人類雙埃柯病毒 (Human Parechovirus, HPeV) 為一種新現型的人類致病性病毒，在分類上屬於微小RNA病毒科 (Picornaviridae)。以往被歸類於腸病毒屬，但後來發現它們在複製與轉譯的特性上仍有明顯差異，因此被獨立出來自成一屬。感染大多是產生較輕微的腸胃道或呼吸道症狀，但也可能會造成腦（膜）炎或新生兒敗血症等嚴重的疾病。高雄榮總於2007年在一名病患的臨床檢體分離出一株本土型的HPeV病毒株，經鑑定後發現與一株第一型人類雙埃柯病毒的德國株（HPeV1 strain 7555312）在基因序列的相似度最高，後經確認這株病毒歸類於HPeV1 1B群型，將之命名為HPeV1 KVP6，並將其完整的核苷酸序列公開歸檔於NCBI GenBank資料庫中，編號KC769584。與其他型別比較後，發現KVP6在基因序列中P2與P3片段發生了型別間重組（intertypic recombination）情形。此基因重組現象與 HPeV1 病毒演化及其致病性之關連性，仍待進一步探討。在 HPeV1的感染特性研究中，我們針對 HPeV1 感染細胞後所誘發的先天性免疫反應（innate immunity）進行分析，以瞭解 HPeV1 與宿主抗病毒反應之間的調控機制。結果發現 HPeV1 可以感染腸道腫瘤細胞（T84 cells）、肺臟腫瘤細胞（A549 cells） 與神經膠質腫瘤細胞（DBTRG-5MG），此實驗室分析結果與臨床上這株病毒常造成腸胃道、呼吸道以及中樞神經疾病的情況吻合。在病毒增殖分析實驗中，發現 HPeV1 在細胞中連續增殖培養數代之後，其細胞毒性（cytopathic effect）及病毒量會增加，並且有基因點突變（point mutation）的產生。進一步分析 HPeV1 對溫度的敏感性，可發現在攝氏 39 度這種高於人體正常生理溫度的培養環境下，不利於HPeV1病毒粒子的合成。在宿主抗病毒機制方面，A549 細胞經 HPeV1 感染後，干擾素調控轉錄因子interferon regulatory factor 3（IRF3）會被磷酸化，使第一型干擾素（type I interferon，type I IFN）的表達增加。第一型干擾素抗病毒訊息進一步活化後，可能使 HPeV1病毒在細胞內的複製獲得控制；然而我們同時發現 T84 細胞對第一型干擾素的刺激較不敏感，在T84細胞觀察不到類似第一型干擾素抗病毒機制的活化現象，顯示 HPeV1 誘發之宿主抗病毒機轉具有細胞特異性。此外我們也觀察到，受 HPeV1 感染的細胞，對外加第一型干擾素的刺激已無法有效反應，此現象顯示 HPeV1 具有迴避第一型干擾素之機制，並抑制細胞抗病毒之能力。透過本研究，我們對新興病毒 HPeV1 的感染特性有了更進一步的認識，對往後臨床上的診斷、預防與治療將有很大的幫助。

Human parechoviruses (HPeVs), members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis. A new contemporary strain of HPeV1, KVP6 (accession no. KC769584), was isolated from a clinical specimen. Full-genome alignment revealed that HPeV1 KVP6 shares high genome homology with the German strain of HPeV1, 7555312 (accession no. FM178558) and could be classified in the clade 1B group. An intertypic recombination was shown within the P2-P3 genome regions of HPeV1. Cell-type tropism test showed that T84 cells (colon carcinoma cells), A549 cells (lung carcinoma cells) and DBTRG-5MG cells (glioblastoma cells) were susceptible to HPeV1 infection, which might be relevant clinically. A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages. HPeV1 is sensitive to elevated temperature because 39C incubation impaired virion production. HPeV1 induced innate immunity with phosphorylation of interferon (IFN) regulatory transcription factor 3 and production of type I IFN in A549 but not T84 cells. Furthermore, type I IFN inhibited HPeV1 production in A549 cells but not T84 cells; T84 cells may be less responsive to type I IFN stimulation. Moreover, HPeV1-infected cells showed downregulated type I IFN activation, which indicated a type I IFN evasion mechanism. The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.

中文摘要 i
英文摘要 iii
目錄 iv
圖次目錄 vi
表次目錄 vii
Chapter 1. Introduction 1
1.1 Historical background and classification of human parechovirus 1
1.2 Biology, receptors and cell entry of HPeV 1
1.3 Clinical significance of HPeV 3
1.4 Research experience obtained from the study of Aichi virus 4
Chapter 2. Materials and Methods 6
2.1 Virus, cell lines and reagents 6
2.2 Viral titration 6
2.3 RNA extraction, viral genome sequence and quantitative real-time PCR 7
2.4 Phylogenetic analysis and similarity plots 9
2.5 Antibodies 10
2.6 Immunofluorescence assay 10
2.7 Immunoblotting analysis 10
2.8 Cell proliferation assay 11
2.9 Luciferase reporter assay 11
Chapter 3. Results 12
3.1 HPeV1 KVP6 genome sequence and analysis 12
3.2 HPeV3 VGHKS-2007 genome sequence and analysis 13
3.3 Cell type tropism of HPeV1 14
3.4 HPeV1 progeny analysis and the kinetics of replication 14
3.5 The effect of incubation temperature on HPeV1 replication 16
3.6 Cell type-dependence of efficiency of type I IFN against HPeV1 17
3.7 HPeV1 attenuates type I IFN signaling 18
Chapter 4. Discussion 20
4.1 HPeV1 KVP6 genome recombination 20
4.2 HPeV1 replication kinetics is affected by incubation temperature 21
4.3 The innate immune response of HPeV1-infected cells 22
4.4 Comparisons between HPeV1 and Aichi virus. 23
Figures and figure legends 25
References 47
Supplemental materials 56

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