Comparative inhibitory activity of the stilbenes resveratrol and oxyresveratrol on African swine fever virus replication.

Stilbenols are polyphenolic phytoalexins produced by plants in response to biotic or abiotic stress. These compounds have received much attention because of their significant biological effects. One of these is their antiviral action, which has previously been documented for two members of this class, namely resveratrol and oxyresveratrol. Here we tested the antiviral effect of these two compounds on African swine fever virus, the only member of the newly created family Asfarviridae and a serious limitation to porcine production worldwide. Our results show a potent, dose-dependent antiviral effect of resveratrol and oxyresveratrol in vitro. Interestingly, this antiviral activity was found for these synthetic compounds and also for oxyresveratrol extracted from new natural sources (mulberry twigs). The antiviral effect of these two drugs was demonstrated at concentrations that do not induce cytotoxicity in cultured cells. Moreover, these antivirals achieved a 98-100% reduction in viral titers. Both compounds allowed early protein synthesis but inhibited viral DNA replication, late viral protein synthesis and viral factory formation.

Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants.

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

High-yield expression of a viral peptide vaccine in transgenic plants.

A high-yield production of a peptide vaccine in transgenic plants is described here. A 21-mer peptide, which confers protection to dogs against challenge with virulent canine parvovirus, has been expressed in transgenic plants as an amino-terminal translational fusion with the GUS gene. Transformants were selected on the basis of their GUS activities, showing expression levels of the recombinant protein up to 3% of the total leaf soluble protein, a production yield comparable to that obtained with the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant-derived peptide was demonstrated in mice immunized either intraperitoneally or orally with transgenic plant extracts, providing the suitability of the GUS fusions approach for low-cost production of peptide vaccines.

Determinación de la permitividad y conductividad eléctricas a la frecuencia de microondas de varios tejidos de rata tratadas con cadmio / Determination of permittivity and conductivity in several rat tissues treated with cadmium

Los campos electromagnéticos ambientales cada vez más presentes en el entorno, pueden afectar a los propios seres humanos y, según su intensidad (magnetoterapia), activar o modular procesos fisiológicos o inducir a posibles patologías. En este trabajo se pretende determinar las características fisicoquímicas (los parámetros permitividad, (e) y conductividad eléctrica (s)) de varios tejidos de rata, mediante la aplicación de microondas de radiofrecuencia, ante variaciones en la concentración de cadmio (Cd) y comprobar si se pueden utilizar como indicadores del efecto específico de la presencia de cadmio, evaluar su efecto por la contaminación del entorno y cuantificar su acúmulo. Por consiguiente, se pretende aportar nuevos datos sobre los efectos de la toxicidad del cadmio en diversos órganos sistémicos y comprobar si existe sinergismo entre el efecto tóxico del cadmio (contaminante ambiental) y el de los campos electromagnéticos. Se utilizan dos grupos de ratas macho Wistar, uno control y otro tratado con Cd (CdCl2 ), con doce dosis (una por día) por vía intraperitoneal desde 0,1 a 1 mg Cd/Kg rata/día. Se extrae la sangre bajo anestesia con éter por punción cardíaca hasta su muerte, y se diseccionan los órganos. Se analizan diversos parámetros sanguíneos, y se congelan los órganos siguientes: hígado, riñones, pulmones, corteza cerebral, testículos, páncreas y músculo, en los que se determina la permitividad y conductividad. Se determinan los coeicientes de reflexión y transmisión de estas muestras a la frecuencia industrial de 2,45 GHz. Esta frecuencia está comprendida dentro del rango de las frecuencias de microondas. Los coeficientes de reflexión y transmisión se miden mediante un analizador de redes, se usa un estimulador HFSS de HP que determina la permitividad de un tejido que produjera los mismos valores de los coeficientes de reflexión y transmisión medidos experimentalmente. Los resultados muestran que en el caso del riñón e hígado los valores de e (permitividad) y s (conductividad) son menores que en el control, lo que se puede explicar porque ambos órganos son los principales tejidos diana de la toxicidad del cadmio. Este elemento provoca disfunción en los túbulos proximales del riñón, y en el caso del hígado, el cadmio también se acumula en este órgano provocando daño hepático, un descenso de la integridad estructural de los hepatocitos y liberación de las transaminasas en el suero. Del análisis de sangre se observa que el cadmio provoca un estado anémico, de forma que se produce una disminución de los valores de hematocrito y hemoglobina con respecto a los controles (AU)

Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus.

Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV.

Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants.

It has been reported recently that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. The structural protein VP1 of foot-and-mouth disease virus (FMDV), which has frequently been shown to contain critical epitopes, has been expressed in different vectors and shown to induce virus-neutralizing antibodies and protection in experimental and natural hosts. Here we report the production of transformed plants (Arabidopsis thaliana) expressing VP1. Mice immunized with leaf plant extracts elicited specific antibody responses to synthetic peptides representing amino acid residues 135 to 160 of VP1, to VP1 itself, and to intact FMDV particles. Additionally, all of the immunized mice were protected against challenge with virulent FMDV. To our knowledge, this is the first study showing protection against a viral disease by immunization with an antigen expressed in a transgenic plant.

Characterization of the IgA and subclass IgG responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus.

In this study, we have investigated the characteristics of secreted IgA and other classes of Ig induced after vaccination of sows with transmissible gastroenteritis virus (TGEV) or the antigenically related porcine respiratory coronavirus (PRCV). Both viruses induced the secretion of neutralizing antibodies of different classes in the sows' milk, but these protected suckling piglets against TGEV to different degrees. Quantitative differences in the induction of IgA by both viruses were found among the different viral antigenic sites and subsites of glycoprotein S. In TGEV-vaccinated sows, antigenic subsite A was the best inducer of IgA, followed by antigenic site D. After vaccination with PRCV, lower levels of IgA were detected on colostrum and milk, antigenic site D and subsite Ab being the immunodominant sites. This quantitative difference in epitope recognition could explain the differences in newborn piglet protection found using Ig classes purified from the milk of sows immunized with both viruses. Apparently only IgA recognizing at least antigenic sites A and D confers good protection in vivo, whereas any Ig class recognizing only one antigenic site may neutralize the virus in cell culture. These results indicate that the formulation of a subunit vaccine against TGEV has to consider the inclusion of more than one antigenic site involved in virus neutralization.