在台灣的HIV-1感染者以男同性戀者為主，感染之病毒亞型主要為B亞型；靜脈注射毒癮者次之，感染之病毒亞型以CRF07_BC為主；而異性戀者感染之病毒亞型有B亞型、CRF07_BC和CRF01_AE。由於有多種病毒亞型在台灣同時流行，感染者體內存在不同之病毒株或多重感染的機會也相對提高。過去由於技術上的限制，當HIV-1感染者體內存在兩種或以上病毒時，很難以傳統桑格氏定序偵測。本研究期以具更高敏感性之次世代定序系統，來了解台灣是否有多重感染發生及其在抗藥性基因型檢測的應用性。
本研究首先評估次世代定序系統之引子在不同亞型與不同循環重組模式中是否適用，再評估引子在台大醫院HIV抗藥性篩檢資料庫中不同亞型是否適用，最後以臨床病人檢體進行實際測試。當病人之HIV蛋白酶和反轉錄酶區域的桑格氏定序結果，分別被定義為不同的基因型，便被認為可能是共感染或多重感染或經過重組。我們提供三位疑似多重感染患者與九名其餘亞型之患者，並使用羅氏454次世代定序系統調查他們是否是共感染或多重感染，接著在針對一多重感染患者做長達6年之回溯性研究。
結果顯示次世代系統引子對於全球部份亞型及CRF可能不適用，而對台灣的B亞型與CRF07_BC適用，對CRF01_AE有較差適用性。不論是否懷疑為多重感染，部份HIV病患確實同時感染不只一種的HIV亞型。因為共同感染，一些重組病毒株確實存在於部分檢體中，證實重組確實會發生在這些感染了不同病毒株的病人體中。有部份檢體之次族群被分析出具有與主要族群不同之抗藥性變化。而在長時間的回溯性研究，可以看到病人體內不同亞型族群的改變，重組病毒株的消長，及抗藥性族群的出現與置換。本研究顯示此次世代系統適用於台灣不同亞型之抗藥性檢測與多重感染之偵測，而台灣多重感染的發生率可能被低估，需要進一步評估。

In Taiwan, the major transmisstion routes for HIV infection are men who have sex with men (MSM), injecting drug use (IDU), and hetersexual sexual contact. The major HIV subtypes in these three populations are subtype B, CRF07_BC, and subtypeB/CRF07_BC/CRF01_AE, respectively. The cocirculation of different subtypes in Taiwan has raised the concern of super-infection or co-infection by different HIV subtypes.
In the past, due to the technical limitations of Sanger sequencing, co-infection or super-infection patients were difficult to be identified. Here we used the deep sequencing to investigate the presence of super-infection or co-infection in Taiwan and to evaluate its application in the genotypic drug resistance testing.
First, we evaluated the applicability of next generation sequencing (NGS) primers in different subtypes from the database and the epidemic strains amplified in Taiwan. We selected three patients whose protease and reverse transcriptase sequences were previously defined as different genotypes to evaluate whether they were infected by different HIV subtypes. Nine patients infected with dominant HIV-1 subtypes in Taiwan were selected for comparison. We used Roche 454 NGS to investigate whether these patients had super-infection, and we determined the genotypic resistance of a patient with super-infection in a longitudinal follow-up study.
Our data showed that the primers designed for NGS might not be suitable for all HIV subtypes. Super-infection (or co-infection) was observed in some cases. Because of multiple infections with HIV, recombinant strains did exist. Some undetected drug resistance mutants appeared in a minor viral population. In the longitudinal study, we observed the shift of different virus population, the rise and fall of recombinant strains, and the replacement of wild type by resistance strains.
In conclusion, the NGS can be applied to detect the genetypic drug resistance and super-infection. Furthermore, the rate of super-infection in Taiwan may have been underestimated.

目錄
致謝 VIII
中文摘要 IX
英文摘要 X
第一章 前言 1
1-1人類免疫缺乏病毒 1
1-1-1病毒簡介 1
1-1-2 一般病毒亞型與循環重組形式 1
1-1-3 病毒傳染模式與危險族群 2
1-1-4 台灣HIV感染歷史與現況 2
1-2高效率抗反轉錄病毒藥物治療 3
1-2-1 高效率抗反轉錄病毒治療藥物簡介 3
1-2-2 核苷酸類反轉錄酶抑制劑（NRTI） 3
1-2-3 非核苷酸反轉錄酶抑制劑（NNRTI） 4
1-2-4 蛋白酶抑制劑（PI） 4
1-2-5 其餘抗反轉錄病毒藥物 4
1-2-6 治療指引 5
1-2-7 抗藥性基因突變介紹 5
1-3 共感染或多重感染人類免疫缺乏病毒 6
1-3-1 共感染或多重感染HIV簡介 6
1-3-2 共感染或多重感染之結果 6
1-3-3 共感染或多重感染之偵測 7
1-4 次世代定序系統 8
1-4-1 定序系統之發展 8
1-4-2 次世代定序系統之簡介 8
1-4-3 次世代定序系統在HIV的應用 10
第二章 實驗動機與目的 12
第三章 實驗方法與材料 13
3-1 實驗材料 13
3-1-1 檢體 13
3-1-2 試劑 13
3-1-3 特殊實驗用具 13
3-1-4 實驗儀器 13
3-2 實驗方法 14
3-2-1 病毒RNA抽取 14
3-2-2 病毒RNA反轉錄反應 14
3-2-3 擴增子生成反應 15
3-2-4 擴增子的純化 16
3-2-5 以Picogreen螢光染料定量DNA濃度 16
3-2-6 委託羅氏藥廠進行最終反應 17
3-2-7 統計方法與分析軟體 18
第四章 實驗結果 21
4-1 引子的適用性分析 21
4-2 檢體的挑選 22
4-3 檢體前處理之結果 22
4-4 懷疑為多重感染之檢體分析結果 23
4-5 B亞型之檢體分析結果 25
4-6 CRF01_AE之檢體分析結果 26
4-7 CRF07_BC之檢體分析結果 27
4-8 CRF07_BC之重複檢體分析結果 29
4-9 單一檢體之回溯性研究 29
第五章 實驗討論 32
第六章 參考文獻 38

 
圖目錄

圖一、 次世代定序系統檢體編號1-1之(A)第一片段(RTP-I)與(B)第二片段(RTP-II) 50
圖二、 次世代定序系統檢體編號1-1之(A)第三片段(RTP-III)與(B)第四片段(RTP-IV) 51
圖三、 次世代定序系統檢體編號1-3之(A)第一片段(RTP-I)與(B)第二片段(RTP-II) 52
圖四、 次世代定序系統檢體編號1-3之(A)第三片段(RTP-III)與(B)第四片段(RTP-IV)與(C)第四片段中的CRF01_AE 53
圖五、 次世代定序系統檢體編號3之第三與第四片段重疊序列比較 54
圖六、 次世代定序系統檢體編號1-10之(A)第一片段(RTP-I)與(B)第二片段(RTP-II)與(C)第三片段(RTP-III)與(D)第四片段(RTP-IV) 55
圖七、 次世代定序系統檢體編號1-6之(A)第一片段(RTP-I)與(B)第三片段(RTP-III)與(C)第四片段(RTP-IV) 56
圖八、 次世代定序系統檢體編號1-9之(A)第一片段(RTP-I)與(B)第三片段(RTP-III)與(C)第四片段(RTP-IV) 57
圖九、 次世代定序系統檢體編號2-1之(A)第一片段(RTP-I)與(B)第一片段(RTP-II) 58
圖十、 次世代定序系統檢體編號1-4之(A)第一片段(RTP-I)與(B)第二片段(RTP-II)與(C)第三片段(RTP-III)與(D)第四片段(RTP-IV) 59
圖十一、 次世代定序系統檢體編號1-5之(A)第一片段(RTP-I)與(B)第二片段(RTP-II)與(C)第三片段(RTP-III)與(D)第四片段(RTP-IV) 60
圖十二、 次世代定序系統檢體編號1-2之(A)第一片段(RTP-I)與(B)第三片段(RTP-III)與(C)第四片段(RTP-IV) 61
圖十三、 次世代定序系統檢體編號1-7之(A)第一片段(RTP-I)與(B)第二片段(RTP-II)與(C)第三片段(RTP-III)與(D)第四片段(RTP-IV) 62
圖十四、 次世代定序系統檢體編號1-8之(A)第一片段(RTP-I)與(B)第二片段(RTP-II)與(C)第四片段(RTP-IV) 63
圖十五、 次世代定序系統檢體編號2-5之(A)第一片段(RTP-I)與(B)第二片段(RTP-II) 64
圖十六、 次世代定序系統檢體編號2-6之(A)第一片段(RTP-I)與(B)第二片段(RTP-II) 65
圖十七、 次世代定序系統檢體編號2-6之(C)第三片段(RTP-III)與(D)第四片段(RTP-IV) 66
圖十八、 次世代定序系統檢體編號2-7之(A)第一片段(RTP-I)與(B)第二片段(RTP-II)與(C)第三片段(RTP-III)與(D)第四片段(RTP-IV) 67
圖十九、 次世代定序系統檢體編號2-8之(A)第一片段(RTP-I)與(B)第二片段(RTP-II) 68
圖二十、 次世代定序系統檢體編號2-8之(A)第三片段(RTP-III)與(B)第四片段(RTP-IV) 69
圖二十一、 次世代定序系統檢體編號2-9之(A)第三片段(RTP-III)與(B)第四片段(RTP-IV) 70
圖二十二、 次世代定序系統檢體編號2-10之(A)第三片段(RTP-III)與(B)第四片段(RTP-IV) 71
圖二十三、 次世代定序系統檢體編號1-7與2-2之重複檢體第一片段比較 72
圖二十四、 次世代定序系統檢體編號1-7與2-2之重複檢體第二片段比較 73
圖二十五、 次世代定序系統檢體編號1-7與2-2之重複檢體第三片段比較 74
圖二十六、 次世代定序系統檢體編號1-7與2-2之重複檢體第四片段比較 75
圖二十七、 次世代定序系統檢體編號2-3之第二片段(RTP-II) 76
圖二十八、 次世代定序系統檢體編號1-8與2-4之重複檢體第一片段比較 77
圖二十九、 次世代定序系統檢體編號1-8與2-4之重複檢體第二片段比較 78
圖三十、 檢體1-7號之病毒量、CD4細胞數與用藥資訊，以及檢體時間點相關性 79
圖三十一、 長時間回溯性實驗第一片段之病毒族群分布 80
圖三十二、 長時間回溯性實驗第一片段之重組分析 81
圖三十三、 長時間回溯性實驗第二片段之病毒族群分布 82
圖三十四、 長時間回溯性實驗第三片段之病毒族群分布 83
圖三十五、 長時間回溯性實驗第四片段之病毒族群分布 84
圖三十六、 長時間回溯性實驗體內之亞型分布 85

 
表目錄

表一、黑色螢光盤之檢體對應孔 86
表二、主要類別之高效率反轉錄病毒藥物 87
表三、其餘類別之高效率反轉錄病毒藥物 88
表四、次世代定序系統擴增子生成盤引子序列 89
表五、HIV病患之數目與其基因型分布表 90
表六、所有基因型與引子結合位之差異：第一片段 91
表七、所有基因型與引子結合位之差異：第二片段 92
表八、所有基因型與引子結合位之差異：第三片段 93
表九、所有基因型與引子結合位之差異：第四片段 94
表十、次世代定序系統引子與台大醫院不同HIV亞型之引子結合位比較：第一片段 95
表十一、次世代定序系統引子與台大醫院不同HIV亞型之引子結合位比較：第二片段 96
表十二、次世代定序系統引子與台大醫院不同HIV亞型之引子結合位比較：第三片段 97
表十三、次世代定序系統引子與台大醫院不同HIV亞型之引子結合位比較：第四片段 98
表十四、第一片段不同HIV亞型之引子結合位差異分佈 99
表十五、第二片段不同HIV亞型之引子結合位差異分佈 100
表十六、第三片段不同HIV亞型之引子結合位差異分佈 101
表十七、第四片段不同HIV亞型之引子結合位差異分佈 102
表十八、次世代定序系統之檢體清單 103

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