臺灣博碩士論文加值系統

星狀病毒(Astrovirus)首次於下痢兒童檢體發現，可感染人類及動物，多與腸胃道疾病相關。自 2012 年起，陸續報導發現實驗小鼠星狀病毒(murine astrovirus, MuAstV)的自然感染病例。依國外數據顯示，此病毒在實驗小鼠的 感染率很高，但對 MuAstV 在國內實驗小鼠的感染情形卻並不清楚。本研究針 對台灣實驗小鼠族群的 MuAstV 感染情形與台灣 MuAstV 病毒株特性作進一步 的調查與研究。本研究中，依據國外已發表實驗小鼠星狀病毒的基因序列設計 引子，以 RT-PCR 進行初步調查台灣的實驗小鼠是否有感染星狀病毒，經由病 毒基因複製與序列分析，共發現 18 株台灣實驗小鼠星狀病毒株(MuAstV TWs; TW1 - 18)。並將所發現的台灣實驗小鼠星狀病毒株(MuAstV TWs)，經由轉 譯酶與鞘膜蛋白的核苷酸片段序列演化樹分析發現，台灣的 MuAstV 分為兩個 群組，其中一群包括部分台灣病毒株以及大部分的美國病毒株，另外一群則以 台灣病毒株為主，因此建議台灣應有一群新的實驗小鼠星狀病毒變異株。進一 步利用基因重組分析時，更發現病毒株 MuAstV TW3 有病毒基因重組交換 (intra-genotypic recombination)的現象，此乃首篇 MuAstV 病毒基因重組的報 導。將具有完整病毒基因序列的 TW1 與 TW2 兩株病毒進行核苷酸與預期氨基 酸序列比對分析，顯示 TW2 與以前已發現的 MuAstV 較相似，然而 TW1 卻與過去發現的 MuAstV 差異性較大，建議 TW1 病毒株應歸為 MuAstV 的新亞型 (novel subtype)。本研究為首篇於美國以外地區發現小鼠星狀病毒完整基因序 列的報告，且是首次發現 MuAstV 有病毒基因重組的證據;同時調查資料顯示 台灣實驗小鼠的星狀病毒感染率可高達 45.7%(48/105)，指出小鼠星狀病毒 是一個台灣實驗動物界應正視的新興傳染性疾病問題。

Astroviruses are non-enveloped, positive-sense, single-stranded RNA viruses and often associated with gastrointestinal diseases. In 2011, murine astroviruses (MuAstV) were first detected in laboratory mouse colonies. Although infected mice do not present significant clinical symptoms, viruses might interfere the research results. So, monitoring the infectious status of murine astroviruses in laboratory mice is very important now. Recently, the surveillance data indicates that murine astroviruses are highly prevalent in laboratory mice in U.S. and Japan. The aims of our study are to identify the novel murine astroviruses in laboratory mice in Taiwan, to compare the genome sequences with previously identified astroviruses, and to survey the MuAstV infection rate in laboratory mouse colonies in Taiwan. In this study, a total of 18 MuAstV variants (MuAstV TWs) were identified in laboratory mice. The phylogenetic trees of ORF1b and ORF2 nucleotide sequences of MuAstVs identified in laboratory mice revealed that 18 MuAstV TWs were separated into two subclusters. One subcluster was closely related to MuAstVs identified in U.S. and the other subcluster mainly contained MuAstVs identified in Taiwan and considered as a new subtype of MuAstV variants. The evidence of an intra-genotypic recombination among two subclusters of MuAstV TWs was recognized in one MuAstV TW variant and this is the first report of murine astrovirus with the intra-genotypic recombination. Two completely characterized murine astroviruses in Taiwan, MuAstV TW1 and MuAstV TW2 exhibited 89% and 94% nucleotide identities with the MuAstV published in 2012, respectively. Molecular surveillance of MuAstV infection in Taiwan revealed that 45.7% mice (48/105) were positive and 7 out of 9 colonies (77.8%) were contaminated. The present study has described the molecular characterization of novel subtype of murine astroviruses, revealed the first recombination event of MuAstV, and demonstrated a distinctly high infection rate of MuAstV in the laboratory mouse colonies in Taiwan.

ACKNOWLEDGMENTS ........................................................................................... I ABSTRACT IN CHINESE ........................................................................................II ABSTRACT IN ENGLISH ...................................................................................... IV TABLE OF CONTENTS ......................................................................................... VI LIST OF FIGURES .................................................................................................VII LIST OF TABLES ................................................................................................. VIII INTRODUCTION........................................................................................................1
MATERIALS AND METHODS ..............................................................................10 Animals .............................................................................................................................. 10 Oligonucleotide primers ................................................................................................... 11 RNA extraction and reverse transcription ..................................................................... 12
DNA amplification by PCR .............................................................................................. 13
DNA sequencing and analysis .......................................................................................... 14
RESULTS ...................................................................................................................20
Detection of the newly identified MuAstV ...................................................................... 20
Molecular characterization of MuAstV TW1 and MuAstV TW2 ................................ 23 The surveillance of MuAstV of laboratory mice in Taiwan. ......................................... 26
DISCUSSION .............................................................................................................36 Astroviruses in rodents ..................................................................................................... 36 Recombination of astrovirus genome .............................................................................. 39
Genomic comparison of astroviruses in mammals......................................................... 40
Significance of subclinical viral infection in laboratory mice ....................................... 43
The concern of MuAstV infection in laboratory colonies.............................................. 45 Conclusion.......................................................................................................................... 47
REFERENCES...........................................................................................................51 APPENDIX.................................................................................................................65

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