1. Summer Research Inhibition of Type I Interferon Signal Transduction Pathways by JEV infection 中央研究院生物醫學科學研究所 林宜玲 老師 University of California, Los Angeles Summer Student: 陳勃睿 清華大學生科 05 級 2003/12/5

2. Introduction Flaviviruses Journal of Infection (2001) 42, 104–115

3. Introduction Flaviviruses  Enveloped virus  Single-strand, positive sense RNA as genome  One ORF encoding a polyprotein C prM/M E NS1 NS2A NS2B NS3 NS4ANS4B NS5 : host signalase : viral protease NS2B-3 : furin or other golgi-localized protease : unknown protease * * * : glycosylation

4. Introduction Interferons (IFNs) are a family of cytokines that proceed antiviral activity. -Type I (viral IFNs)  include IFN-α, IFN-β, IFN-ω, IFN-τ  produced by most body cells -Type II (immune IFNs)  include IFN-γ  produced by certain immune cells only such as CD4 + T help cells

5. Introduction Signal Transduction Pathways

6. Introduction Viral interference in the IFN system

7. Material Japanese Encephalitis Virus - E domain II, prM/M, E pIRES-hrGFP, r21-TE12Q Competent Cells: Top10, DH5α Transfection: BHK-21 (mouse)

8. Method Molecular Cloning Technique Transfection Immunofluorescence Assay (IFA) Western Blotting Polymerse Chain Reaction (PCR) Deletion with one primer in single step

9. Molecular Cloning JE-II/pSecTag B Nhe I T4 DNA polymerase Sal I 養菌，抽 plasmid Gel Extraction pIRES-hrGFP Sma I Sal I CIP (AP) Ligation Transformation

10. Transfection Lipofectamine  artificial membrane Sindbis Virus  interferon-sensitive virus

11. Immunofluorescence Assay DAPI

12. Immunofluorescence Assay DAPI

13. Western Blotting Failed...

14. Single Primer Deletion higher efficiency than primer pairs (although less colonies) DpnI (5’-G m6 ATA-3’) cost is reduced BioTechniques Vol.29, No.5, 970-972

15. Lab Members

16. Thank you!