臺灣藍舌病第二血清型病毒株BTV2/KM/2003引起牛隻免疫細胞第一型過敏反應之探討

Abstract

Bluetongue (BT) causes fatal infectious hemorrhagic disease in ruminants, so it is listed as a notifiable disease under the Office international des epizooties (OIE) Terrestrial Animal Health Code. Bluetongue virus (BTV), belongs to genus Orbivirus of the Reoviridae family, is transmitted via Culicoides insect vector. Thus far 29 serotypes of BTV have been recognized worldwide, and two strains, BTV2/KM/2003 and BTV12/PT/2003, were isolated in Taiwan. We hypothesized that various BTV strains differ in their ability to induce clinical signs of variable severities. The aim of this study was to compare the interactions between BTVs and bovine immune cells, to demonstrate the strain differences in immunological perspective. In chapter two, replication of BTV and cytokines expression profile in bovine peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM) and macrophages reconstituted with autologous lymphocytes (MDM-Lymphocyte) were monitored. The replication curves of BTV2 were similar and without significant difference among PBMC, MDM and MDM-Lymphocyte. In PBMC and MDM-Lymphocyte, the expression of IL-4 mRNA was earlier than the cytokines of innate immunity and cell-mediated immunity (CMI). The IL-4 protein and IgE antibodies in the supernatant indicated that type I hypersensitivity was involved in BTV pathogenesis. However, the IL-1β, TNF-α and IL-12p40 mRNA increased in MDM (no lymphocytes) without inhibition. This result supports that lymphocytes and IL-4 mediated negative feedback in innate immunity and CMI. Noteworthily, the pathogenesis among BTV serotypes was also different, even in the same animal species. In chapter three, BTV2 and BTV12 were used to investigate the differential immunological effects on PBMC. BTV2 preferentially activated IL-4 (T helper 2 pathway) and likely feedback to inhibit the innate immunity. In contrast, BTV12 preferentially activated the innate immunity (TNF-α and IL-1β), with only minimal subsequent IL-4. The nonstructural protein 3 antibody (anti-BTV-NS3) fluorescent signals showed that monocytes in PBMC and MDM were the essential targets of BTV replication. Bioinformatics analysis revealed that the capability to induce IL-4 were attributed to the tip region of the VP2 protein three-dimensional structure, wherein a higher number of predicted peptide segments on BTVs were positively correlated with the allergy reported in cattle. Synthetic peptides of BTV2-VP2 induced significant IL-4 and IgE within 12-48 h post-infection (hpi) in PBMC, whereas those of BTV12 did not, consistent with the bioinformatics prediction. Bovine PBMC and synthetic peptides together seem to serve as a good model for pursuing the BTV-induced IL-4 and IgE activity that precedes the development of an allergic reaction. This method may facilitate the study on pathogenesis of other viral proteins and the development of vaccine in the future.