T helper 1-type cytokine transcription in peripheral blood mononuclear cells of pseudorabies virus (Suid herpesvirus 1)-primed swine indicates efficient immunization. (1/25)

The induction of porcine cytokines, which are believed to be important for the regulation of T helper (Th)1- and Th2-specific immune responses of pigs, was analysed after in vitro restimulation with a herpesvirus, Suid herpes 1 (pseudorabies virus [PRV]), in peripheral blood mononuclear cells (PBMC). To this end, quantitative, competitive reverse transcription-polymerase chain reaction (RT-qcPCR) was established using constructed heterologous DNA MIMICS, which contain cytokine- or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primer-binding sites. This is a simple method that allows reliable determination of the differing regulation of cytokine mRNAs specific for porcine interleukin (IL)-2, -4 and -10, interferon gamma (IFN-gamma) and the housekeeping gene, GAPDH, as an endogenous control. PBMC derived from naive (innate response) and PRV-primed (memory response) outbred swine were analysed comparatively. The results demonstrated that restimulation with PRV significantly enhanced the transcription of Th1-type cytokines (IL-2 and IFN-gamma) but not of Th2-type cytokines (IL-4 and IL-10). This virus-specific cytokine response was only found with PBMC from swine protected against lethal PRV challenge infection, but not with naive PBMC or with PBMC from pigs immunized with plasmid DNA encoding PRV glycoprotein gC. Notably, PBMC derived from immune and naive pigs constitutively produced relatively high amounts of IL-10-specific mRNA, exceeding that of GAPDH mRNA, independently of the addition of viral antigen or the mitogen concanavalin A (Con A). The results of this work should help to provide a better understanding of the effector cell/cytokine network response to infection with, or vaccination against, PRV. Additionally, the simple, reliable and sensitive RT-qcPCR, when used to determine the porcine cytokine pattern, might be of prognostic value for the induction of protective immunity.  (+info)

An analysis of a presumed major outbreak of pseudorabies virus in a vaccinated sow herd. (2/25)

We describe a major outbreak of pseudorabies virus (PRV) in a sow herd in which the sows were vaccinated simultaneously three times a year with a vaccine containing Bartha strain. Also in the associated rearing herd in which the gilts were vaccinated twice an outbreak of PRV occurred. The outbreak was analysed with mathematical models, statistical methods and Monte-Carlo simulation. Under the assumption that the outbreak started with one introduction of virus the reproduction ratio R(ind)--as a measure of transmission of PRV between individuals--in the sow herd was estimated with a Generalized Linear Model to be 1.6. Also under the assumption of one introduction of virus R(ind) in the rearing herd was estimated with a martingale estimator to be 1.7. Both estimates were significantly larger than 1. Mathematical analysis showed that heterogeneity in the sow herd, because of the presence of not-optimally immunized replacement sows could not be the only cause of the observed outbreak in the sow herd. With Monte-Carlo simulations, the duration of an outbreak after a single introduction of virus and R(ind) = 1.6 did not mimic the data and thus the hypothesis of a single introduction with R(ind) = 1.6 could also be rejected and R(ind) is thus, not necessarily above 1. Moreover, with statistical analysis, endemicity in the combination of herds as a cause for the observed outbreak could be rejected. Endemicity in the rearing herd alone could not be excluded. Therefore, multiple introductions from outside and most probably from the rearing herd were possibly the cause of the observed outbreak(s). The implications for eradication of pseudorabies virus were discussed.  (+info)

Type I IFN modulates the immune response induced by DNA vaccination to pseudorabies virus glycoprotein C. (3/25)

DNA vaccines have the capacity to induce strong Th1-biased immune responses that are of major importance to providing protection against intracellular pathogens. In the present study we have focused on the role played by type I IFN in immune responses induced after DNA vaccination. Mice lacking the IFNAR1 chain of the type I IFN receptor (IFNAR K/O mice) were immunized with a plasmid encoding glycoprotein C of pseudorabies virus (PRV-gC). After DNA vaccination, wild-type (WT) mice showed features characteristic of Th1 immune responses, such as high IgG2a:IgG1 anti-PRV Ab ratio and antigen-specific IFN-gamma production by spleen cells. In contrast, IFNAR K/O mice showed a significantly lower IgG2a:IgG1 Ab ratio and IFN-gamma production. In addition, the percentage of CD8(+) and B lymph-node cells expressing CD69 after PRV-gC DNA vaccination was lower in IFNAR K/O than in WT mice. These results support a major role played by type I IFN in shaping Th1 immune responses after DNA vaccination. Codelivery of plasmids encoding IL-12 and IL-18 along with the plasmid encoding PRV-gC restored Th1 responses in IFNAR K/O mice.  (+info)

Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection. (4/25)

The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar insertions in the gG locus of the wild-type PRV strain Becker had no effect on the ability of virus infection to spread between cells. Insertions in the gG locus of the virulent NIA-3 strain gave results similar to those found with the Bartha strain. To examine the role of gG in cell-to-cell spread, a nonsense mutation in the gG signal sequence was constructed and crossed into the Bartha strain. This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain. These data indicated that, while insertions in the gG locus result in decreased cell-to-cell spread, the phenotype was not due to loss of gG expression as first predicted. Analysis of gene expression upstream and downstream of gG revealed that expression of the upstream Us3 protein is reduced by insertion of lacZ or egfp at the gG locus. By contrast, expression of the gene immediately downstream of gG, Us6, which encodes glycoprotein gD, was not affected by insertions in gG. These data indicate that DNA insertions in gG have polar effects and suggest that the serine/threonine kinase encoded by the Us3 gene, and not gG, functions in the spread of viral infection between cells.  (+info)

Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant. (5/25)

To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfolipo-cyclodextrin in squalene in water. We demonstrate that a DNA vaccine combined with DDA, but not with the other adjuvants, induced significantly stronger immune responses than plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection. This is the first study to demonstrate that DDA, a conventional adjuvant, enhances DNA vaccine-induced antiviral immunity.  (+info)

A fusion protein of IgG fc and mouse-derived antigen on the surface of pseudorabies virus particles does not accelerate production of harmful auto-reactive antibodies. (6/25)

Previously we reported that immunization with pseudorabies virus (PRV), harboring chimeric Fc on the surface of the virus particles (PRV/Fc), induced higher immune responses than normal PRV particles. The chimeric Fc was fused with mouse transferrin receptor of transmembrane domain (mTR) and the Fc region of immunoglobulin G1. Since it has been reported that some chimeric protein of Fc and self-antigen induce auto-reactive antibodies, in this present study, we examined whether PRV/Fc induces auto-reactive antibodies that react with mTR. PRV/Fc immunized mice produced higher levels of anti-PRV antibodies and antibodies that reacted with mouse-derived 3T3/A31 cells (A31 cell), compared to normal PRV immunized mice. However, antibodies that reacted with mTR in A31 cells were not detected in both Western blot analyses and indirect immunofluorescence assay. The antibodies reacted with an antigen of approximately 16 kDa in A31 cells, but this antigen has a different molecular mass from that of mTR. The antibody also reacted with the antigen of approximately 16 kDa in RK13 cells in which the virus had been propagated. In addition, antibodies induced by immunization with normal PRV also reacted with the same antigen in A31 and RK13 cells. Moreover, neither kidney disorders, in which high levels of mTR were expressed, nor clinical symptoms of autoimmune diseases were observed in mice immunized with either PRV or PRV/Fc. These results indicated that the antibodies were not induced by mTR-Fc, but were instead induced by trace amounts of RK13 derived antigens contained in PRV or PRV/Fc preparations, and cross-reacted with equivalent molecules in mouse derived A31 cells. Therefore, this study confirmed that immunization with PRV/Fc did not induce harmful auto-reactive antibodies.  (+info)

Limited protection conferred by a DNA vaccine against a lethal pseudorabies virus infection at day 5 postvaccination. (7/25)

No pseudorabies virus (PRV)-specific neutralizing or immunoglobulin G1-type antibodies were detected in sera 5 days after injection of a DNA vaccine against PRV infection in pigs. PRV-stimulated peripheral blood mononuclear cells produced gamma interferon mRNA in vitro. Two out of five pigs recovered from lethal PRV infection without attenuation of nasal viral excretion.  (+info)

Porcine circovirus type 2-induced interleukin-10 modulates recall antigen responses. (8/25)

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