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

Type I Hypersensitivity in Bovine Immune Cells Induced by Bluetongue Virus Taiwan Isolate BTV2/KM/2003

楊佳玲(Jia-Ling Yang)

指導教授 ： 王汎熒

共同指導教授 ： 王家琪(Chia-Chi Wang)

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2021年)

https://doi.org/10.6342/NTU202101179

摘要

藍舌病(Bluetongue, BT)因造成反芻獸出血性疾病，對畜牧產業衝擊嚴重。世界動物衛生組織(OIE)將BT列為須通報疾病，許多國家重視BT的防治與疫苗開發。藍舌病病毒(bluetongue virus, BTV)屬於呼腸孤病毒科(Reoviridae)的Orbivirus屬，通過昆蟲媒介傳播。迄今全世界已經確認了29種BTV血清型。西元2003年，台灣也分離出兩種BTV毒株，分別為BTV2 / KM / 2003和BTV12 / PT / 2003。由於BTV毒株間差異可能引發不同強度的臨床症狀，因此本論文旨在比較該兩種病毒株與其宿主免疫細胞的交互作用，觀察各病毒株間的差異。在第二章，我們將BTV2分別接種牛隻週邊血液單核細胞(peripheral blood mononuclear cell, PBMC)、單核球來源巨噬細胞(monocyte-derived macrophage, MDM)及巨噬細胞重建自體淋巴球細胞(MDM-Lymphocyte)後，觀察BTV的複製及細胞素的變化。此三類細胞群分別代表了BTV進入宿主體內，會遭遇的不同細胞族群。BTV2的複製曲線在PBMC、MDM與MDM-Lymphocyte中，三者相似且無顯著差異。在PBMC與MDM-Lymphocyte中，IL-4 mRNA的表現早於先天免疫和細胞介導免疫的細胞素。培養細胞上清液中IL-4蛋白的穩定存在與IgE抗體的出現，顯示第I型過敏反應參與了致病機轉。然而在MDM(無淋巴細胞)中，IL-1β，TNF-α和IL-12p40的mRNA表現均未受抑制。此結果支持了淋巴球與IL-4可能對先天免疫與細胞介導免疫的抑制作用。值得注意的是，即使在相同物種中，各BTV血清型之間的致病機轉也不同，因此在第三章，我們比較了BTV2和BTV12在PBMC中免疫路徑的差異。BTV2優先刺激IL-4的早期表現(T輔助2路徑)，並可能反饋抑制先天免疫。相反地，BTV12優先刺激先天免疫，以誘導TNF-α和IL-1β。BTV非結構蛋白3抗體(抗BTV-NS3)螢光信號顯示PBMC和MDM中的單核球是BTV複製的首選細胞。生物資訊學分析顯示誘導IL-4的能力可能位於VP2蛋白三維結構的尖端區域(tip region)。其中VP2尖端區上，預測肽段的數量與BTV感染中報導過的過敏反應呈正相關。BTV2-VP2的合成肽可於12-48小時，誘導IL-4及IgE表現，而BTV12的合成肽則無，與生物資訊學預測一致。合成肽刺激牛PBMC似乎是BTV誘導過敏反應的良好模型，此方法應可加速病毒蛋白致病機轉和發展疫苗的應用研究。

關鍵字

藍舌病 ； 週邊血液單核細胞 ； 巨噬細胞 ； 細胞素 ； 致病機轉 ； 生物資訊學

並列摘要

Bluetongue (BT) causes fatal infectious hemorrhagic disease in ruminants, so it is listed as a notifiable disease under the Office international des épizooties (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.

並列關鍵字

bluetongue virus ； peripheral blood mononuclear cell ； monocyte-derived macrophage ； cytokines ； pathogenesis ； bioinformatics

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