Development of a recombinant antigen-based ELISA for the sero-detection of porcine lymphotropic herpesviruses.

BACKGROUND: Xenotransplantation using pigs as donor species carries a risk for the activation of latent porcine herpesviruses and potential transmission to the human recipient. The porcine lymphotropic herpesviruses (PLHV-1, -2, -3) are widespread in domestic pigs and closely related to the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, causing lymphoproliferative disorders. PLHV-1 has been associated with a porcine post-transplantation lymphoproliferative disorder (PTLD), affecting miniature swine after experimental transplantation. In human xenotransplantation, PLHV might be transferred to the transplant recipient and cause PTLD or related diseases. The elimination of PLHV from donor pigs is therefore necessary, and requires the availability of nucleic-acid- and antibody-based detection methods. METHODS: The N- and C-terminal parts (gB1 and gB2) of the glycoprotein B gene of PLHV-1, -2 and -3 were cloned and expressed in Escherichia coli. Antisera were raised in mice. PLHV PCR was performed as published earlier. RESULTS: An ELISA was developed, using recombinant glycoprotein B of PLHV-1 as the antigen, and used for the analysis of groups of pigs, differing by age and origin. Seropositivity ranged from 38% (piglets) to 90% (gilts) and 100% (breeding sows, miniature pigs and pigs for slaughter). In comparison, PCR products of PLHV were found in the blood of 0 to 80% of the pig groups. Additionally, a group of 12 piglets was tested repeatedly after birth until the age of 156 days. A decline of antibodies was found during the first 3 weeks after birth, followed by a rise in most pigs during the weeks thereafter. PLHV PCR products in the blood were only observed later than 3 weeks after birth. CONCLUSION: Newborn pigs may be passively protected by maternal antibodies against PLHV infection during the first 3 weeks post partum. The rise of antibody titers thereafter and the appearance of PLHV sequences in the blood possibly indicates de novo infection by contact to the infected mother sow. The PLHV-ELISA may aid in breeding PLHV-free pigs.

Dual specificity phosphatase 1 knockout mice show enhanced susceptibility to anaphylaxis but are sensitive to glucocorticoids.

Dual specificity phosphatase DUSP1 (otherwise known as mitogen-activated phosphatase 1 or MKP-1) dephosphorylates MAPKs, particularly p38, and negatively regulates innate immunity. Recent studies have shown that the DUSP1 gene is transcriptionally up-regulated by glucocorticoids (GCs) and that the antiinflammatory action of GCs is impaired in DUSP1-/- mice. Here we show that GC-mediated dephosphorylation of ERK-1 and ERK-2 activated by IgE receptor cross-linking is unimpaired in bone marrow-derived mast cells (BMMCs) of DUSP1-/- mice. Dephosphorylation of phospho-p38 MAPK is impaired but only at early times of GC treatment. Proinflammatory cytokine and chemokine gene expression (CCL2, IL-6, TNFalpha) is still down-regulated by GCs in BMMCs from DUSP1-/- mice, suggesting a compensatory mechanism for the GC action in these mice. In both DUSP1+/+ and DUSP1-/- BMMCs, GC up-regulated the expression of several phosphatase genes (DUSP2, DUSP4, DUSP9, and PEST domain-enriched tyrosine phosphatase). DUSP1-/- mice show enhanced mast cell degranulation and are highly susceptible to anaphylaxis, but these effects are still down-regulated by GCs. GCs also repressed other inflammatory responses such as dinitrofluorobenzene-induced contact hypersensitivity and lipopolysaccharide-induced mortality in DUSP1-/- mice. Thus GC-mediated antiinflammatory action is largely independent of DUSP1.

A novel porcine gammaherpesvirus.

A novel porcine gammaherpesvirus was detected in the blood of domestic pigs by PCR. With degenerate-primer PCR and subsequent long-distance PCR approaches a 60-kbp genome stretch was amplified. Sequence analysis revealed the presence of the gammaherpesvirus ORFs 03 to 46 as well as a putative chemokine receptor and a v-bcl-2 gene. The 60-kbp sequence was compared with the corresponding sequence of the porcine lymphotropic herpesvirus 1 (PLHV-1) published recently and the sequence of PLHV-2, which was amplified from porcine tonsil. Considerable sequence differences (amino acid identities: 49-89%) were found between the novel virus and PLHV-1 as well as PLHV-2, which were very closely related to each other (amino acid identities: 85-98%). The novel virus had essentially the same genome organization as PLHV-1 and -2 and was therefore designated PLHV-3. Like PLHV-1 and -2, PLHV-3 was frequently found in the blood and in lymphoid organs of domestic and feral pigs from different geographic locations. In the blood, the PLHVs were detected predominantly in B-cells. Indication for latent as well as productive PLHV-3 infection was found in the porcine B-cell line L23. It can be concluded that the PLHVs are widespread and are likely to cause a persistent B-lymphotropic infection. Since PLHV-1 has been implicated in the development of porcine posttransplantation lymphoproliferative disease, all porcine lymphotropic gammaherpesviruses are of concern when pigs are used as donors in xenotransplantation.