RESUMEN
A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.
Asunto(s)
Enterovirus Humano B/aislamiento & purificación , Virus de la Fiebre Aftosa/aislamiento & purificación , Análisis por Micromatrices/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virus del Exantema Vesicular del Cerdo/aislamiento & purificación , Animales , Bovinos , Enterovirus Humano B/clasificación , Enterovirus Humano B/genética , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/clasificación , Virus de la Fiebre Aftosa/genética , Sensibilidad y Especificidad , Ovinos , Porcinos , Enfermedad Vesicular Porcina/diagnóstico , Enfermedad Vesicular Porcina/virología , Exantema Vesicular del Cerdo/diagnóstico , Exantema Vesicular del Cerdo/virología , Virus del Exantema Vesicular del Cerdo/clasificación , Virus del Exantema Vesicular del Cerdo/genéticaAsunto(s)
Aborto Veterinario/virología , Infecciones por Caliciviridae/veterinaria , Caliciviridae/aislamiento & purificación , Enfermedades de los Bovinos/diagnóstico , Feto/virología , Animales , Caliciviridae/clasificación , Caliciviridae/genética , Caliciviridae/ultraestructura , Infecciones por Caliciviridae/diagnóstico , Infecciones por Caliciviridae/virología , Bovinos , Enfermedades de los Bovinos/virología , Diagnóstico Diferencial , Femenino , Enfermedades Fetales/diagnóstico , Enfermedades Fetales/veterinaria , Enfermedades Fetales/virología , Pulmón/embriología , Pulmón/ultraestructura , Pulmón/virología , Microscopía Electrónica/veterinaria , Filogenia , ARN Viral/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Homología de Secuencia , Exantema Vesicular del Cerdo/diagnóstico , Exantema Vesicular del Cerdo/virología , Virus del Exantema Vesicular del Cerdo/clasificación , Virus del Exantema Vesicular del Cerdo/aislamiento & purificaciónRESUMEN
The San Miguel sea-lion viruses (SMSV) and vesicular exanthema of swine viruses (VESV) are members of the calicivirus family and aetiologic agents of vesicular disease in susceptible hosts. These two virus groups have been shown by several serological methods to be closely related antigenically. To further examine their relatedness, two sets of non-degenerate oligonucleotide primers were designed for the specific amplification of two distinct regions of the SMSV and VESV genomes using a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol. The sequence of the primers were based on the nucleotide sequence of SMSV serotypes 1 and 4. The RNAs from a number of SMSV serotypes and a single VESV isolate were used as template in this study. These included SMSV serotypes 1, 2, 4, 5, 6, 7, 13 and 14 and VESV serotype A48. Also included in this study were Tillamook calicivirus (Bos-1 calicivirus, BCV) and a recently isolated skunk calicivirus (SCV). The first primer set amplified a 357-bp fragment from the 2C-like or RNA-helicase-encoding region (11 of 11 viruses) and the second set amplified a fragment from the RNA-dependent RNA polymerase region (520 bp, 9 of 11 viruses). These primer sets did not amplify product from either feline calicivirus or mink calicivirus. The results of this study demonstrate the genetic relatedness of SMSV and VESV and the potential usefulness of RT-PCR to detect and identify these viruses in diagnostic and routine screening applications.
Asunto(s)
Caliciviridae/genética , Caliciviridae/aislamiento & purificación , Cartilla de ADN , ADN Viral/química , Genoma Viral , Reacción en Cadena de la Polimerasa/métodos , Leones Marinos/virología , Exantema Vesicular del Cerdo/diagnóstico , Virus del Exantema Vesicular del Cerdo/genética , Virus del Exantema Vesicular del Cerdo/aislamiento & purificación , Animales , Secuencia de Bases , Caliciviridae/clasificación , Gatos , Bovinos , Mephitidae , Visón , Datos de Secuencia Molecular , ARN Viral/aislamiento & purificación , ARN Polimerasa Dependiente del ARN/biosíntesis , ARN Polimerasa Dependiente del ARN/genética , Serotipificación , Porcinos , Virus del Exantema Vesicular del Cerdo/clasificaciónRESUMEN
The clinical signs, diagnosis and epizootiology of swine vesicular disease (SVD) are described. The clinical appearance is illustrated by photographs of experimentally and naturally infected pigs. Special attention is paid to differences between SVD and foot-and-mouth disease (FMD) and to the choice of disinfectants.