RESUMO
Type I interferons (IFN-I) play a pivotal role in vertebrate innate immunity against viruses. This study is an analysis of IFN-I genes in an updated version of the Atlantic salmon genome published in 2021 (version Ssal_v3.1), revealing 47 IFN-I genes in the Atlantic salmon genome. The GH1 locus of chromosome (Chr) 3 harbors 9 IFNa genes, 5 IFNb genes, 6 IFNc genes, 11 IFNe genes and 1 IFNf gene. The GH2 locus on Chr6 contains 1 IFNa gene, 12 IFNc genes and 1 IFNf gene while Chr19 carries a single IFNd gene. Intraperitoneal injection of Atlantic salmon presmolts with poly I:C, a mimic of virus double-stranded RNA, significantly up-regulated IFNc genes from both Chr3 and Chr6 in heart, with lower expression in head kidney. IFNe expression increased in the heart, but not in the head kidney while IFNf was strongly up-regulated in both tissues. Antiviral activity of selected IFNs was assessed by transfection of salmon cells with IFN-expressing plasmids followed by infectious pancreatic necrosis virus infection, and by injection of fish with IFN-plasmids followed by measuring expression of the antiviral Mx1 gene. The results demonstrated that IFNc from both Chr3 and Chr6 provided full protection of cells against virus infection, whereas IFNe and IFNf showed lesser protection. IFNc from Chr3 and Chr6 along with IFNe and IFNf, up-regulated the Mx1 gene in the muscle, while only the IFNcs caused induction of Mx1 in liver. Overall, this study reveals that Atlantic salmon possesses an even more potent innate immune defense against viruses than previously understood.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Imunidade Inata , Interferon Tipo I , Poli I-C , Salmo salar , Animais , Salmo salar/genética , Salmo salar/imunologia , Doenças dos Peixes/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata/genética , Poli I-C/farmacologia , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/genética , Vírus da Necrose Pancreática Infecciosa/fisiologia , Regulação da Expressão Gênica/imunologia , Perfilação da Expressão Gênica/veterináriaRESUMO
Infectious pancreatic necrosis virus (IPNV) is an important pathogen that is threatening the worldwide salmon and trout industry. But there is no therapeutic drug available for now. In this study, we demonstrate that MK-0608 is highly efficient against IPNV and low cytotoxic, with a 50 % effective concentration (EC50) of 0.20 µM and selectivity index (SI) of about 268. Time of addition assay illustrated that MK-0608 targeted the early stage of IPNV life cycle. Furthermore, we found that MK-0608 blocked IPNV attachment on the premise of sufficient pre-incubation time but MK-0608 did not influence viral internalization and release. MK-0608 could inhibit IPNV genome synthesis, and combination with ribavirin enhanced the inhibition effect, which might be functional via binding to IPNV RNA dependent RNA polymerase (RdRp), which was predicted by using molecular docking methods. In vivo test showed that IPNV was extremely suppressed in the rainbow trout (Oncorhynchus mykiss) with one single dose of MK-0608, and the higher dosage of 50 mg/kg could cause 3 log decrease of IPNV loads in fish tissues.
Assuntos
Antivirais , Infecções por Birnaviridae , Doenças dos Peixes , Vírus da Necrose Pancreática Infecciosa , Oncorhynchus mykiss , Replicação Viral , Vírus da Necrose Pancreática Infecciosa/fisiologia , Vírus da Necrose Pancreática Infecciosa/efeitos dos fármacos , Animais , Doenças dos Peixes/virologia , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/virologia , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , RNA Viral/genética , Replicação do RNARESUMO
The mitochondrial anti-viral signaling (MAVS) protein is an intermediary adaptor protein of retinoic acid-inducible gene-1 (RIG-I) like receptor (RLR) signaling, which activates the transcription factor interferon (IFN) regulatory factor 3 (IRF3) and NF-kB to produce type I IFNs. MAVS expression has been reported in different fish species, but few studies have shown its functional role in anti-viral responses to fish viruses. In this study, we used the transcription activator-like effector nuclease (TALEN) as a gene editing tool to disrupt the function of MAVS in Chinook salmon (Oncorhynchus tshawytscha) embryonic cells (CHSE) to understand its role in induction of interferon I responses to infections with the (+) RNA virus salmonid alphavirus subtype 3 (SAV-3), and the dsRNA virus infectious pancreatic necrosis virus (IPNV) infection. A MAVS-disrupted CHSE clone with a 7-aa polypeptide (GVFVSRV) deletion mutation at the N-terminal of the CARD domain infected with SAV-3 resulted in significantly lower expression of IRF3, IFNa, and ISGs and increased viral titer (1.5 log10) compared to wild-type. In contrast, the IPNV titer in MAVS-disrupted cells was not different from the wild-type. Furthermore, overexpression of salmon MAVS in MAVS-disrupted CHSE cells rescued the impaired type I IFN-mediated anti-viral effect against SAV-3.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Infecções por Alphavirus , Alphavirus , Doenças dos Peixes , Vírus da Necrose Pancreática Infecciosa , Transdução de Sinais , Replicação Viral , Animais , Vírus da Necrose Pancreática Infecciosa/fisiologia , Vírus da Necrose Pancreática Infecciosa/imunologia , Alphavirus/imunologia , Alphavirus/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , Salmão/virologia , Salmão/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/veterinária , Infecções por Birnaviridae/virologiaRESUMO
Antimicrobial peptides (AMPs) are considered a novel approach to stimulate fish antiviral mechanisms for defense against a broad range of viral infections by enhancing immunomodulatory activities. Octominin is an AMP derived from the defense proteins of Octopus minor. In this study, preliminary screening of octominin against viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV) was carried out. Moreover, immune responses upon octominin treatment and IHNV challenge were investigated using fathead minnow (FHM) cells. The CC50s of octominin for FHM and Chinook salmon embryo-214 (CHSE-214) cells were 2146.2 and 1865.2 µg/mL, respectively. With octominin treatment, EC50 resulted in 732.8, 435.1, and 925.9 µg/mL for VHSV, IHNV, and IPNV, respectively. The selectivity indices were 2.9, 4.9, and 2.0, respectively. The transcriptional analysis results demonstrated the induced transcription factors (Irf3; 143-fold, Irf7; 105-fold, and NF-κB; 8-fold), stress response gene (HspB8; 2-fold), and apoptosis functional gene (p53; 3-fold) in octominin treated (500 µg/mL) FHM cells for 48 h. Moreover, IHNV viral copy number was slightly decreased with the octominin treatment (500 µg/mL) in FHM cells. Overall results suggest that octominin could be a potential antiviral agent, although further studies are necessary to understand its mode of action and the mechanism of its antiviral activity.
Assuntos
Cyprinidae , Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Vírus da Necrose Pancreática Infecciosa , Animais , Linhagem Celular , Peptídeos Antimicrobianos , Vírus da Necrose Pancreática Infecciosa/fisiologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Antivirais/farmacologia , ImunidadeRESUMO
Infectious pancreatic necrosis virus (IPNV) has proven to effectively evade the host antiviral responses. This study clarifies whether the modulation of the antiviral immune response exerted by IPNV involves epigenetic mechanisms. An in-silico characterization of the rainbow trout IFN1 and IFNγ2 promoters was performed, identifying the islands or sequences rich in CpG dinucleotides and the putative transcription factor binding sites (TBS) for both gene promoters. RTS11 cells (rainbow trout monocyte/macrophage) were infected with IPNV, and the course of viral infection was followed up to 48 h post infection (hpi). Infected cells showed increased IFN1 and IFNγ2 transcriptional expression at 6 and 24 hpi, respectively. IPNV infection caused increases and decreases in global IFNγ2 promoter methylation at 6 and 24 hpi, respectively. The CpG dinucleotides at positions -392 and + 38 of this promoter were the most sensitive to methylation changes. The IFN1 promoter remained fully unmethylated during the course of the infection, similar to the control. The changes in the methylation pattern observed for the IFNγ2 promoter were coincident with the changes in DNA methyltransferase (DNMT) expression levels, increasing at 6 hpi and decreasing below basal level at 24 hpi. Similarly, the H4 histones associated with the IFN1 and IFNγ2 promoters were hyperacetylated at 6 hpi, subsequently decreasing their acetylation below basal levels at 24 hpi, in both promoters. Coincidentally with the above, overexpression of histone acetyltransferase (HAT) was observed at 6 hpi and of histone deacetylase (HDAC) at 24 hpi, with return to baseline of HAT. These results suggest that IPNV would epigenetically modulate the expression of IFN1 by changing acetylation levels of the histones H4 associated with its promoter. Also, the modulation of the expression of IFNy2 would be by switching methylation/demethylation levels of its promoter, in addition to changes in acetylation levels of histones H4 associated with this promoter. This study is the first to demonstrate the effect of epigenetic reprogramming after IPNV infection in salmonid cells, demonstrating that promoter methylation/demethylation level and changes in the histone code associated with promoters may play a role in the modulation of the immune response induced by the virus.
Assuntos
Infecções por Birnaviridae , Doenças dos Peixes , Vírus da Necrose Pancreática Infecciosa , Oncorhynchus mykiss , Animais , Vírus da Necrose Pancreática Infecciosa/fisiologia , Histonas/genética , Antivirais , Epigênese Genética , Infecções por Birnaviridae/veterináriaRESUMO
Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.
Assuntos
Vacinas contra Adenovirus , Infecções por Birnaviridae , Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Vírus da Necrose Pancreática Infecciosa , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Vacinas Virais , Animais , Vírus da Necrose Pancreática Infecciosa/fisiologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Vacinas Sintéticas , Adenoviridae/genética , Infecções por Rhabdoviridae/prevenção & controle , Infecções por Rhabdoviridae/veterinária , Infecções por Birnaviridae/prevenção & controle , Infecções por Birnaviridae/veterináriaRESUMO
Infectious hematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) are the most common viral diseases of salmon in aquaculture worldwide. The co-infection of rainbow trout (Oncorhynchus mykiss) with IHN virus (IHNV) and IPN virus (IPNV) is known to occur. To determine the influence of IPNV on IHNV in co-infection, rainbow trout were intraperitoneally (i.p.) injected with IPNV at different time intervals prior to, simultaneously to, or after IHNV infection. The replication of IHNV in the brain, gill, heart, liver, spleen, and head kidney was detected by real-time quantitative polymerase chain reaction (qRT-PCR). The results showed that when rainbow trout were i.p. injected with IPNV prior to, simultaneously to, or after IHNV on 2 day (d), IHNV replication was inhibited (p < 0.05) in all collected tissues. Nevertheless, when rainbow trout were i.p. injected with IPNV after IHNV on 7 d (H7P), IHNV replication was only inhibited (p < 0.05) in the liver 14 d post-IHNV infection. Moreover, stronger antiviral responses occurred in all challenge groups. Our results suggest that IPNV can inhibit IHNV replication before or simultaneously with IHNV infection, and induce a stronger antiviral response, and that this inhibition is most sensitive in the liver. Early i.p. injection of IPNV can significantly reduce the mortality of rainbow trout, compared with the group only injected with IHNV.
Assuntos
Infecções por Birnaviridae , Coinfecção , Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Vírus da Necrose Pancreática Infecciosa , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Animais , Antivirais/farmacologia , Infecções por Birnaviridae/veterinária , Coinfecção/veterinária , Vírus da Necrose Pancreática Infecciosa/fisiologia , Infecções por Rhabdoviridae/veterináriaRESUMO
BACKGROUND: The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms' thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified. METHODS: We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes' functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues. RESULTS: We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors' activity and key anti-inflammatory factors such as cytokine Il10 and Tgfß in target tissues. CONCLUSION: These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural-immune adaptation to infection and its protective relevance in ectotherm organisms.
Assuntos
Comportamento Animal , Infecções por Birnaviridae/complicações , Febre/patologia , Imunidade , Vírus da Necrose Pancreática Infecciosa/fisiologia , Inflamação/patologia , Reflexo , Animais , Infecções por Birnaviridae/virologia , Regulação da Temperatura Corporal , Citocinas/metabolismo , Febre/etiologia , Peixes , Inflamação/etiologiaRESUMO
Infectious pancreatic necrosis virus (IPNV) infection has been a major problem in salmonid aquaculture. Marker-assisted selection of individuals with resistant genotype at the major IPN quantitative trait locus (IPN-QTL) has significantly reduced mortality in recent years. We have identified host miRNAs that respond to IPNV challenge in salmon fry that were either homozygous resistant (RR) or homozygous susceptible (SS) for the IPN-QTL. Small RNA-sequenced control samples were compared to samples collected at 1, 7, and 20 days post challenge (dpc). This revealed 72 differentially expressed miRNAs (DE miRNAs). Viral load (VL) was lower in RR vs. SS individuals at 7 and 20 dpc. However, analysis of miRNA expression changes revealed no differences between RR vs. SS individuals in controls, at 1 or 7 dpc, while 38 "high viral load responding" miRNAs (HVL-DE miRNAs) were identified at 20 dpc. Most of the HVL-DE miRNAs showed changes that were more pronounced in the high VL SS group than in the low VL RR group when compared to the controls. The absence of differences between QTL groups in controls, 1 and 7 dpc indicates that the QTL genotype does not affect miRNA expression in healthy fish or their first response to viral infections. The miRNA differences at 20 dpc were associated with the QTL genotype and could, possibly, contribute to differences in resistance/susceptibility at the later stage of infection. In silico target gene predictions revealed that 180 immune genes were putative targets, and enrichment analysis indicated that the miRNAs may regulate several major immune system pathways. Among the targets of HVL-DE miRNAs were IRF3, STAT4, NFKB2, MYD88, and IKKA. Interestingly, TNF-alpha paralogs were targeted by different DE miRNAs. Most DE miRNAs were from conserved miRNA families that respond to viral infections in teleost (e.g., miR-21, miR-146, miR-181, miR-192, miR-221, miR-462, miR-731, and miR-8159), while eight were species specific. The miRNAs showed dynamic temporal changes implying they would affect their target genes differently throughout disease progression. This shows that miRNAs are sensitive to VL and disease progression, and may act as fine-tuners of both immediate immune response activation and the later inflammatory processes.
Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Necrose Pancreática Infecciosa/fisiologia , MicroRNAs/genética , Salmo salar/genética , Animais , Sequência de Bases , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/virologia , Simulação por Computador , Progressão da Doença , Resistência à Doença , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Genótipo , Interações Hospedeiro-Patógeno/imunologia , Locos de Características Quantitativas , RNA Viral/análise , RNA-Seq , Salmo salar/crescimento & desenvolvimento , Salmo salar/imunologia , Salmo salar/virologia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Análise Serial de Tecidos , Carga ViralRESUMO
Gilthead seabream (Sparus aurata) is among the most important cultured fish species in the Mediterranean area and pathogen diseases one of the bottlenecks to the aquaculture sector. For this reason, generation of laboratory tools for diagnostic and research applications would be beneficial to improve the seabream aquaculture. In this sense, we aimed to generate a seabream cell line for biological studies. Thus, we have obtained a brain-derived cell line (SaB-1) that is continuously growing for more than 4 years. Cellular characterization of the SaB-1 cells shows that they express both neural and glial cell markers, suggesting they are neural-stem cells, have a neuron-like morphology and show a rapid growth in culture. We evaluated their susceptibility to the main fish viruses: nervous necrosis virus (NNV), spring viremia carp virus (SVCV), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). SaB-1 cells are susceptible to all the tested viruses. In addition, the transcription of genes related to the type I interferon (IFN) is greatly up-regulated by the NNV infection whilst the viral infection with SVCV, IPNV or VHSV failed to do so. These data demonstrate that the seabream SaB-1 cell line is continuous, stable and could be useful, at least, for fish virology and immunity applications.
Assuntos
Infecções por Birnaviridae/veterinária , Suscetibilidade a Doenças/veterinária , Doenças dos Peixes/virologia , Infecções por Rhabdoviridae/veterinária , Dourada , Animais , Aquabirnavirus/fisiologia , Infecções por Birnaviridae/virologia , Encéfalo , Linhagem Celular , Suscetibilidade a Doenças/virologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Novirhabdovirus/fisiologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/virologiaRESUMO
Salmonids can be co-infected by infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) under natural or experimental conditions. To reveal the influence of IPNV on IHNV in co-infections, CHSE-214 cells were inoculated with IPNV at different time intervals prior to or after IHNV infection. Propagation of IHNV was determined by an immunofluorescence antibody test, real-time quantitative polymerase chain reaction, flow cytometry, and virus titration. The results showed that when cells were inoculated with IPNV prior to IHNV, IHNV multiplication was inhibited. This inhibitory effect became stronger with increasing time intervals (P < 0.05). When cells were inoculated with IPNV after IHNV, the inhibitory effect became weaker with increasing time intervals (P < 0.05), and no significant inhibition was observed at 12 h (P > 0.05) compared with the single IHNV infection group. The findings suggest that IHNV is inhibited at the early stage of infection by IPNV and in a time dependent manner during co-infection. Furthermore, the effect of IPNV on IHNV entry and expression of IHNV entry-related genes clathrin, dynamin-2, adaptor protein 2, and vacuolar protein sorting 35 were also determined. The results showed that IPNV did not affect the amount of IHNV entering the cells. However, the expression levels of clathrin and dynamin-2 were significantly lower in co-infection than those in single IHNV infection, which suggests that IPNV likely inhibits IHNV by affecting IHNV invasion via downregulating IHNV entry-related genes clathrin and dynamin-2.
Assuntos
Infecções por Birnaviridae/veterinária , Coinfecção/veterinária , Doenças dos Peixes/imunologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Infecções por Rhabdoviridae/veterinária , Salmão , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/virologia , Linhagem Celular , Coinfecção/imunologia , Coinfecção/virologia , Regulação para Baixo , Embrião não Mamífero , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologiaRESUMO
Infectious pancreatic necrosis virus (IPNV) is a common pathogen that causes huge economic losses for the salmonid aquaculture industry. Autophagy plays an important regulatory role in the invasion of pathogenic microorganisms. In this study, we explored the relationship between IPNV infection and autophagy in Chinook salmon embryo (CHSE-214) cells using standard methods. Transmission electron microscopy showed that IPNV infection produced typical structures of autophagosomes in CHSE-214 cells. Transformation of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II protein, a marker of autophagy, was observed in IPNV-infected cells using confocal fluorescence microscopy and western blot analysis. Western blotting also showed that expression of the autophagy substrate p62 was significantly decreased in IPNV-infected cells. The influence of autophagy on IPNV multiplication was further clarified with cell culture experiments using autophagy inducer rapamycin and autophagy inhibitor 3-methyladenine. Rapamycin promoted IPNV multiplication at both the nucleic acid and protein levels, which led to higher IPNV yields; 3-methyladenine treatment had the opposite effect. This study has demonstrated that IPNV can induce autophagy, and that autophagy promotes the multiplication of IPNV in CHSE-214 cells.
Assuntos
Autofagia , Infecções por Birnaviridae/veterinária , Doenças dos Peixes/virologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Salmão , Replicação Viral , Animais , Autofagossomos/ultraestrutura , Autofagossomos/virologia , Infecções por Birnaviridae/virologia , Linhagem Celular , Embrião não Mamífero/virologia , Microscopia Eletrônica de Transmissão/veterinária , Salmão/embriologiaRESUMO
Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.
Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/mortalidade , Vírus da Necrose Pancreática Infecciosa/fisiologia , Oncorhynchus mykiss , Carga Viral/veterinária , Animais , Infecções por Birnaviridae/mortalidade , Infecções por Birnaviridae/virologia , Chile/epidemiologia , Doenças dos Peixes/virologia , Genótipo , Vírus da Necrose Pancreática Infecciosa/genética , FilogeniaRESUMO
Infectious pancreatic necrosis (IPN) is a viral disease with considerable negative impact on the rainbow trout (Oncorhynchus mykiss) aquaculture industry. The aim of the present work was to detect genomic regions that explain resistance to infectious pancreatic necrosis virus (IPNV) in rainbow trout. A total of 2,278 fish from 58 full-sib families were challenged with IPNV and 768 individuals were genotyped (488 resistant and 280 susceptible), using a 57K SNP panel Axiom, Affymetrix. A genome-wide association study (GWAS) was performed using the phenotypes time to death (TD) and binary survival (BS), along with the genotypes of the challenged fish using a Bayesian model (Bayes C). Heritabilities for resistance to IPNV estimated using genomic information, were 0.53 and 0.82 for TD and BS, respectively. The Bayesian GWAS detected a SNP located on chromosome 5 explaining 19% of the genetic variance for TD. The proximity of Sentrin-specific protease 5 (SENP5) to this SNP makes it a candidate gene for resistance against IPNV. In case of BS, a SNP located on chromosome 23 was detected explaining 9% of the genetic variance. However, the moderate-low proportion of variance explained by the detected marker leads to the conclusion that the incorporation of all genomic information, through genomic selection, would be the most appropriate approach to accelerate genetic progress for the improvement of resistance against IPNV in rainbow trout.
Assuntos
Resistência à Doença/genética , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Vírus da Necrose Pancreática Infecciosa/fisiologia , Oncorhynchus mykiss/genética , Animais , Teorema de Bayes , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/mortalidade , Infecções por Birnaviridae/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/mortalidade , Proteínas de Peixes/imunologia , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno/genética , Vírus da Necrose Pancreática Infecciosa/patogenicidade , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/virologia , Polimorfismo de Nucleotídeo Único , Replicação Viral/fisiologiaRESUMO
We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE-214 by macropinocytosis. In this study, we have extended our investigation into SHK-1 cells, a macrophage-like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK-1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK-1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK-1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis-mediated entry of IPNV into its target cells.
Assuntos
Vírus da Necrose Pancreática Infecciosa/fisiologia , Macrófagos/virologia , Pinocitose , Salmão/virologia , Internalização do Vírus , Actinas/metabolismo , Animais , Infecções por Birnaviridae/virologia , Linhagem Celular , Doenças dos Peixes/virologia , Rim Cefálico/virologia , Macrófagos/citologiaRESUMO
Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout. Their co-infection is also common, which causes great economic loss in juvenile salmon species. Although vaccines against IHNV and IPNV have been commercialized in many countries, the prevalence of IHNV and IPNV is still widespread in modern aquaculture. In the present study, two IHNV recombinant viruses displaying IPNV VP2 protein (rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE) were generated using the RNA polymerase â ¡ system to explore the immunogenicity of IHNV and IPNV. The recombinant IHNV viruses were stable, which was confirmed by sequencing, indirect immunofluorescence assay, western blotting, transmission electron microscopy and viral growth curve assay. IHNV and IPNV challenge showed that the recombinant viruses had high protection rates against IHNV and IPNV with approximately 65% relative percent survival rates. Rainbow trout (mean weight 20â¯g) vaccinated with these two recombinant viruses showed a high level of antibodies against IHNV and IPNV infection. Taken together, our findings demonstrate that rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE might be promising vaccine candidates against IHNV and IPNV.
Assuntos
Doenças dos Peixes/imunologia , Oncorhynchus mykiss/imunologia , Proteínas Estruturais Virais/farmacologia , Vacinas Virais/farmacologia , Animais , Infecções por Birnaviridae/imunologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Distribuição Aleatória , Infecções por Rhabdoviridae/imunologia , Vacinas Sintéticas/farmacologiaRESUMO
Maintaining fish health is one of the most important aims in aquaculture. Prevention of fish diseases therefore is crucial and can be achieved by various different strategies, including most often a combination of different methods such as optimal feed and fish density, as well as strengthening the immune system. Understanding the fish innate immune system and developing methods to activate it, in an effort to prevent infections in the first place, has been a goal in recent years. In this study we choose different inducers of the innate immune system and examined their effects in vitro on the salmon cell line CHSE-214. We found that the butyrate derivatives 4-phenyl butyrate (PBA) and ß-hydroxy-ß-methyl butyrate (HMB) induce the expression of various innate immune genes differentially over 24-72 h. Similarly, lipids generated from fish oils were found to have an effect on the expression of the antimicrobial peptides cathelicidin and hepcidin, as well as iNOS and the viral receptor RIG-1. Interestingly we found that vitamin D3, similar as in mammals, was able to increase cathelicidin expression in fish cells. The observed induction of these different innate immune factors correlated with antibacterial activity against Aeromonas salmonicida and antiviral activity against IPNV and ISAV in vitro. To relate this data to the in vivo situation we examined cathelicidin expression in juvenile salmon and found that salmon families vary greatly in their basal cathelicidin levels. Examining cathelicidin levels in families known to be resistant to IPNV showed that these QTL-families had lower basal levels of cathelicidin in gills, than non QTL-families. Feeding fish with HMB caused a robust increase in cathelicidin expression in gills, but not skin and this was independent of the fish being resistant to IPNV. These findings support the use of fish cell lines as a tool to develop new inducers of the fish innate immune system, but also highlight the importance of the tissue studied in vivo. Understanding the response of the innate immune system in different tissues and what effect this might have on infections and downstream cellular pathways is an interesting research topic for the future.
Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata , Salmo salar/genética , Salmo salar/imunologia , Aeromonas salmonicida/fisiologia , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/veterinária , Linhagem Celular , Colecalciferol/administração & dosagem , Colecalciferol/metabolismo , Furunculose/imunologia , Expressão Gênica , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Vírus da Necrose Pancreática Infecciosa/fisiologia , Lipídeos/administração & dosagem , Fenilbutiratos/administração & dosagem , Fenilbutiratos/metabolismo , Valeratos/administração & dosagem , Valeratos/metabolismoRESUMO
The pathogenic infectious pancreatic necrosis virus (IPNV) causes high economic losses in fish farming. This virus can modulate several cellular processes during infection, but little is known about the infection mechanism. To investigate gene activation in response to IPNV, CHSE/F and SHK-1 cell line were infected with a cytopathic Sp field isolate of IPNV, and the expression profiles of proinflammatory, antiviral cytokine, and extracellular matrix markers were analyzed. IPNV induced the production of perlecan, fibulin-1, matrix metalloproteinase-2, 14-3-3ß, interleukin-1ß, Mx1, and interferon regulatory factors-1, -3, and -9. Interestingly, IPNV-mediated activity was blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus-target motifs, suggest that IPNV regulates gene expressions in fish through the activation of several key transcription factors. Collectively, these data indicate that IPNV is a viral regulator of expression for extracellular-matrix and immune markers, even during early infection. Finally, this is the first report in fish to find IPNV modulating the activation of interleukin-1ß production primarily through the NF-κB pathway.
Assuntos
Matriz Extracelular/virologia , Doenças dos Peixes/virologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Animais , Biomarcadores/metabolismo , Linhagem Celular , Matriz Extracelular/metabolismo , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , NF-kappa B/metabolismo , Perciformes , Salmo salarRESUMO
Infectious pancreatic necrosis virus (IPNV) is a non-enveloped virus belonging to the Birnaviridae family. IPNV produces an acute disease in salmon fingerlings, with high mortality rates and persistent infection in survivors. Although there are reports of IPNV binding to various cells, the viral receptor and entry pathways remain unknown. The aim of this study was to determine the endocytic pathway that allows for IPNV entry. We observed that IPNV stimulated fluid uptake and virus particles co-localysed with the uptake marker dextran in intracellular compartments, suggesting a role for macropinocytosis in viral entry. Consistent with this idea, viral infection was significantly reduced when the Na+/H+ exchanger NHE1 was inhibited with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). Neither chlorpromazine nor filipin complex I affected IPNV infection. To examine the role of macropinocytosis regulators, additional inhibitors were tested. Inhibitors of the EGFR pathway and the effectors Pak1, Rac1 and PKC reduced viral infection. Together, our results indicate that IPNV is mainly internalized into CHSE-214 cells by macropinocytosis.
Assuntos
Vírus da Necrose Pancreática Infecciosa/fisiologia , Pinocitose , Internalização do Vírus , Animais , Infecções por Birnaviridae/virologia , Caveolinas/metabolismo , Linhagem Celular , Clorpromazina/farmacologia , Dinaminas/metabolismo , Endocitose , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/virologia , Microdomínios da Membrana , Pinocitose/efeitos dos fármacos , Salmão/virologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Infectious pancreatic necrosis virus (IPNV) is the etiological agent of a contagious viral disease causing remarkable mortalities in different fish species. Despite the availability of commercial vaccines against IPN, the disease still constitutes one of the main threats to the aquaculture industry worldwide. In this study, we developed a DNA vaccine encoding the VP2 gene of IPNV and evaluated its ability to induce protective immunity in rainbow trout fry (3 g) at doses of 10 and 25 µg/fish and boosting with the same doses two weeks later through the oral route using chitosan/tripolyphosphate (CS-TPP) nanoparticles and alginate microparticles incorporated into fish feed. The distribution of the administered vaccines in different organs and transcription of VP2 gene were confirmed by RT-PCR assay at day 30 post boost-vaccination. Transcript levels of IFN-1, Mx-1, IgM, IgT and CD4 genes was dependent on vaccine dose and was significantly up-regulated in head kidney of all orally vaccinated fish groups compared to controls (pcDNA3.1). Cumulative mortalities post-challenge with virulent isolate of the virus were lower in the vaccinated fish and a relative percentage survival (RPS) of 59% and 82% were obtained for the 10 and 25 µg/fish pcDNA3.1-VP2 groups, respectively. Vaccination with the same amount of pcDNA3.1-VP2 encapsulated with CS-TPP nanoparticles resulted in RPS of 47 %and 70%, respectively. Detectable anti-IPNV antibodies were shown until 90 days postvaccination. The orally administrated vaccines significantly decreased VP4 transcripts thus contributing to reducing viral load in surviving fish on day 45 post-challenge. In conclusion, these results show good to high protection post-vaccination alongside with significant up-regulation of key immune genes and detectable levels of circulating antibodies after oral administration of the DNA vaccine formulated in CS-TPP nanoparticles and alginate microparticles in fish feed.