| Summary: | 碩士 === 臺灣大學 === 臨床醫學研究所 === 95 === Background. Type A influenza virus infects 20% of global population every year, and genetic reassortment between different strains could cause pandemic outbreak. Vaccine is the only available strategy for type A influenza prevention, however, the identity between vaccine and circulating strain is only 52 to 82% in Taiwan. Anti-influenza drugs are not reliable due to highly mutant character of type A influenza virus. Therefore, a new strategy for prophylaxis and treatment for type A influenza virus should be studied. Small interfering RNAs (siRNAs), which target the more conserved sequence of type A influenza virus, could knockdown the gene expression and virus replication. Plasmid or lentivirus vectors carriage with short hairpin RNA cassette might more persistently express the shRNA and inhibit the virus.
Materials and Methods. The target genes (NS1 gene, PA gene and NP gene) of two type A influenza virus strains, A/WSN/33 (H1N1) and A/Panama/1/68(H3N2), are aligned for the siRNA target sequences. MDCK-ATCC and p69 cell lines are then transfected with quantitative shRNA plasmid, followed by influenza A virus infection. Pseudotyped lentivirus with shRNA sequence is used as an alternative tool for cell transduction. The viral load of supernatant are analyzed by hemagglutiation assay (HA assay), real-time PCR and plaque assay. If several potently inhibitory sequences could be found, the inhibitory effect of co-transfection is also studied.
Results. For MDCK-ATCC cell, the transfection efficiencies of jetPEI and Lipofectamine 2000 are 20-30% and 34%, respectively, which were inverse to the passage number. Large amount NP-1496 siRNA (500pmol) via jetPEI delivery is related to influenza virus inhibition (50-75% HA unit decrease). In H3N2 (A/Panama/1/68) shRNA study, PA-1’ is the most powerful sequence for influenza virus inhibition, which reveals 50% and 67% influenza virus decrease, respectively. PA-2,NP-3 and PA-4’ also have some inhibitory effects. The co-transfection reveal no synergistic effect. In H1N1 (A/WSN/33) shRNA study, PA-1’ and NP-1496 sequences could achieve 50-75% HA unit decrease after transfection to MDCK-p69 cell. The transfection efficiency could increase to 74% after pseudotyped lentivrus transduction and puromycin selection. PA-1’ and NP-3 sequences reveal more obvious influenza virus inhibition (50-88% HA unit and 57-91% plaques decrease). There is no relationship between the virus inhibition and secondary structure of 3’ end of target sequences in this study.
Conclusion. PA-1’ sequence is a more effective sequence in type A influenza virus inhibition. However, some sequences reveal diverse conclusion. No more effect could be achieved after changing the loop sequence of shRNA. The knockdown effect of shRNA plasmid is related to the transfection efficiency, cell passage number and timing of virus infection. The transfection efficiency is better in lentivirus system than in liposome system. The inhibition effect could be completely different between two nearby sequences. The relationship between secondary structure of 3’ end of target sequence and virus inhibition should be further surveyed.
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