HIV-1 分子流行病學，傳染來源辨別，及發展一個即時病毒追蹤系統之研究

Molecular epidemiology, source-identification and development of a real-time virion-tracking system for HIV-1 infection

李威佑(Wei-You Li)

指導教授 ： 陳宜民

高雄醫學大學/醫學院/醫學研究所博士班/博士(2020年)

摘要

第一型人類免疫缺乏病毒 (type 1 human immunodeficiency virus, HIV-1) 自1981年被公布於世至今，已成為當代最為嚴重的傳染疾病之一。即使在高效能抗反轉錄病毒療法已問世並大為普及的現代，HIV-1與 AIDS (acquired immune deficiency syndrome) 依然在不同社會與健康議題層面上有許多尚待解決的問題。據此，本研究欲分別從分子流行病學、鑑識科學與方法學等各面向進行HIV-1 相關議題的探討。在流行病學監測的部分，我們於2013-2015年間從大台北與高雄地區參與匿名篩檢的男男間性行為者中進行個案招募。共計有 4,675 位個案參與，HIV-1總盛行率為 4.3% (201/4675)，並發現盛行率與發生率有逐年下降的趨勢，且與保護性行為因子之變化顯著相關。系統發生樹分析結果顯示不同風險族群於國內及國際間的交流有助長HIV-1傳播的可能。在傳染方向性鑑定的研究部分，其為國內首次將maximum likelihood 與 Bayesian系統發生樹分析結果作為鑑識證據呈現於法庭之案例，共有三個來自不同法庭之法律案件參與。我們亦比較了傳統以分子轉殖方式與高通量焦磷酸測序 (ultra-deep pyrosequencing, UDPS) 技術取得病毒基因序列在分析上的差異。結果顯示分子轉殖取得序列之結果可清楚呈現案件ㄧ與三之方向性，惟案件二因採血時間點延遲，需結合UDPS技術取得序列分析方可進一步輔助來源鑑定。我們的結果亦呈現了UDPS在方向性分析應用上的限制與不足，有待進一步改進。在單一病毒顆粒追蹤系統的研究中，我們意圖發展ㄧ以量子點 (Quantum-dots, QDs) 為基礎之病毒標記模型，並應用於3D多解析度即時單一顆粒追蹤系統中。此模型基於ㄧ帶有8個組胺酸重複標記之pCMVΔR8.91質體，並使其在病毒自組裝過程中與QDs結合，以偵測及紀錄其 3D 移動軌跡，最後與雙光子顯微鏡即時掃描影像所建模的宿主細胞微環境模型疊合，便可呈現病毒與宿主細胞的交互作用。截至當前，我們已取得ㄧ完整的單病毒軌跡，其在自由擴散狀態、細胞貼附期，以及病毒配體與細胞表面受器錨定後之移動過程皆已被成功記錄。

關鍵字

第一型人類免疫缺乏病毒 ； 愛滋病 ； 男男間性行為者 ； 分子流行病學 ； 風險因子 ； 盛行率 ； 鑑識學方法 ； 傳染來源鑑定 ； 系統發生學分析 ； 高通量焦磷 酸測序 ； 量子點 ； 單病毒追蹤 ； 三維多解析顯微鏡

並列摘要

Since it has been revealed and reported to the human society at 1981, HIV-1 has become one of the most serious transmitted diseases of all time worldwide. Even with the emergence of highly active antiretroviral therapy, several issues have been implicated with HIV-1 on different levels. This study investigated HIV-1 in distinct aspects, including molecular epidemiology, forensic science, and virological methodology. In the surveillance part, we recruited men who have sex with men (MSM) subjects in northern and southern Taiwan for anonymous HIV-1 screening during 2013–2015. In total, 4675 subjects were enrolled. The annual prevalence and incidence of HIV-1 showed a downward trend, which was associated with protective factors. Phylogenetic tree analysis showed that the cross-regional and international interactions of the local MSM population may have facilitated the transmission of HIV-1. The second part introduced phylogenetic tree results as forensic evidence in a trial in Taiwan for the first time. Three lawsuit cases were involved. We identified the source of transmission in each lawsuit based on the maximum likelihood and Bayesian phylogenetic tree analyses using the sequences from molecular cloning and ultra-deep pyrosequencing (UDPS). The results of phylogenetic analysis using molecular cloning sequences were clear in lawsuits 1 and 3. Due to the delayed sampling, the transmission direction of lawsuit 2 could not be confirmed unless it was combined with the UDPS sequences results. However, the validity and evidential effects of the UDPS method application were still limited and need further optimization. In the third part, we aimed to develop a quantum dots (QD)-based virion labeling method that is trackable with living cells via a novel 3D multi-resolution real-time single particle tracking system. A QD- pCMVΔR8.91 plasmid that can be labeled was cloned. Currently, a single-virion trajectory containing free diffusion, cell membrane attachment, and docking-like process between the virion and the host cell has been recorded successfully.

並列關鍵字

HIV-1 ； AIDS ； MSM ； molecular epidemiology ； risk factors ； prevalence rate ； forensic method ； intentional transmission ； source identification ； phylogenetic analysis ； UDPS ； QDs ； single virus tracking ； 3D multi resolution microscope

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