Molecular characterization of Brazilian isolates of orf virus.

Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.

Development of snake antivenom antibodies in chickens and their purification from yolk.

Adult white leghorn hens hyperimmunised with Brazilian snake venoms of the genus Bothrops and/or Crotalus produced antibodies capable of recognising, combining with and neutralising the toxic and lethal components of the venoms. The antibodies were first detected by an enzyme-linked immunosorbent assay two weeks after starting the immunisation schedule, reached the highest titres by the third week and remained high for at least 24 weeks. These antibodies are transferred to the egg yolk from which they were isolated as enriched IgY preparations by a combination of methods using positive and negative precipitation with sodium sulphate and/or caprylic acid. The yolk-derived IgY preparations contained antibodies which blocked the phospholipase A2-dependent haemolytic activity of both venoms and the haemorrhagic activity of Bothrops venom, and neutralised the toxic lethal activities of the venoms with good efficacy. The median effective dose (ED50) of the IgY anti-Bothrops venom was 592.5 microliters/2LD50 and, 1.0 ml neutralised 0.0675 mg of venom. The ED50 of the IgY anti-Crotalus venom was 457.5 microliters/3LD50 and 1.0 ml neutralised 0.075 mg of venom.

Desarrollo de una prueba ELISA para identificar anticuerpos antivirus de estomatitis vesicular Indiana-3

La prueba ELISA de competición en fase líquida fue adaptada para la identificación de anticuerpos antivirus de estomatitis vesicular serotipo Indiana-3, usando como antígeno la glicoproteína viral. La validez y repetibilidad de la prueba se determinó por el estudio de 533 sueros de animales negativos y 305 sueros de animales positivos, obteniéndose una sensibilidad de 99 por ciento y una especificidad de 100 por ciento. No se observaron diferencias significativas entre las repeticiones de la prueba. Los valores predictivos de los resultados, positivo y negativo, y el valor global de la prueba estudiada indican que la ELISA es un método satisfactorio para identificar anticuerpos de estomatitis vesicular.

The competitive ELISA in liquid phase was adapted to identify antibodies to vesicular stomatitis Indiana-3 virus useing the viral glycoprotein as antigen. Test validity and reproducibility were determined by testing 553 sera from negative animals and 305 sera from positive animals, obtaining values of 99% sensitiviy and 100% specificity. No significant differences were observed among repeated tests. Prodictive values for positive and negative results, and the overall value of the technique studied indicate that it is a satisfactory method for use in the identification of antibodies to vesicular stomatitis virus.

Anticuerpos monoclonales específicos para herpesvirus bovino tipo-1 (cepa Cooper)

Anticuerpos monoclonales fueron preparados contra herpesvirus bovino tipo 1 (BHV-1) purificado y se seleccionaron varios de acuerdo con su habilidad de neutralizar la infectividad viral. El virus fue purificado por dos métodos alternativos de concentración, con polietilenglicol seguido de ultracentrifugación sobre un colchón de sacarosa al 25 por ciento (p/p) o usando un gradiente linear de tartrato de potasio. De un total de 204 líneas celulares que segregaban anticuerpos para el virus, obtenidas en dos fusiones, se seleccionaron y expandieron clonalmente 39 hibridomas. La selección se basó en su reactividad específica por ELISA. De éstos, 11 anticuerpos monoclonales fueron capaces de neutralizar BHV-1 total o parcialmente, con o sin el agregado de complemento. Siete anticuerpos monoclonales fueron producidos (en la forma de fluido ascítico) y purificados por precipitación en sulfato de amonio. De éstos, dos fueron capaces de prevenir la penetración de virión luego de su adherencia a la células. Asimismo, tres anticuerpos monoclonales fueron seleccionados para ser usados en las pruebas de inmunoperoxidasa para la detección del BHV-1 en cultivos celulares infectados.

Monoclonal antibodies (McAbs) were prepared against purified Bovine Herpesvirus Type-(BHV-1) and selected for their ability to neutralize viral infectivity. Viral purification was performed by polyethylene glycol concentration and ultracentrifugation on a 25% (w/w) sucrose cushion, and by potassium tartrate linear gradient. Out of 204 cell lines expressing antobodies to the virus, obtained in two fusions, 39 hybridomas were selected and cloned based on enzyme-linked immunosorbent assay (ELISA) reactivity. Eleven McAbs were able to neutralize BHV-1 partially or totally, with or without complement. Seven McAbs were produced as ascitic fluid, and ammonium sulfate-purified. Of these, two were able to prevent virion penetration into the cell after attachment. Three neutralizing McAbs were selected for use in immunoperoxidase tests for detecting BHV-1 in infected cell cultures.

Producción de vacuna antiaftosa: filtrabilidad del suero bovino tratado con polietilenglicol y su efecto en el crecimiento celular

Se analizó el efecto del suero bovino con anticuerpos para el virus de la fiebre aftosa sin tratamiento y después de tratado com 2, 4, 6 y 8% de polietilenglicol (PEG), en el crecimiento de células BHK-21, cutivadas en suspención. También se investigó la capacidad filtrante a través de menbranas de 0,22 del medio de crecimiento suplementado con suero de bovino previamente tratado com PEG. Se comprobó que el más bajo porcentaje de PEG que aún mantenía el crecimiento celular y una filtrabilidad de alto redimiento era del 2%.

Molecular analysis of contagious pustular dermatitis virus: a simplified method for viral DNA extraction from scab material.

A new simplified method of DNA extraction of contagious pustular dermatitis virus directly from scab material of natural and experimental infections is described. Scabs are suspended in buffer solution and an enriched core suspension is obtained after treatment with detergent, quelants and centrifugation. DNA is isolated after proteinase digestion and phenolchloroform extraction. Viral DNA and fragments with sizes ranging from 23-25 kb were observed by agarose gel electrophoresis. This DNA was used for digestion with several restriction endonucleases which produced parapoxvirus-specific patterns. Southern blots with the TK gene of vaccinia virus as probe confirmed the virus as being poxvirus-related and allowed a preliminary TK gene location for our isolates. The method was developed in order to allow a quick epidemiological survey of contagious pustular dermatitis virus in Brazil, eliminating the need for time-consuming and expensive viral propagation in cell culture and purification.