Screening for anti-influenza virus compounds

• Main Authors: Jo-Yu Hung, 洪若瑜
• Other Authors: Chi-Ju Chen
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/96990418172582078576

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 104 === Influenza A viruses are members of the family Orthomyxoviridae. They cause contagious respiratory disease resulting in hospitalization and even death in high risk populations. Currently available drugs for influenza infection are Oseltamivir and Zanamivir, neuraminidase blockers, and Favipiravir, polymerase inhibitors. However, rapid emergence of drug-resistant mutants restrains their application due to minimum proofreading activity of viral RNA-dependent RNA polymerase (RdRp). In this study, we screened marketed drugs for their anti-influenza activity and tested selected ones for their efficacy at lower dosages. To screen anti-viral drugs, we established cell lines that stably express influenza reporter system. In these cells, RNA polymerase Ι recognized reporter construct and produced virus-like RNA. When RdRp was produced, virus-like RNA would be transcribed into mRNA, and then translated into luciferase protein. Subsequently, luminescence reading reflect RdRp activity. The first reporter established was based on the UTR sequence of A/Puerto Rico/8/1934 H1N1 (PR8) and tested using RdRp from the same virus (PR8).To optimize reporter system for 2009 pandemic H1N1 (2009pdm), we established another one based on the UTR sequence of 2009pdm. However, 2009pdm -based reporter system did not work as effective as PR8-based one. Thus in this study we used PR8-based reporter as our screening system. Once we have candidate drugs, we will test their antiviral efficacy against various strains of influenza virus. We screened US Food and Drug Administration (FDA)-approved 780 drugs for the RdRp inhibiting effect, and selected 20 compounds. We then examined their abilities to reduce cytolytic effect caused by infection as well as viral replication. At the end, we selected 6 drugs and tested minimum effective dose. In the future, we hope to find combinations of drugs at low doses that can treat influenza effectively.