- 學位論文
豬流行性感冒病毒之偵測及其分型方法之建立
Detection and Typing of Swine Influenza Virus
摘要
豬隻在A型流行性感冒病毒傳播上扮演一個重要的角色。由於豬隻的氣管上皮細胞同時具有哺乳類動物和禽類動物來源之流感病毒接受器，所以豬隻被認為在哺乳類動物和禽類動物來源之流感病毒間扮演一個Mixing vessel的角色，其可能促進病毒進行基因重排（Genetic reassortment）。近年來豬呼吸道疾病複合症（Porcine respiratory disease complex，PRDC）已嚴重影響台灣的養豬產業，而豬流行性感冒病毒也是其中常見的病原之一。但在豬流感病毒感染時，其病毒血症的存在時間通常十分短暫，病毒很快便會被宿主免疫系統所清除，而導致於田間分離或檢測病毒上的困難。因此，對於臨床上流感病毒的檢測，或許直接檢測在相關細胞內的病毒核酸及抗原會更有利於檢出。故在本研究中，嘗試著去開發對於豬流行性感冒病毒更快速的檢測及分型方法，同時也希望探討病毒分佈與肺臟病灶之間的相關性。在本研究中利用ISH和IHC之方法檢測福馬林固定石蠟包埋組織蠟塊中病毒核酸與抗原的存在，同時也利用RT-PCR對野外收集的肺臟乳劑進行檢測。利用NP探針進行ISH，可於支氣管和細支氣管上皮細胞或於增生、壞死區之單核炎症細胞中檢測到SIV核酸存在。而RT-PCR已可成功用於區分台灣常見三種病毒亞型（分別為H1N1, H3N2 和 H1N2）。而利用抗NP、H1和H3三種單株抗體進行IHC，其結果顯示病毒抗原之分佈位置與ISH之結果相似，但在IHC中於支氣管周圍的漿液、黏液腺中也偵測到陽性訊號存在。而利用IHC對20個SIV ISH陽性的病例做回溯性的檢測，發現台灣混合H1和H3兩種亞型的共同感染可能普遍存在，其陽性率達75% (15/20)。
關鍵字
豬流行性感冒病毒並列摘要
Swine play an important role in the transmission of influenza A virus. Trachea epithelium of swine have both mammalian and avian receptors for influenza viral infection, so swine are though to be a mixing vessel for genetic reassortment between human and avian influenza. Recently, porcine respiratory disease complex (Ellis et al.) has heavily impacted the pig industry in Taiwan; and the swine influenza virus is one of the pathogens commonly involved. The viremic stage of influenza viral infection in the pigs is usually short or transient, which hamper the detection or isolation of this virus from the field herds. Consequently, detection of cell associated viral nucleic acid and antigens may be more helpful than isolation and evaluation of the clinical viral infected cases and advancing subtyping. In this study, we tried to develop more rapid diagnostic assays for detections and subtyping specific subtype of swine influenza viruses, additionally, to correlate viral distribution and pneumonic lesions in the field cases. In situ hybridization (ISH) and immunohistochemisty (IHC) for the formalin-fixed, paraffin-embedded lung tissues and RT-PCR for homogenates of lung tissues were applied for this purpose. ISH probe of NP gene has successfully detected influenza virus infection, and positive signals are present in the bronchial and bronchiolar epithelium or mononuclear inflammatory cells in the necrotic and proliferative areas. Subtyping RT-PCR for H1, H3, N1 and N2 which could distinguish the common subtypes (H1N1, H3N2 and H1N2) of swine influenza virus in Taiwan has been established simultaneously. The monoclonal antibodies for NP, H1 and H3 have applied to detect and subtype influenza virus by IHC. The distributions of viral antigens revealed by IHC matched well with that ISH, but positive signals of seormucinous glands only revealed by IHC. The results of IHC retrospective study of 20 ISH positive cases indicated that H1 and H3 subtype SIV may infect together commonly in Taiwan.