登革病毒（Dengue virus）屬於黃病毒科（Flaviviridae）中的黃病毒屬（Flavivirus）。目前全球約有36億人處於受登革病毒感染的威脅，全世界每年估計約有3.9億人受到感染登革病毒感染。2015年台灣台南市登革病毒感染之通報案例為22,777例。本次研究中，建立並分析兩種優化登革病毒培養標準操作方法。兩種方法的陽性率於統計上無顯著差異，但操作方法一所需孵育時間較方法二短，方法一非常適合用於臨床診斷。本次研究，利用C6/36細胞從628份病患血清中分離出129株登革病毒。將其中45株登革病毒毒株的C，prM和E基因進行序列定序，並與GenBank代表性序列進行序列比對。以C基因至 E基因序列區域進行親緣演化分析（phylogenetic analysis），其結果顯示2015年台南爆發之登革熱疫情之病毒株均被歸類為登革病毒血清第二型，而基因型為世界性基因型（Cosmopolitan genotype），此結果與僅用E基因序列區域進行親緣演化分析結果一致。此45株病毒株之C基因至 E基因序列區域中沒有鑑定出正向和負向選擇性位點（positive and negative selection sites）。與其他基因型序列相比，我們發現了幾個序列變異位點。主要變異位點為C-A314G（K73R），並建立了反向遺傳病毒（reverse-genetics virus）。在BHK-21和C6 / 36細胞中，野生型病毒（wildtype virus）之生長速率比C-A314G（K73R）變異病毒株更快。登革病毒之準種分析實驗中，我們從一名感染登革病毒患者收集三個不同時間點之血清，並利用次世代定序（next-generation sequencing, NGS）將血清中登革病毒定序。依據定序結果發現，登革病毒感染患者血清中之單倍體病毒株（haplotype）的數量隨時間降低。在感染後第5天，病患血清內產生兩個新的單倍體，這兩個新的單倍體序列變異皆位於NS4A基因第115位點，其胺基酸由亮氨酸突變為異亮氨酸。在我們對登革病毒準種分析（quasispecies analysis）中，發現核苷酸變異位點都位於基因組之非結構區域（non-structural region），包含NS2B、NS3和NS4A基因，其基因轉譯出之相關蛋白酶功能對於病毒生命週期中是不可或缺的且重要的。總結來說，以C至E基因序列之親緣性演化分析結果顯示45株登革病毒株之核苷酸序列是高度保守的，具有作為登革病毒候選疫苗株的潛力。所有結果顯示登革病毒在宿主間和宿主內透過RNA序列變異持續適應及對抗宿主的免疫反應。

Dengue virus (DENV) belongs to the genus Flavivirus in the Flaviviridae family. Approximately 3.6 billion people are now at risk for dengue, and there are an estimated 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. Two optimized standard operation protocol for dengue virus culture were established and analyzed in this study. Although the positive rates of the two protocols did not have a significant difference, protocol-I with a shorter incubation time is very suitable for use in clinical diagnosis. There were 129 DENV isolated from 628 sera in C6/36 cells up to now. Partial sequences of C, prM, and E genes from 45 DENV strains were obtained and aligned with representative sequences from GenBank. Phylogenetic analysis based on the sequences of C to E genes revealed that all strains from the 2015 Tainan epidemic were classified as DENV serotype 2 Cosmopolitan genotype, which was consistent with the phylogenetic analysis of E gene only. There were no positive and negative selection sites identified in these sequences. Compared with other genotypes, several variations were found. The major variation was C-A314G (K73R) and a reverse-genetics virus was established. In BHK-21 and C6/36 cells, the wildtype virus had a higher replication rate than C-A314G (K73R). Three sera were collected from one DENV infected patient at different time-points and sequenced by Next Generation Sequencing (NGS). The number of haplotypes decreased with time in the DENV-infected patient. On the fifth day post-infection, two new haplotypes emerged whereby leucine mutated to isoleucine and was identified in NS4A-115. In our quasispecies analysis of DENV, the variations were all located in the nonstructural region of the genome, namely NS2B, NS3, and NS4A which are important with respect to various enzymatic functions needed during the viral life cycle. In conclusion, genetic analysis of C to E gene showed that nucleotide sequences of 45 DENV strains were highly conserved with the potential for being a vaccine candidate. All results demonstrated that DENV continually adapts between inter-and intra-host.

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