登革病毒(dengue virus)經由蚊子傳播，造成登革熱出血熱(dengue hemorrhagic fever)以及登革熱休克症候群(dengue shock syndrome)。我們已發現登革病毒感染會誘發自噬反應(autophagy)並促進病毒複製。他人之研究指出登革病毒感染會增加糖解作用(glycolysis)，也會促進病毒複製。本研究的主旨為探討登革病毒第二型感染誘發之自噬反應和糖解作用兩者之關係及在細胞及小鼠中對病毒複製及致病性之影響。本研究使用肺癌A549，神經細胞瘤SK-N-SH和肝癌Huh7細胞進行病毒感染。在飢餓條件下，與對照和紫外光去活化病毒的細胞組相比，活病毒感染可增加葡萄糖螢光類似物2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG)的攝取量，以及自噬LC3-II蛋白和己糖激酶-2(hexokinase-2, HK2)之表現量。進一步利用免疫螢光染色方式觀察，在登革病毒感染的細胞中，可見到自噬點(autophagic puncta)和HK2激酶表現量均增加。然而，感染的細胞中乳酸(lactate)生產沒有變化，顯示DENV2感染促進糖解流量(glycolytic flux) 不經由乳酸途徑。此外，登革病毒感染可誘導葡萄糖轉運蛋白-1（GLUT1）的表達，但對糖解酵素HK2的mRNA表達沒有影響，顯示其影響是在後轉錄階段。進一步證實，使用細胞自噬抑制劑spautin-1或在Atg5基因表現弱化A549細胞中(使用lentivirus載體)，細胞內葡萄糖之攝取、糖解酵素HK2表現量和病毒產量均下降。同樣的，使用自噬誘發劑(amiodarone)可促進病毒感染之細胞內葡萄糖的攝取，糖解酵素表現量和病毒產量。此結果揭示細胞自噬可調控糖解作用和病毒複製。再使用葡萄糖類似物2-Deoxy-D-glucose(2DG)來阻斷糖解作用，如所預期2DG可降低葡萄糖之攝取，伴隨著糖解酵素HK2的表現量和病毒量之下降，意外的是細胞內之自噬活性卻增加，可能是由於葡萄糖缺乏造成。以顱內接種6日齡的ICR哺乳小鼠證實登革病毒第二型(strain PL046)能夠有效的感染小鼠，在接種後第6天小鼠體重降低及疾病症狀增加，並於第10天死亡。最後使用自噬抑製劑(3MA)和2DG抑制DENV2感染的小鼠體內之自噬和糖解作用。結果顯示LC3-II和HK2的蛋白表達均下降，而且藥物處理隻小鼠存活率較高。綜述之，本研究揭示登革病毒感染細胞或小鼠可同時增加宿主自噬反應、葡萄糖之吸收及HK2之表現量，這些反應可增加病毒複製量及小鼠之病情。而且糖解代謝過程受到在自噬反應之調控。

Dengue virus (DENV) transmits diseases through mosquitoes. Severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) mostly occurs in the individuals during secondary infection of a different DENV serotype. We have reported that DENV infection induces autophagic activity, which further promotes viral replication. Others reported that DENV infection increases glycolysis, which also promotes viral replication. This study intends to clarify the relationship among DENV induced autophagy, glycolysis, viral replication and pathogenesis both in vivo and in vitro. Here, we used lung cancer A549, neuroblastoma SK-N-SH and hepatoma Huh7 cells for DENV infection. Under starvation conditions, DENV2 infection of the cells increased fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) uptake, the protein levels of autophagy LC3 type II and the rate limiting glycolytic kinase hexokinase-2 (HK2), compared to the mock- and UV inactivated virus infected cells. DENV2 infected cells showed increased autophagic puncta and HK2 kinase by immunofluorescence staining. However, the lactate production did not change in DENV2-infected cells, indicating that DENV2 infection promoted glycolytic flux is lactate pathway independent. Furthermore, DENV2 infection increased the expression of glucose transporter-1 (GLUT1) protein, but showed no effect on mRNA expression of the glycolytic kinase HK2. We further demonstrated that the glucose uptake, glycolytic kinase levels and viral titer decreased by the autophagy inhibitor spautin-1, or in the Atg5 gene silenced A549 cells (using lentivirus-shAtg5). In contrast, autophagy inducer amiodarone increased glucose uptake, glycolytic kinases expression and viral titer. These data imply that autophagy regulates glycolysis and viral replication. We then used 2-Deoxy-D-glucose (2DG), a glucose analog, to block glycolysis, and revele that 2DG decreased glucose uptake, the expression of glycolytic kinases HK2 and viral titer. However, autophagic activity increased because of deficiency of glucose. We demonstrated that DENV2 (strain PL046) can effectively infect the mice six day old suckling, and showed that disease symptoms increased accompanied with decreased body weight at day 6. All mice died at day 10 post inoculation. We used autophagy inhibitor 3-methyladenine (3MA) and glycolysis inhibitor 2DG to study viral replication and pathogenesis of DENV2-infected mice. Our reveals showed that the survival rate of the mice was prolonged, and protein expression of LC3-II and HK2 decreased compared to the untreated mice. Altogether, this study reveal that DENV2 infection induces autophagy which regulate glucose uptake and HK2 protein expression. These events lead to increased viral titer and disease symptoms.

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