RESUMEN
We compared 18 salmonid alphaviruses (SAV) including the reference F93-125 salmon pancreas disease virus (SPDV) and S49p sleeping disease virus (SDV) isolates by nucleotide sequence analyses of regions within the E1, nsP4 and nsP3 genes, and found these to comprise 3 distinct groups, which we have designated Subtypes 1, 2 and 3: Subtype 1, which comprised SAVs with sequences closely similar to the reference SPDV isolate, included SAVs from pancreas disease (PD) outbreaks in farmed salmon in Ireland and Scotland over a 10 yr period; viruses from recent outbreaks of sleeping disease (SD) in freshwater-reared trout farmed in England, Scotland and France were closely similar to and were grouped with the reference SDV isolate in Subtype 2; 3 viruses isolated from PD-affected salmon in Norway were genetically different from viruses belonging to Subtypes 1 and 2 and have been assigned to Subtype 3; 1 virus isolated from PD-affected salmon in the Western Isles, Scotland, in 2003 showed consistent nucleotide sequence differences from SAV Subtypes 1, 2 and 3, but was more closely related to the Subtype 1 SAVs. The occurrence of the different subtype SAVs appeared to have a geographical basis, which may prove useful in future molecular epidemiology studies of SAV-induced disease outbreaks.
Asunto(s)
Infecciones por Alphavirus/veterinaria , Alphavirus/genética , Brotes de Enfermedades/veterinaria , Enfermedades de los Peces/epidemiología , Enfermedades de los Peces/virología , Variación Genética , Salmo salar , Infecciones por Alphavirus/epidemiología , Animales , Acuicultura , Secuencia de Bases , Análisis por Conglomerados , Cartilla de ADN , Europa (Continente)/epidemiología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN/veterinaria , Proteínas del Envoltorio Viral/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Longitudinal serological surveys for salmon pancreas disease virus (SPDV), the causal agent of pancreas disease (PD), were conducted on multiple caged populations of Atlantic salmon, Salmo salar L., on two farms over a 77-week period (farm 1, freshwater and marine stages) and a 36-week period (farm 2, marine stage only), using a microtitre-based virus neutralization (VN) assay. Collected sera were also screened for viraemia with SPDV, and pancreas, heart and muscle tissues were examined for lesions consistent with PD. Outbreaks of PD occurred during the marine phase on both farms, as demonstrated by seroconversion, the isolation of virus and progressive histopathological changes consistent with a PD outbreak. All populations monitored showed a progressive increase in seroprevalence of 90-100%, typically accompanied by rises in geometric mean antibody titres. With the exception of one caged population, which showed a marked biphasic seroprevalence pattern, the seroprevalence figures in the remaining four monitored populations remained high (> or =70%) until the end of the study period. Peak VN titres of > or =1/1280 were detected on both farms. The results provide essential baseline information for the interpretation of SPDV VN serology results, and indicate that this methodology is suited to both the diagnosis and seroepidemiology of SPDV infections.
Asunto(s)
Alphavirus/aislamiento & purificación , Técnicas para Inmunoenzimas/métodos , Pruebas de Neutralización/métodos , Salmo salar/virología , Animales , Acuicultura , Recolección de Datos , Estudios Longitudinales , Estudios SeroepidemiológicosRESUMEN
A simple method of detecting the presence of the salmonid alphaviruses (SAVs), salmon pancreas disease virus (SPDV) and sleeping disease virus (SDV), from serum samples is described. Using a 96-well tissue-culture plate format, test sera are diluted in medium and added to chinook salmon embryo (CHSE-214) cells. After incubation for 3 days at 15 degrees C, plates are fixed and stained using a monoclonal antibody (mAb)-based immunoperoxidase (IPX) detection system, and virus-infected cells are observed microscopically by white light. Application of this screening test, which is now used routinely in our laboratory in conjunction with an IPX-based virus neutralization (IPX-VN) test for detecting antibodies to SAVs, has resulted in the recovery of 12 additional isolates from salmon sera and four additional isolates from trout sera. A low level of antigenic variation was detected when these SAV isolates were investigated by indirect immunofluorescence using a panel of mAbs raised to reference SPDV and SDV isolates.
Asunto(s)
Infecciones por Alphavirus/veterinaria , Alphavirus/aislamiento & purificación , Acuicultura/métodos , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/virología , Técnicas para Inmunoenzimas/veterinaria , Alphavirus/inmunología , Infecciones por Alphavirus/diagnóstico , Animales , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Enfermedades de los Peces/sangre , Técnica del Anticuerpo Fluorescente , Técnicas para Inmunoenzimas/métodos , Oncorhynchus mykiss , Salmo salarRESUMEN
A modified virus neutralization (VN) assay was developed to replace an existing assay read on the presence or absence of virus-induced cytopathic effect (CPE). The modified assay used a monoclonal antibody to salmon pancreas disease virus as the first layer of an immunoperoxidase (IPX)-based immunostaining technique to detect viral growth. The IPX-based VN assay required only 3 days to perform, and the adoption of a 96-well microtitre format facilitated a high throughput of samples requiring small volumes of serum, cells and virus. When 352 sera from farmed salmon and 302 sera from farmed trout were tested by both the modified and the original CPE-based assays, overall correlations of 97.72 and 96.03% were, respectively, obtained (96.94% combined). When the modified assay was used to test 188 sera collected from wild salmonids in freshwater river systems in Northern Ireland, no positive results were recorded.
Asunto(s)
Infecciones por Alphavirus/diagnóstico , Infecciones por Alphavirus/veterinaria , Alphavirus/aislamiento & purificación , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/virología , Técnicas para Inmunoenzimas/métodos , Técnicas para Inmunoenzimas/veterinaria , Alphavirus/inmunología , Infecciones por Alphavirus/epidemiología , Infecciones por Alphavirus/inmunología , Animales , Línea Celular , Enfermedades de los Peces/epidemiología , Enfermedades de los Peces/inmunología , Pruebas de Neutralización/métodos , Pruebas de Neutralización/veterinaria , Irlanda del Norte/epidemiología , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/virología , Salmo salar/inmunología , Salmo salar/virología , Estudios SeroepidemiológicosRESUMEN
Six mouse monoclonal antibodies (mAbs) specific to salmon pancreas disease virus (SPDV) were produced following immunisation with purified virus preparations. These mAbs and 2 mAbs resulting from an earlier investigation were characterised. None of the mAbs possessed virus neutralising activity but all reacted with 4 geographically different SPDV isolates as determined by indirect immunofluorescence. Three mAbs produced positive immunostaining with Western blots of SPDV proteins. The 4H1 mAb reacted with the 53 kDa structural E1 glycoprotein present in virus-infected cells and in gradient-purified virus. Two mAbs, 5A5 and 7B2, which exhibited unusual immunofluorescence staining of the nuclear margin, reacted with a 35 kDa protein, which is present in gradient-purified virus and which is considered to be the capsid protein. A sandwich ELISA, based on the use of mAb 2D9 for capture and a biotinylated conjugate of mAb 7A2 for detection, detected SPDV antigen in virus-infected Chinook salmon embryo-214 cells and gradient-purified virus. These mAbs may be of use in pathogenesis studies and in diagnostic test development.
Asunto(s)
Infecciones por Alphavirus/veterinaria , Alphavirus/inmunología , Anticuerpos Monoclonales/biosíntesis , Enfermedades de los Peces/inmunología , Enfermedades Pancreáticas/veterinaria , Salmonidae , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/virología , Animales , Anticuerpos Monoclonales/clasificación , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/clasificación , Anticuerpos Antivirales/inmunología , Western Blotting/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de los Peces/virología , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Hibridomas , Ratones , Pruebas de Neutralización/veterinaria , Enfermedades Pancreáticas/inmunología , Enfermedades Pancreáticas/virologíaRESUMEN
We report the development of a competitive enzyme-linked immunosorbent assay (c-ELISA) for the detection of antibodies to porcine circovirus type 2 (PCV2), the agent associated with the recently described postweaning multisystemic wasting syndrome in pigs. At present, no method has been published describing a c-ELISA for the detection of antibodies to PCV2, and currently employed tests are impractical for use in some laboratories. The assay described here uses a cell culture isolate of porcine circovirus type 2 as antigen and a PCV2-specific monoclonal antibody as the competing reagent. Evaluation of the ELISA was performed by comparison with results obtained using an indirect immunofluorescent test on 484 sera from pig herds in the United Kingdom, Canada, France, and the USA and serial bleeds from pigs experimentally infected with porcine circoviruses. The sensitivity and specificity of the ELISA were determined as 99.58% and 97.14%, respectively, at 2 standard deviations (SD) from the mean or 95.81% and 100% at 3 SD from the mean. Using this ELISA, a serologic survey of 461 sera collected from commercial pig herds in Northern Ireland between 1973 and 1999 was undertaken. Analysis of the results of this survey demonstrated that the number of ELISA-positive sera detected in an individual year during this period ranged from 55% to 100%. This c-ELISA has applications for large-scale rapid diagnosis of PCV2 infection in pig populations worldwide and for immunoscreening of sera from other species for antibodies to PCV2.
Asunto(s)
Infecciones por Circoviridae/veterinaria , Circovirus/inmunología , Enfermedades de los Porcinos/diagnóstico , Animales , Anticuerpos Antivirales/análisis , Antígenos Virales/inmunología , Infecciones por Circoviridae/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Sensibilidad y Especificidad , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , PorcinosRESUMEN
Porcine circovirus (PCV) was initially recognized as a contaminant of continuous pig kidney cell lines and was not thought to be pathogenic. Antibodies reactive to the cell culture isolate of PCV (PCV PK-15) are prevalent in the swine population worldwide. Recently, PCV PK-15-like antigen and nucleic acid were demonstrated in lesions associated with wasting syndromes in pigs in North America and Europe. Monoclonal antibodies raised to circoviruses isolated from pigs with wasting syndromes highlighted differences between these circoviruses and the PCV PK-15 cell culture isolate. This has led to speculation that a new pathogenic PCV may have emerged in the swine populations of several countries. We report the cloning and characterization of novel circovirus DNAs purified from virus isolates made from tissues of North American and European pigs with wasting syndromes. These North American and European circoviruses form a closely related group at the nucleotide sequence level (> 96% intra-group nucleotide sequence identity) but exhibit < 80% nucleotide sequence identity with the PCV PK-15 cell culture isolate. This report provides evidence for a new type of possibly pathogenic PCV. We propose that these new circoviruses should be referred to as PCV2 as opposed to the original PK-15 cell culture isolate, which should be referred to as PCV1.