登革病毒屬於Flaviviridae 的一員。它的傳染途徑是經由Aedes aegypti和Aedes albopictus 的叮咬而使人類感染。一般而言，感染登革病毒會造成發燒(dengue fever, DF)、出血性發燒(dengue hemorrhagic fever, DHF)以及休克症狀(dengue shock syndrome, DSS)。由於登革熱病毒有四種不同的血清型，而這四種形式的病毒在臨床上交叉感染會造成嚴重的出血及休克等症狀，所以在設計疫苗方面是非常困難的。RNA干擾術(RNA interference,RNAi)為當內源性mRNA 編碼區與某段導入細胞的雙股RNA (double-stranded RNA, dsRNA)序列同源時，dsRNA 會被一種名為Dicer的酵素分解成許多小片斷，這些小片斷的RNA (Short interfering RNAs，簡稱siRNAs)結合到同源序列的mRNA 上時，會引發該mRNA 的降解，使mRNA 無法轉譯蛋白質，造成該特定基因沈默(gene silencing)而失去功能。登革熱病毒為一正股RNA 病毒，僅含有一個RNA 基因體，其基因的表現是先轉譯出一段多蛋白質前驅物，再經由酵素切割成數種具功能性的病毒蛋白；因此，只要能抑制RNA 基因體的複製，便能使得下游所有的病毒蛋白不產生，而達到抑制病毒表現與增殖之目的。所以於本實驗中設計出一套針對登革病毒的RNAi 表現系統，抑制登革病毒感染哺乳類動物細胞後，病毒蛋白的表現與基因的複製。首先，分別針對登革二型病毒(DENV-2)的透膜醣蛋白(Membrane glycoprotein, MG)、表面外套蛋白(Envelope glycoprotein, E)及非結構性蛋白1 (Nonstructural protein 1, NS1)設計小片段干擾性RNA (siRNAs)，再把加強型綠色螢光基因(EGFP)接在RNAi 表現載體pSUPER 上以偵測siRNAs 在細胞內的表現情形，並且分別將MG、E與NS1 基因銜接螢火蟲冷光酶(firefly luciferase)基因形成一個融合性基因，作為受siRNAs 作用之目標基因，將目標基因載體與RNAi 載體共同送入細胞以迅速篩選出有效的標的序列。之後將篩選出的有效siRNAs 過渡性轉染至BHK-21 (baby hamster kidney-21)細胞株，經24 小時後以登革二型病毒進行感染，再過24 小時後以流氏細胞儀分析結果顯示，有效的siRNAs 可防止登革病毒蛋白在受感染的細胞中表現。若將細胞固定於玻片上以免疫螢光染色法染病毒蛋白，能觀察到有siRNAs 表現的細胞內病毒蛋白NS1 與E 的表現量受到壓制，甚至沒有表現。此外，分別收取細胞上清液以空斑形成單位試驗
(Plaque-forming unit assay)來看釋出之病毒量，則發現病毒量有降低的趨勢。同樣的，有siRNAs 表現的細胞於病毒感染後細胞病變(Cytopathic effect)的情形有緩和的趨勢，而病毒感染後細胞凋亡的情形也得以改善。綜合多項結果顯示，利用RNA 干擾技術來抑制登革熱病毒在哺乳類動物細胞內的表現與複製是可行的，同時也開啟了一個對付登革病毒感染的醫療新領域。

　　The Dengue virus is a member of the virus family Flaviviridae and is transmitted to people through the bite of the mosquitos Aedes aegypti and Aedes albopictus. It can cause dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome　(DSS). Dengue vaccine is difficult to produce, because there are four serotypes of dengue virus, and the cross-infection of different serotype will have a high risk at the DHF/DSS development. So far, the most important aspect in treatment of DHF is to prevent further fluid loss. RNA interference (RNAi) is a key mechanism of post-transcriptional gene silencing (PTGS) in which double-stranded, small interfering RNAs trigger a sequence-specific gene-silencing process. Dengue virus is a positive-strand RNA virus, and contains a single RNA genome and translates only a single polyprotein precursor. In this study, we introduce RNAi system against the polyprotein precursor, which can　sufficiently inhibit dengue virus replication in mammalian cells. We constructed enhanced green fluorescence protein (EGFP) gene on pSUPER vector. EGFP signal indicates that the cells were transfected with siRNA vectors successfully and pSUPER vector directs siRNAs synthesis in mammalian cells. The designed siRNAs were derived from the genome of dengue virus type 2 (DENV-2) membrane glycoprotein (MG), envelope protein (E), and non-structural protein 1 (NS1) regions. First, we used the MG, E or NS1-firefly luciferase fusion to screen effective siRNA targeting sequences　separately. Subsequently, BHK-21 (baby hamster kidney-21) cells were transfected with effective siRNA vectors and challenged with DENV-2 for 24 hours. Then we assayed NS1 antigen content of the EGFP-expressing cells by flow cytometry and bserved that the percentage of positive cells was significant reduction. Moreover, we fixed the　DENV-2-infected BHK-21 cells on the slides for immunofluorescence staining and saw IV that EGFP-expressing cells had little virus NS1 and E antigens. We also collected the supernatants separately for plague-forming unit (PFU) assay and calculated that the virus titers decreased. Besides, transfecting effective siRNA vectors into BHK-21 cells could reduce the virus-induced cytopathic effect (CPE) and cell apoptosis. These results suggest that RNAi can block dengue virus replication in mammalian cells and offer a hope for the treatment of dengue virus infection on the therapeutic promise of RNAi.

參考文獻
Adelman, Z.N., Sanchez-Vargas, I., Travanty, E.A., Carlson, J.O., Beaty, B.J.,
Blair, C.D., Olson, K.E. (2002). RNA silencing of dengue virus type 2 replication
in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived
from the virus genome. J Virol. 76:12925-12933.
Avirutnan, P., Malasit, P., Seliger, B., Bhakdi, S., Husmann, M. (1998). Dengue
virus infection of human endothelial cells leads to chemokine production,
complement activation, and apoptosis. J Immunol. 161:6338-6346.
Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function.
Bhamarapravati, N. (1989). Hemostatic defects in dengue hemorrhagic fever. Am
Bhamarapravati, N., Sutee, Y. (2000). Live attenuated tetravalent dengue vaccine.
Brummelkamp, T.R., Bernards, R., Agami, R. (2002). A system for stable
expression of short interfering RNAs in mammalian cells. Science. 296:550-553.
Caplen, N.J., Parrish, S., Imani, F., Fire, A., Morgan, R.A. (2001). Specific
inhibition of gene expression by small double-stranded RNAs in invertebrate and
vertebrate systems. Proc Natl Acad Sci U S A. 98:9742-9747.
Carrington, J.C., Ambros, V. (2003). Role of microRNAs in plant and animal
development. Science. 301:336-338.
Chen, W.J., Chen, S.L., Chien, L.J., Chen, C.C., King, C.C., Harn, M.R.,
Hwang, K.P., Fang, J.H. (1996). Silent transmission of the dengue virus in
southern Taiwan. Am J Trop Med Hyg. 55:12-16.
Chen, Y., Maguire, T., Hileman, R.E., Fromm, J.R., Esko, J.D., Linhardt, R.J.,
Marks, R.M. (1997). Dengue virus infectivity depends on envelope protein
binding to target cell heparan sulfate. Nat Med. 3:866-871.
Cullen, B.R. (2002). RNA interference: antiviral defense and genetic tool. Nat
Immunol. 3:597-599.
Dougherty, J.P., Samanta, H., Farrell, P.J., Lengyel, P. (1980). Interferon,
double-stranded RNA, and RNA degradation. Isolation of homogeneous
pppA(2'p5'A)n-1 synthetase from Ehrlich ascites tumor cells. J Biol Chem.
255:3813-3816.
Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T.
(2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
Mammalian cells. Nature. 411:494-498.
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C.
(1998). Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegan. Nature. 391:806-811.
Gubler, D.J. (1998). Dengue and dengue hemorrhagic fever. Clin Microbiol Rev.
11:480-496.
Guirakhoo, F., Pugachev, K., Zhang, Z., Myers, G., Levenbook, I., Draper, K.,
Lang, J., Ocran, S., Mitchell, F., Parsons, M., Brown, N., Brandler, S.,
Fournier, C., Barrere, B., Rizvi, F., Travassos, A., Nichols, R., Trent, D.,
Monath, T. (2004). Safety and efficacy of chimeric yellow fever-dengue virus
tetravalent vaccine formulations in nonhuman primates. J Virol. 78:4761-4775.
Guo, S., Kemphues, K.J. (1995). Par-1, a gene required for establishing polarity
in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically
distributed. Cell. 81:611-620.
Halstead, S.B., O'Rourke, E.J. (1977). Antibody-enhanced dengue virus
infection in primate leukocytes. Nature. 265:739-741.
Hamilton, A.J., Baulcombe, D.C. (1999). A species of small antisense RNA in
post-transcriptional gene silencing in plants. Science. 286:950-952.
Hammond, S.M., Caudy, A.A., Hannon, G.J. (2001). Post-transcriptional gene
silencing by double-stranded RNA. Nat Rev Genet. 2:110-119.
Hannon, G.J. (2002). RNA interference. Nature. 418:244-251.
Henchal, E.A., Putnak, J.R. (1990). The dengue viruses. Clin Microbiol Rev.
3:376-396.
Huang, K.J., Li, S.Y., Chen, S.C., Liu, H.S., Lin, Y.S., Yeh, T.M., Liu, C.C., Lei,
H.Y. (2000). Manifestation of thrombocytopenia in dengue-2-virus-infected mice.
J Gen Virol. 81:2177-2182.
Huang, Y.H., Lei, H.Y., Liu, H.S., Lin, Y.S., Liu, C.C., Yeh, T.M. (2000).
Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production.
Am J Trop Med Hyg. 63:71-75.
Hung, S.L., Lee, P.L., Chen, H.W., Chen, L.K., Kao, C.L., King, C.C. (1999).
Analysis of the steps involved in dengue virus entry into host cells. Virology.
257:156-167.
Kalayanarooj, S., Vaughn, D.W., Nimmannitya, S., Green, S., Suntayakorn, S.,
Kunentrasai, N., Viramitrachai, W., Ratanachu-eke, S., Kiatpolpoj, S., Innis,
B.L., Rothman, A.L., Nisalak, A., Ennis, F.A. (1997). Early clinical and
laboratory indicators of acute dengue illness. J Infect Dis. 176:313-321.
Kennerdell, J.R., Carthew, R.W. (2000). Heritable gene silencing in Drosophila
using double-stranded RNA. Nat Biotechnol. 18:896-898.
Kuwabara, T., Hsieh, J., Nakashima, K., Taira, K., Gage, F.H. (2004). A small
modulatory dsRNA specifies the fate of adult neural stem cells. Cell. 116:779-793.
Lei, H.Y., Yeh, T.M., Liu, H.S., Lin, Y.S., Chen, S.H., Liu, C.C. (2001). Immuno
-pathogenesis of dengue virus infection. J Biomed Sci. 8:377-388.
Lipardi, C., Wei, Q., Paterson, B.M. (2001). RNAi as random degradative PCR,
siRNA primers convert mRNA into dsRNA that are degraded to generate new
siRNAs. Cell. 107:297-307.
Littaua, R., Kurane, I., Ennis, F.A. (1990). Human IgG Fc receptor II mediates
antibody-dependent enhancement of dengue virus infection. J Immunol. 144:3183
-3186.
Milhavet, O., Gary, D.S., Mattson, M.P. (2003). RNA interference in biology and
medicine. Pharmacol Rev. 55:629-648.
Miyagishi, M., Taira, K. (2002). U6-promoter-driven siRNA with four uridine 3’
overhangs efficiently suppress target gene expression in mammalian cells. Nat
Biotechnol. 20:497-500.
Monath, T.P. (1994) Dengue: the risk to developed and developing countries. Proc
Natl Acad Sci U S A. 91:2395-2400.
Murgue, B., Cassar, O., Guigon, M., Chungue, E. (1997). Dengue virus inhibits
human hematopoietic progenitor growth in vitro. J Infect Dis. 175:1497-1501.
Naito, Y., Yamada, T., Ui-Tei, K., Morishita, S., Saigo, K. (2004) siDirect:
highly effective, target-specific siRNA design software for mammalian RNA
interference. Nucleic Acids Res. 32:W124-W129.
Napoli, C., Lemieux, C., Jorgensen, R. (1990). Introduction of a chalcone
synthase gene into Petunia results in reversible co-suppression of homologous
genes in trans. Plant Cell. 2:279-289.
Nishikura, K. (2001). A short primer on RNAi: RNA-directed RNA polymerase
acts as a key catalyst. Cell. 107:415-418.
Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., Conklin, D.S. (2002).
Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian
cells. Genes Dev. 16:948-958.
Rubinson, D.A., Dillon, C.P., Kwiatkowski, A.V., Sievers, C., Yang, L., Kopinja,
J., Rooney, D.L., Ihrig, M.M., McManus, M.T., Gertler, F.B., Scott, M.L., Van
Parijs, L. (2003). A lentivirus-based system to functionally silence genes in
primary mammalian cells, stem cells and transgenic mice by RNA interference.
Nat Genet. 33:401-406.
Sabin, A.B. (1952). Research on dengue during World War II. Am J Trop Med
Hyg. 1:30-50.
Sanchez-Vargas, I., Travanty, E.A., Keene, K.M., Franz, A.W., Beaty, B.J.,
Blair, C.D., Olson, K.E. (2004). RNA interference, arthropod-borne viruses, and
mosquitoes. Virus Res. 102:65-74.
Sharp, P.A. (2001). RNA interference─2001. Genes Dev. 15:485-490.
Song, E., Lee, S.K., Dykxhoorn, D.M., Novina, C., Zhang, D., Crawford, K.,
Cerny, J., Sharp, P.A., Lieberman, J., Manjunath, N., Shankar, P. (2003).
Sustained small interfering RNA -mediated human immunodeficiency virus type 1
inhibition in primary macrophages. J Virol. 77:7174-7181.
Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. (1995). A
novel assay for apoptosis. Flow cytometric detection of phosphatidylserine
expression on early apoptotic cells using fluorescein labelled Annexin V. J
Immunol Methods. 184:39-51.
Yang, G., Cai, K.Q., Thompson-Lanza, J.A., Bast, R.C. Jr., Liu, J. (2004).
Inhibition of breast and ovarian tumor growth through multiple signaling pathways
by using retrovirus-mediated small interfering RNA against Her-2/neu gene
expression. J Biol Chem. 279:4339-4345.
Yokota, T., Sakamoto, N., Enomoto, N., Tanabe, Y., Miyagishi, M., Maekawa,
S., Yi, L., Kurosaki, M., Taira, K., Watanabe, M., Mizusawa, H. (2003).
Inhibition of intracellular hepatitis C virus replication by synthetic and
vector-derived small interfering RNAs. EMBO 4:602-608.
Yu, J.Y., DeRuiter, S.L., Turner, D.L. (2002). RNA interference by expression of
short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci
U S A. 99:6047-6052.
Zamore, P.D., Tuschl, T., Sharp, P.A., Bartel, D.P. (2000). RNAi:
double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23
nucleotide intervals. Cell. 101:25-33.
Zhang, J., Yamada, O., Sakamoto, T., Yoshida, H., Iwai, T., Matsushita, Y.,
Shimamura, H., Araki, H., Shimotohno, K. (2004). Down-regulation of viral
replication by adenoviral-mediated expression of siRNA against cellular cofactors
for hepatitis C virus. Virology. 320:135-143.