Evaluation Of A Recombinant Influenza Virus Expressing Gaussia Luciferase And Its Application In Screening For Antiviral Traditional Chinese Medicines

Influenza A virus(IAV)is a major cause of respiratory diseases,with great morbidity and mortality worldwide every year.At present,antiviral drugs and influenza vaccines could be used to counteract influenza virus infection.However,the effectiveness of vaccines was limited by the high variability of influenza viruses,and drug resistance emerged rapidly.Therefore,development of novel antivirals is urgently needed.This study established a system for antiviral screen and therapy evaluation,using a recombinant influenza virus expressing Gaussia luciferase(Gluc),PR8-NS1-Gluc.Subsequently,Traditional Chinese Medicines(TCMs)were screened for antiviral activities.Chapter 1.Introduction.In this chapter,the biology of IAV,the prevention and therapy of influenza virus infection,as well as the application and advantages of TCMs in anti-influenza were reviewed.Chapter 2.Evaluation of recombinant influenza virus PR8-NS1-Gluc.In this study,a recombinant IAV carrying Gaussia luciferase gene PR8-NS1-Gluc was generated,and it was demonstrated that this recombinant virus is replication-competent in tissue culture and pathogenic in mice.Luciferase expression correlated well with virus propagation both in vitro and in vivo,providing a simple measure for viral replication in tissue culture and in mouse lungs.To demonstrate the utility of this virus,ribavirin and oseltamivir phosphate were used to treat the IAV-infected cells and mice,and the dose-dependent inhibition of viral replication was determined by a luciferase assay.Moreover,the decreased luciferase expression in the infected lungs could predict the protective efficacy of antiviral interventions as early as day 2 post virus challenge.In summary,this study provides a new and quantitative approach to evaluate antivirals against IAV.Chapter 3.Screen for antiviral Traditional Chinese Medicines.Based on the reporter IAV PR8-NS1-Gluc,a screening system for antivirals was established.Then 352 TCMs were screened,and 12 TCMs were found to possess antiviral activities.Among the 12 TCMs,8 of which showed good selectivity index(SI>20).The 15 compounds from unripe pods of T.chebula and ripe pods of T.chebula were screened.The Mechanism of action of the chebulinic acid and chebulagic acid were further investigated using one-cycle infection inhibition assay,virus release inhibition assay,neuraminidase(NA)inhibition assay.Chebulinic acid and chebulagic acid showed high inhibitory effect on Oseltamivir-resistance influenza A/H1N1pdm(09),wild-type H1N1 and H3N2.Chapter 4.Conclusion and discussion.In summary,this study characterized a reporter IAV PR8-NS1-Gluc both in vitro and in vivo,and determined its potential as a tool for antiviral screen and therapy evaluation.Using the reporter virus,an antiviral screen system was established and 352 TCMs were screened,of which 8 showed high antiviral activities.The chebulinic acid and chebulagic acid were selected from ripe pods and unripe pods of T.chebula.This study provides a simple and robust approach for antiviral discovery and evaluation,and it will be interesting to further identify the active components of the 8 positive TCMs.