登革病毒感染近年來在熱帶及亞熱帶地區成為一個日益嚴重的公共衛生問題。在台灣，2015年台南市及高雄市更是遭逢大規模的登革熱疫情，總共累積了43,784登革熱病例，其中包含674例發展成重症及224例死亡。死亡率高於往年流行疫情。因此，我們假設，有可能是在登革病毒基因組發生了和疾病嚴重性相關之突變，而導致2015的嚴重疫情。我們收集了在基因銀行（GenBank）上所有台灣的登革病毒第二型（Dengue virus type 2）序列（27株），並拿它們和先前我們實驗室從2015年爆發收集並定序了的病毒株進行比較。我們看到了在2015年的病毒株中總共有11個胺基酸被取代，其中包括膜蛋白上的K73R，非結構蛋白NS1的P73Q、H224N、K272R、D278，NS2A的S39T、T104、K166R、I171T，NS3的V395I以及NS5的K387R。而此研究則聚焦於NS1蛋白進行近一步的分析，探討NS1基因的突變是否會對蛋白質和登革病毒的生物學特性造成影響。我們利用點突變建構了帶有NS1突變的蛋白，並於293T細胞中進行表達。另外，我們也利用了反基因法（reverse genetics）建構了帶有NS1突變的登革病毒。結果中，我們發現NS1的突變雖不會影響到登革病毒NS1誘導的內皮通透性，但其增加了在哺乳動物細胞中的病毒生長。總之，本研究中我們看到了在登革病毒株發生了不同於以往的胺基酸取代，而其中在NS1上的突變可能在2015年疾病嚴重程度上扮演了部分角色作用。

Dengue virus (DENV) infection is an increasing problem in tropical and subtropical regions. In 2015, there was a large DENV-2 outbreak, which occurred in southern Taiwan, caused a total of 43,784 dengue fever (DF) cases, included 674 dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS) cases with 224 deaths. A higher fatality was observed in the 2015 outbreak. We hypothesized that there may be mutations which occurred in the DENV viral genome that is associated with increased disease severity during the outbreak. Thus, we collected all Taiwan DENV-2 sequences (27 DENV-2 strains) in GenBank and compared them to the 22 samples we previously sequenced from the 2015 outbreak. A total of 11 amino acid substitutions were identified, which were located in the envelope protein (K73R), nonstructural proteins NS1 (P73Q, H224N, K272R, D278E), NS2A (S39T, T104A, K166R, I171T), NS3 (V395I) and NS5 (K387R). In this study, we focused on the NS1 protein gene for further analysis and examined whether these substitutions on NS1 gene affect biological properties of NS1 protein and DENV. By site-directed mutagenesis, we constructed four NS1 mutant plasmids and expressed in 293T cells. Also, viruses with NS1 substitutions were generated by reverse genetics system, and conducted further analysis. The results showed that these substitutions have no effect on the level of DENV NS1-induced endothelial hyperpermeability, which is one of the important characteristics of severe dengue. However, they increase the viral growth in BHK-21 cell line. In conclusion, consistent mutations were observed in 2015 outbreak, and these NS1 substitutions may play a role in DENV disease severity.

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