RESUMEN
Foot and mouth disease virus RNA was visualized in infected primary tissue culture cells by in situ PCR incorporating digoxigenin-labeled dUTP. The viral RNA polymerase gene was used as a target for amplification. Infected cells revealed cytoplasmic staining, predominantly perinuclear. The intensity of staining was in proportion to the degree of cytopathology observed and similar to the results obtained using immunoperoxidase staining. The in situ PCR technique for FMDV detection could be applied to formalin-fixed samples and be useful for the study of persistent infections.
Asunto(s)
Aphthovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/análisis , Animales , Aphthovirus/genética , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Riñón/citología , Datos de Secuencia Molecular , OvinosRESUMEN
Foot-and-mouth disease virus (FMDV), by nature of its RNA genome, possesses a high rate of mutation during replication. This results in extensive genetic polymorphism of virus populations in nature. The emergence of FMDV variants during replication has been reported. Genetic changes in the viral capsid protein (VP1) gene can result in amino acid changes affecting the immunodominant epitopes of FMDV. The genetic heterogeneity of FMDV in the field and the antigenic variants observed after cell culture isolation has been investigated by PCR sequencing and reactivity with monoclonal antibodies. These methods were applied to viruses causing two different outbreaks of FMD before and after replication in cell culture and in the animal host. The VP1 region of the genome was amplified by PCR and sequenced to reveal variant sequences identified after passage and to determine their presence in the original field tissue. In one case, reactivity with monoclonal antibodies was lost after passage as a result of an amino acid change in the subpopulation. These findings suggest that host cells can select specific virus genetic and antigenic subpopulations during virus isolation and propagation.
Asunto(s)
Antígenos Virales/genética , Aphthovirus/genética , Cápside/genética , Fiebre Aftosa/microbiología , Variación Genética/genética , Secuencia de Aminoácidos , Animales , Antígenos Virales/análisis , Aphthovirus/aislamiento & purificación , Aphthovirus/fisiología , Secuencia de Bases , Brasil/epidemiología , Cápside/análisis , Proteínas de la Cápside , Bovinos , Brotes de Enfermedades/veterinaria , Epitelio/microbiología , Fiebre Aftosa/epidemiología , Italia/epidemiología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Análisis de Secuencia de ADN , Porcinos , Cultivo de Virus , Replicación Viral/genéticaRESUMEN
This study was undertaken in order to explore possible sites of foot-and-mouth disease virus (FMDV) persistence during the carrier state. Tissue samples taken from experimentally infected animals at different times post-infection (p.i.) were examined by conventional viral isolation and the polymerase chain reaction (PCR) technique. The analysis of samples from several organs taken from 17 bovines between 3 and 270 days p.i. allowed the following conclusions: 1) Virus present in oesophageal-pharyngeal fluids (OPF) during the carrier state originates in the pharynx as shown by the detection of antisense FMDV RNA by PCR, 2) PCR is more sensitive than standard virus isolation techniques and may be used for the rapid detection of FMDV in specimens obtained during the acute stage of FMD and for identification of persistently infected cattle.