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1.
PLoS Pathog ; 16(3): e1008429, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32208449

RESUMO

Chromatin dynamics regulated by epigenetic modification is crucial in genome stability and gene expression. Various epigenetic mechanisms have been identified in the pathogenesis of human diseases. Here, we examined the effects of ten epigenetic agents on pseudorabies virus (PRV) infection by using GFP-reporter assays. Inhibitors of bromodomain protein 4 (BRD4), which receives much more attention in cancer than viral infection, was found to exhibit substantial anti-viral activity against PRV as well as a range of DNA and RNA viruses. We further demonstrated that BRD4 inhibition boosted a robust innate immune response. BRD4 inhibition also de-compacted chromatin structure and induced the DNA damage response, thereby triggering the activation of cGAS-mediated innate immunity and increasing host resistance to viral infection both in vitro and in vivo. Mechanistically, the inhibitory effect of BRD4 inhibition on viral infection was mainly attributed to the attenuation of viral attachment. Our findings reveal a unique mechanism through which BRD4 inhibition restrains viral infection and points to its potent therapeutic value for viral infectious diseases.


Assuntos
Proteínas de Ciclo Celular/imunologia , Dano ao DNA/imunologia , Vírus de DNA/imunologia , Imunidade Inata , Proteínas Nucleares/imunologia , Vírus de RNA/imunologia , Fatores de Transcrição/imunologia , Células A549 , Animais , Chlorocebus aethiops , Infecções por Vírus de DNA/imunologia , Cães , Feminino , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Células RAW 264.7 , Infecções por Vírus de RNA/imunologia , Suínos , Células Vero
2.
PLoS One ; 15(1): e0227670, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31917803

RESUMO

Torque teno virus (TTV) is an unenveloped, circular, single stranded DNA virus with a genome size of approximately 3.8 kb. Previous studies have demonstrated varying grades of association between TTV DNA levels and immune deficiencies related to age, chronic infections and cancer. Alzheimer's disease (AD) has been related to persistent viral infections such as HSV-1 and CMV, but it is not known whether TTV viral load could serve as a functional biomarker of cellular immunity in this setting. Therefore, the objective of this study was to investigate whether TTV infection and viral load is related to AD status, CMV immunity, systemic inflammation or HLA types connected to anti-viral immunity. A total of 50 AD subjects and 51 non-demented controls were included in the study. AD subjects were diagnosed according to NINCDS-ADRDA and DSM-IV criteria and neuroradiologic findings were consistent with the diagnosis. TTV viral load was analyzed in plasma samples using a quantitative real-time PCR. Using a cut-off for TTV status at 200 copies/ml, 88% (89/101) of the study subjects were classified as TTV positive. TTV viral load significantly increased with age (beta 0.049 per year, p<0.001) but significantly decreased in relation to CMV IgG levels (beta -0.022 per 1000 units, p = 0.005) and HLA-B27 positivity (beta -0.53, p = 0.023). In conclusion, TTV immune control is not significantly affected by AD status, but appears related to age, CMV humoral immune response and HLA type.


Assuntos
Doença de Alzheimer/imunologia , Doença de Alzheimer/virologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/virologia , Torque teno virus/patogenicidade , Carga Viral , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/complicações , Estudos de Casos e Controles , Infecções por Citomegalovirus/complicações , Infecções por Vírus de DNA/complicações , Feminino , Teste de Histocompatibilidade , Humanos , Imunidade Celular , Masculino , Torque teno virus/genética , Torque teno virus/imunologia , Carga Viral/imunologia
3.
Fish Shellfish Immunol ; 96: 311-318, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31830568

RESUMO

C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein kinase (MAP) kinase kinase, which can initiate a mitogen-activated protein kinase (MAPK) cascade by phosphorylating the dual-specific MAP kinase kinases (MEK1/2), and in turn activate the extracellular signal-regulated kinases (ERK1/2). To study the function of c-Raf in teleost fish, a c-Raf cDNA sequence from orange-spotted grouper (Epinephelus coioides) was cloned. Ecc-Raf shared 81%-99% amino acid identity with other vertebrate c-Raf molecules, and shared the highest amino acid identity (99%) with Lates calcarifer c-Raf. Genomic structure analysis revealed that grouper c-Raf shared a conserved exon structure with other vertebrates. Tissue distribution showed that Ecc-Raf was mainly transcribed in systemic immune organs. Ecc-Raf was distributed throughout the cytoplasm of transfected GS cells and the overexpression of Ecc-Raf only slightly enhanced the activation of Activator protein 1. The phosphorylation levels of Ecc-Raf can be induced by PMA and H2O2 treatment, in contrast to DMSO or untreated HKLs. Moreover, the phosphorylation level of the Raf-MEK-ERK axis was downregulated after 24 h of SGIV infection. On the other hand, the total level and phosphorylation level of c-Raf significantly increased post C. irritans infection and showed an enhanced level post immunization. The results of this study suggested that the Raf-MEK-ERK cascade was involved in the response to viral or parasitic infections.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/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 , Sistema Imunitário/metabolismo , Filogenia , Proteínas Proto-Oncogênicas c-raf/química , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
4.
J Appl Microbiol ; 128(1): 41-53, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31529740

RESUMO

AIMS: To determine the immune gene expression response of gilthead seabream (Sparus aurata) that is experimentally infected with the lymphocystivirus LCDV-Sa. METHODS AND RESULTS: Viral DNA and transcripts were detected by qPCR in all samples from fish injected with LCDV-Sa, demonstrating that the virus establish a systemic and asymptomatic infection. The expression of 23 immune-related genes was also analysed by RT-qPCR in the head kidney (HK) and intestine at several times post-infection (dpi). In HK, the expression of five type I interferon (IFN)-related genes (ifn, irf3, mx2, mx3 and isg15), il10 and ck10 was upregulated at 1-3 dpi, while genes related to the inflammation process (tnfα, il1ß, il6, casp1) were not differentially expressed or even downregulated. The expression profile in the intestine was different regarding type I INF-related genes. An upregulated c3 and ighm expression was observed in both HK and intestine at 3-8 dpi. Finally, the transcription of nccrp1 and mhcIIα was induced in HK, whereas tcrß expression was downregulated in both organs. CONCLUSIONS: LCDV-Sa seems to trigger an immune response in gilthead seabream characterized by a partial activation of type I IFN system and a lack of systemic inflammatory response which may be related to viral persistence. SIGNIFICANCE AND IMPACT OF THE STUDY: The immune response observed in gilthead seabream infected by LCDV-Sa could be implicated in the establishment of an asymptomatic persistent infection.


Assuntos
Infecções Assintomáticas , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Iridoviridae/fisiologia , Dourada/imunologia , Animais , Citocinas/genética , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Rim Cefálico/imunologia , Intestinos/imunologia , Dourada/genética , Dourada/virologia
5.
Int J Mol Sci ; 20(22)2019 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-31717495

RESUMO

Hepcidin is a liver-derived peptide hormone that is related to iron balance and immunity in humans. However, its function in Siniperca chuatsi has not been well elucidated. In this study, we analyzed the expression and function of the S. chuatsi hepcidin (Sc-hep) gene. Sc-hep was specifically expressed in the liver and appeared to be one of the most highly expressed genes in the liver. After spleen and kidney necrosis virus (ISKNV) infection and lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C) stimulation, the expression of Sc-hep in the liver increased by approximately 110, 6500, and 225 times, respectively. After ferrous sulfate (FS) injection, the expression of Sc-hep in the liver increased approximately 520-fold. We found that miR-19c-5p could inhibit Sc-hep expression. Five CpG dinucleotides distributed in the promoter region showed no differential methylation between the liver and the stomach, both presenting high methylation rates. After FS or LPS injection, the expression of three iron balance-related genes (FPN1, TFR1, and FTN) and five immune-related cytokine genes (IL-1ß, IL8, TNF-α, TLR22, and SOCS3) significantly changed. These results indicate that Sc-hep participates in the regulation of iron balance and plays an important role in the immune system. Sc-hep increased approximately 52-fold when mandarin fish were domesticated with artificial diets. Sc-hep might be used as a real-time biomarker of mandarin fish liver because its expression markedly varies under different physiological conditions.


Assuntos
Doenças dos Peixes/genética , Proteínas de Peixes/genética , Peixes/genética , Hepcidinas/genética , Animais , Metilação de DNA , Infecções por Vírus de DNA/genética , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Proteínas de Peixes/imunologia , Peixes/imunologia , Peixes/virologia , Regulação da Expressão Gênica , Hepcidinas/imunologia , Imunidade , Iridoviridae/imunologia , Lipopolissacarídeos/imunologia , MicroRNAs/genética , MicroRNAs/imunologia , Filogenia
6.
Fish Shellfish Immunol ; 94: 81-89, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31476389

RESUMO

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), one of the interferon stimulated genes (ISGs), is strongly induced by type I interferon (IFN), double-stranded RNAs and virus infection. To investigate the actions of fish IFIT1 in response to virus infection, we cloned an IFIT1 homolog from orange spotted grouper (EcIFIT1) and clarified its function in this study. The full-length cDNA of EcIFIT1 is 1839 bp, which is composed of 436 amino acid (aa) residues, with 77.8% and 22.8% identity to IFIT1 homolog of yellow perch (Perca flavescens) and humans (homo sapiens), respectively. Sequence alignment analysis showed that EcIFIT1 contained three tetratricopeptide repeats (TPRs). Tissue distribution analysis indicated that EcIFIT1 was abundant in intestine, spleen, liver, and heart. Moreover, EcIFIT1 was significantly up-regulated by Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infection, and polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS) treatment in vitro. Under fluorescence microscopy, EcIFIT1 was found to localize throughout the cytoplasm in transfected cells. EcIFIT1 overexpression significantly suppressed the replication of SGIV and RGNNV, demonstrated by decreasing the cytopathic effect (CPE) severity, viral gene transcription and the virus titers. Further studies showed that the ectopic expression of EcIFIT1 increased the transcription level of IFN related molecules, including IFN regulatory factor (IRF) 3, IRF7, IFN stimulated gene (ISG) 15 and myxovirus resistance gene (MX) I. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcIFIT1. In addition, flow cytometry analysis suggested that EcIFIT1 overexpression affected cell cycle progression by mediating S/G2 transition. Taken together, our results indicated that EcIFIT1 might exert antiviral function against fish virus by up-regulating interferon response or affecting cell cycle.


Assuntos
Bass/genética , Bass/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , 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 , Lipopolissacarídeos/farmacologia , Nodaviridae/fisiologia , Filogenia , Poli I-C/farmacologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
7.
Fish Shellfish Immunol ; 94: 113-121, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31491526

RESUMO

Autophagy related gene 16 (Atg16), which encodes a core protein for autophagosome formation, participates in autophagy activity, the ubiquitin proteasome system and inflammatory response in mammals. In this study, we cloned and characterized an Atg16 homolog from orange-spotted grouper (Epinephelus coioides) (EcAtg16L1). EcAtg16L1 encodes a 656-amino acid polypeptide, which shares 94.22% and 72.65% homology with large yellow croakers (Larimichthys crocea) and humans (Homo sapiens), respectively. EcAtg16L1 contains a conserved Atg16 domain and a WD-repeat-containing domain. Subcellular localization showed that EcAtg16L1 was distributed in the cytoplasm of grouper cells with a dot-like pattern. EcAtg16L1 overexpression promoted Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by the increase in viral gene transcription and viral coat protein. Furthermore, EcAtg16L1 overexpression negatively regulated interferon (IFN)-related molecules and proinflammatory cytokines, and decreased IFN, IFN-stimulated response element, and nuclear factor κB promoter activities. Taken together, aside from its function in autophagosome formation, EcAtg16L1 also plays role in promoting SGIV and RGNNV replication and the pro-viral effect might involve its down regulation to interferon and inflammatory responses.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas Relacionadas à Autofagia/química , 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 , Nodaviridae/fisiologia , Filogenia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
8.
Fish Shellfish Immunol ; 93: 702-710, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421242

RESUMO

Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process used to maintain cell survival and homeostasis. A series of autophagy-related genes (Atgs) are involved in the autophagic pathway. In mammals, a growing number of studies have attributed functions to some Atgs that are distinct from their classical role in autophagosome biogenesis, such as resistance to pathogens. However, little is known about the functions of fish Atgs. In this study, we cloned and characterized an atg12 homolog from orange spotted grouper (Epinephelus coioides) (Ecatg12). Ecatg12 encodes a 117 amino acid protein that shares 94.0% and 76.8% identity with gourami (Anabas_testudineus) and humans (Homo sapiens), respectively. The transcription level of Ecatg12 was lower in cells infected with Singapore grouper iridovirus (SGIV) than in non-infected cells. Fluorescence microscopy revealed that EcAtg12 localized in the cytoplasm and nucleus in grouper spleen cells. Overexpression of EcAtg12 significantly increased the replication of SGIV, as evidenced by increased severity of the cytopathic effect, transcription levels of viral genes, levels of viral proteins, and progeny virus yield. Further studies showed that EcAtg12 overexpression decreased the expression levels of interferon (IFN) related molecules and pro-inflammatory factors and inhibited the promoter activity of IFN-3, interferon-stimulated response element, and nuclear factor-κB. Together, these results demonstrate that EcAtg12 plays crucial roles in SGIV replication by downregulating antiviral immune responses.


Assuntos
Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/imunologia , Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteína 12 Relacionada à Autofagia/química , 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 , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
9.
Fish Shellfish Immunol ; 93: 406-415, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31369857

RESUMO

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , 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 , Iridoviridae/fisiologia , Filogenia , Poli I-C/farmacologia , Polidesoxirribonucleotídeos/farmacologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária , Acetato de Tetradecanoilforbol/farmacologia , Proteína 1 de Ligação a Y-Box/química
10.
Fish Shellfish Immunol ; 94: 38-49, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31470135

RESUMO

Peroxisome proliferator-activated receptor δ (PPAR-δ), also called PPAR-ß or PPAR-ß/δ, is a member of the peroxisome proliferator-activated receptor (PPAR) family, which belongs to the nuclear steroid receptor superfamily. Activated PPARs participate in the regulation of lipid and glucose metabolism and also affect cellular proliferation, differentiation, and apoptosis, and the immune responses. To investigate the roles of PPAR-δ in Singapore grouper iridovirus (SGIV) infection, we cloned and characterized the gene encoding a PPAR-δ homologue from the orange-spotted grouper, Epinephelus coioides (EcPPAR-δ). EcPPAR-δ encodes a 514-amino-acid polypeptide, with 95.29% and 74.76% homologue to the Seriola dumerili and human proteins, respectively. EcPPAR-δ contains a typical DNA-binding domain and a ligand-binding domain. Its expression was induced by SGIV infection in vitro. A subcellular localization analysis showed that EcPPAR-δ localizes throughout the cytoplasm and nucleus, with a diffuse intracellular expression pattern. SGIV replication was reduced by EcPPAR-δ overexpression, which was evident in the reduced severity of the cytopathic effect, reduced viral gene transcription, and the reduced expression of the viral capsid protein. The replication of SGIV increased with the knockdown of EcPPAR-δ. The overexpression and silencing of EcPPAR-δ in grouper spleen cells showed that EcPPAR-δ plays a positive role in the regulation of the interferon signaling pathway, but has an anti-inflammatory effect on the inflammatory response. The anti-inflammatory effect of EcPPAR-δ may be related to its function in maintaining cell homeostasis. Because the interferon signaling pathway plays an important role in antiviral immune responses, we speculate that the activation of the interferon signaling pathway by EcPPAR-δ overexpression underlies its inhibitory effect on SGIV replication. Together, our data greatly extend our understanding of the roles of the EcPPAR-δ family members in the pathogenesis of fish viruses.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , PPAR delta/genética , PPAR delta/imunologia , Sequência de Aminoácidos , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , PPAR delta/química , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
11.
Fish Shellfish Immunol ; 93: 50-54, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31276790

RESUMO

Atypical chemokine receptor 4 (ACKR4) is regulated by cytokines, binds chemokines and regulates the chemokine gradient. We verified the cDNA sequence by confirming ACKR4 from red sea bream (PmACKR4) by next generation sequencing (NGS) and analysed the molecular characteristics and gene expression profile. In the analysis using the predicted amino acid sequence of PmACKR4, a highly conserved G protein-coupled receptor 1 region and two cysteine residues were identified and included in the ACKR4 teleost cluster in the phylogenetic analysis. In healthy red sea bream, PmACKR4 mRNA was expressed at the highest levels in head kidney and was upregulated in all immune -related tissues used in the experiment after challenges with Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV). These results suggest that ACKR4 is highly conserved in red sea bream and may play an important role in the immune system as previously reported. It is thought that ACKR4 acts as a regulator of immune -related cells via immune reactions after pathogenic infection.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Receptores CCR4/genética , Dourada/imunologia , Sequência de Aminoácidos , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Perfilação da Expressão Gênica/veterinária , Iridoviridae/fisiologia , Filogenia , Receptores CCR4/metabolismo , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus iniae/fisiologia
12.
Fish Shellfish Immunol ; 92: 889-896, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299465

RESUMO

Ranaviruses belong to the family Iridoviridae, and have become a serious threat to both farmed and natural populations of fish and amphibians. Previous reports showed that ranaviruses could encode viral Bcl-2 family-like proteins (vBcl-2), which play a critical role in the regulation of cell apoptosis. However, the mechanism of ranaviruses vBcl-2 interactions with host protein in mediating apoptosis remains unknown. Tiger frog virus (TFV) belonging to the genus Ranavirus has been isolated from infected tadpoles of Rana tigrina rugulosa, and it causes a high mortality rate among tiger frog tadpoles cultured in southern China. This study elucidated the molecular mechanism underlying the interaction of TFV ORF104R with the VDAC2 protein to regulate cell apoptosis. TFV ORF104R is highly similar to other ranaviruses vBcl-2 and host Mcl-1 proteins, indicating that TFV ORF104R is a postulate vBcl-2 protein. Transcription and protein expression levels showed that TFV orf104r was a late viral gene. Western blot results suggested that TFV ORF104R was a viral structural protein. Subcellular localization analysis indicated that TFV ORF104R was predominantly colocalized with the mitochondria. Overexpressed TFV ORF104R could suppress the release of cytochrome C and the activities of caspase-9 and caspase-3. These results indicated that TFV ORF104R might play an important role in anti-apoptosis. Furthermore, the interaction between TFV ORF104R and VDAC2 was detected by co-immunoprecipitation in vitro. The above observations suggest that the molecular mechanism of TFV-regulated anti-apoptosis is through the interaction of TFV ORF104R with the VDAC2 protein. Our study provided a mechanistic basis for the ranaviruses vBcl-2-mediated inhibition of apoptosis and improved the understanding on how TFV subverts host defense mechanisms in vivo.


Assuntos
Apoptose/imunologia , Cyprinidae , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Genes Virais , Ranavirus/fisiologia , Canal de Ânion 2 Dependente de Voltagem/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Fases de Leitura Aberta , Canal de Ânion 2 Dependente de Voltagem/genética
13.
Fish Shellfish Immunol ; 92: 649-654, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31265911

RESUMO

Singapore grouper iridovirus (SGIV) is the main grouper-infecting virus in southern China that causes serious economic losses. However, there is no effective way to control this viral disease. In this study, SGIV ORF19R (SGIV-19R) encoding a viral membrane protein was constructed into pcDNA3.1-HA and then was used to evaluate the immune protective effects in grouper Epinephelus coioides. Subcellular localization showed that SGIV-19R distributed in the cytoplasm and co-localization analysis indicated the protein partially co-localized with the endoplasmic reticulum (ER). RT-PCR and Western blot analyses confirmed the expression of the vaccine plasmids in grouper muscle tissues. Moreover, the transcription levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), myxovirus resistance 1 (Mx1) and immunoglobulin M (IgM) genes were significantly up-regulated in the spleen, liver and kidney of vaccinated groupers. SGIV challenge experiments showed the relative percent survival (RPS) was significantly enhanced in fish with 49.9% at the DNA dose of 45 µg pcDNA3.1-19R, while 75.0% RPS when using 90 µg pcDNA3.1-19R. Meanwhile, vaccination with pcDNA3.1-19R significantly reduced the virus replication, evidenced by a low viral load in the spleen of survivals groupers after SGIV challenge. These results imply that pcDNA3.1-19R could induce protective immunity in grouper, and might be a potential vaccine candidate for controlling SGIV disease.


Assuntos
Imunidade Adaptativa , Bass/imunologia , Doenças dos Peixes/prevenção & controle , Imunidade Inata , Ranavirus/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/prevenção & controle , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Injeções Intramusculares/veterinária , Iridovirus/fisiologia , Distribuição Aleatória , Proteínas da Matriz Viral/imunologia
14.
Fish Shellfish Immunol ; 93: 183-190, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31330254

RESUMO

In mammals, a matricellular protein, thrombospondin 2 (Thbs2) has been reported to play important roles in modulating cell-matrix interactions, vascular integrity and thrombosis formation. However, the role of gene, thbs2 has not yet been studied in teleost. In the present study, this novel fish gene from Japanese flounder was cloned and its function in resistant to lymphocystis disease virus was elucidated. The Japanese flounder thbs2 encoded a 1176-amino acid protein with 91% identity to medaka. Amino acid sequence indicated that Japanese flounder Thbs2 contained 10 typical conserved domains. The thbs2 was expressed in all stages of embryo development, and in hatched larva stage, its expression was significantly higher than that in other stages (P < 0.05). The relative expression level of thbs2 was significantly higher in the head kidney, liver, blood, gill, and heart of the lymphocystis disease virus resistant fish than in sensitive fish (P < 0.05); and in muscle, this difference was at highly significant (P < 0.01). Additionally, the distribution of Thbs2 in tissue was evaluated by immunohistochemical staining. Subcellular localization analysis showed that Thbs2 was distributed throughout the cytoplasm of the cells. Taken together, our results provide new basic data for thbs2 function, especially its role in anti-lymphocystis disease virus immune response.


Assuntos
Doenças dos Peixes/imunologia , Linguados/genética , Linguados/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Trombospondinas/genética , Trombospondinas/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , 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 , Iridoviridae/fisiologia , Filogenia , Alinhamento de Sequência/veterinária , Trombospondinas/química
15.
Science ; 365(6454)2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31320558

RESUMO

DNA viruses typically eject genomic DNA into the nuclei of host cells after entry. It is unclear, however, how nuclear pathogen-derived DNA triggers innate immune responses. We report that heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) recognizes pathogenic DNA and amplifies interferon-α/ß (IFN-α/ß) production. Upon DNA virus infection, nuclear-localized hnRNPA2B1 senses viral DNA, homodimerizes, and is then demethylated at arginine-226 by the arginine demethylase JMJD6. This results in hnRNPA2B1 translocation to the cytoplasm where it activates the TANK-binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway, leading to IFN-α/ß production. Additionally, hnRNPA2B1 facilitates N 6-methyladenosine (m6A) modification and nucleocytoplasmic trafficking of CGAS, IFI16, and STING messenger RNAs. This, in turn, amplifies the activation of cytoplasmic TBK1-IRF3 mediated by these factors. Thus, hnRNPA2B1 plays important roles in initiating IFN-α/ß production and enhancing stimulator of interferon genes (STING)-dependent cytoplasmic antiviral signaling.


Assuntos
Núcleo Celular/imunologia , Núcleo Celular/virologia , Infecções por Vírus de DNA/imunologia , DNA Viral/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Imunidade Inata , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Citoplasma/metabolismo , Células HEK293 , Herpesvirus Humano 1/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Fator Regulador 3 de Interferon , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7
16.
Fish Shellfish Immunol ; 93: 208-215, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31306760

RESUMO

Cathepsin Z (CTSZ) is a lysosomal cysteine protease that is known to be involved in the maintenance of homeostasis and the biological mechanisms of immune cells. In this study, we have confirmed the tissue specific expression of the cathepsin Z (PmCTSZ) gene in Pagrus major, and confirmed its biological function after producing recombinant protein using Escherichia coli (E. coli). Multiple sequence alignment analysis revealed that the active site of the cysteine proteases and three N-glycosylation sites of the deduced protein sequence were highly conserved among all of the organisms. Phylogenetic analysis revealed that PmCTSZ was included in the clusters of CTSZ and the cysteine proteases of other bony fish and is most closely related to Japanese flounder CTSZ. PmCTSZ was distributed in all of the tissues from healthy red sea bream that were used in the experiment and was most abundantly found in the spleen and gill. Analysis of mRNA expression after bacterial (Edwardsiella piscicida: E. piscicida and Streptococcus iniae: S. iniae) or viral (red seabream iridovirus: RSIV) challenge showed significant gene expression regulation in immune-related tissues, but they maintained relatively normal levels of expression. We produced recombinant PmCTSZ (rPmCTSZ) using an E. coli expression system and confirmed the biological function of extracellular rPmCTSZ in vitro. We found that bacterial proliferation was significantly inhibited by rPmCTSZ, and the leukocytes of red sea bream also induced apoptosis and viability reduction.


Assuntos
Catepsina Z/genética , Catepsina Z/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Dourada/genética , Dourada/imunologia , Sequência de Aminoácidos , Animais , Catepsina Z/química , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Edwardsiella/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Iridoviridae/fisiologia , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus iniae/fisiologia
17.
Fish Shellfish Immunol ; 92: 500-507, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31247318

RESUMO

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/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 , MAP Quinase Quinase 6/química , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio alginolyticus/fisiologia
18.
Fish Shellfish Immunol ; 92: 133-140, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31173860

RESUMO

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which lead to significant economic loss on mandarin fish in China. There is no effective drug or vaccine against this fatal disease at present. Meanwhile, many drugs and vaccines had no effect in many cases account of several impenetrable barriers (cell, skin and gastrointestinal tract). Here we reported an immersion subunit vaccine system (SWCNTs-MCP) encoding MCP gene of ISKNV based on single-walled carbon nanotubes (SWCNTs). To evaluate its efficacy against ISKNV, we found a stronger and longer duration immune response (serum antibody production, enzyme activities and immune-related genes expression) can be induced in fish vaccinated with SWCNTs-MCP in comparison with those vaccinated with MCP alone. Importantly, SWCNTs can increase the immune protective effect of naked subunit vaccine by ca. 23.8%. Thereby, this study demonstrates that SWCNTs as a promising carrier for subunit vaccine might be used to vaccinate large-scale juvenile mandarin fish by bath administration approach.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata , Iridoviridae/imunologia , Nanotubos de Carbono , Perciformes/imunologia , Vacinação/veterinária , Vacinas Virais/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Vacinas de Subunidades/imunologia
19.
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
20.
Fish Shellfish Immunol ; 91: 40-49, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31082519

RESUMO

DEAD (Asp-Glu-Ala-Asp)-box polypeptide 41 (DDX41) is a member of the DEXDc family of helicases, that has recently been identified to be a crucial intracellular DNA sensor that triggers multiple signaling molecules to activate the type I interferon response. However, the precise function of DDX41 in fish during a viral infection remains unknown. In the present study, the DDX41 homolog from orange spotted grouper, Epinephelus coioides (EcDDX41), was cloned and its potential role in the immune response to a fish viral infection were investigated. EcDDX41 encodes a putative protein of 614 amino acid residues that contained two conserved domains: 1) DEADc domain; and 2) HELICc domain. The sequence analysis indicated that EcDDX41 shared 99%, 94%, and 86% identity with Asian seabass (Lates calcarifer), zebrafish (Danio rerio), and humans (Homo sapiens), respectively. EcDDX41 mRNA was present in all of the detected tissues, with the highest level of expression in the gills. The level of EcDDX41 expression was up-regulated following infection with Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) in grouper spleen (GS) cell cultures, suggesting that EcDDX41 may be involved in fish virus infection. Furthermore, EcDDX41 overexpression in GS cells significantly inhibited SGIV and RGNNV replication. EcDDX41 overexpression significantly increased the expression of antiviral and inflammatory cytokine genes, including interferon regulatory factor genes (e.g., IRF1, IRF2, IRF3, and IRF7), interferon induced genes (e.g., ISG15, ISG56, IFP35, Viperin, and MXI), and pro-inflammatory cytokine genes (e.g., TNFα, IL-1ß, and IL-8). Moreover, EcDDX41 positively regulated the mitochondrial antiviral-signaling protein (MAVS) and TANK-binding kinase 1 (TBK1)-induced interferon immune response, but did mediate IRF3 activation (MITA) to evoke an interferon immune response in unstimulated cells. Together, our results provide novel insight into the role of fish DDX41 in the antiviral innate immune response.


Assuntos
Bass/genética , Bass/imunologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , RNA Helicases DEAD-box/química , Infecções por Vírus de DNA/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Nodaviridae/fisiologia , Filogenia , Infecções por Vírus de RNA/imunologia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária
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