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Differences in the kinetics of expression and cell distribution among FMDV non-structural proteins (NSPs) have been observed in BHK-21-infected cells. 3D(pol) was the first protein detected by immunofluorescence (1.5 h p.i.), showing a perinuclear distribution. At 2-2.5 h p.i., 2B, 2C, 3B and 3C were detected, mostly exhibiting a punctuated, scattered pattern, while 3A and 3D(pol) appeared concentrated at one side of the nucleus. This distribution was exhibited by all the NSPs from 3 h p.i., being 2C and, to a lesser extent, precursors 2BC and 3ABBB, the only proteins detected by Western blotting at that infection time. From 4 h p.i., all mature NSPs as well as precursors 2BC, 3ABBB, 3ABB, 3AB and 3CD(pol) were detected by this technique. In spite of their similar immunofluorescence patterns, 2C and 3A co-localized partially by confocal microscopy at 3.5 h p.i., and 3A, but not 2C, co-localized with the ER marker calreticulin, suggesting differences in the distribution of these proteins and/or their precursors as infection proceeded. Transient expression of 2C and 3AB resulted in punctuated fluorescence patterns similar to those found in early infected cells, while 3A showed a more diffuse distribution. A shift towards a fibrous pattern was noticed for 3ABB, while a major change was observed in cells expressing 3ABBB, which displayed a perinuclear fibrous distribution. Interestingly, when co-expressed with 3D(pol), the pattern observed for 3ABBB fluorescence was altered, resembling that exhibited by cells transfected with 3AB. Transient expression of 3D(pol) showed a homogeneous cell distribution that included, as determined by confocal microscopy, the nucleus. This was confirmed by the detection of 3D(pol) in nuclear fractions of transfected cells. 3D(pol) and its precursor 3CD(pol) were also detected in nuclear fractions of infected cells, suggesting that these proteins can directly interact with the nucleus during FMDV infection.

The importance of the induction of virus neutralizing antibodies to provide protection against foot-and-mouth disease virus (FMDV) infection is well established. However, recent studies with recombinant adenovirus expressing the precursor polypeptide of the viral capsid (P1) indicate that cattle inoculated with this recombinant vector developed partial protection against FMDV infection, in the absence of a detectable specific humoral response. Other viral vectors have been widely used to induce protective immunity against many pathogens, and it has been reported that the use of different vectors for priming and boosting injections can provide a synergistic effect on this response. In this work, we determined the immunogenicity of two recombinant viruses (adenovirus and vaccinia) expressing P1-FMDV, administered either individually or sequentially, and the protection that they induced against FMDV challenge in pigs. A double immunization with the adeno-P1 virus was the most effective strategy at inducing protective immunity. In contrast to previous reports, the use of two different vectors for priming and boosting did not show a synergistic effect on the protection induced against FMD. Interestingly, immunized pigs developed FMDV-specific T cell responses but not detectable antibodies. Thus, the protection observed was likely to be mediated by a cellular immune response.

CD163 is a member of the family of proteins with scavenger receptor cysteine-rich domains, whose expression is restricted to monocytes and macrophages. It functions as a receptor for haemoglobin/haptoglobin complexes and it has also been implicated in the regulation of inflammatory processes. Treatment of monocytes/macrophages with phorbol esters, LPS or cross-linking of Fc gamma receptors, causes shedding of CD163 from cell surface by a protease-dependent mechanism. The level of soluble CD163 (sCD163) in serum and other body fluids has been considered a useful marker of macrophage activation. In addition, porcine CD163 has been shown to play a role in the infection of monocytes/macrophages by the African swine fever virus. The porcine CD163 cDNA has been cloned, and expressed in transfected CHO cells. Clones of CHO cells stably expressing porcine CD163 have been used for the characterization of three new mAbs against porcine CD163. Using two of these mAbs, that bind to different epitopes on CD163 molecule, a sandwich enzyme-linked immunoassay (ELISA) has been developed to measure levels of sCD163 in porcine sera and biological fluids. The assay was calibrated using lysates of CD163 transfectants, showing a sensitivity of 10 5 cells mL -1 , that allowed to detect sCD163 in sera and in culture supernatants of activated alveolar macrophages. This assay can be a useful method of monitoring the degree of activation of macrophages in a variety of inflammatory and infectious diseases of swine.Additional key words: macrophage, SRCR family, swine. ResumenClonación y expresión de CD163 porcino: aplicación en la caracterización de anticuerpos monoclonales frente a CD163 porcino, y desarrollo de un ELISA para cuantificar CD163 soluble en fluidos biológicos CD163 es un miembro de la familia de proteínas con dominios de receptores «scavenger» ricos en cisteína, con una expresión restringida a células del linaje monocítico. Se ha demostrado su función como receptor de complejos hemoglobina/haptoglobina, y se le ha implicado en la regulación de procesos inflamatorios. El tratamiento de monocitos/macrófagos con ésteres de forbol, LPS, o mediante entrecruzamiento de receptores Fc gamma, provoca la liberación de CD163 de la superficie celular por un mecanismo dependiente de proteasas. La cantidad de CD163 soluble (sCD163) en suero y otros fluidos corporales se considera un indicador de la activación de los macrófagos. Recientemente, nuestro grupo demostró que CD163 está implicado en la infección de monocitos/macrófagos porcinos por el virus de la peste porcina africana. En este trabajo se describe la clonación del cDNA de CD163 porcino, y su expresión en células CHO transfectadas. Se obtuvieron clones de células CHO transfectadas que expresan de forma estable la molécula CD163 porcina, y se utilizaron para caracterizar tres nuevos anticuerpos monoclonales que reconocen específicamente CD163 porcino. El uso de dos de estos anticuerpos, que reconocen epítopos diferentes en CD163 porcino, ha permitido desarrollar un ensayo i...

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An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP 1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter. Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia virus recombinant vTF1-6,2 resulted in expression of chimeric proteins efficiently reactive with both anti-FMDV and anti-VSV G antibodies. However, in vitro translation of transcripts of these VSV-G/FMDV-ASA chimeric plasmids resulted in proteins that were recognized by anti-G serum but not by anti-FMDV serum, indicating a requirement for in vivo conformation to expose the ASA antigenic determinant. Insertion of DNA coding for a dimer of the ASA unidecapeptide between the VSV-NJ G gene region coding for amino acids 160 and 161 gave rise to a chimeric ASA-dimer protein designated GF2d, which reacted twice as strongly with anti-FMDV antibody as did chimeric proteins in which the ASA monomer was inserted in the same position or two other G-gene positions. For even greater expression of chimeric VSV-G/FMDV-ASA proteins, plasmid pGF2d and a deletion mutant p⌬GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and ⌬GF2d-bac produced in Sf9 insect cells. Mice vaccinated with three booster injections of 30 g each of partially purified GF2d-bac protein responded by enzyme-linked immunosorbent assay with FMDV antibody titers of 1,000 units, and those injected with equivalent amounts of ⌬GF2d-bac protein showed serum titers of up to 10,000 units. Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with ⌬GF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-g doses of native FMDV virions. Despite excellent reactivity with native FMDV, the anti-⌬GF2d-bac antibody present in vaccinated mouse serum showed no capacity to bind to sodium dodecyl sulfate (SDS)-denatured FMDV virions and only minimal reactivity with VP 1 protein by Western blotting (immunoblotting) after SDS-polyacrylamide gel electrophoresis. It was also shown in a competitive binding assay that a synthetic ASA unidecapeptide, up to concentrations of 200 g/ml, was quite limited in its ability to inhibit binding of anti-⌬GF2-bac antibody to native FMDV virions. These results suggest that the chimeric VSV-G/FMDV-ASA proteins mimic the capacity of FMDV to raise and react with neutralizing antibodies to a restricted number of ASA conformations present on the surface of native FMDV particles.