Detection of Bluetongue Virus in Tissues of Experimentally Infected Sheep

藍舌病是一種由昆蟲媒介的病毒性疾病。主要存在於馴養及野生反芻獸，如: 綿羊、山羊、牛及大部分的非洲羚羊和各種偶蹄類動物，但以綿羊最具感受性。典型症狀一開始為持續性發熱伴隨口、鼻腔黏膜充、出血，甚至潰瘍。嚴重病例可見舌頭發紺及出血，因此被稱為”藍舌病”。本病是由藍舌病病毒（Bluetongue virus, BTV）所造成，隸屬於里奧病毒科（Reoviridae）中的環狀病毒屬（Orbivirus），為一雙股RNA病毒，目前已分離鑑定出至少24種血清型，分佈於全世界。台灣於2003年首次分離到本病毒，分別為由金門的山羊所分離血清第2型的BTV2-KM-2003株；與由屏東的乳牛所分離血清第12型的BTV12-PT-2003株。惟目前台灣尚無臨床病例。了解台灣分離株之致病性，使用BTV2-KM-2003進行人工皮下接種於抗體陰性之柯麗黛綿羊，並分別於感染後七天及十一天犧牲。除發燒外並未引起明顯的臨床症狀。舌頭固有層及耳翼真皮層可見輕度多發點狀出血，心臟、腎臟及淋巴節亦可見輕微出血，但藍舌病典型之肺動脈基部出血並不明顯。 本研究之目的是以免疫組織化學染色及原位雜交偵測藍舌病病毒與病變的相關性，以排除人工因素並做為鑑別診斷，並觀察病毒於體內分佈之情形，作為了解其致病機制之參考。疫組織化學染色顯示病毒主要分佈於耳朵、舌頭及臉部皮膚之發炎、水腫處及各淋巴組織中。在脾臟，病毒分佈位置依犧牲時間的不同而有所改變，感染後七天，其訊號主要存在於白髓的邊緣區(marginal zone)及隨機分佈於紅髓，感染後十一天則出現於淋巴濾泡中。根據形態學及訊號分佈位置，推測感染細胞以單核吞噬細胞及T淋巴球為主。原位雜交結果顯示病毒分佈位置與免疫組織化學染色所得結果相符，但另外發現訊號於白髓邊緣區的B淋巴球。依目前結果判斷，台灣之分離株應屬於弱毒株，其在脾臟分佈位置的變化與抗原處理、呈現及抗體產生有關。Bluetongue virus (BTV), a member of the Orbivirus genus of the family Reoviridae, is the causative agent of a noncontagious, insect-borne disease. Bluetongue virus infection involves domestic and wild ruminants, and causes thrombo-hemorrhagic fevers mainly in sheep. The BTVs were first isolated from domestic ruminants in Taiwan in year 2003, respectively BTV2-KM-2003 from goats and BTV12-PT-2003 from dairy cattle. So far, there is no clinical outbreak of BTV in Taiwan. n experimental infection of BTV2-KM-2003 had been conducted for evaluating its virulence on Corriedale sheep, one of the predominant breeds in Taiwan, and then euthanatized at 7 and 11 days post-infection (DPI). No obvious clinical signs were noted, except for a mild fever at DPI 4 or 5. Histological lesions were characterized by multifocal hemorrhage with mild to moderate infiltration of mononuclear cells and edema in the contralateral ear pinna, tongue, and facial skin, but the characteristic ecchymotic hemorrhage at the base of pulmonary artery of virulent BTV was absent.he purpose of this study was to detect the BTV in experimentally infected Corriedale sheep by immunohistochemistry (IHC) and in-situ hybridization (ISH) in order to correlate viral distributions with the lesions, and to further the understanding of pathogenesis. Viral antigen signals were detected by IHC in the spleen, lymph node, tonsil, contralateral ear pinna, tongue, and facial skin. The presence of BTV antigen signals in the inflammatory areas correlated with the lesions on sites. While in the spleen, strong BTV signals were chiefly located in macrophages of the marginal zone and T lymphocytes of the red pulp on DPI 7, by DPI 11 most BTV signals shifted to macrophages and small lymphocytes within the general center, presumbably coincident with the antigen capture and stimulation for antibody synthesis. The results of ISH were similar to those of IHC, except that B lymphocytes of the marginal zone were also detected. Based on the clinical signs, gross and histological findings, the Taiwan isolate BTV2-KM-2003 was able to induce mild petechiated hemorrhages and to establish non-lethal infection, and is thus of low virulence to Corriedale sheep.中文摘要 Ibstract II錄 IVist of Tables VIist of Figures VIIhapter I Introduction 1hapter II Literature review 4.1 Etiology 4.2 Clinical signs, Pathology and Pathogenesis 6.2.1 Clinical signs 6.2.2 Gross findings 7.2.3 Histological findings 8.2.4 Pathogenesis 8.3 Immune responses against BTV 10.3.1 Humoral immunity 10.3.2 Cellular immunity 10.4 Situation of BTV in Taiwan 11hapter III Materials and methods 12.1 Sample collections 12.1.1 Tissues of experimental sheep 12.1.2 Cell culture slides for positive control 12.1.3 Formalin-fixed paraffin-embedded (FFPE) agarose cell blocks for positive control 13.2 Immunohistochemistry (IHC) 14.2.1 Search for applicable anti-BTV antibodies (primary antibodies) 14.2.2 IHC procedures 15.3 Amplification of the VP7 gene by two-step RT-PCR and cloning 16.3.1 Reverse transcription 16.3.2 Polymerase chain reaction 16.4 Cloning and sequencing 17.5 In situ hybridization 17.5.1 Preparation of probe 17.5.2 Dot-blot hybridization 18.5.3 Hybridization procedures 18hapter IV Results 20.1 Histological lesions 20.2 Establishment of known positive and negative controls 20.3 Detection of BTV antigen in tissues by IHC 22.4 RT-PCR 24.5 Detection of BTV RNA in cells and tissues by ISH 24hapter V Discussion 26ources and Manufacturers 30ables 31igures 35eferences 4