RESUMEN
Infectious pancreatic necrosis virus (IPNV) is the etiological agent of a highly contagious disease that is endemic to salmon farming in Chile and causes great economic losses to the industry. Here we compared different diagnostic methods to detect IPNV in field samples, including 3 real-time reverse transcription PCR (qRT-PCR) assays, cell culture isolation, and indirect fluorescent antibody test (IFAT). Additionally, we performed a phylogenetic analysis to investigate the genogroups prevailing in Chile, as well as their geographic distribution and virulence. The 3 qRT-PCR assays used primers that targeted regions of the VP2 and VP1 genes of the virus and were tested in 46 samples, presenting a fair agreement within their results. All samples were positive for at least 2 of the qRT-PCR assays, 29 were positive for cell culture, and 23 for IFAT, showing less sensitivity for these latter 2 methods. For the phylogenetic analysis, portions of 1180 and 523 bp of the VP2 region of segment A were amplified by RT-PCR, sequenced and compared with sequences from reference strains and from isolates reported by previous studies carried out in Chile. Most of the sequenced isolates belonged to genogroup 5 (European origin), and 5 were classified within genogroup 1 (American origin). Chilean isolates formed clusters within each of the genogroups found, evidencing a clear differentiation from the reference strains. To our knowledge, this is the most extensive study completed for IPNV in Chile, covering isolates from sea- and freshwater salmon farms and showing a high prevalence of this virus in the country.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Enfermedades de los Peces/virología , Virus de la Necrosis Pancreática Infecciosa/genética , Oncorhynchus mykiss , Filogenia , Salmo salar , Animales , Infecciones por Birnaviridae/epidemiología , Infecciones por Birnaviridae/virología , Línea Celular , Chile/epidemiología , Enfermedades de los Peces/epidemiología , Técnica del Anticuerpo Fluorescente Indirecta , Regulación Viral de la Expresión Génica , Virus de la Necrosis Pancreática Infecciosa/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismoRESUMEN
The structural proteins of infectious pancreatic necrosis virus (IPNV) have been analyzed. Two-dimensional gel electrophoresis showed that IPNV proteins are slightly acidic with apparent pIs ranging from 5.8 to 6.6. To identify the IPNV surface-located proteins, purified virus was labelled either with fluorescein isothiocyanate (FITC) or with Na 125I. After analysis by SDS-PAGE, only the major viral protein, VP2, was labelled by either procedure. The accessibility of VP2 to these reagents suggests that this protein is externally located. In addition, using Concanavalin A conjugated with FITC and IPNV labelling with 3H-mannose, evidence is present that VP2 contains carbohydrate residues.
Asunto(s)
Fluoresceína-5-Isotiocianato/análogos & derivados , Proteínas Estructurales Virales/análisis , Virus/análisis , Animales , Cápside/análisis , Proteínas de la Cápside , Concanavalina A/análogos & derivados , Fluoresceínas , Glicosilación , Punto Isoeléctrico , Necrosis , Enfermedades Pancreáticas/microbiología , Enfermedades Pancreáticas/patología , SalmonidaeRESUMEN
An improved method for the isolation and purification of infectious pancreatic necrosis virus (IPNV) is described. Virions released into the clarified growth medium are adsorbed to an anion exchange resin of diethylaminoethyl cellulose at pH 8.1. IPNV together with the likewise released and accumulated excess pool of the precursor to the major capsid protein, ICP62, are eluted at a salt concentration between 100 and 125 mM NaCl. The bovine serum albumin content of the growth medium supplement also elutes close to this position. Upon one step of combined sucrose- and CsCl-gradient centrifugation the recovered viruses display lower levels of aggregation, higher specific nucleic acid contents and an approximately 350% higher specific infectivity as compared with pools of viruses processed in parallel and isolated according to the established method relying on precipitation with poly(ethylene glycol).
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Virus de la Necrosis Pancreática Infecciosa/aislamiento & purificación , Virología/métodos , Animales , Células Cultivadas , Fibroblastos , Virus de la Necrosis Pancreática Infecciosa/patogenicidad , Oncorhynchus mykissAsunto(s)
Iminoácidos/metabolismo , Nitrógeno/metabolismo , Saccharomyces/metabolismo , Sarcosina/metabolismo , Acridinas/farmacología , Aerobiosis , Anaerobiosis , Azetinas/metabolismo , Transporte Biológico , Radioisótopos de Carbono , Cicloheximida/farmacología , Diaminas/farmacología , Proteínas Fúngicas/biosíntesis , Hidroxiprolina/metabolismo , Isoleucina/metabolismo , Leucina/metabolismo , Fenilalanina/metabolismo , Prolina/metabolismo , ARN Mensajero/biosíntesis , Saccharomyces/efectos de los fármacos , Factores de Tiempo , Tritio , Triptófano/farmacologíaAsunto(s)
Saccharomyces/metabolismo , Sarcosina/metabolismo , Aminoácidos/farmacología , Azetinas/farmacología , Azidas/farmacología , Transporte Biológico Activo , Isótopos de Carbono , Radioisótopos de Carbono , Cromatografía en Papel , Dinitrofenoles/farmacología , Cinética , Prolina/metabolismo , Prolina/farmacología , Saccharomyces/efectos de los fármacos , Relación Estructura-Actividad , Temperatura , Tiazoles/farmacología , Factores de TiempoRESUMEN
The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.
Asunto(s)
Enfermedades de los Peces/microbiología , Enfermedades Pancreáticas/veterinaria , Infecciones por Reoviridae/veterinaria , Reoviridae/patogenicidad , Salmón , Animales , Línea Celular , Endocitosis , Endosomas/microbiología , Ferritinas/metabolismoRESUMEN
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
The multiplication of IPNV in CHSE cells is inhibited by ammonium chloride. This inhibition is complete if NH4Cl is added early in the infective cycle. Immunofluorescence analysis shows that NH4Cl reduces the number of virus producing cells and thus suggests it may act by inhibiting virus internalization. In addition, NH4Cl can produce partial inhibition of IPNV multiplication if added later in the replicative cycle. Analysis of 3H-uridine incorporation into viral RNA shows that this later effect could be an inhibition of viral RNA synthesis.
Asunto(s)
Cloruro de Amonio/farmacología , Virus ARN/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Cloroquina/farmacología , Depresión Química , Virus ARN/fisiología , ARN Viral/biosíntesis , Factores de TiempoRESUMEN
Nucleic acid and protein electrophoretic analyses of the Chilean isolate of infectious pancreatic necrosis virus showed that genomic RNA, as well as the major proteins, were indistinguishable from those of the VR-299 serotype. The data are in good agreement with the theory that the virus was introduced into Chile from North America.
Asunto(s)
Enfermedades de los Peces/microbiología , Virus ARN/aislamiento & purificación , Animales , Chile , Peso Molecular , Páncreas/microbiología , Virus ARN/análisis , Virus ARN/clasificación , ARN Bicatenario/análisis , ARN Viral/análisis , Proteínas Virales/análisisRESUMEN
The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm.
Asunto(s)
Cápside/análisis , Virus de la Necrosis Pancreática Infecciosa/química , Animales , Proteínas de la Cápside , Células Cultivadas , Retículo Endoplásmico/química , Glicosilación , Aparato de Golgi/química , Salmón , Replicación ViralRESUMEN
Infectious pancreatic necrosis virus (IPNV) attaches to CHSE-214 cells through two types of cell components: specific and non-specific ones. Competition experiments with inactivated IPNV showed that IPNV requires specific components to productively infect cells. Just a low amount of adsorbed IPNV enters the cell. After 20 minutes, part of the adsorbed IPNV was internalized into acid compartments. Also, the viruses adsorbed on the cell surface require similar periods of time to escape from the neutralization of antibodies.
Asunto(s)
Infecciones por Birnaviridae/fisiopatología , Birnaviridae/crecimiento & desarrollo , Peces/microbiología , Animales , Células Cultivadas , Endocitosis , Pruebas de Neutralización , ARN Viral/biosíntesis , Factores de TiempoRESUMEN
Several rifamycin derivatives inhibited the DNA-dependent RNA polymerase of African swine fever (ASF) virus particles. The inhibition was similar to that found with vaccinia virus RNA polymerase. Coumermycin A1, an inhibitor of type II DNA topoisomerases, inhibited strongly RNA synthesis in vitro by ASF virus particles. This suggests that transcription of ASF virus DNA requires a DNA topoisomerase.
Asunto(s)
Virus de la Fiebre Porcina Africana/efectos de los fármacos , Antibacterianos/farmacología , Cumarinas/farmacología , Iridoviridae/efectos de los fármacos , ARN Viral/antagonistas & inhibidores , Rifamicinas/farmacología , Virus de la Fiebre Porcina Africana/metabolismo , Aminocumarinas , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Virión/efectos de los fármacos , Virión/metabolismoRESUMEN
African swine fever virus contains nucleoside triphosphate phosphohydrolase activity which releases 32P phosphate from gamma-32P ATP at a rate of about 1 mumol/h mg of virus protein. The hydrolase activity is slightly stimulated by adding nucleic acids to the reaction mixture and under conditions of RNA synthesis. A study of the rate of ATP hydrolysis at different concentrations of ATP suggests the existence of two phosphohydrolase activities with apparent Km values of about 0.04 and 1 mM.