Detection of Hepatitis E virus (HEV) Infection in Farm Pigs, Zoo Animals and Environmental Rodents in Taiwan

E型肝炎病毒 (Hepatitis E virus；HEV) 是經糞口途徑傳播，為許多發展中國家和工業化國家非A及非B型肝炎中的主要病因。美國及日本可在豬隻及野生動物身上發現HEV感染的病例，因此HEV在台灣地區可能也是一個重要的人畜共通疾病。因此本研究針對豬隻、動物園動物及環境野鼠進行HEV檢測。本研究收集2006年至2007年台灣南部地區病死或病弱豬隻196例，以RT nested- PCR檢測其HEV陽性率為7.65% (15/196)，在不同的檢測樣本中，以糞便樣本檢出率較高，為10.00% (7/70)，肝臟樣本陽性率為5.22% (7/134)，膽汁樣本陽性率為2.11% (2/95)。若以感染豬隻年齡進行分析，主要以4至9週齡豬隻的陽性比例較高，約為9.65% (11/114)，0至4週齡陽性比例為4.26% (2/47)，9至16週齡陽性比例為6.67% (2/30)，至於在16週齡以上豬隻的陽性比例為0% (0/5)。將陽性PCR產物經核苷酸定序，並與5株台灣HEV分離株的基因序列比對結果，其相似性達80.8%∼93.7%，與其他國家HEV genotype 4相似性達84.0%~88.9%，與genotype 2及genotype 3的序列相似性分別為66.6%∼68.6%及71.8%∼76.3%。本研究之動物園動物及野鼠肝臟樣本均無陽性病例檢出 (0/144 及0/12)。利用原位雜交法 (In-situ hybridization；ISH) 和免疫組織化學染色 (Immunohistochemistry；IHC) 偵測HEV核酸及抗原分佈位置，在肝細胞及庫氏細胞 (Kupffer cells) 之細胞質內可見陽性訊號，IHC可在膽管上皮細胞表面發現陽性訊號。本實驗結果顯示台灣豬隻已經有HEV感染病例出現，其中又以4至9週齡豬隻的陽性比例較高，另外，核酸序列分析結果也證實台灣HEV應屬於一種本土特異株。Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries and is also endemic in many industrialized countries. The recent discovery of HEV in domestic pigs and wild animals in United States and Japan, respectively, has suggested that HEV may be an important zoonotic issue in Taiwan. Thus, the aim of this study was to investigate HEV infection in farm pigs, zoo animals and environmental rodents in Taiwan. Between 2006 and 2007, we collected 17 serum samples, 136 liver samples, 102 bile samples and 104 fecal samples from pig farms; 13 liver samples of primates, 52 mammals' liver samples, 45 reptiles' liver samples and 34 avian liver samples from Taipei city zoo and 14 liver samples from environmental rodents. HEV RNA was detected by reverse transcriptase nested polymerase chain reaction (RT nested-PCR) from homogenates of the samples. HEV positive signals were detected in 7 of 134 (5.22%) liver samples, 2 of 95 (2.11%) bile samples and 7 of 70 (10.00%) fecal samples from pig farms. The porcine serum samples and all liver samples from zoo animal and environmental rodent were all negative. The highest HEV positive rate (9.65%) was in pigs of 4 to 9 weeks age, followed by 6.67% and 4.26% in pigs of 9 to 16 weeks and 0 to 4 weeks age, respectively; and none in pigs older than 16 weeks age. The identity of nucleotide sequences of PCR products was 80.8%∼93.7% between swine and human HEV strains in Taiwan, 84.0%~88.9% between genotype 4 strains from different countries, and 66.6%∼68.6% and 71.8%∼76.3% between genotype 2 and genotype 3 strains. By in-situ hybridization (ISH) and immunohistochemistry (IHC), the positive signals were detected in cytoplasm of hepatocytes and kupffer cells. In addition, the positive signals were also detected on the luminal surface of bile epithelial cells by IHC. The HEV infections of pigs in Taiwan are confirmed in this study. The most susceptible age is from 4 to 9 weeks old. Besides, the result of sequence analysis also supports that HEV of Taiwan isolated belongs to an unique local or regional strain.目錄 中文摘要------------------------------------------------------------------------------ I 英文摘要------------------------------------------------------------------------------ II 目錄------------------------------------------------------------------------------------ IV 表次------------------------------------------------------------------------------------ VI 圖次------------------------------------------------------------------------------------ VI 第一章 序言------------------------------------------------------------------------ 1 第二章 文獻探討------------------------------------------------------------------ 3 第一節 前言----------------------------------------------------------------------- 3 1-1 肝臟的一般正常結構、型態與生理-------------------------------- 3 1-2 肝炎之分類-------------------------------------------------------------- 5 1-2-1 A型肝炎----------------------------------------------------------------- 5 1-2-2 B型肝炎----------------------------------------------------------------- 6 1-2-3 C型肝炎----------------------------------------------------------------- 6 1-2-4 D型肝炎----------------------------------------------------------------- 6 1-2-5 E型肝炎----------------------------------------------------------------- 7 第二節 E型肝炎病毒----------------------------------------------------------- 7 2-1 E型肝炎病毒之特性、型態及構造-------------------------------- 7 2-2 E型肝炎病毒之臨床症狀-------------------------------------------- 9 2-3 E型肝炎病毒之流行病學-------------------------------------------- 11 2-3-1 人之流行病學---------------------------------------------------------- 11 2-3-2 豬之流行病學---------------------------------------------------------- 13 2-3-3 其他動物之流行病學-------------------------------------------------- 15 第三節 E型肝炎診斷方法----------------------------------------------------- 17 3-1 巢式聚合酶鏈鎖反應-------------------------------------------------- 17 3-2 原位雜交----------------------------------------------------------------- 18 3-3 免疫組織化學染色----------------------------------------------------- 19 第三章 材料與方法-------------------------------------------------------------- 22 第一節 實驗設計及流程圖------------------------------------------------------ 22 第二節 實驗材料------------------------------------------------------------------ 23 2-1 材料收集------------------------------------------------------------------ 23 2-2 材料處理------------------------------------------------------------------ 23 第三節 實驗方法------------------------------------------------------------------ 23 3-1 反轉錄聚合酶鍊鎖反應------------------------------------------------ 23 3-1-1 RNA之萃取-------------------------------------------------------------- 23 3-1-2 反轉錄反應--------------------------------------------------------------- 24 3-1-3 G3PDH之聚合酶鏈鎖反應溶液成分及條件----------------------- 24 3-1-4 G3PDH之聚合酶鏈鎖反應產物電泳-------------------------------- 25 3-1-5 HEV之巢式聚合酶鏈鎖反應溶液成分及條件--------------------- 26 3-1-6 HEV之巢式聚合酶鏈鎖反應產物電泳------------------------------ 26 3-1-7 巢式聚合酶鏈鎖反應產物的直接核苷酸定序--------------------- 27 3-2 HEV探針之合成-------------------------------------------------------- 27 3-3 免疫膜點法--------------------------------------------------------------- 27 3-4 原位雜交------------------------------------------------------------------ 28 3-5 免疫組織化學染色------------------------------------------------------ 29 第四章 結果----------------------------------------------------------------------- 31 第一節 實驗動物之背景----------------------------------------------------- 31 1-1 豬隻樣本------------------------------------------------------------- 31 1-2 動物園野生動物樣本----------------------------------------------- 31 1-3 環境中野鼠樣本----------------------------------------------------- 32 第二節 聚合酶鏈鎖反應結果------------------------------------------------ 36 2-1 G3PDH之聚合酶鏈鎖反應之結果-------------------------------- 36 2-2 HEV之巢式聚合酶鏈鎖反應之結果------------------------------ 36 2-3 HEV陽性豬隻年齡分佈分析-------------------------------------- 37 2-4 巢式聚合酶鏈鎖反應產物直接核苷酸定序之結果--------------- 43 第三節 HEV探針製備之結果----------------------------------------------- 45 第四節 原位雜交與免疫組織化學染色結果------------------------------- 45 4-1 HEV原位雜交結果------------------------------------------------- 45 4-2 HEV免疫組織化學染色結果-------------------------------------- 45 第五章 討論------------------------------------------------------------------ 50 第六章 參考文獻------------------------------------------------------------ 59 附錄------------------------------------------------------------------------------------ 69 附錄一 豬隻各檢體G3PDH及HEV RT-PCR/ RT nested-PCR結果---- 69 附錄二 引子及其可檢出各型HEV之分析----------------------------------- 79 表次 Table 4-1 Various species of primate samples collected from the zoo ------ 33 Table 4-2 Various species of mammal samples collected from the zoo------ 33 Table 4-3 Various species of avian samples collected from the zoo---------- 34 Table 4-4 Various species of reptile and amphibian samples collected from the zoo-------------------------------------------------------------------- 35 Table 4-5 Detection of G3PDH in liver, bile and feces samples from pigs.-- 38 Table 4-6 Detection of G3PDH in liver sample of primate, mammal and environmental rodent from zoo.--------------------------------------- 38 Table 4-7 Nested-PCR results of HEV in the sample of liver, bile, feces and serum from pigs.--------------------------------------------------- 38 Table 4-8 Detection of hepatitis E virus (HEV) in the sample of liver, bile and feces from pigs of various ages.---------------------------------- 39 Table 4-9 Detection of hepatitis E virus (HEV) from pigs of various ages.- 39 圖次 Fig 2-1 Time course of hepatitis E virus infection ------------------------- 10 Fig 2-2 Histopathological examination-------------------------------------- 14 Fig 3-1 Experiment design and its flow chart------------------------------- 22 Fig 4-1 RT-PCR result of G3PDH from selected porcine samples.-------- 40 Fig 4-2 Detection of HEV RNA from pig samples by PCR.--------------- 41 Fig 4-3 Detection of HEV RNA from pig samples by nested PCR.------- 42 Fig 4-4 Sequene distances of swine and human HEVs based on the nucleotide sequences of the ORF2 gene.--------------------------- 44 Fig 4-5 Phylogenetic analysis of swine and human HEVs based on the nucleotide sequences of the ORF2 gene.--------------------------- 44 Fig 4-6 Electrophoresis results of HEV plasmid by RT-PCR.-------------- 46 Fig 4-7 Electrophoresis results of HEV probe by DIG-labeling PCR.----- 46 Fig 4-8 Detection of HEV nucleic acid in the liver of HEV positive pigs by in situ hybriduzation (ISH)----------------------------------------- 47 Fig 4-9 Detection of HEV antigen in the liver of HEV positive pigs by immunohistochenistry (IHC)------------------------------------------ 48 Fig 4-10 Detection of HEV antigen in the liver of HEV positive pigs by immunohistochenistry (IHC)------------------------------------------ 4