2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647360摘要：抗藥性病毒株的產生及一些可持續存在病人體內的抗藥性基因突變如K103N，不僅會導致雞尾酒治療失敗，也會加快HIV疾病進程。在臺灣，自從疾管署開始實施「抗HIV藥品處方使用規範」，建議初次治療病患使用非核苷酸反轉錄酶抑制劑(nNRTI)，我們即觀察到未接受治療病患間的nNRTI抗藥性盛行率大幅提升。在這期間我們雖然觀察到一些nNRTI抗藥性的群聚感染，但並不足以解釋nNRTI抗藥性盛行率的短期升高。進一步分析病人的病毒基因序列，我們發現到一些少見的nNRTI抗藥性基因突變，且其出現時間與nNRTI抗藥性盛行率相符。因此本研究首先想分析這些突變是否會透過提高病毒複製速率及生長合宜性來增加其傳播速率。此外，也將從中和實驗及病人治療效果來評估這些突變對病毒藥物感受性的影響。第二、我們想藉由次世代基因序列分析了解這些突變在新感染病人體內的持續存在時間及演化方向，並將突變的組合及其出現時間與第一目標的結果相互比較。第三、我們想探討這些突變如何影響nNRTI與核苷酸反轉錄酶抑制劑(NRTI)的藥物交互作用及其作用機制。第四、我們希望藉由結合限制脢切口複製反應及螢光標幟分子信標來建立一個快速偵測抗藥性基因突變的方法並評估其表現。我們希望研究成果可協助建立下一世代抗病毒藥物的篩選平台。<br> Abstract: Despite the introduction of highly active antiretroviral therapy (HAART) has significantly prolonged survival of patient infected with human immunodeficiency virus type 1 (HIV-1), the emergence of antiretroviral drug resistance was subsequently reported. A increasing trend of transmitted drug resistance (TDR) in treatment-naive patients has compromised the initial treatment responses, especially in countries with limited drug selection. The persistence of resistant mutations, such as non-nucleoside-analogues reverse transcriptase inhibitors (nNRTIs)-related mutation K103N, in primary HIV-infected patients was shown to result in treatment failure and clinical progression upon initiation of HAART. In Taiwan, after the implementation of regulations on the prescription of HAART to antiretroviral-naive HIV-infected patients on 1st June 2012, an increasing trend of TDR to nNRTIs was observed. Although a transmission cluster was identified among these patients, it can not explain for the robust increase of TDR to nNRTIs. In detailed sequence analysis, we found K103N only accounts for 14.9% of the detected mutations. Other less frequently reported nNRTI-related mutations were observed instead. Among these, L100V is predominant (28%) and the increasing trend of L100V in infected patients is coincident with the increasing prevalence of nNRTI-resistance. These observations prompt us to investigate the influences of these mutations on viral susceptibility to reverse transcriptase inhibitors, viral replication capacity, and clinical progression in patients with acute HIV infection. First, we plan to determine the impacts of these TDR mutations on viral replication, fitness, and susceptibility to antiretroviral drugs. The impact of L100V, along with other non-polymorphic mutations will be examined by site-directed mutagenesis or swapping of patient-derived viral reverse transcriptase fragments into the HXB2 backbone. The treatment responses of patients harboring these mutations will be monitored and the contribution of minor virus population will be determined by next generation sequencing (NGS). Second, the early establishment of virus population and the temporal evolution of resistant viruses acquired upon primary HIV infection will be characterized. The sequence variations during the follow-up after acute HIV infection will be determined by NGS. The viral evolution will be evaluated by calculating the p-distance and the ratio of the proportions of synonymous and nonsynonymous substitutions (dS/dN ratio). Third, the interactions between NRTI- and nNRTI-related mutations and their influences on viral susceptibility to antiretroviral drugs will be determined. The roles of non-polymorphic mutations which preferentially coincident with TDR mutations will be determined by site-directed mutagenesis. Their impact on the degree of NRTI and nNRTI interactions will be characterized by isobole curves and fractional inhibitory concentration index method. Forth, a rapid diagnosis assay for detection of TDR mutations will be established by combing the nicking endonuclease amplification reaction (NEAR) technique and fluorescently labeled molecular beacons. The performance of the established assay will be evaluated by using the the same RNA preparation previously analyzed in Aims 1-3. Finally, we hope our study results will help to elucidate the contributions and interactions between RTI-related mutations and non-polymorphic mutations, which can provide a platform for screening of the next-generation antiretroviral drugs in the future.