Bovine herpesvirus 1 (BoHV-1) is an important pathogen of domestic and wild cattle responsible for major economic losses in dairy and beef industries throughout the world. Inhibition of viral entry plays a crucial role in the control of BoHV-1 infection and aptamers have been reported to inhibit viral replication. In this study, nine DNA aptamers that target BoHV-1 were generated using systemic evolution of ligands by exponential enrichment. Of the nine candidates, aptamer IBRV-A4 exhibited the highest affinity and specificity for BoHV-1, which bound to BoHV-1 with a Kd value of 3.519 nM and demonstrated the greatest virus binding as shown by fluorescence imaging. The neutralizing ability of aptamer IBRV-A4 was determined using neutralization assays and real time PCR in BoHV-1 infected Madin-darby bovine kidney cells. Virus titration, immunofluorescence and confocal laser scanning microscopy showed virus replication significantly decreased when aptamer IBRV-A4 was added to BoHV-1 infected MDBK cells at 0 and 0.5 hours post-infection, whereas no change was seen when IBRV-A4 was added 2 hours post-infection. This concludes that aptamer IBRV-A4 efficiently inhibits viral entry of BoHV-1 in MDBK cells and is therefore a novel tool for diagnosis and treatment of BoHV-1 infection in cattle.

ABSTRACT. We investigated the immune response effects of porcine circovirus type 2 (PCV2) on cells inoculated with pseudorabies attenuated vaccine (PRV). Real-time PCR was used to detect the mRNA expression levels of the regulatory cytokines IL-4, IL-10, IL-12p40, and IFN-γ in pig peripheral blood mononuclear cells, after in vitro single vaccination and co-inoculation with PCV2 and the PRV. We found that PRV causes upregulation of IL-4, IL-12p40, and IFN-γ mRNA expression, while PCV2 causes mRNA upregulation of IL-4, IL-10, and IL-12p40. Moreover, PCV2 inhibited PRV-induced upregulation of IL-4, IL-12p40, and IFN-γ mRNA expression; IFN-γ mRNA expression was significantly inhibited. We conclude that PCV2 can reduce the cellular immune response to PRV.

BackgroundPorcine circovirus (PCV) disease caused by PCV type 2 (PCV2) is mainly attributed to immunosuppression and immune damage. PCV2 can infect vascular endothelial cells and induce high expression of endothelial IL-8. Dendritic cells (DCs), as professional antigen-presenting cells, can not only present antigens but also activate naïve T-cells, causing an immune response.MethodsTo demonstrate whether endothelial IL-8 is the main factor inhibiting the maturation and related functions of dendritic cells during PCV2 infection, monocyte-derived DCs (MoDCs) and porcine iliac artery endothelial cells (PIECs) processed by different methods were co-cultured in two ways. Flow cytometry, molecular probe labeling, fluorescence quantitative PCR, and the MTS assay were used to detect the changes in related functions and molecules of MoDCs.ResultsCompared to those in the PIEC-DC group, the endothelial IL-8 upregulation co-culture group showed significantly lower double-positive rates for CD80/86 and MHC-II of MoDCs and significantly increased endocytosis of MoDCs. Meanwhile, the adhesion rate and average fluorescence intensity of MoDCs were significantly downregulated in migration and adhesion experiments. Furthermore, the MHC-I and LAMP7 mRNA levels in MoDCs and the proliferation of MoDC-stimulated T-cells were markedly reduced. However, the changes in MoDCs of the endothelial IL-8 downregulation co-culture group were the opposite.ConclusionsPCV2-induced endothelial IL-8 reduces the adhesion and migration ability of MoDCs, resulting in a decreased maturation rate of MoDCs, and further inhibits antigen presentation by DCs. These results may explain the immunosuppressive mechanism of PCV2 from the perspective of the interaction between endothelial cells and DCs in vitro.

The efficiency of immune responses and host defense against pathogens largely depends on the function of dendritic cells (DCs). Porcine circovirus type 2 (PCV2) infection causes viremia and extensive modulation of immune activities in the blood. The objective of the present study was to investigate the effects of PCV2 infection in vivo on the immunological function of DCs induced from peripheral blood monocytes (MoDCs). At different points after infection with PCV2, peripheral blood monocytes from PCV2-infected pigs were used to induce differentiation of DCs in vitro. Flow cytometry and quantitative real-time reverse transcription PCR were conducted to detect mRNA expression of surface markers related to antigen presentation and inflammatory/immunosuppressive cytokines of the induced MoDCs. The ability of induced MoDCs to stimulate T cells was measured using an MTS assay. In the early phase of infection at 3 days post-inoculation (DPI), IL-10, IL-8 and MIP-1β in MoDCs were upregulated significantly. By the peak of virus proliferation at 7 DPI, antigen presentation molecules SLA-DR (MHC II) and CD80/86 together with cytokines IL-12 and IL-10 had decreased, accompanied by a rapid reduction of IL-8 and MIP-1β. The T cell stimulation index of induced MoDCs in PCV2 groups after different infection times declined to some extent, with a significant difference at 7 DPI. PCV2 infection in vivo functionally reduced the antigen presentation capability of induced MoDCs from peripheral blood and modified expression of inflammatory/immunosuppressive cytokines that may be related to PCV2-induced immunosuppression.

Porcine circovirus-associated disease is caused by porcine circovirus type 2 (PCV2) infection, which targets iliac artery endothelial cells (PIECs); it leads to severe immunopathologies and is associated with major economic losses in the porcine industry. Here, we report that in vitro PCV2 infection of PIECs causes cell injury, which affects DC function as well as adaptive immunity. Specifically, PCV2 infection downregulated PIEC antigen-presenting molecule expression, upregulated cytokines involved in the immune and inflammatory response causing cell damage and repair, and altered the migratory capacity of PIECs. In addition, PCV2-infected PIECs inhibited DC maturation, enhanced the endocytic ability of DCs, and weakened the stimulatory effect of DCs on T lymphocytes. Together, these findings indicate that profound functional impairment of DCs in the presence of PCV2-infected PIECs may be a potential pathogenic mechanism associated with PCV2-induced porcine disease.