The Establishment And Application Of A Reporter Gene Assay System For Detecting And Quantifying Influenza A Virus Replication

Influenza virus is the causative agents of influenza which is commonly referred to as the flu.In the seasonal epidemics of flu,the infection rate of influenza virus may be 15-20% across the whole population.It is estimated that more than 500,000 person globally died from influenza virus infection or related complication every year.The influenza pandemics were much more devastating then seasonal flu.Four influenza pandemics occurred in the 20th century killed tens of millions of people as well as caused great economic losses.The prevalence of highly pathogenic avian influenza virus in recent years and the current widely spread of H1N1 influenza virus A in multi-country highlight the importance and the urgency of studies on the influenza virus again.In view of the limitations of the commonly-used methods for detection of influenza virus, and the urgent request for a simple,fast and specific cell-based quantitative detection system of influenza virus when performing the high-throughput anti-influenza virus drugs screens and influenza virus related RNAi screens,we here set up a reporter gene assay system which can quantify influenza A virus replication efficiently and specificly. The reporter plasmid pHH21-Luc has an eukaryotic RNA polymeraseâ… promoter.In the cell,a single-stranded RNA segment can be transcribed from this reporter plasmid.The structure of this RNA segment is similar to that of the segment 5 of influenza virus' RNAs,in which the NP gene was replaced by a reporter gene.With plasmid pHH21-Luc, we successfully detected and quantified influenza A virus PR/8/34 both in 293T and A549 cells.Infection with influenza virus at different PFUs,we defined the linear range of quantitation.In this range,the expression of luciferase can reflect the viral replication. To make the detecting more intuitionistic and convenient,we cloned EGFP open reading frames to replace the luciferase gene.The new plasmid was named as pHH21-EGFP. pHH21-EGFP plasmid also works in 293T and A549 cells for influenza virus strain PR/8/34.This reporter-gene-based influenza virus detection platform was successfully used to show the anti-virus function of a well-known anti-influenza drug,adamantane.With this system,we also found that the inhibitor of mTOR(mammalian Target of Rapamycin) can inhibit the replication of influenza virus,which indicate a potential role of TOR on influenza virus infection.We also performed a RNAi experiment with the EGFP-targeted siRNA encoding plasmid.The results showed that this virus detecting system can be used in RNAi studies.This study laid the bases for the future RNAi screen studies to resolve the host factors which are important for influenza virus in our lab. In conclusion,the reporter gene assay system for detecting and quantifying influenza A virus replication established in this study will be a powerful tool in the cell-based high-throughput anti-influenza drugs screens or the pathogenesis study of influenza virus.