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研究生:劉安庭
研究生(外文):An-Ting Liou
論文名稱:以免疫健全及免疫缺陷小鼠為模型,研究腸病毒71型感染之致病機轉與治療策略
論文名稱(外文):Studies on pathogenesis and therapy of Enterovirus 71 infection in immunocompetent and immunodeficient mouse models
指導教授:施嘉和
指導教授(外文):Chiaho, Shih
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:60
中文關鍵詞:腸病毒71型動物模型免疫缺陷免疫健全
外文關鍵詞:Enterovirus 71animal modelImmunodeficientImmunocompetent
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腸病毒71型對於全球的孩童而言,是一重要且具威脅性的感染疾病,被病毒感染的兒童,輕則無明顯的臨床症狀,或是出現手足口症;重則引起腦炎、癱瘓、肺水腫,乃至於死亡。對於研究腸病毒71型病理機制而言,更方便操作且結果顯著的動物模型是必要的。
在本研究中,我們建立了三種不同可被臨床病毒株感染的動物模型。第一種動物模型已發表於Journal of Virology。在此模型中,我們發現了stat-1免疫缺陷小鼠被腸病毒71型感染後,會引起癱瘓以及死亡的現象。我們收集了肌肉組織、腦部、脊髓等組織,比較了發病前、中、後期在小鼠體內VP1 RNA和蛋白質的表現,發現了stat-1免疫缺陷小鼠中,腸病毒71型有以下特殊的病理現象: (1) 在中樞神經系統表現出高度神經特異性 (2) 病毒蛋白VP1在小腦中的Purkinje layer、橋腦、腦幹以脊髓組織中大量表現 (3) 微膠細胞的大量增生。雖然stat-1免疫缺陷小鼠可被腸病毒71型感染成功,但其發病率只有30%;因此,我們想建立更強大的動物模型,以提供研究者進行腸病毒71型的病理機制研究。第二種動物模型也已發表於Scientific Reports。第二種模型建立的的策略是透過雜交SCARB2基因轉殖鼠及stat-1免疫缺陷小鼠,建立全新的雜交動物模型(hSCARB2 +/+/ stat-1 -/-),並比較此動物模型與種鼠之間對於腸病毒71型的易感性及病理機制。我們在雜交小鼠模型中發現腦部和脊隨有病毒蛋白VP1 沿著神經纖維分布的現象;此外, 雜交小鼠可在兩週齡時,以腹腔注射的感染途徑,被不同基因型的腸病毒71感染,且濃度只需種鼠的千分之一倍即可成功。被腸病毒71型感染的雜交小鼠模型,比起種鼠更早發生中樞神經的病變,同時有更高的比例產生肢體癱瘓及死亡。上述雜交小鼠的優點,我們認為此動物模型,無論在基礎研究、臨床前期治療或病理研究上,均可符合腸病毒71型領域的急迫需要。
第三種動物模型是以免疫健全的老鼠為主,關於此部分的結果由於尚在進行投稿的階段,故在論文初稿中,實驗結果暫不展示,待定稿後再公開。關於此篇研究的標題以及英文摘要,附於本稿中的第v頁及第vi頁。
Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis.
In this study, we established three animal models which support clinical isolates of EV71 in vivo infection. The first animal model was published on the Journal of Virology, titled “Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of Enterovirus 71 (Liao and Liou, et al.). ” First, we demonstrated that EV71 infect stat-1 knockout (KO) mice successfully and develop paralysis and death. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared in stat-1 KO models before, during, and after disease onset. The characteristic pathology of the stat-1 KO model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells. Since we found that stat-1 KO mice can be infected with clinical isolates of EV71, the rate of disease manifestation was only 30%. A more robust animal model was still required for investigating EV71 pathogenesis. Therefore, we established the second animal model -- a hybrid (hSCARB2 +/+/ stat-1 -/-) mouse strain, which was published on Scientific Reports, titled “A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71” (Liou, et al., 2016). This hybrid strain was generated from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. In the hybrid strain, VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an i.p. route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis.
The third mouse model was an immunocompetent mouse model. The manuscript of this animal model is submitted with title “High therapeutic efficacy with interferon-alpha against clinical isolate of enterovirus 71 in a novel immune competent mouse model”. The title and abstract was attached on page v and vi.
Acknowledgments ---------------------------------- i
Chinese Abstract --------------------------------- ii
English Abstract ----------------------------------iii
List of Figures ---------------------------------- vii
List of Tables -----------------------------------ix
Chapter 1 Introduction ------------------------------1
1.1 Epidemiology of EV71 infection --------------------1
1.2 Human receptors support EV71 infection ----------- 1
1.3 Small animal models in EV71 infection ------------ 2
1.4 The objective of this study ---------------------- 4
Chapter 2 Results ------------------------------------ 5
2.1 stat-1 KO mice infected with clinical isolates of EV71 successfully------------------------------------------ 5
2.2 stat-1 KO mice developed CNS pathological changes infected with EV71------------------------------------ 5
2.3 Inflammatory cytokines were secreted in various tissues ---------------------------------------------- 7
2.4 Generation of hSCARB2 +/+/stat-1-/- hybrid mouse model
----------------------------------------------------7
2.5 Comparing infection efficacy among parental mouse strains and hybrid mouse ---------------------------- 8
2.6 Hybrid strain mice were successfully infected with clinical isolates of EV71 at an older age -------------- 9
2.7 CNS inflammation in EV71-infected hybrid mouse model
---------------------------------------------------- 10
2.8 IFNs or SCARB2 expression affected susceptibility of EV71 in hybrid mouse model ?
-----------------------------------------------------12
Chapter 3 Discussion
3.1 Non-transgenic mouse models supported EV71 in vivo infection. -------------------------------------------13
3.2 Immunodeficiency or mouse genetic background is important to EV71 infection? ------------ 13
3.3 Pathogenesis in the stat-1 KO mouse model. Limb paralysis due to myositis or CNS involvement?
--------------------------------- 13
3.4 Viral spread pathways. --------------------- 14
3.5 Failed in oral infection via a natural enteric route in stat-1 KO mouse model. --------------------- 14
3.6 Advantage of hybrid mouse strain. -------------15
3.7 Age dependent in parental and hybrid strain. ------ 16
3.8 Neurotropism of EV71 in hybrid mouse model. ------- 16
Chapter 4 Methods
4.1 Mouse model -------------------------------- 18
4.2 Cell and virus preparation ---------------------- 20
4.3 Pathological Examination -------------------------21
4.4 Real time reverse transcription—PCR ------------- 21
4.5 Cytokine quantification ------------------------- 22
4.6 Immunoblotting ---------------------------------- 22
4.7 Statistical analysis ---------------------------- 23
References--------------------------------------------55



List of Figures
Figure 1. Clinical score of stat-1 KO mice infected with EV71 through intraperitoneal (ip) or subcutaneous (sc) injection.-------------------------------------------- 24
Figure 2. Limb paralysis occurred in stat-stat 1 KO mice infected with EV71. -----------------------------------25
Figure 3. Viral VP1 RNA was detected in CNS in stat-1 KO mice infected with EV71.------------------------------ 26
Figure 4. Histopathological examination of brain, spinal cord, intestine, muscle, and spleen, by H&E and Masson’s staining. ---------------------------------------------27
Figure 5. VP1 protein was expressed in brain of EV71 infected mice detected by IHC staining. -------------- 28
Figure 6. Preferential VP1 expression in spinal cord.-29
Figure 7. Detection of microglia marker, IBA1, in CNS.-30
Figure 8. Side-by-side kinetic comparisons of viral RNA, infectivity and VP1 protein expression in muscle, brain, and spinal cord, between experimental infections of NOD/SCID and stat-1 KO mouse models.----------------- 31
Figure 9 The construct of human SCARB2 (hSCARB2) cDNA in generating transgenic mouse.---------------------------33
Figure 10. Generation of a hybrid mouse model SCARB2 /stat-1 KO by crossbreeding hSCARB2 transgenic mice and stat-1 KO mice. ----------------------------------- 34
Figure 11. The hybrid mouse strain hSCARB2+/+/stat-1 -/- is most susceptible to infection and pathogenesis.--- 36
Figure 12. Young age is critical for the clinical score and survival rate in the hybrid mouse model infected with EV71. ----------------------------------------------- 38
Figure 13. Histopathological examination of CNS sections in three different mouse models. -------------------- 39
Figure 14. Histopathological comparisons of sectioned muscle in parental and hybrid mice with or without EV71 infection. ------------------------------------------ 41
Figure 15. VP1 streaking in nerve fibers of brain and spinal cord. ---------------------------------------- 42
Figure 16. Loss of Nissl Bodies in infected spinal cord of hybrid mice. -------------------------------------- 43
Figure 17. IBA1, a microglial cell marker was increased in EV71-infected spinal cord. ----------------------- 44
Figure 18. Expression profiles of EV71 specific RNA, protein in CNS of infected hybrid strain mice before, during, and after onset of limb paralysis. ---------- 45
Figure 19. Expression profiles of cellular cytokines in CNS of infected hybrid strain mice before, during, and after onset of limb paralysis.----------------------- 47
Figure 20. Comparisons of VP1, hSCARB2 and interferon expressions between 2-week- and 3-week-old hybrid mice infected with EV71. -------------------------------- 48
Figure 21. Histopathological comparisons of sectioned muscle in parental and hybrid mice with or without EV71 infection. ------------------------------------------ 50


List of Tables
Table 1 Studies on various immunodeficient mice infected with a clinical isolate of EV71 strain F23 (genotype B5).
----------------------------------------------------- 52

Table 2 Tissue cytokine profiles in EV71-infected stat-1 KO mouse model. --------------------------------------53

Table 3 Summary of disease manifestations in three different animal models. ----------------------------- 54
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