property of a GCSF homologue, PoGCSF, from Japanese flounder (Paralichthys olivaceus). Structural analysis showed that PoGCSF possesses conserved structural characteristics of GCSF proteins, including a signal peptide and a typical IL-6 domain. The expression of PoGCSF was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoGCSF increased the capability of host cells to defense against pathogen infection and enhanced the expression of immune related genes. The knockdown of PoGCSF attenuated the ability of host cells to eliminate pathogenic bacteria. In vivo results showed that overexpression of PoGCSF promoted the host defense against invading pathogenic microorganism. Collectively, this study is the first report about the immunoregulatory property and anti-infectious immunity of GCSF in teleost. These findings suggested that PoGCSF serves as an immune-related cytokine and plays an important role in the immune defense system of Japanese flounder.


Assuntos
Doenças dos Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/veterinária , Fenômenos Fisiológicos Bacterianos , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Fator Estimulador de Colônias de Granulócitos/química , Iridoviridae/fisiologia , Alinhamento de Sequência/veterinária
8.
Fish Shellfish Immunol ; 88: 391-402, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30853655

RESUMO

Tripartite motif (TRIM) proteins have been demonstrated to exhibit critical functions in multiple cellular processes, including development, carcinogenesis, and programmed cell death, and are also widely recognized to be important antiviral restriction factors or modulators of immune and inflammatory signaling pathways. However, in teleosts, additional TRIM members have been identified and their functions remain largely unknown. Here, a novel finTRIM gene from orange spotted grouper (EcfinTRIM82) was cloned and characterized. Sequence analysis indicated that EcfinTRIM82 encoded a 575 amino acid peptide which shared 94% and 82% identity with Asian sea bass (Lates calcarifer), and zebrafish (Danio rerio) finTRIM82, respectively. EcfinTRIM82 contained three conserved domains, including a RING, B-Box, and SPRY domain. Using fluorescence microscopy, we found that green fluorescence aggregates were observed in the cytoplasm of EcfinTRIM82-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcfinTRIM82 transcription was significantly upregulated in Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infected GS cells. This suggests that EcfinTRIM82 might be involved in fish virus infection. The in vitro overexpression of EcfinTRIM82 in GS cells significantly enhanced the replication of SGIV and RGNNV, evidenced by increased expression of viral genes, including the SGIV major capsid protein (MCP), VP19, ICP-18, RGNNV coat protein (CP), and RNA-dependent RNA polymerase (RdRp). Furthermore, the ectopic expression of EcfinTRIM82 significantly decreased the expression of interferon (IFN)-related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), ISG56, IFP35, and myxovirus resistance gene (MXI), suggesting that EcfinTRIM82 regulated viral replication via the negative regulation of the host IFN response. In addition, EcfinTRIM82 overexpression substantially decreased the level of proinflammatory cytokine transcription. Furthermore, the ectopic expression of EcfinTRIM82 significantly weakened the melanoma differentiation-associated protein 5 (MDA5), mediator of IRF3 activation (MITA) and mitochondrial antiviral-signaling (MAVS) protein-induced IFN response by detecting the transcription of interferon related cytokines and the promoter activity of IFN. Together, our results demonstrate that finTRIM82 negatively regulates the innate antiviral immune response against grouper virus infection.


Assuntos
Bass/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/imunologia , Imunidade Inata , Interferons/imunologia , Proteínas com Motivo Tripartido/imunologia , Animais , Bass/virologia , Clonagem Molecular , Infecções por Vírus de DNA/imunologia , DNA Complementar , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Iridovirus/imunologia , Filogenia , RNA Mensageiro , Alinhamento de Sequência , Análise de Sequência de DNA , Baço/citologia , Baço/virologia , Proteínas com Motivo Tripartido/genética , Peixe-Zebra/imunologia
9.
Fish Shellfish Immunol ; 88: 217-224, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30807858

RESUMO

Chemokine receptors are a superfamily of seven-transmembrane domain G-coupled receptors and have important roles in immune surveillance, inflammation, and development. In previous studies, a series of CXCRs in grouper (Epinephelus coioides) was identified; however, the function of CXCR in viral infection has not been studied. To better understand the effect of the CXCR family on the fish immune response, full-length CXCR1a was cloned, and its immune response to Singapore grouper iridovirus (SGIV) was investigated. Grouper CXCR1a shared a seven-transmembrane (7-TM) region and a G protein-coupled receptor (GPCR) family 1 that contained a triaa stretch (DRY motif). Phylogenetic analysis indicated that CXCR1a showed the nearest relationship to Takifugu rubripes, followed by other fish, bird and mammal species. Fluorescence microscopy revealed that CXCR1a was expressed predominantly in the cytoplasm. Overexpression of CXCR1a in grouper cells significantly inhibited the replication of SGIV, demonstrating that CXCR1a delayed the occurrence of cytopathic effects (CPE) induced by SGIV infection and inhibited viral gene transcription. Furthermore, our results also showed that CXCR1a overexpression significantly increased the expression of interferon-related cytokines and activated ISRE and IFN promoter activities. Taken together, the results demonstrated that CXCR1a might have an antiviral function against SGIV infection.


Assuntos
Bass/genética , Bass/imunologia , Infecções por Vírus de DNA/veterinária , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Animais , Citocinas/metabolismo , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Microscopia de Fluorescência , Filogenia , Ranavirus/fisiologia , Replicação Viral/imunologia
10.
Immunity ; 50(1): 51-63.e5, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30635239

RESUMO

Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Infecções por Vírus de DNA/imunologia , Vírus de DNA/fisiologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , 2',5'-Oligoadenilato Sintetase/genética , Animais , AMP Cíclico/metabolismo , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Células THP-1 , Replicação Viral
11.
Virology ; 528: 37-47, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30554072

RESUMO

In vertebrates, pyroptosis is an intense inflammatory form of programmed cell death. This death pathway is critical for controlling pathogenic infection. In invertebrates, however, due to the lack of adaptive immune response, it is still elusive whether Caspase 1-dependent cell death pathway exists. In this study, our data showed that Caspase 1-mediated cell death was activated by white spot syndrome virus to counteract virus infection. Caspase 1 had a higher expression in hemocytes and lymphoid-like organ in shrimp and WSSV infection was promoted upon the inhibition of Caspase 1 enzymatic activity. IL-1ß-like protein was identified as the substrate of Caspase 1 and its interaction with Caspase 1 was validated ectopically and endogenously. Moreover, IL-1ß like protein was released into extracellular contents under WSSV infection and Prophenoloxidase system was activated, resulting in the reduction of WSSV. Our data unraveled a previously unidentified mechanism through which Caspase 1-dependent cell death controlled virus infection in shrimp.


Assuntos
Caspase 1/metabolismo , Morte Celular , Infecções por Vírus de DNA/veterinária , Hemócitos/virologia , Penaeidae/virologia , Animais , Caspase 1/genética , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Infecções por Vírus de DNA/imunologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Hemócitos/patologia , Penaeidae/imunologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Vírus da Síndrome da Mancha Branca 1
12.
Dev Comp Immunol ; 90: 1-9, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30031870

RESUMO

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


Assuntos
Agrocybe/genética , Antígenos de Fungos/genética , Infecções por Vírus de DNA/imunologia , Lectinas/genética , Penaeidae/imunologia , Transgenes/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Antivirais/metabolismo , Clonagem Molecular , Agregação Eritrocítica , Imunidade Inata , Lectinas/metabolismo , Penaeidae/virologia , Ligação Proteica , Proteínas Virais/metabolismo
13.
Dev Comp Immunol ; 90: 10-20, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30165083

RESUMO

Chemokines comprise a group of small molecular weight (6-14 kDa) cytokines; chemokine receptors are a superfamily of seven transmembrane domain G-coupled receptors. Both chemokines and their receptors have important roles in immune surveillance, inflammation, and development. Recently, 9 CXC chemokine ligands (CXCLs) and 8 CXC chemokine receptors (CXCRs) were identified and cloned from orange-spotted grouper (Epinephelus coioides) and annotated by phylogenetic and syntenic analyses. We detected mRNA transcripts for CXCLs and CXCRs in healthy tissues of E. coioides. Our data show that CXCL genes are highly expressed in the spleen, kidney and liver and that CXCR genes are ubiquitously expressed, rather than being expressed only in immune organs. Analysis of gene expression after Singapore grouper iridovirus infection indicated that CXCL and CXCR genes are regulated in a gene-specific manner. CXCL8 and CXCL12a were significantly upregulated in the spleen, kidney and liver of resistant fish, indicating potential roles in immunity against the pathogen. Additionally, CXCR4a was upregulated in all three organs in resistant fish, suggesting that CXCL8 or CXCL12a may participate in the immune response via interaction with CXCR4a. In addition, the new orange-spotted grouper receptor CXCR1b was found to be upregulated in the spleen and kidney of resistant fish, indicating that this receptor plays an important role in immune responses to viral infection. These results are valuable for comparative immunological studies and provide insight into the roles of these genes in viral infection.


Assuntos
Quimiocina CXCL12/genética , Infecções por Vírus de DNA/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Interleucina-8/genética , Iridovirus/fisiologia , Perciformes/imunologia , Receptores CXCR4/genética , Receptores de Interleucina-8A/genética , Animais , Quimiocina CXCL12/metabolismo , Clonagem Molecular , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Imunidade Inata/genética , Interleucina-8/metabolismo , Filogenia , Receptores CXCR4/metabolismo , Receptores de Interleucina-8A/metabolismo , Transcriptoma
14.
Dev Comp Immunol ; 90: 70-79, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30195709

RESUMO

In this study, the transcriptional response of grouper to Singapore grouper iridovirus (SGIV) stimulation was characterized using RNA sequencing. Transcriptome sequencing of three test groups in the grouper was performed using the Illumina MiSeq platform. The three test groups were a control group, which was injected with PBS buffer; a high-susceptible (HS) group, which died shortly after the SGIV injection; and a high-resistance (HR) group, which survived the SGIV injection. In total, 38,253 unigenes were generated. When the HS group was compared with the control group, 885 unigenes were upregulated and 487 unigenes were downregulated. When the HR and control groups were compared, 1114 unigenes were upregulated and 420 were downregulated, and when the HR and HS groups were compared, 1010 unigenes were upregulated and 375 were downregulated. In the KEGG analysis, two immune-related pathways, the p53 and peroxisome proliferator-activated receptor pathways, were detected with highly significant enrichment. In addition, 7465 microsatellites and 22,1569 candidate single nucleotide polymorphisms were identified from our transcriptome data. The results suggested several pathways that are associated with traits of disease susceptibility or disease resistance, and provided extensive information about novel gene sequences, gene expression profiles, and genetic markers. This may contribute to vaccine research and a breeding program against SGIV infection in grouper.


Assuntos
Infecções por Vírus de DNA/imunologia , Iridovirus/fisiologia , Perciformes/imunologia , Animais , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Estudos de Associação Genética , Imunidade Inata/genética , Repetições de Microssatélites/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Dev Comp Immunol ; 91: 17-25, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30278186

RESUMO

The oyster's immune system is capable of adapting upon exposure to a pathogen-associated molecular pattern (PAMP) to have an enhanced secondary response against the same type of pathogen. This has been demonstrated using poly(I:C) to elicit an antiviral response in the Pacific oyster (Crassostrea gigas) against Ostreid herpesvirus (OsHV-1). Improved survival following exposure to poly(I:C) has been found in later life stages (within-generational immune priming) and in the next generation (transgenerational immune priming). The mechanism that the oyster uses to transfer immunity to the next generation is unknown. Here we show that oyster larvae have higher survival to OsHV-1 when their mothers, but not their fathers, are exposed to poly(I:C) prior to spawning. RNA-seq provided no evidence to suggest that parental exposure to poly(I:C) reconfigures antiviral gene expression in unchallenged larvae. We conclude that the improved survival of larvae might occur via maternal provisioning of antiviral compounds in the eggs.


Assuntos
Crassostrea/imunologia , Infecções por Vírus de DNA/imunologia , Vírus de DNA/fisiologia , Doenças dos Peixes/imunologia , Exposição Materna , Poli I-C/imunologia , Vacinas Virais/imunologia , Animais , Antivirais , Feminino , Imunidade Inata , Imunidade Materno-Adquirida , Larva , Masculino , Óvulo/imunologia , Óvulo/virologia , Exposição Paterna
16.
Dev Comp Immunol ; 91: 37-49, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30336173

RESUMO

To elucidate the proteomic responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection at the proteome level, a quantitative shotgun proteomic analysis was performed to detect differentially synthesized proteins in infected hemocytes of white shrimp (Litopenaeus vannamei). We identified 1528 proteins associated to 203 gene ontology (GO) categories. The most representative GO categories were regulation of cellular processes, organic substance metabolic processes and nitrogen compound metabolic processes. Most of the 83 detected up-regulated proteins are involved in DNA regulation and organization and cell signaling. In contrast, most of the 40 down-regulated proteins were related to immune defense processes, protein folding, and development. Differentially induced proteins were further analyzed at the transcript level by RT-qPCR to validate the results. This work provides new insights into the alterations of L. vannamei hemocytes at the protein level at 12 h post-infection with WSSV. Interestingly, several of the up-regulated proteins are allergy-related proteins in humans. Based on our results, we suggest a deeper analysis of the effects of this interaction on the regulation of allergy related-proteins as their up-regulation during WSSV could represent a threat to human health.


Assuntos
Proteínas de Artrópodes/metabolismo , Infecções por Vírus de DNA/imunologia , Hemócitos/fisiologia , Hipersensibilidade/metabolismo , Penaeidae/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Proteínas de Artrópodes/genética , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Hipersensibilidade/genética , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/virologia , Proteoma
17.
Dev Comp Immunol ; 91: 50-61, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30339874

RESUMO

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


Assuntos
Antivirais/imunologia , Proteínas de Artrópodes/imunologia , Infecções por Vírus de DNA/imunologia , Hemocianinas/imunologia , Penaeidae/imunologia , Peptídeos/imunologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Clonagem Molecular , Imunidade Inata , Penaeidae/virologia , Ligação Proteica , Ativação Transcricional , Proteínas do Envelope Viral/metabolismo , Replicação Viral
18.
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
19.
Fish Shellfish Immunol ; 86: 1026-1034, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30584907

RESUMO

Virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible (viperin), is an antiviral protein, induced by interferon (IFN), poly(I:C) and viral infection to exert antiviral function. To investigate the roles of viperin during fish virus infection, a viperin homolog from orange spotted grouper (Epinephelus coioides) (Ecviperin) was cloned and characterized in this study. Ecviperin encoded a 361-aa protein which shared 87% and 69% identity with Siniperca undulata and Homo sapiens, respectively. Amino acid alignment analysis showed that Ecviperin contained a conserved radical-SAM domain (aa73-281). Phylogenetic analysis indicated that Ecviperin showed the nearest relationship with S. undulata. In healthy grouper, Ecviperin was distributed in all tissues, and the expression of Ecviperin was the highest in kidney and spleen. In vitro, the mRNA expression of Ecviperin was significantly up-regulated in response to Singaporean grouper iridovirus (SGIV) infection. Subcellular localization analysis showed that Ecviperin was distributed in the cytoplasm and co-localized with endoplasmic reticulum (ER). The ectopic expression of Ecviperin significantly inhibited the replication of SGIV. Furthermore, overexpression of Ecviperin positively regulated the interferon related molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), myxovirus resistance gene I (MXI), interferon-induced 35-kDa protein (IFP35), and TNF receptor-associated factor 6 (TRAF6). In addition, the expression of pro-inflammation cytokines was differently regulated by Ecviperin overexpression. Furthermore, reporter gene analysis showed that the overexpression of Ecviperin enhanced the activity of nuclear factor of kappa B (NF-κB), IFN-1 and interferon-stimulated response element (ISRE) promoter, suggesting that Ecviperin might restrict SGIV replication by the positive regulation of interferon and inflammatory response. Taken together, our results demonstrated that Ecviperin encoded an ER-localized protein, and exerted antiviral function against fish DNA virus by up-regulating interferon and pro-inflammatory response.


Assuntos
Bass/imunologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Proteínas de Peixes/imunologia , Iridovirus , Sequência de Aminoácidos , Animais , Infecções por Vírus de DNA/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Imunidade Inata , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Interferons/imunologia , Interferons/metabolismo , Filogenia , Alinhamento de Sequência/veterinária
20.
Fish Shellfish Immunol ; 86: 1088-1095, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30593901

RESUMO

Protein SUMOylation (SUMO is small ubiquitin-related modifier) is a dynamic process that is strictly regulated under physiological and pathological conditions. We previously cloned and characterized two SUMO homologue genes (EcSUMO1 and EcSUMO2) from orange-spotted grouper (Epinephelus coioides). In the present study, the SUMO3 homologue from E. coioides (EcSUMO3) was cloned and its possible roles in fish immunity were analyzed. The open reading frame of EcSUMO3 contains 285 base pairs encoding a 94 amino acid protein with a predicted molecular mass of 10.73 kDa. The protein sequence of EcSUMO3 revealed similar domains with mammals, including the UBQ (ubiquitin-like proteins) domain, the hydrophobic surface, the Ulp1-Smt3 interaction sites, a VKTE motif and the C-terminal Gly residues. EcSUMO3 shares 46.83% and 89.58% identity with EcSUMO1 and EcSUMO2, respectively, and it shares 94%, 98%, and 98% identity with SUMO3 from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. Quantitative real-time polymerase chain reaction analysis indicated that EcSUMO3 was constitutively expressed in all of the analyzed tissues in healthy grouper. EcSUMO3 expression levels were remarkably (p < 0.01) up-regulated in grouper spleen (GS) cells in response to stimulation with red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV). EcSUMO3 was distributed in both the cytoplasm and nucleus in GS cells. EcSUMO3 enhanced SGIV and RGNNV replication during viral infection in vitro. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.


Assuntos
Bass/genética , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Infecções por Vírus de RNA/veterinária , Proteína SUMO-1/genética , Sequência de Aminoácidos , Animais , Bass/imunologia , Infecções por Vírus de DNA/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata/genética , Iridovirus/fisiologia , Nodaviridae/fisiologia , Proteína SUMO-1/imunologia , Ubiquitinas/metabolismo
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