RESUMEN
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.
Asunto(s)
Infecciones por Birnaviridae , Enfermedades de los Peces , Virus de la Necrosis Pancreática Infecciosa , Oncorhynchus mykiss , Animales , Virus de la Necrosis Pancreática Infecciosa/fisiología , Histonas/genética , Antivirales , Epigénesis Genética , Infecciones por Birnaviridae/veterinariaRESUMEN
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.
Asunto(s)
Conducta Animal , Infecciones por Birnaviridae/complicaciones , Fiebre/patología , Inmunidad , Virus de la Necrosis Pancreática Infecciosa/fisiología , Inflamación/patología , Reflejo , Animales , Infecciones por Birnaviridae/virología , Regulación de la Temperatura Corporal , Citocinas/metabolismo , Fiebre/etiología , Peces , Inflamación/etiologíaRESUMEN
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.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Enfermedades de los Peces/mortalidad , Virus de la Necrosis Pancreática Infecciosa/fisiología , Oncorhynchus mykiss , Carga Viral/veterinaria , Animales , Infecciones por Birnaviridae/mortalidad , Infecciones por Birnaviridae/virología , Chile/epidemiología , Enfermedades de los Peces/virología , Genotipo , Virus de la Necrosis Pancreática Infecciosa/genética , FilogeniaRESUMEN
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.
Asunto(s)
Resistencia a la Enfermedad/genética , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Virus de la Necrosis Pancreática Infecciosa/fisiología , Oncorhynchus mykiss/genética , Animales , Teorema de Bayes , Infecciones por Birnaviridae/genética , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/mortalidad , Infecciones por Birnaviridae/veterinaria , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/mortalidad , Proteínas de Peces/inmunología , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno/genética , Virus de la Necrosis Pancreática Infecciosa/patogenicidad , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/virología , Polimorfismo de Nucleótido Simple , Replicación Viral/fisiologíaRESUMEN
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.
Asunto(s)
Virus de la Necrosis Pancreática Infecciosa/fisiología , Macrófagos/virología , Pinocitosis , Salmón/virología , Internalización del Virus , Actinas/metabolismo , Animales , Infecciones por Birnaviridae/virología , Línea Celular , Enfermedades de los Peces/virología , Riñón Cefálico/virología , Macrófagos/citologíaRESUMEN
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.
Asunto(s)
Matriz Extracelular/virología , Enfermedades de los Peces/virología , Virus de la Necrosis Pancreática Infecciosa/fisiología , Animales , Biomarcadores/metabolismo , Línea Celular , Matriz Extracelular/metabolismo , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , FN-kappa B/metabolismo , Perciformes , Salmo salarRESUMEN
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.
Asunto(s)
Virus de la Necrosis Pancreática Infecciosa/fisiología , Pinocitosis , Internalización del Virus , Animales , Infecciones por Birnaviridae/virología , Caveolinas/metabolismo , Línea Celular , Clorpromazina/farmacología , Dinaminas/metabolismo , Endocitosis , Enfermedades de los Peces/tratamiento farmacológico , Enfermedades de los Peces/virología , Microdominios de Membrana , Pinocitosis/efectos de los fármacos , Salmón/virología , Internalización del Virus/efectos de los fármacosRESUMEN
Innate pathway activation is fundamental for early anti-viral defense in fish, but currently there is insufficient understanding of how salmonid fish identify viral molecules and activate these pathways. The Toll-like receptor (TLR) is believed to play a crucial role in host defense of pathogenic microbes in the innate immune system. In the present study, the full-length cDNA of Salmo salar TLR3 (ssTLR3) was cloned. The ssTLR3 cDNA sequence was 6071 bp long, containing an open reading frame of 2754 bp and encoding 971 amino acids. The TLR group motifs, such as leucine-rich repeat (LRR) domains and Toll-interleukin-1 receptor (TIR) domains, were maintained in ssTLR3, with sixteen LRR domains and one TIR domain. In contrast to descriptions of the TLR3 in rainbow trout and the murine (TATA-less), we found a putative TATA box in the proximal promoter region 29 bp upstream of the transcription start point of ssTLR3. Multiple-sequence alignment analysis of the ssTLR3 protein-coding sequence with other known TLR3 sequences showed the sequence to be conserved among all species analyzed, implying that the function of the TLR3 had been sustained throughout evolution. The ssTLR3 mRNA expression patterns were measured using real-time PCR. The results revealed that TLR3 is widely expressed in various healthy tissues. Individuals challenged with infectious pancreatic necrosis virus and immunostimulated with polyinosinic:polycytidylic acid exhibited increased expression of TLR3 at the mRNA level, indicating that ssTLR3 may be involved in pathogen recognition in the early innate immune system.
Asunto(s)
Clonación Molecular/métodos , Inmunidad Innata , Salmo salar/genética , Salmo salar/inmunología , Receptor Toll-Like 3/genética , Animales , Resistencia a la Enfermedad , Evolución Molecular , Virus de la Necrosis Pancreática Infecciosa/fisiología , Filogenia , Poli I-C/farmacología , Salmo salar/virología , Análisis de Secuencia de ADN , Receptor Toll-Like 3/químicaRESUMEN
Infectious pancreatic necrosis virus (IPNV) is an economically important pathogen of the salmonid aquaculture industry. Selective breeding has been employed to improve resistance to this infectious disease, and it is of importance to investigate the expression profile of immune genes of Atlantic salmon with different genetic background in response to this virus. This study examined the immune modulation response of eight candidate genes in head kidney tissue in two families of Atlantic salmon with high and low mortalities, after challenge with IPNV. The results showed that the expression pattern of target genes differed in the two families. Generally, higher expression of antiviral, pro-inflammatory genes and transcription factors such as tripartite motif, NF-κB, IFNI, STAT1, protein kinase R, and Vig-2 in the resistant family were observed at the same time point. One may speculate the functional importance of these putative candidate genes in the characterization of the IPNV-resistant (low mortalities) immune phenotype. Therefore, on our findings, we suggest that future salmonids studies aiming to identify candidate genes/pathway or vaccines evaluation should consider validating detected genes/pathway across different genetic backgrounds or immune phenotype.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Enfermedades de los Peces/virología , Interacciones Huésped-Patógeno/inmunología , Virus de la Necrosis Pancreática Infecciosa/fisiología , Salmo salar/virología , Animales , Infecciones por Birnaviridae/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Interacciones Huésped-Patógeno/genética , Interferón-alfa/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT1/metabolismo , Salmo salar/fisiología , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1ß, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1ß and IL-8 did not rise in the spleen although an increase of IL-1ß, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1ß and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1ß and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Citocinas/metabolismo , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica , Virus de la Necrosis Pancreática Infecciosa/fisiología , Salmo salar , Animales , Infecciones por Birnaviridae/inmunología , Línea Celular , Enfermedades de los Peces/virología , Perfilación de la Expresión Génica , Branquias/inmunología , Riñón Cefálico/inmunologíaRESUMEN
Investigating the capacity of the infectious pancreatic necrosis virus (IPNV) to promote apoptosis with a TUNEL kit, designed to visualize fragmented DNA, a striking labeling pattern was found. In addition to the fluorescence observed in the nucleus of cells with fragmented DNA, the infected cells showed an intense particulate fluorescence in their cytoplasm. The cytoplasmic labeling seems to be concomitant with the timing of virus life cycle because labeled viral proteins coexist with TUNEL cytoplasmic fluorescence at different times during infection. In principle the terminal deoxynucleotidyl transferase (TdT) used in TUNEL assays should not be able to label substrates other than DNA, however, our results are consistent with the idea that viral structures are being labeled. We propose that the TdT enzyme label the 3'-hydroxyl ends of viral RNAs during its polymerization. This kind of reaction could be possible because it has been reported that TdT is an enzyme that can use rNTPs as substrates and the 3'-hydroxyl priming end can be provided by a ribonucleotide as well. Thus, allowing the visualization of virus RNA-containing intermediates during IPNV replication.
Asunto(s)
Etiquetado Corte-Fin in Situ , Virus de la Necrosis Pancreática Infecciosa/fisiología , ARN Viral/fisiología , Replicación Viral/fisiología , Animales , Apoptosis/fisiología , Línea CelularRESUMEN
Infectious pancreatic necrosis is a disease caused by a birnavirus affecting several wild and commercial aquatic organisms. This infectious disease results in significant losses in the farming industry and therefore effective therapeutic agents are needed to control outbreaks caused by this pathogen. Our goal was to evaluate in vitro antiviral effect of a group of natural compounds (geranyl aromatic derivatives) isolated from the resinous exudate of the plant Heliotropium filifolium (Heliotropiaceae), semi-synthetics compounds obtained from them, and the resinous exudate, on CHSE-214 cell line infected with infectious pancreatic necrosis virus (IPNV) using a virus plaque inhibition assay at various concentrations. The compound ester filifolinyl senecionate was the best antiviral with EC(50) 160 microg/mL and a cytotoxic concentration required to reduce cell viability by 50% up to 400 microg/mL. In order to obtain information about the mechanism of the antiviral action, was evaluated the influence of ester filifolinyl senecionate on the viral RNA synthesis. This compound produced inhibition of the synthesis of viral genomic RNA, suggesting that the ester could be interacting with the viral RNA during the viral cycle. Additionally, a preliminary study of the interaction between ester and a sample of single-stranded RNA was studied at the level of theory Restricted Hartree Fock PM3 method. The results showed that the ester formed hydrogen bonds mainly with nitrogenous bases but not with ribose and phosphate. These results allow propose that the ester filifolinyl senecionate is a good candidate for used as antiviral therapy for IPN virus in salmon fry.
Asunto(s)
Antivirales/farmacología , Heliotropium/química , Virus de la Necrosis Pancreática Infecciosa/efectos de los fármacos , Virus de la Necrosis Pancreática Infecciosa/fisiología , Exudados de Plantas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , Exudados de Plantas/química , ARN/metabolismo , SalmónRESUMEN
Infectious Pancreatic Necrosis Virus (IPNV) is a bisegmented, double-stranded RNA virus, which belongs to the Birnaviridae family. In the current study, we have analyzed the RNA replication intermediates (RI) purified throughout the viral replication cycle in cultured cells. Equilibrium ultracentrifugation of infected cellular lysates resulted in two major peaks of viral components. The first peak, at a buoyant density of 1.33 g/cm(3), contained assembled IPNV viral particles A and B, whereas the second peak, located at buoyant densities >1.4 g/cm(3), contained a higher molecular weight viral ribonucleoprotein complex composed of, at least, VPg/VP1 and a heterogeneous population of single- and double-stranded viral RNA species. Interestingly, analyses of these dsRNA RI indicated that they contain single-stranded segments of incompletely synthesized positive-strands of RNA. Northern blot experiments of total RNA isolated from infected cells confirmed our proposed configuration of the RNA RI, where the full-length negative-strand of RNA is used as the template for the synthesis of several 3'-truncated forms of the positive-strand of the viral RNA. Together, our results indicate that IPNV utilizes the negative-strand of RNA as template for genome replication.
Asunto(s)
Virus de la Necrosis Pancreática Infecciosa/fisiología , ARN Viral/aislamiento & purificación , ARN Viral/metabolismo , Replicación Viral , Animales , Línea Celular , Centrifugación Isopicnica , Sustancias Macromoleculares/aislamiento & purificación , SalmónRESUMEN
Infectious pancreatic necrosis virus (IPNV)-infected cells were labeled with Annexin V and propidium iodide in order to determine the proportion of cells, which developed necrosis and/or apoptosis during the time course of infection. Contrasting with earlier reports, we found that at any time during IPNV multiplication cycle, the percentage of apoptotic cells never exceeded the 12% of the whole population of the infected cells. In addition, the percentage of necrotic cells increased continuously until reaching the 75% of the infected cells at 15 h post infection. Apoptotic cells were also identified by in situ terminal deoxynucleotidyl transferase-mediated BrdUTP nick end labeling (TUNEL). Our results are in accordance with the idea that apoptosis rarely precedes necrosis.
Asunto(s)
Apoptosis , Efecto Citopatogénico Viral , Virus de la Necrosis Pancreática Infecciosa/fisiología , Necrosis , Animales , Anexina A5/metabolismo , Línea Celular , Etiquetado Corte-Fin in Situ , Propidio/metabolismo , Salmón , Coloración y Etiquetado , Ensamble de VirusRESUMEN
The early events in the infection of unenveloped viruses are still rather unknown and puzzling. However, as in the case of enveloped viruses, the acid pH of endosomes can be important to trigger the virus entry into the cytosol. To test if the infectious pancreatic necrosis virus (IPNV), an unenveloped virus, requires acid endosomal pH to infect cells, we assayed the effect of Bafilomycin A1 on IPNV replication. Concentrations of the antibiotic which inhibited the endosomal acidification of the host cells were unable to affect IPNV replication in CHSE-214 cells; therefore, the acid pH of endosomes seems not to be a mandatory condition for the entry of IPNV into cells.
Asunto(s)
Virus de la Necrosis Pancreática Infecciosa/fisiología , Macrólidos , Animales , Antibacterianos/farmacología , Línea Celular , Concentración de Iones de Hidrógeno , Virus de la Necrosis Pancreática Infecciosa/efectos de los fármacos , Virus de la Necrosis Pancreática Infecciosa/crecimiento & desarrollo , ATPasas de Translocación de Protón/antagonistas & inhibidores , SalmónRESUMEN
In attempts to detect inhibitors of infectious pancreatic necrosis virus (IPNV) replication, we have evaluated, by an IPNV plaque inhibition assay, a group of compounds that have broad spectrum antiviral activity for both single- and double-stranded RNA viruses. The inosine monophosphate dehydrogenase (IMP dehydrogenase) inhibitors 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and the orotidine monophosphate decarboxylase (OMP decarboxylase) inhibitor 4-hydroxy-3-beta-D-ribofuranosylpyrazole-5-carboxamide (pyrazofurin), were found to inhibit IPNV replication. For EICAR and pyrazofurin the concentrations that inhibited the IPNV plaque formation by 50% (EC50) were 0.01 micrograms/ml and 0.5 micrograms/ml, respectively. The cytotoxic concentrations required to reduce cell viability by 50% (CC50) were 50 micrograms/ml and 100 micrograms/ml, respectively, and the concentrations that reduced [methyl-3H] thymidine incorporation by 50% (IC50) were 0.5-1 and 50 micrograms/ml. Thus, for both compounds the IPNV-inhibitory concentration was 50-100 times lower than the concentration that affected DNA synthesis in growing cells. EICAR and pyrazofurin seem to be good candidates for further evaluation in an in vivo model of IPNV infection.