Bluetongue is an infectious viral hemorrhagic disease of domestic and wild ruminants that has a considerable economic impact on domestic ruminants. There are currently at least 29 serotypes of bluetongue virus (BTV) in the world. Noteworthily, the pathogenesis among BTV serotypes is different, even in the same animal species. In this study, BTV2/KM/2003 and BTV12/PT/2003 were used to investigate the differential immunological effects on bovine peripheral blood mononuclear cells (PBMCs). The BTV viral load and the expression of cytokine messenger RNA (mRNA) in PBMCs were measured by fluorescence-based real-time reverse-transcription PCR (qRT-PCR). The immunofluorescence assay (IFA) was applied to detect BTV sign... More
Bluetongue is an infectious viral hemorrhagic disease of domestic and wild ruminants that has a considerable economic impact on domestic ruminants. There are currently at least 29 serotypes of bluetongue virus (BTV) in the world. Noteworthily, the pathogenesis among BTV serotypes is different, even in the same animal species. In this study, BTV2/KM/2003 and BTV12/PT/2003 were used to investigate the differential immunological effects on bovine peripheral blood mononuclear cells (PBMCs). The BTV viral load and the expression of cytokine messenger RNA (mRNA) in PBMCs were measured by fluorescence-based real-time reverse-transcription PCR (qRT-PCR). The immunofluorescence assay (IFA) was applied to detect BTV signals in monocyte-derived macrophages (MDMs). The SWISS-MODEL and IL-4pred prediction tools were used to predict the interleukin 4 (IL-4)-inducing peptides in BTV-coat protein VP2. Synthetic peptides of VP2 were used to stimulate PBMCs for IL-4-inducing capability. This study demonstrated that the cytokine profiles of BTV-induced PBMCs were significantly different between BTV2/KM/2003 and BTV12/PT/2003. BTV2 preferentially activated the T helper 2 (Th2) pathway, represented by the early induction of IL-4, and likely fed back to inhibit the innate immunity. In contrast, BTV12 preferentially activated the innate immunity, represented by the induction of tumor necrosis factor -α (TNF-α) and interleukin 1 (IL-1), with only minimal subsequent IL-4. The BTV nonstructural protein 3 antibody (anti-BTV-NS3) fluorescent signals demonstrated that monocytes in PBMCs and MDMs were the preferred targets of BTV replication. Bioinformatics analysis revealed that the capability to induce IL-4 was attributed to the tip region of the VP2 protein, wherein a higher number of predicted peptide segments on BTVs were positively correlated with the allergic reaction reported in cattle. Synthetic peptides of BTV2-VP2 induced significant IL-4 within 12-24 h post-infection (hpi) in PBMCs, whereas those of BTV12 did not, consistent with the bioinformatics prediction. Bovine PBMCs and synthetic peptides together seem to serve as a good model for pursuing the BTV-induced IL-4 activity that precedes the development of an allergic reaction, although further optimization of the protocol is warranted.
