Detection of foot-and-mouth disease viral sequences in various fluids and tissues during persistence of the virus in cattle.

OBJECTIVE: To assess whether foot-and-mouth disease virus (FMDV)-specific sequences could be identified in tissues from persistently virus-infected animals. DESIGN: Cattle with experimentally induced persistent FMDV infections were slaughtered at 750 days after viral exposure. Experimentally infected pigs were slaughtered at 28 days after FMDV inoculation. Postmortem specimens were asceptically removed. ANIMALS: Three bovids and 3 pigs were studied, as well as 1 control animal for each species. PROCEDURE: Various tissues were examined for the presence of FMDV-specific sequences by dot-blot hybridization assay, using a molecularly cloned FMDV cDNA corresponding to the polymerase coding region. RESULTS: The FMDV-specific genomic sequences were only detected in RNA from spleen, lung, larynx, tonsils, pancreas, liver, esophagus, and WBC of bovids. CONCLUSIONS: It was established that, at late stages of the persistent infection, when virus isolation was not possible, cattle may carry FMDV-specific sequences in different tissues. Retention of viral sequences could not be demonstrated in specimens from experimentally infected swine, 28 days after viral inoculation.

Ensaios de infecciosidade do vírus da febre aftosa. I. Transmissão entre bovinos infectados e contatos e suínos

Quatro experimentos de infecção por instilação nasal dos vírus O, A e C da febre aftosa em bovinos e sua transmissão para bovinos e suínos foram realizados com a finalidade de estudar vários parâmetros indicativos da infecção. No experimento com vírus A24 onde os bovinos foram inoculados por vía intranasal em grupos com doses infectantes crescentes (10(3), 10(5) e 10(7) as curvas de replicação do vírus na área faríngea estiveram condicionadas ás concentrações do vírus inoculadas, principalmente nas primeiras horas pós-infecção (HPI). De nove bovinos inoculados foi possível detectar viremia em quatro, a qual antecedeu em 24 horas ao aparecimento de lesões clínicas. As lesões clínicas foram detectadas em dois bovinos ás 148 HPI e em dois às 196 HPI evidenciando um retardamento já observado anteriormente em testes com virus A24. O aparecimento das lesões em suínos também esteve relacionado com a dose do vírus inoculada nos bovinos. No teste com o vírus C3 dois animais sem anticorpos, inoculados frente a esse vírus apresentaram curvas de replicação na área faríngea e viremia semelhantes. Em outro bovino que apresentaram curvas de replicação na área da faríngea e viremia semelhantes. Em outro bovino que tinha anticorpos, houve dois ciclo de replicação do vírus na área faríngea,formação de anticorpos neutralizantes e anti-Via, porém não houve a doença clínica. Nos bovinos em contato com os inoculados foi detectado vírus na área faríngea a partir de 48 horas pós-contato (HPC) com aparecimento de lesões clínicas entre 96 e 120 HPC. Nos suínos em contato, as lesões foram evidentes entre 96 e 144 HPC. Nos dois experimentos com vírus O1 as curvas de replicação do vírus na área faríngea e viremia foram similares, sendo detectado vírus na área faríngea dos bovinos inoculados a partir das 5 HPI e após 48 horas nos bovinos contatos. A viremia foi detectada entre 72 e 96 HPI ou HPC antecedendo em um dia ou coincidindo com o aparecimento das lesões clínicas. Os suínos em contato evidenciaram lesões clínicas entre 7 e 9 dias pós-contato (DPC).Embora sendo oriundos de áreas endêmicas, os bovinos apresentaram padrões de replicação do vírus da febre aftosa na área faríngea e viremia, assim como o processo de transmissão entre bovinos e suínos similares àqueles encontrados por outros pesquisadores com animais de áreas livres.

Detección de secuencias genómicas del virus de la fiebre aftosa mediante la hibridación de ácidos nucleicos: un enfoque para el diagnóstico de infección persistente

Se ha desarrollado un ensayo de hibridación capaz de detectar secuencias genómicas del virus de la fiebre aftosa (VFA) en muestras de cultivos de células infectadas o de fluidos esofágico-faríngeo (OP). El ensayo se basa en el uso de una sonda marcada de ADN complementaria a la región que codifica para la polimerasa del VFA. la sonda se obtuvo amplificando en E. coli un plásmido conteniendo el fragmento de ADN complementario (cADN) a la región de 6,3 a 7,8 kilobases del genoma del VFA. Debido al alto grado de homología de la secuencia de ácidos nucleicos en esta parte del genoma, fue posible la detección de los tres serotipos sudamericanos en un único ensayo de hibridación. Los resultados positivos obtenidos con muestras de OP de animales experimentalmente infectados extraídas a tiempos tardíos de la infección, durante los cuales la recuperación de virus era principalmente negativa, revelaron el considerable potencial de este método como complemento de los procedimientos convencionales de diagnóstico de infección persistente.

Desarrollo de un método más simple y rápido para el análisis bidimensional de oligonucleótidios del virus de la fiebre aftosa: minifingerprinting de ARN

El éxito de los programas de inmunización depende, en parte, de una caracterización precisa de las cepas que circulan en el campo y de la evaluación de sus semejanzas con las cepas de los virus que se incluyen en las vacunas. Actualmente se dispone de varias técnicas bioquímicas como fingerprinting de ARN (3, 4), ADN recombinante (8) y secuenciamiento rápido (10) para estudiar, con significativa precisión, las características genéticas de las cepas virales. La técnica de fingerprinting resulta muy adecuada para evaluar las relaciones (5, 6, 7, 12) y en el caso del VFA también se ha probado su utilidad para fines de diagnóstico.

Heterogeneity of the polyribocytidilic acid tract in aphthovirus: changes in the size of the poly(C) of viruses recovered from persistently infected cattle.

A sample of aphthovirus type C3 strain Resende carrying two polyribocytidilic acid [poly(C)] tracts was cloned in tissue culture. One clone with a poly(C)-rich tract of about 145 nucleotides long (clone 3B) and another with a poly(C)-rich tract of about 230 nucleotides long (clone 12) and a mixture of both were injected intralingually into three steers. Samples from all three animals were recovered during the acute phase of the disease, from the blood and from the feet, and at various days after inoculation from the oesophageal-pharyngeal (OP) fluids. Analysis of the viral RNAs of the positive samples by means of RNase T1 maps on one- and two-dimensional gels showed (1) changes in the electrophoretic mobility of the poly(C)-rich tracts of viruses recovered from the OP fluids at various times after infection; (2) selection of virus populations with poly(C)-rich tracts of increased size; (3) later on, changes in the patterns of oligonucleotides of persistent viruses. These variations may lead to the production of new strains with altered biological properties that may contribute to the maintenance and spread of these viruses in the field.

Caracterización epidemiológica, antigénica, inmunogénica y bioquímica del virus de la fiebre aftosa A-81 Argentina/87

Estudios seroepidemiológicos llevados a cabo en muestras de campo, realizados a fines de 1986 por el Laboratorio de Diagnóstico y Referencia para las Américas en el Centro Panamericano de Fiebre Aftosa (CPFA), mostraron una variación antigénica de virus de la fiebre aftosa tipo A en relación con el usual A24 y aislamientos de campo A-79 en la Argentina.

Seroepidemiologic studies constantly carried out on field samples by the Diagnostic Reference Laboratory for te Americas at the Pan American Foot-and-Mouth Disease Center (PAFMDC) revealed, late in 1986, an antigenic variation of FMDV type A in relation to the usual A24 and A-79 field isolates in Argentina.

RNA fingerprinting of South American prototype aphthovirus strains.

Aphthovirus strains used in South America for vaccine production or as reference for diagnostic purposes were analysed by RNA fingerprinting (RNase T1 maps, one- and two-dimensional gels). The results obtained constitute the basis for a data bank containing available information about the genome structure of strains of aphthovirus prevalent in this continent and can be used as an adjunct to serological and immunological information. These data are currently being used in South American countries to assess the genetic stability of strains during vaccine production; to establish possible vaccine origin of field outbreaks and to monitor the origin, behaviour and fate of new strains in the field.

Biochemical characterization of an aphthovirus type 0(1) strain campos attenuated for cattle by serial passages in chicken embryos.

The biochemical properties of a virulent and an attenuated strain of foot-and-mouth disease virus (FMDV) Type 0(1) Campos (0(1)C) were compared in order to establish differences that could account for their altered biological functions. The avirulent strain (0(1)C-O/E) was derived from the virulent strain 0(1)C by serial passages in chicken embryos. Analysis of the RNase T1-generated oligonucleotides of the viral RNA through one- and two-dimensional (2D) gel electrophoresis (fingerprints) revealed a few changes in the genome structure of the 0(1)C-O/E strain compared to the wild type strain. In addition there was a significant decrease in the length of the poly(C) rich tract of the 0(1)C-O/E RNA. All virion structural proteins, except VP4, their precursors, and the viral RNA polymerase (p56a) show charge differences. In addition a significant decrease in the apparent molecular weight of polypeptide p100 (primary translational product from the 3' end region of the genome) of the attenuated strain was observed.

Caracterización bioquímica y serológica de diferentes cepas de campo de virus de la fiebre aftosa aisladas en América del Sur

En esta comunicación, se extendieron los estudios al análisis de varias cepas relevantes del virus de la fiebre aftosa, subtipos O, A y C, aisladas en diferentes épocas en el campo y en distintas regiones de América del Sur.

In the present communication studies are axtended to the analysis of several relevant strains of FMDV srotypes O, A and C isolated at different times in the field in different regions of South America.

Heterogeneity of the polyribocytidylic acid tract in aphthovirus: biochemical and biological studies of viruses carrying polyribocytidylic acid tracts of different lengths.

In this paper we report a study of a sample of foot-and-mouth disease virus carrying two polyribocytidylic acid [poly(C)] tracts of different lengths. By plaque purification in tissue culture, we isolated two populations of particles, one carrying the long poly(C) tract and the other carrying only the short homopolymer. The fingerprints of both viruses were indistinguishable from each other and from that of the virus present in the original sample, suggesting that the main difference between the two types of particles is limited to the poly(C) tracts of their genomic RNAs, to the flanking sequences of the poly(C) tract, or to both. In addition, some biological properties of these viruses are reported, such as stability upon serial passages in different cell lines, plaque size, and pathogenicity for cattle. The results indicate that the size of the poly(C) tract is not directly related to the virulence of these viruses. However, the size of the homopolymer could play a role in determining their efficiency of replication, and it appears that the particles with the short poly(C) tract might have some replicative advantage over those carrying the long one.

Examen histológico en cerdos vacunados con vacuna antiaftosa con adyuvante oleoso

[Introducción] Este trabajo describe los resultados de los exámenes histológicos de muestras colectadas de cerdos inoculados por las vías SC, IM, IP con vacuna antiaftosa de EP o de ED.

[Introduction] The present paper describes the histological examination of samples collected from pigs vaccinated with PE or DE FMD vaccine inoculated by SC, IM or intraperitoneal (IP) routes.

Detección del virus de la fiebre aftosa en material faríngeo de bovinos sacrificados en matadero: comunicación breve

La existencia de bovinos portadores de virus de la fiebre aftosa (FA) fue confirmada por investigadores del Centro Panamericano de Fiebre Aftosa (CPFA), al comienzo de la década de 1960. Desde entonces, la denominada prueba de probang para portadores ha sido extensamente utilizada en programas de importación y exportación de bovinos y ocasionalmente, en encuestas como indicador epidemiológico. Durante los últimos años el CPFA viene utilizando el método para determinar la presencia o ausencia del virus de la FA en rebaños bovinos experimentales o en áreas piloto donde se aplican vacunas antiaftosa con adyuvante oleoso.

The existence of foot-and-mouth disease (FMD) virus carriers in the field was confirmed by workers of the Pan American Foot-and-Mouth Disease Center (PAFMDC), Rio de Janeiro, Brazil, in the early 1960's. Since then the so-called probang test for carriers has been extensively used in import and export programs and occasionally for virus surveys. During the last few years the PAFMDC used the method to determine the absence or presence od FMD viral activity in experimental fams and pilot areas or demonstration areas where oil adjuvanted vaccines were used.

Influence of the degree of dispersion in the aqueous phase on the immunogenicity of oil-adjuvanted foot-and-mouth disease vaccine

Both primary emulsion (water-in-oil) and double emulsion (water-in-oil-water) foot-and-mouth disease vaccines with different viscosities produced exellent persistent levels of neutralizing antibodies. The degree of dispersion of the aqueous antgenic phase in the oily phase, which determined the viscosity of the vaccine, did not have a demonstrable effect on the long-term immunogenicity of the vaccines.

Vacuna antiaftosa con adyuvante oleoso para cerdos. III. Respuesta inmunitaria con vacunas emulsificadas por vibración ultrasónica o por agitación mecánica

La respuesta inmunitaria y la protección de cerdos vacunados por vía intraperitoneal con vacunas antiaftosas de emulsión doble con adyuvante oleoso fue evaluada por comprobación y por examen de anticuerpos. Una vacuna fue emulsificada con un equipo de ultrasonido (Vacuna 1) y la otra con un equipo mecánico (Vacuna 2). Ambas vacunas fueron analizadas por las DP50 en cobayos. Todas las pruebas indicaron que las dos vacunas eran satisfactorias. Sin embargo, la Vacuna 2 fue ligeramente superior a la 1. También se demostró que un índice de seroprotección (ISP) de 3,0 o un título de microneutralización de 2,5 indican un alto grado de protección de los cerdos.

Susceptibility of various FMDV strains to urea.

Resistance to urea in vitro at 37 degrees C varied for each FMDV strain analysed. The urea marker did not correlate with other markers such as resistance to acid, resistance to acidity or size of plaques under agar on BHK21/13 cells. The resistance to urea of subtypes A24 Cruzeiro, O1 Caseros and C3 Resende varied in accordance with their antigenic potency when administered to swine as a trivalent water-in-oil emulsion type vaccine.

Comparación de vacunas con adyuvante oleoso preparadas con Arlacel A y Montanide 80

Para este experimento e prepararon dos series de vacunas antiaftosas inactivadas(1) de una suspensión acuosa trivalente que contenía las cepas de virus de fiebre aftosa O1 Campos, A24 Cruzeiro y C3 Resende emulsificadas con Arlacel A o con Montanide.

Foot-and-mouth disease oil-adjuvanted vaccines for pigs. I. Double emulsion vaccine applied by different routs

[Introduction] In the present young pigs were inoculated with double emulsion oil adjuvanted vaccine using foot-and-mouth disease (FMD) antigens without concentration or purification. Observations were made of the applied by subcutaneous (SC), intramuscular (IM) and intraperitoneal (IP) routes.