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T-cell/transmembrane immunoglobulin and mucin domain-containing (TIM) protein family has attracted particular attention because of their broad immune functions and the response to viral infections. TIM-1, a member of the TIM family, has been demonstrated to play an important role in viral infections. However, its roles during fish nodavirus infection still remained largely unknown. In this study, a homolog of TIM-1 from orange-spotted grouper (Epinephelus coioides) (EcTIM-1) was identified, and characterized. EcTIM-1 encoded a 217-amino acids protein, containing one Immunoglobulin domain. Homology analysis showed that EcTIM-1 shared 98.62 % and 42.99 % identity to giant grouper (E. lanceolatus) and human (Homo sapiens). Quantitative Real-time PCR analyses indicated that EcTIM-1 was expressed in all examined tissues, with higher expression in liver, spleen, skin, and heart, and was significantly up-regulated in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTIM-1 was distributed in the cytoplasm, and partly co-localized with Golgi apparatus and lysosomes in vitro. The ectopic expression of EcTIM-1 promoted RGNNV replication by increasing the level of viral genes transcription and protein synthesis. Besides, overexpression of EcTIM-1 decreased the luciferase activity of type I interferon (IFN1), interferon stimulated response elements (ISRE) and nuclear factor kappa-B (NF-κB) promoters, as well as the transcription of pro-inflammatory factors and interferon related genes. EcTIM-1 significantly suppressed the luciferase activity of IFN1, ISRE and NF-κB promoters evoked by Epinephelus coioides melanoma differentiation-associated gene 5 (EcMDA5), mitochondrial antiviral signaling protein (EcMAVS), stimulator of IFN genes (EcSTING) or TANK-binding kinase 1 (EcTBK1). Collectively, EcTIM-1 negatively regulated interferon and inflammatory response to promote RGNNV infection. These results provide a basis for a better understanding of the innate immune response of TIM-1 in fish.
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NLRP3 has an important role in the immune response and viral infection as an essential inflammasome component. However, it is unclear whether the grouper immune system is regulated by NLRP3 inflammasome. In this study, we cloned the NLRP3 gene from Epinephelus coioides. Ec-NLRP3 encodes 893 amino acids and contains two major structural domains, the NACHT domain (69-234aa) and the LRR domain (477-893aa). Tissue distribution analysis showed that Ec-NLRP3 was expressed in all tissues tested, with the spleen exhibiting the highest expression. Additionally, after being infected with SGIV, the expression of the Ec-NLRP3 gene was significantly increased. The results of subcellular localization revealed that Ec-NLRP3 was distributed throughout GS cells. In addition, Ec-NLRP3 co-localized with Ec-ASC and was observed as a cytosolic speck. Ec-NLRP3 overexpression significantly inhibited SGIV infection, which was further inhibited by co-overexpression of Ec-NLRP3 and Ec-ASC. Further studies revealed that overexpression of Ec-NLRP3 significantly upregulated caspase-1 activity, and co-overexpression of Ec-NLRP3 and Ec-ASC further upregulated caspase-1 activity. In addition, inhibition of Caspase-1 activity with VX-765 significantly increased the infection of SGIV. Furthermore, the NLRP3 inflammasome activator Nigericin was able to inhibit the infection of SGIV significantly. The above findings suggest that Ec-NLRP3 inhibits SGIV infection by upregulating caspase-1 activity.
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The high mortality rate of Singapore grouper iridovirus (SGIV) posing a serious threat to the grouper aquaculture industry and causing significant economic losses. Therefore, finding effective drugs against SGIV is of great significance. Eugenol (C10H12O2) is a phenolic aromatic compound, has been widely studied for its anti-inflammatory, antioxidant and antiviral capacity. In this study, we explored the effect of eugenol on SGIV infection and its possible mechanisms using grouper spleen cells (GS) as an in vitro model. We found that treatment of GS cells with 100 µM eugenol for 4 h exhibited the optimal inhibitory effect on SGIV. Eugenol was able to reduce the expression level of inflammatory factors by inhibiting the activation of MAPK pathway and also inhibited the activity of NF-κB and AP-1 promoter. On the other hand, eugenol attenuated cellular oxidative stress by reducing intracellular ROS and promoted the expression of interferon-related genes. Therefore, we conclude that eugenol inhibits SGIV infection by enhancing cellular immunity through its anti-inflammatory and antioxidant functions.
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Antivirales , Lubina , Infecciones por Virus ADN , Eugenol , Enfermedades de los Peces , Ranavirus , Animales , Eugenol/farmacología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Antivirales/farmacología , Lubina/inmunología , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/tratamiento farmacológico , Ranavirus/fisiología , Bazo/inmunología , Bazo/efectos de los fármacos , Bazo/citología , Células CultivadasRESUMEN
The largemouth bass has become one of the economically fish in China, according to the latest China Fishery Statistical Yearbook. The farming scale is constantly increasing. Salidroside has been found in past studies to have oxidative stress reducing and immune boosting properties. In this study, the addition of six different levels of salidroside supplements were 0ã40ã80ã120ã160 and 200 mg/kg. A 56-day feeding trial was conducted to investigate the effects of salidroside on the intestinal health, immune parameters and intestinal microbiota composition of largemouth bass. Dietary addition of salidroside significantly affected the Keap-1ß/Nrf-2 pathway as well as significantly increased antioxidant enzyme activities resulting in a significant increase in antioxidant capacity of largemouth bass. Dietary SLR significantly reduced feed coefficients. The genes related to tight junction proteins (Occludin, ZO-1, Claudin-4, Claudin-5) were found to be significantly upregulated in the diet supplemented with salidroside, indicating that salidroside can improve the intestinal barrier function (p < 0.05). The dietary administration of salidroside was found to significantly reduce the transcription levels of intestinal tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) (p < 0.05). Furthermore, salidroside was observed to reduce the transcription levels of intestinal apoptosis factor Bcl-2 associated death promoter (BAD) and recombinant Tumor Protein p53 (P53) (p < 0.05). Concomitantly, the beneficial bacteria, Fusobacteriota and Cetobacterium, was significantly increased in the SLR12 group, while that of pathogenic bacteria, Proteobacteria, was significantly decreased (p < 0.05). In conclusion, the medium-sized largemouth bass optimal dosage of salidroside in the diet is 120mg/kg-1.
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Alimentación Animal , Lubina , Dieta , Suplementos Dietéticos , Microbioma Gastrointestinal , Glucósidos , Fenoles , Animales , Lubina/inmunología , Microbioma Gastrointestinal/efectos de los fármacos , Alimentación Animal/análisis , Dieta/veterinaria , Suplementos Dietéticos/análisis , Glucósidos/administración & dosificación , Glucósidos/farmacología , Fenoles/administración & dosificación , Fenoles/farmacología , Intestinos/efectos de los fármacos , Intestinos/inmunología , Intestinos/microbiología , Inmunidad Innata/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Distribución AleatoriaRESUMEN
The treatment of osteoporotic bone defects poses a challenge due to the degradation of the skeletal vascular system and the disruption of local bone metabolism within the osteoporotic microenvironment. However, it is feasible to modulate the disrupted local bone metabolism imbalance through enhanced vascularization, a theory termed "vascularization-bone metabolic balance". This study developed a 3D-printed polycaprolactone (PCL) scaffold modified with EPLQLKM and SVVYGLR peptides (PCL-SE). The EPLQLKM peptide attracts bone marrow-derived mesenchymal stem cells (BMSCs), while the SVVYGLR peptide enhances endothelial progenitor cells (EPCs) vascular differentiation, thus regulating bone metabolism and fostering bone regeneration through the paracrine effects of EPCs. Further mechanistic research demonstrated that PCL-SE promoted the vascularization of EPCs, activating the Notch signaling pathway in BMSCs, leading to the upregulation of osteogenesis-related genes and the downregulation of osteoclast-related genes, thereby restoring bone metabolic balance. Furthermore, PCL-SE facilitated the differentiation of EPCs into "H"-type vessels and the recruitment of BMSCs to synergistically enhance osteogenesis, resulting in the regeneration of normal microvessels and bone tissues in cases of femoral condylar bone defects in osteoporotic SD rats. This study suggests that PCL-SE supports in-situ vascularization, remodels bone metabolic translational balance, and offers a promising therapeutic regimen for osteoporotic bone defects.
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Regeneración Ósea , Homeostasis , Células Madre Mesenquimatosas , Neovascularización Fisiológica , Osteogénesis , Osteoporosis , Impresión Tridimensional , Ratas Sprague-Dawley , Andamios del Tejido , Animales , Regeneración Ósea/efectos de los fármacos , Osteoporosis/metabolismo , Osteoporosis/terapia , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Andamios del Tejido/química , Osteogénesis/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Poliésteres/química , Diferenciación Celular/efectos de los fármacos , Femenino , Ratas , Células Progenitoras Endoteliales/metabolismo , Huesos/metabolismoRESUMEN
Singapore grouper iridovirus (SGIV) belongs to the family Iridoviridae and the genus Ranavirus, which is a large cytoplasmic DNA virus. Infection of grouper with SGIV can cause hemorrhage and swelling of the spleen of the fish. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. In the present study, the protein encoded by SGIV ORF128 (VP128) was identified. VP128 is predominantly localized within the endoplasmic reticulum (ER). Overexpression of VP128 significantly promoted SGIV replication. VP128 inhibited the interferon (IFN)-3 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), and TANK-binding kinase 1 (EcTBK1). Moreover, VP128 interacted with EcSTING and EcTBK1. The interaction between VP128 and EcSTING was independent of any specific structural domain of EcSTING. Together, our results demonstrated that SGIV VP128 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion.
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Infecciones por Virus ADN , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Ranavirus , Transducción de Señal , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Ranavirus/fisiología , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Transducción de Señal/inmunología , Inmunidad Innata/genética , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Evasión Inmune , Lubina/inmunología , Regulación de la Expresión Génica/inmunología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de AminoácidosRESUMEN
Exocyst, a protein complex, plays a crucial role in various cellular functions, including cell polarization, migration, invasion, cytokinesis, and autophagy. Sec3, known as Exoc1, is a key subunit of the Exocyst complex and can be involved in cell survival and apoptosis. In this study, two subtypes of Sec3 were isolated from Epinephelus coioides, an important marine fish in China. The role of E. coioides Sec3 was explored during Singapore grouper iridovirus (SGIV) infection, an important pathogen of marine fish which could induce 90 % mortality. E. coioides Sec3 sequences showed a high similarity with that from other species, indicating the presence of a conserved Sec3 superfamily domain. E. coioides Sec3 mRNA could be detected in all examined tissues, albeit at varying expression levels. SGIV infection could upregulate E. coioides Sec3 mRNA. Upregulated Sec3 significantly promoted SGIV-induced CPE, and the expressions of viral key genes. E. coioides Sec3 could inhibit the activation of NF-κB and AP-1, as well as SGIV-induced cell apoptosis. The results illustrated that E. coioides Sec3 promotes SGIV infection by regulating the innate immune response.
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Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Filogenia , Ranavirus , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Inmunidad Innata/genética , Lubina/inmunología , Ranavirus/fisiología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , Regulación de la Expresión Génica/inmunología , Alineación de Secuencia/veterinaria , Secuencia de Aminoácidos , Perfilación de la Expresión Génica/veterinariaRESUMEN
Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that has caused significant economic losses to the grouper aquaculture industry. So far, the structure and function of SGIV proteins have been successively reported. In the present paper, the protein of SGIV VP146 was cloned and identified. VP146 was whole-cell distributed in GS cells. VP146 promoted SGIV replication and inhibited the transcription of interferon-related genes as well as pro-inflammatory cytokines in GS cells. In addition, VP146 was involved in the regulation of the cGAS-STING signaling pathway, and decreased cGAS-STING induced the promoter of ISRE and NF-κB. VP146 interacted with the proteins of cGAS, STING, TBK1, and IRF3 from grouper, but did not affect the binding of grouper STING to grouper TBK1 and grouper IRF3. Interestingly, grouper STING was able to affect the intracellular localization of VP146. Four segment structural domains of grouper STING were constructed, and grouper STING-CTT could affect the intracellular localization of VP146. VP146 had no effect on the self-binding of EcSITNG, nor on the binding of EcSTING to EcTBK1 and EcIRF3. Together, the results demonstrated that SGIV VP146 modulated the cGAS-STING signaling pathway to escape the interferon immune response.
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Proteínas Adaptadoras Transductoras de Señales , Lubina , Iridovirus , Nucleotidiltransferasas , Transducción de Señal , Iridovirus/inmunología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Transducción de Señal/inmunología , Lubina/genética , Lubina/inmunología , Lubina/virología , Línea Celular , Bazo/citología , Regulación de la Expresión Génica/inmunología , Replicación Viral/inmunología , Interferones/genética , Interferones/inmunología , Proteínas de Peces/inmunología , AnimalesRESUMEN
Red-spotted grouper nervous necrosis virus (RGNNV) is a major viral pathogen of grouper and is able to antagonize interferon responses through multiple strategies, particularly evading host immune responses by inhibiting interferon responses. Ovarian tumor (OTU) family proteins are an important class of DUBs and the underlying mechanisms used to inhibit interferon pathway activation are unknown. In the present study, primers were designed based on the transcriptome data, and the ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) and OTUB2 genes of Epinephelus coioides (EcOTUB1 and EcOTUB2) were cloned and characterized. The homology alignment showed that both EcOTUB1 and EcOTUB2 were most closely related to E. lanceolatus with 98 % identity. Both EcOTUB1 and EcOTUB2 were distributed to varying degrees in grouper tissues, and the transcript levels were significantly up-regulated following RGNNV stimulation. Both EcOTUB1 and EcOTUB2 promoted replication of RGNNV in vitro, and inhibited the promoter activities of interferon stimulated response element (ISRE), nuclear transcription factors kappaB (NF-κB) and IFN3, and the expression levels of interferon related genes and proinflammatory factors. Co-immunoprecipitation experiments showed that both EcOTUB1 and EcOTUB2 could interact with TRAF3 and TRAF6, indicating that EcOTUB1 and EcOTUB2 may play important roles in interferon signaling pathway. The results will provide a theoretical reference for the development of novel disease prevention and control techniques.
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Lubina , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Nodaviridae , Infecciones por Virus ARN , Replicación Viral , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Inmunidad Innata/genética , Nodaviridae/fisiología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/veterinaria , Lubina/inmunología , Filogenia , Regulación de la Expresión Génica/inmunología , Secuencia de Aminoácidos , Alineación de Secuencia/veterinaria , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/inmunología , Perfilación de la Expresión Génica/veterinariaRESUMEN
In the context of burgeoning global aquaculture, its environmental repercussions, particularly in marine ecosystems, have gained significant attentions. Cage aquaculture, a prominent method, has been observed to significantly influence marine environments by discharging substantial amounts of organic materials and pollutants. It is also one of the important reasons for water eutrophication. This study investigated the impacts of cage aquaculture on microbial diversity and functional potential using metagenomics. Specifically, a comparison was made of the physicochemical indicators and microbial diversity between three grouper aquaculture cage nets in Lingshui Xincun Port and three nearby non-aquaculture area surface waters. We found that compared to non-aquaculture areas, the eutrophication indicators in aquaculture environments significantly increased, and the abundances of Vibrio and Pseudoalteromonas in aquaculture environments significantly rose. Additionally, microbial functional genes related to carbon, nitrogen, and sulfur metabolisms were also found to be significantly affected by aquaculture activities. The correlation analysis between microbial populations and environmental factors revealed that the abundances of most microbial taxa showed positive correlations with dissolved inorganic nitrogen, soluble reactive phosphorus, NH4+, and negative correlations with dissolved oxygen. Overall, this study elucidated the significant impacts of aquaculture-induced eutrophication on the diversity and functions of planktonic bacterial communities.
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During virus-host co-evolution, viruses have developed multiple strategies to dampen IFN response and prevent its antiviral activity in host cells. To date, the interactions between host IFN response and the immune evasion strategies exploited by fish iridoviruses still remain largely uncertain. Here, a potential immune evasion protein candidate of Singapore grouper iridovirus (SGIV), VP82 (encoded by SGIV ORF82) was screened and its roles during viral replication were investigated in detail. Firstly, VP82 overexpression dramatically decreased IFN or ISRE promoter activity and the transcription levels of IFN stimulated genes (ISGs) stimulated by grouper cyclic GMP-AMP synthase (EccGAS)/stimulator of interferon genes (EcSTING), TANK-binding kinase 1 (EcTBK1), IFN regulatory factor 3 (EcIRF3)and EcIRF7. Secondly, Co-IP assays indicated that VP82 interacted with EcIRF3 and EcIRF7, but not EcSTING and EcTBK1, which was consistent with the co-localization between VP82 and EcIRF3 or EcIRF7. Furthermore, VP82 promoted the degradation of EcIRF3 and EcIRF7 in a dose-dependent manner via the autophagy pathway. Finally, VP82 overexpression accelerated SGIV replication, evidenced by the increased transcriptions of viral core genes and viral production. Moreover, the antiviral action of EcIRF3 or EcIRF7 was significantly depressed in VP82 overexpressed cells. Together, VP82 was speculated to exert crucial roles for SGIV replication by inhibiting the IFN response via the degradation of IRF3 and IRF7. Our findings provided new insights into understanding the immune evasion strategies utilized by fish iridovirus through IFN regulation.
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Infecciones por Virus ADN , Enfermedades de los Peces , Proteínas de Peces , Factor 3 Regulador del Interferón , Factor 7 Regulador del Interferón , Ranavirus , Proteínas Virales , Animales , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/metabolismo , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/veterinaria , Ranavirus/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Inmunidad Innata/genética , Interferones/genética , Interferones/inmunología , Interferones/metabolismo , Evasión Inmune , Lubina/inmunología , Lubina/genética , Replicación Viral , Proteínas de Pez Cebra , Factores Reguladores del InterferónRESUMEN
The vertebrate central nervous system (CNS) is the most complex system of the body. The CNS, especially the brain, is generally regarded as immune-privileged. However, the specialized immune strategies in the brain and how immune cells, specifically macrophages in the brain, respond to virus invasion remain poorly understood. Therefore, this study aimed to examine the potential immune response of macrophages in the brain of orange-spotted groupers (Epinephelus coioides) following red-spotted grouper nervous necrosis virus (RGNNV) infection. We observed that RGNNV induced macrophages to produce an inflammatory response in the brain of orange-spotted grouper, and the macrophages exhibited M1-type polarization after RGNNV infection. In addition, we found RGNNV-induced macrophage M1 polarization via the CXCR3.2- CXCL11 pathway. Furthermore, we observed that RGNNV triggered M1 polarization in macrophages, resulting in substantial proinflammatory cytokine production and subsequent damage to brain tissue. These findings reveal a unique mechanism for brain macrophage polarization, emphasizing their role in contributing to nervous tissue damage following viral infection in the CNS.
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Encéfalo , Enfermedades de los Peces , Macrófagos , Nodaviridae , Infecciones por Virus ARN , Animales , Macrófagos/inmunología , Macrófagos/virología , Enfermedades de los Peces/virología , Enfermedades de los Peces/inmunología , Encéfalo/virología , Encéfalo/inmunología , Encéfalo/patología , Nodaviridae/fisiología , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Quimiocina CXCL11 , Receptores CXCR3/metabolismo , Lubina/inmunología , Lubina/virología , Transducción de Señal , Citocinas/metabolismo , Citocinas/inmunología , Proteínas de Peces/inmunología , Proteínas de Peces/genéticaRESUMEN
Iridovirus poses a substantial threat to global aquaculture due to its high mortality rate; however, the molecular mechanisms underpinning its pathogenesis are not well elucidated. Here, a multi-omics approach was applied to groupers infected with Singapore grouper iridovirus (SGIV), focusing on the roles of key metabolites. Results showed that SGIV induced obvious histopathological damage and changes in metabolic enzymes within the liver. Furthermore, SGIV significantly reduced the contents of lipid droplets, triglycerides, cholesterol, and lipoproteins. Metabolomic analysis indicated that the altered metabolites were enriched in 19 pathways, with a notable down-regulation of lipid metabolites such as glycerophosphates and alpha-linolenic acid (ALA), consistent with disturbed lipid homeostasis in the liver. Integration of transcriptomic and metabolomic data revealed that the top enriched pathways were related to cell growth and death and nucleotide, carbohydrate, amino acid, and lipid metabolism, supporting the conclusion that SGIV infection induced liver metabolic reprogramming. Further integrative transcriptomic and proteomic analysis indicated that SGIV infection activated crucial molecular events in a phagosome-immune depression-metabolism dysregulation-necrosis signaling cascade. Of note, integrative multi-omics analysis demonstrated the consumption of ALA and linoleic acid (LA) metabolites, and the accumulation of L-glutamic acid (GA), accompanied by alterations in immune, inflammation, and cell death-related genes. Further experimental data showed that ALA, but not GA, suppressed SGIV replication by activating antioxidant and anti-inflammatory responses in the host. Collectively, these findings provide a comprehensive resource for understanding host response dynamics during fish iridovirus infection and highlight the antiviral potential of ALA in the prevention and treatment of iridoviral diseases.
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Enfermedades de los Peces , Iridovirus , Hígado , Ácido alfa-Linolénico , Animales , Ácido alfa-Linolénico/metabolismo , Enfermedades de los Peces/virología , Enfermedades de los Peces/metabolismo , Hígado/metabolismo , Hígado/virología , Iridovirus/fisiología , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/virología , Metabolómica , Antivirales/farmacología , Transcriptoma , Reprogramación Metabólica , MultiómicaRESUMEN
The elongation of very long chain fatty acids (ELOVL) proteins are key rate-limiting enzymes that catalyze fatty acid synthesis to form long chain fatty acids. ELOVLs also play regulatory roles in the lipid metabolic reprogramming induced by mammalian viruses. However, little is known about the roles of fish ELOVLs during virus infection. Here, a homolog of ELOVL7 was cloned from Epinephelus coioides (EcELOVL7a), and its roles in red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection were investigated. The transcription level of EcELOVL7a was significantly increased upon RGNNV and SGIV infection or other pathogen-associated molecular patterns stimulation in grouper spleen (GS) cells. Subcellular localization analysis showed that EcELOVL7a encoded an endoplasmic reticulum (ER) related protein. Overexpression of EcELOVL7a promoted the viral production and virus release during SGIV and RGNNV infection. Furthermore, the lipidome profiling showed that EcELOVL7a overexpression reprogrammed cellular lipid components in vitro, evidenced by the increase of glycerophospholipids, sphingolipids and glycerides components. In addition, VLCFAs including FFA (20:2), FFA (20:4), FFA (22:4), FFA (22:5) and FFA (24:0), were enriched in EcELOVL7a overexpressed cells. Consistently, EcELOVL7a overexpression upregulated the transcription level of the key lipid metabolic enzymes, including fatty acid synthase (FASN), phospholipase A 2α (PLA 2α), and cyclooxygenases -2 (COX-2), LPIN1, and diacylglycerol acyltransferase 1α (DGAT1α). Together, our results firstly provided the evidence that fish ELOVL7a played an essential role in SGIV and RGNNV replication by reprogramming lipid metabolism.
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Lubina , Infecciones por Virus ADN , Elongasas de Ácidos Grasos , Enfermedades de los Peces , Proteínas de Peces , Metabolismo de los Lípidos , Replicación Viral , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/metabolismo , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/inmunología , Lubina/inmunología , Lubina/genética , Elongasas de Ácidos Grasos/genética , Nodaviridae/fisiología , Regulación de la Expresión Génica , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Infecciones por Birnaviridae/veterinaria , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/virología , Perfilación de la Expresión Génica/veterinaria , Iridoviridae/fisiología , Iridovirus/fisiología , Filogenia , Alineación de Secuencia/veterinaria , Secuencia de Aminoácidos , Reprogramación MetabólicaRESUMEN
Singapore grouper iridovirus (SGIV) is one of the major infectious diseases responsible for high mortality and huge economic losses in the grouper aquaculture industry. Berberine (BBR), a naturally occurring plant alkaloid, is a phytochemical having a variety of biological properties, such as antiviral, antioxidant, and anti-inflammatory effects. In this work, we used an in vitro model based on Western blot, ROS fluorescence probe, and real-time quantitative PCR (qRT-PCR) to examine the antiviral qualities of BBR against SGIV. The outcomes demonstrated that varying BBR concentrations could significantly inhibit the replication of SGIV. In addition, BBR greatly inhibited the production of genes associated with pro-inflammatory cytokines in SGIV-infected or SGIV-uninfected GS cells based on qRT-PCR data. Subsequent investigations demonstrated that BBR suppressed the expression of the promoter activity of NF-κB and NF-κB-p65 protein. Additionally, BBR reduced the phosphorylation of ERK 1/2, JNK, and p38. Furthermore, BBR also inhibits SGIV-induced ROS production by upregulating the expression of antioxidant-related genes. In conclusion, BBR is a viable therapy option for SGIV infection due to its antiviral properties.
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Berberina , Enfermedades de los Peces , Estrés Oxidativo , Replicación Viral , Berberina/farmacología , Animales , Estrés Oxidativo/efectos de los fármacos , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Replicación Viral/efectos de los fármacos , Inflamación/inmunología , Inflamación/veterinaria , Antivirales/farmacología , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/inmunología , Ranavirus/fisiología , Línea CelularRESUMEN
Stimulator of interferon gene (STING) plays a crucial role in the innate immune response against viral and bacterial pathogens. However, its function in largemouth bass iridovirus (LMBV) infection remains uncertain. Here, a STING homolog (MsSTING) from largemouth bass (Micropterus salmoides) was cloned and characterized. MsSTING encoded a 407-amino-acid polypeptide, which shared 84.08% and 41.45% identity with golden perch (Perca flavescens) and human (Homo sapiens) homologs, respectively. MsSTING contained four transmembrane domains and a conserved C-terminal domain. The mRNA level of MsSTING was significantly increased in response to LMBV infection in vitro. Subcellular localization observation indicated that MsSTING encoded a cytoplasmic protein, which co-localized predominantly with endoplasmic reticulum (ER) and partially with mitochondria. Moreover, its accurate localization was dependent on the N-terminal transmembrane motif (TM) domains. MsSTING was able to activate interferon (IFN) response, evidenced by the activation of IFN1, IFN3 and ISRE promoters by its overexpression in vitro. Mutant analysis showed that both the N-terminal and C-terminal domain of MsSTING were essential for its activation on IFN response. In addition, overexpression of MsSTING inhibited the transcription and protein levels of viral core genes, indicating that MsSTING exerted antiviral action against LMBV. Consistently, the inhibitory effects were significantly attenuated when the N-terminal or C-terminal domains of MsSTING was deleted. Furthermore, MsSTING overexpression upregulated the transcriptions of interferon-related genes and pro-inflammatory factors, including TANK-binding kinase 1(TBK1), interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), interferon stimulated exonuclease gene 20 (ISG20), interferon-induced transmembrane protein 1(IFITM1), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interleukin 6 (IL-6). Together, MsSTING exerted antiviral action upon LMBV infection through positive regulation the innate immune response.
Asunto(s)
Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Iridovirus , Ranavirus , Humanos , Animales , Secuencia de Aminoácidos , Proteínas de Peces/química , Inmunidad Innata/genética , Interferón gamma , Antivirales , Ranavirus/fisiologíaRESUMEN
The origin of T cells in the teleost's brain is unclear. While viewing the central nervous system (CNS) as immune privileged has been widely accepted, previous studies suggest that T cells residing in the thymus but not in the spleen of the teleost play an essential role in communicating with the peripheral organs. Here, we identified nine T cell subpopulations in the thymus and spleen of orange-spotted grouper (Epinephelus coioices) through single-cell RNA-sequencing analysis. After viral CNS infection with red-spotted grouper nervous necrosis virus (RGNNV), the number of slc43a2+ T cells synchronously increased in the spleen and brain. During the infection tests in asplenic zebrafish (tlx1â² zebrafish model), no increase in the number of slc43a2+ T cells was observed in the brain. Single-cell transcriptomic analysis indicated that slc43a2+ T cells mature and functionally differentiate within the spleen and then migrate into the brain to trigger an immune response. This study suggests a novel route for T cell migration from the spleen to the brain during viral infection in fish.
Asunto(s)
Enfermedades de los Peces , Nodaviridae , Animales , Inmunidad Innata , Bazo , Pez Cebra , Secuencia de Aminoácidos , Alineación de Secuencia , Linfocitos T , Encéfalo , Nodaviridae/fisiología , Proteínas de Peces/genéticaRESUMEN
Cytoplasmic vacuolization is commonly induced by bacteria and viruses, reflecting the complex interactions between pathogens and the host. However, their characteristics and formation remain unclear. Nervous necrosis virus (NNV) infects more than 100 global fish species, causing enormous economic losses. Vacuolization is a hallmark of NNV infection in host cells, but remains a mystery. In this study, we developed a simple aptamer labelling technique to identify red-spotted grouper NNV (RGNNV) particles in fixed and live cells to explore RGNNV-induced vacuolization. We observed that RGNNV-induced vacuolization was positively associated with the infection time and virus uptake. During infection, most RGNNV particles, as well as viral genes, colocalized with vacuoles, but not giant vacuoles > 3 µm in diameter. Although the capsid protein (CP) is the only structural protein of RGNNV, its overexpression did not induce vacuolization, suggesting that vacuole formation probably requires virus entry and replication. Given that small Rab proteins and the cytoskeleton are key factors in regulating cellular vesicles, we further investigated their roles in RGNNV-induced vacuolization. Using live cell imaging, Rab5, a marker of early endosomes, was continuously located in vacuoles bearing RGNNV during giant vacuole formation. Rab5 is required for vacuole formation and interacts with CP according to siRNA interference and Co-IP analysis. Furthermore, actin formed distinct rings around small vacuoles, while vacuoles were located near microtubules. Actin, but not microtubules, plays an important role in vacuole formation using chemical inhibitors. These results provide valuable insights into the pathogenesis and control of RGNNV infections.
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Enfermedades de los Peces , Infecciones por Virus ARN , Animales , Actinas , Enfermedades de los Peces/genética , Infecciones por Virus ARN/genética , Proteínas de la Cápside , NecrosisRESUMEN
Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, is a highly pathogenic agent and causes heavy economic losses in the global grouper aquaculture. Recent studies demonstrated that SGIV infection attenuated antiviral immune and inflammatory response induced by poly (I:C) in vitro. However, little was known about the potential functions of the immune regulatory proteins encoded by SGIV. Here, we identified the detailed roles of VP20 and clarified the potential mechanism underlying its immune regulatory function during SGIV infection. Our results showed that VP20 was an IE gene, and partially co-localized with Golgi apparatus and lysosomes in grouper cells. Overexpression of VP20 enhanced SGIV replication, demonstrated by the increase in the transcription levels of viral core genes and the protein synthesis of MCP. Reporter gene assays showed that SGIV VP20 overexpression significantly reduced the IFN promoter activity induced by poly (I:C), grouper stimulator of interferon genes (EcSTING) and TANK-binding kinase 1 (EcTBK1). Consistently, the transcription levels of IFN related genes were significantly decreased in VP20 overexpressing cells compared to those in control cells. Co-IP assay and confocal microscopy observations indicated that VP20 co-localized and interacted with EcTBK1 and EcIRF3, but not EcSTING. In addition, VP20 was able to degrade EcIRF3 and attenuate the antiviral action of EcIRF3, while had no effect on EcTBK1. Together, SGIV VP20 was speculated to promote viral replication through attenuating the IFN response mediated by TBK1-IRF3 in vitro. Our findings provided new insights into the immune regulatory function of SGIV encoded unknown proteins.
Asunto(s)
Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Iridovirus , Ranavirus , Animales , Interferones , Ranavirus/fisiología , Inmunidad Innata/genética , Singapur , Secuencia de Aminoácidos , Proteínas de Peces/genética , Alineación de SecuenciaRESUMEN
Iridoviruses are large DNA viruses that infect a wide range of invertebrates and lower vertebrates, causing serious threats to ecological security and aquaculture industry worldwide. However, the mechanisms underlying intracellular transport of iridovirus remain unknown. In this study, the transport of Singapore grouper iridovirus (SGIV) in early endosomes (EEs) and late endosomes (LEs) was explored by single-particle tracking technology. SGIV employs EEs to move rapidly from the cell membrane to the nucleus, and this long-range transport is divided into "slow-fast-slow" stages. SGIV within LEs mainly underwent oscillatory movements near the nucleus. Furthermore, SGIV entered newly formed EEs and LEs, respectively, possibly based on the interaction between the viral major capsid protein and Rab5/Rab7. Importantly, interruption of EEs and LEs by the dominant negative mutants of Rab5 and Rab7 significantly inhibited the movement of SGIV, suggesting the important roles of Rab5 and Rab7 in virus transport. In addition, it seems that SGIV needs to enter clathrin-coated vesicles to move from actin to microtubules before EEs carry the virus moving along microtubules. Together, our results for the first time provide a model whereby iridovirus transport depending on EEs and LEs, helping to clarify the mechanism underlying iridovirus infection, and provide a convenient tactic to investigate the dynamic infection of large DNA virus.