| Summary: | 碩士 === 國立東華大學 === 生命科學系 === 102 === Dengue virus (DENV), becomes one of the reemerging infectious pathogens worldwide. Currently there are still no effective vaccines or anti-viral drugs in clinical. Thus, current researches in developing vaccines and antiviral drugs against dengue infection would be an urgent issue. Marine natural products have been emerging as a new era in drug development, and this study focus on searching the natural compounds from soft coral and then evaluating their effects to inhibit dengue virus infection. Twelve marine compounds were initially screened and compounds named WS 9-5 and WS 9-7 are potent in inhibiting dengue virus infection. The cell cytotoxicity (CC50) of the two terpenoids was evaluated by MTT assay. WS 9-5 is evaluated to be around 21.3 ± 6.42 μM, and WS 9-7 is 47.6 ± 8.82 μM. Immunofluorescence staining evaluations have concluded that WS 9-5 inhibits DENV protein expression (Non-structure protein 1, NS1) with an effective concentration (EC50) of approximately 8.3 μM and its double-stranded RNA (dsRNA) expression with EC50 around 6.8 μM. In addition, compound WS9-5 and WS9-7 were also able to show their impact on DENV virus particle production in which WS 9-5 shows better inhibition rate as compared to WS 9-7. Further, WS 9-5 and WS 9-7 had no inhibitory effect on DENV replication and translation as the luciferase activity of DENV sub-genomic replicon (DENV replicon) has not been suppressed. It is well known that upregulation of innate immune molecules, such as IFN-β and NF-κB, could show an immense down-regulation of DENV infection. However, the inhibitory effects exerted by WS 9-5 and WS 9-7 did not involve the innate antiviral activities as type I interferon-β and NF-κB were not activated in promoter reporter assay. Unexpectedly, premixing WS 9-5 and WS9-7 with In order to know the inhibitory mechanism mediated by WS 9-5 and WS 9-7, pretreatment of cells with compounds 1 hr before infection show no inhibitory effect, but longer incubation for 3 or 6 hrs before infection substantially inhibits virus infection. These results indicate WS 9-5 and WS 9-7 may interfere with the early steps in virus infection. However, compounds will not affect the expression of DV receptors on host cells. Unexpectedly, premixing WS 9-5 and WS 9-7 with virus not only effectively neutralizes virus binding to Huh7 and BHK cells but also suppresses virus infection. This neutralizing activity of WS 9-5 and WS 9-7 was also demonstrated in Japanese Encephalitis Virus infection. Collectively, results suggest that the marine compounds WS 9-5 and WS 9-7 are capable of inhibiting the dengue virus infection through the reduction in attachment, entry, and uncoating, thereby affecting the following translation and replication. Hence this data demonstrate that WS 9-5 WS 9-7 are contemplated to be the effective drugs with high potency in inhibiting the dengue virus infection.
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