Detection and Typing of Swine Influenza Virus

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)。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.目錄 中文摘要-------------------------------------------------------------------------- I 英文摘要-------------------------------------------------------------------------- II 目錄-------------------------------------------------------------------------------- V 表次-------------------------------------------------------------------------------- IV 圖次-------------------------------------------------------------------------------- VI 第一章 序言-------------------------------------------------------------------- 1 第二章 文獻探討-------------------------------------------------------------- 3 第一節 豬流行性感冒病毒------------------------------------------------ 3 1-1 病毒特性--------------------------------------------------------------- 3 1-2 歷史背景及經濟重要性--------------------------------------------- 4 1-3 致病機制--------------------------------------------------------------- 6 1-4 臨床症狀及病理變化------------------------------------------------ 8 第二節 病毒傳播和流行病學上的重要性--------------------------------- 10 2-1 宿主範圍及跨物種間的傳播--------------------------------------- 10 2-2 病毒抗原性改變的方式--------------------------------------------- 13 2-3 豬流行性感冒病毒於公共衛生上的影響------------------------ 14 第三節 原位雜交--------------------------------------------------------------- 16 第四節 免疫組織化學染色--------------------------------------------------- 18 第三章 材料與方法----------------------------------------------------------- 20 第一節 實驗設計及流程圖--------------------------------------------------- 20 第二節 實驗材料--------------------------------------------------------------- 21 2-1 材料收集--------------------------------------------------------------- 21 2-2 材料處理--------------------------------------------------------------- 21 第三節 實驗方法--------------------------------------------------------------- 21 3-1 反轉錄聚合酶鍊鎖反應--------------------------------------------- 21 3-1-1 RNA之萃取----------------------------------------------------------- 21 3-1-2 反轉錄反應------------------------------------------------------------ 22 3-1-3 G3PDH之PCR反應溶液成分及條件---------------------------- 23 3-1-4 SIV之PCR反應溶液成分及條件---------------------------------- 23 3-1-5 聚合酶鏈鎖反應產物電泳------------------------------------------ 25 3-1-6 PCR產物的直接核苷酸定序--------------------------------------- 26 3-2 SIV探針之合成------------------------------------------------------- 26 3-3 免疫膜點法 (Immuno-blot)----------------------------------------- 26 3-4 原位雜交 (In-situ hybridization；ISH)---------------------------- 27 3-5 免疫組織化學染色 (Immunohistochemistry；IHC)------------ 28 3-5-1 IHC 結果和肺臟病變嚴重度之評估方式 28 第四章 結果--------------------------------------------------------------------- 30 第一節 實驗動物之背景------------------------------------------------------ 30 1-1 豬隻樣本--------------------------------------------------------------- 30 1-2 實驗所採用之標準病毒株------------------------------------------ 30 第二節 聚合酶鏈鎖反應結果------------------------------------------------ 31 2-1 G3PDH之聚合酶鏈鎖反應之結果-------------------------------- 31 2-2 NP之聚合酶鏈鎖反應之結果-------------------------------------- 32 2-3 SIV之PCR分型之結果--------------------------------------------- 32 第三節 探針製備結果--------------------------------------------------------- 37 3-1 SIV 之NP探針製備------------------------------------------------- 37 3-2 SIV之H3探針製備-------------------------------------------------- 37 第四節 原位雜交之結果------------------------------------------------------ 40 4-1 NP探針原位雜交結果----------------------------------------------- 40 第五節 免疫組織化學染色之結果------------------------------------------ 42 5-1 NP之免疫組織化學染色結果-------------------------------------- 42 5-2 H1之免疫組織化學染色結果-------------------------------------- 43 5-3 H3之免疫組織化學染色結果-------------------------------------- 44 5-4 H3N1攻毒豬隻其NP、H1及H3 IHC之結果------------------ 44 第五章 討論---------------------------------------------------------------- 53 第六章 參考文獻---------------------------------------------------------- 61 Appendix ---------------------------------------------------------------------- 69 Table 1 NP IHC evaluation of 20 cases------------------------------------- 69 Table 2 H1 IHC evaluation of 20 cases------------------------------------- 70 Table 3 H3 IHC evaluation of 20 cases------------------------------------- 71 Table 4 H1 IHC evaluation--------------------------------------------------- 72 Table 5 H3 IHC evaluation--------------------------------------------------- 72 表次 Table 4-1 Date base of the SIV strains isolated from Taiwan----------------- 31 Table 4-2 Distributions and percentage of positive signals of NP, H1 and H3 IHC in 20 porcine lung samples----------------------------------- 46 圖次 Fig 4-1 Electrophoresis results of G3PDH PCR--------------------------- 33 Fig 4-2 Electrophoresis results of one-step & two-step RT-PCR for NP gene------------------------------------------------------------------- 34 Fig 4-3 Electrophoresis results of one-step RT-PCR for NP gene to detect SIV infection in clinical samples---------------------------- 35 Fig 4-4 Electrophoresis results of subtyping PCR for SIV strains in Taiwan-------------------------------------------------------------------- 36 Fig 4-5 Electrophoresis results of NP PCR for SIV positive samples-- 38 Fig 4-6 Electrophoresis results of DIG-labeling PCR for NP probe--- 38 Fig 4-7 Electrophoresis results of H3 PCR for SIV positive samples-- 39 Fig 4-8 Electrophoresis results of DIG-labeling PCR for H3 probe--- 39 Fig 4-9 In situ hybridization results of NP probe A----------------------- 41 Fig 4-10 Comparison of NP IHC results with H&E stain----------------- 47 Fig 4-11 Comparison of NP IHC results with H&E stain----------------- 48 Fig 4-12 Comparison of H1 IHC results with NP IHC results------------ 49 Fig 4-13 Comparison of H3 IHC results with NP IHC results------------ 50 Fig 4-14 The IHC results of NP, H1 and H3 in the H3N1 subtype inoculated pig------------------------------------------------------------ 51 Fig 4-15 The IHC results of H1 and H3 in the H3N1 subtype inoculated pig------------------------------------------------------------ 5