RESUMEN
In the present work, the establishment and biological characterization of a new cell line, SSP-9, derived from the pronephros of the Atlantic salmon Salmo salar, are reported. These cells grew well in Leibovitz's (L15) medium supplemented with 10% foetal calf serum at temperatures from 15 to 25° C, and they have been sub-cultured over 100 passages to produce a continuous cell line with an epithelial-like morphology. The SSP-9 cells attached and spread efficiently at different plating densities, retaining 80% of cell viability after storage in liquid nitrogen. When karyotyped, the cells had 40-52 chromosomes, with a modal number of 48. Viral susceptibility tests showed that SSP-9 cells were susceptible to infectious pancreatic necrosis virus and infectious haematopoietic necrosis virus, producing infectious virus and regular cytopathic effects. Moreover, these cells could be stimulated by poly I:C, showing significant up-regulation in the expression of the genes that regulate immune responses, such as ifn and mx-1. SSP-9 cells constitutively express genes characteristic of macrophages, such as major histocompatibility complex (mhc-II) and interleukin 12b (il-12b), and flow cytometry assays confirmed that SSP-9 cells can be permanently transfected with plasmids expressing a reporter gene. Accordingly, this new cell line is apparently suitable for transgenic manipulation, and to study host cell-virus interactions and immune processes.
Asunto(s)
Línea Celular , Interferón Tipo I/genética , Pronefro/citología , Salmo salar , Animales , Proliferación Celular , Criopreservación , CariotipoRESUMEN
Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon-farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk-derived proteins as bovine caseins or casein-derived peptides at different stages during the course of IHNV infection. The results indicate that the 3-h fraction of casein and α(S2) -casein hydrolysates reduced the yield of infectious IHNV in a dose-dependent manner and impaired the production of IHNV-specific antigens. Hydrolysates of total casein and α(S2) -casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein-treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV-inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.
Asunto(s)
Antivirales/farmacología , Caseínas/farmacología , Enfermedades de los Peces/prevención & control , Virus de la Necrosis Hematopoyética Infecciosa/efectos de los fármacos , Infecciones por Rhabdoviridae/veterinaria , Trucha , Animales , Bovinos , Línea Celular , Cromatografía Líquida de Alta Presión , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Virus de la Necrosis Hematopoyética Infecciosa/inmunología , Perciformes , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/prevención & control , Infecciones por Rhabdoviridae/virología , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
DNA vaccines and oral DNA-based immunotherapy against infectious pancreatic necrosis virus (IPNV) have scarcely been studied in salmonid fish. Here, a vector with the capsid VP2 gene inserted was encapsulated in alginate microspheres to avoid the aggressive gastrointestinal conditions experienced following oral administration. Alginate microspheres were effective to protect the pDNA encoding VP2, which was expressed early in different organs of the vaccinated trout and that persisted for at least 60 days. The vaccine induces innate immune responses, raising the expression of IFN more than 10-fold relative to the fish vaccinated with the empty plasmid, at 7 and 15 days post-vaccination. Likewise, maximal expression of the IFN-induced antiviral Mx protein was recorded 15 days post-vaccination and neutralizing antibodies were also detected after 15 days, although their titre rose further at 21 days post-vaccination. Protection was high in the immunized fish, which showed around an 80% relative survival when challenged 15 and 30 days after vaccine delivery. Very low viral load with respect to the control group was detected in the vaccinated fish that survived 45 days after challenge. Thus, this study demonstrates the potential of the encapsulation technique for IPNV-DNA vaccine delivery and the relevance of the IPNV-VP2 gene for future plasmid constructs.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Enfermedades de los Peces/prevención & control , Virus de la Necrosis Pancreática Infecciosa/fisiología , Vacunas Virales/inmunología , Administración Oral , Alginatos/química , Animales , Anticuerpos Neutralizantes/sangre , Infecciones por Birnaviridae/mortalidad , Infecciones por Birnaviridae/prevención & control , Enfermedades de los Peces/mortalidad , Enfermedades de los Peces/virología , Proteínas de Unión al GTP/inmunología , Perfilación de la Expresión Génica , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Interferones/inmunología , Microesferas , Proteínas de Resistencia a Mixovirus , Oncorhynchus mykiss , Reacción en Cadena de la Polimerasa , Análisis de Supervivencia , Trucha , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.
Asunto(s)
Infecciones por Virus ARN/veterinaria , Virus ARN/fisiología , Salmonidae/virología , Animales , Línea Celular , Hidrolasas/farmacología , Virus de la Necrosis Hematopoyética Infecciosa/efectos de los fármacos , Virus de la Necrosis Hematopoyética Infecciosa/fisiología , Virus de la Necrosis Pancreática Infecciosa/efectos de los fármacos , Virus de la Necrosis Pancreática Infecciosa/fisiología , Novirhabdovirus/efectos de los fármacos , Novirhabdovirus/fisiología , Infecciones por Virus ARN/patología , Infecciones por Virus ARN/virología , Virus ARN/efectos de los fármacos , Acoplamiento Viral/efectos de los fármacosRESUMEN
The outcomes of a coinfection of rainbow trout ( Oncorhynchus mykiss) with Infectious hematopoietic necrosis virus (IHNV) strain S46 and Infectious pancreatic necrosis virus (IPNV) strain S46 was determined after waterborne infection. Trout infected with the IHNV/IPNV.S46 sample, (a mixed sample containing equal infectious titers of the viruses) showed 50% less mortality than fish infected with either of the reference viruses alone. Forty-five days after the coinfection, IPNV antigens were detected by flow cytometry in 49 to 63% of the leukocytes from the surviving trout; whereas, only 9-15.6% of the leukocytes expressed IHNV viral antigens. IPNV was easily detected by reverse transcription-polymerase chain reaction (RT-PCR), whereas, for IHNV, a second step of amplification of a 753 bp fragment corresponding to the internal sequences of the IHNV G gene was necessary to optimize viral detection. The sequence of the IHNV gene involved in virulence, the glycoprotein (G) gene, was determined for the IHNV.S46 and compared with other sequences available in the GenBank. Changes found were not located in the antigenic domains of the glycoprotein and were considered not significant.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Virus de la Necrosis Hematopoyética Infecciosa/patogenicidad , Virus de la Necrosis Pancreática Infecciosa/patogenicidad , Oncorhynchus mykiss/virología , Infecciones por Rhabdoviridae/veterinaria , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Infecciones por Birnaviridae/mortalidad , Infecciones por Birnaviridae/virología , Línea Celular , Enfermedades de los Peces/mortalidad , Enfermedades de los Peces/virología , Citometría de Flujo , Virus de la Necrosis Hematopoyética Infecciosa/aislamiento & purificación , Virus de la Necrosis Pancreática Infecciosa/aislamiento & purificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Rhabdoviridae/mortalidad , Infecciones por Rhabdoviridae/virología , Análisis de Secuencia de ADN , Proteínas del Envoltorio Viral/genética , VirulenciaRESUMEN
In the present study, six diagnostic methods for the detection of infectious pancreatic necrosis virus (IPNV) (indirect immunofluorescence, flow cytometry, immunoperoxidase, immunodot blot, immunostaphylococcus-protein A, and RT-PCR) have been comparatively evaluated using the seroneutralization as the reference assay, and 83 Spanish isolates and 3 reference strains. The most reliable methods were flow cytometry and RT-PCR which could detect virus at titers of 1x10(2) and 1x10(3) TCID50/ml, respectively. At a multiplicity of infection of 50, both assays allowed the earliest detection of IPNV at 4 h post-inoculation. Indirect immunofluorescence and immunoperoxidase assays required at least 6 h post-inoculation to detect viral antigens. The immunodot blot assay possesses low sensitivity and the immunostaphylococcus-protein A test cannot be applied for routine examination of IPNV. Positive reactions were obtained in 100% of the samples tested by seroneutralization and RT-PCR, 90.4% by the flow cytometry, 80.7% by the indirect immunofluorescence assay, 67.5% by the immunoperoxidase, 62.6% by the immunodot blot, and only 27.7% by immunostaphylococcus-protein A test. Therefore, RT-PCR and flow cytometry were the most appropriate and sensitive methods for the routine detection of IPNV from affected fish.
Asunto(s)
Virus de la Necrosis Pancreática Infecciosa/clasificación , Virus de la Necrosis Pancreática Infecciosa/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salmón/virología , Animales , Infecciones por Birnaviridae/diagnóstico , Infecciones por Birnaviridae/veterinaria , Infecciones por Birnaviridae/virología , Células Cultivadas , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/virología , Citometría de Flujo , Virus de la Necrosis Pancreática Infecciosa/genética , ARN Viral/genética , Sensibilidad y Especificidad , SerotipificaciónRESUMEN
The recently reported SAF-1 cell line from fins of gilt-head seabream was evaluated for susceptibility to lymphocystis disease virus (LDV) and to several salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and several strains of infectious pancreatic necrosis virus (IPNV). LDV, VHSV and IHNV replicated well in the cultured fin cells as demonstrated by cell lysis and increases in viral titer. The potential use of this cell line to detect viruses from fish marine species is discussed.