Generation And Evaluation Of Reporter Influenza Subtype H3N2 Virus Expression Gaussia Luciferase

Influenza A reporter virus(IAV) system carrying fluorescent or luminous genes provides a powerful tool for studying in vivo or in vitro pathogenic mechanisms of influenza A viruses,screening of antiviral drug targets and mechanisms of action,testing for neutralizing antibodies,and imaging of live animals Powerful tools.According to surveys,most articles show that luciferase-reported viruses based on influenza H1N1 subtype virus A/Puerto Rico/8/1934(PR8)or A/WSN/1933 virus have been widely used,but currently we have not found any reports of H3N2 subtype luciferase reporter virus.Because the IAV H3N2 subtype and H1N1 subtype appear in the common transmission of humans and the proportion of H3N2 subtype influenza viruses shows a significant upward trend in the annual "flu outbreak",the reported H3N2 influenza luciferase report virus will have a high value.In this study,three different methods were used to try to construct the H3N2 influenza luciferase reporter virus,and finally a recombinant virus,NY-r19-Gluc,could be successfully used to evaluate antiviral drugs.Experiments have shown that NY-r19-Gluc recombinant virus will become a powerful tool for evaluating new vaccines and screening antiviral drugs.Chapter 1.Introduction.This chapter introduces the relevant theoretical knowledge and biological characteristics of influenza A virus.It also introduces the types of existing reporter genes and their respective characteristics in detail.At the same time,it also reviews the research progress of recombinant influenza viruses carrying reporter genes..Chapter 2.Construction and evaluation of influenza H3N2 subtype NY-Gluc virus.This chapter is a comprehensive description of the first construction method in this study.Starting from the gene level,based on the NY-NS fragment,the pDZ-NY/NS-Gluc plasmid was successfully constructed by mutation and PCR technology,and then the A/NY-NS-Gluc recombinant virus was constructed.In vitro evaluation,The experiment proved that the pDZ-NY/NS-Gluc plasmid successfully generated the type A H3N2 influenza luciferase reporter virus,but with the passage of the recombinant virus in MDCK cells,the expression level of the reporter gene Gluc decreased rapidly,The Gluc signal was hardly detected after two passages,which indicates that the A/NY-NS-Gluc virus cannot be stably inherited in vitro.Later,we introduced mutations in the NCR region of the NY-NS-Gluc fragment in hopes of optimizing the Gluc expression level of the recombinant virus but ultimately failed to rescue it.Chapter 3.Construction and evaluation of influenza H3N2 subtype NY-r18-Gluc virus.This chapter is a comprehensive account of the second construction method in this study.This method is based on the original 8 gene fragments of A/NY virus,replacing pDZ-NY-NS fragment with pDZ-PR8-NS-Gluc fragment,and then constructing H3N2 recombinant influenza fluorescein capable of expressing Gluc gene Enzyme reporter virus,and named it A/NY-r18-Gluc.Through in vitro replication evaluation of the A/NY-r18-Gluc virus,we know that the virus can stably express high levels of Gluc signal during continuous passage at the cell level,but unfortunately,A/NY-r18-Gluc During the fourth passage of the virus,there was a phenomenon of specific CPE changes in the infected MDCK cells.For this reason,we considered that this phenomenon may be caused by the mutation of HA gene in the virus and carried out preliminary analysis and exploration.The mutation site has not yet been determined,but the recombinant virus is no longer suitable as a research tool for subsequent antiviral studies.Chapter 4.Construction and evaluation of influenza H3N2 subtype NY-r19-Gluc virus.This chapter is a comprehensive account of the third construction method in this study.The method is constructed in the form of a 6: 2 genetic reassortant,which is based on the original 8 gene fragments of the A/PR8-Gluc virus,and the pDZ-PR8-HA and pDZ-PR8-NA The two fragments were replaced with pDZ-NY-HA,pDZ-NY-NA fragments,and then the recombinant influenza luciferase reporter virus encoding A/NY virus HA and NA was obtained by reverse genetics technology-A/NY-r19-Gluc recombinant virus.Similarly,we evaluated the in vitro replication of the A/NY-r19-Gluc recombinant virus and found that the virus was able to stably express high levels of Gluc levels during successive passages at the MDCK cell level,and infected MDCK cells there is no specific CPE phenomenon.Therefore,we conducted experiments on the virus growth kinetics,luminescence kinetics,and antiviral detection of the virus to evaluate its antiviral ability.The results showed that the H3N2 influenza luciferase reporter virus constructed by this method conforms to this experiment the final requirements can provide a powerful research tool for subsequent antiviral experiments and related research.Chapter 5.Conclusion and discussion.This chapter focuses on a comprehensive description of the full text of the research work,and summarizes some of the unresolved problems and subsequent research ideas that appear in this article.In the above experiments in this paper,we introduced in detail the H3N2 influenza luciferase reporter virus constructed under the guidance of three different ideas,completed the in vitro evaluation of the three recombinant reporter viruses,and finally determined the A/NY-r19-Gluc recombinant virus it can be used as a successful tool for subsequent antiviral research.This subject as a basic research laid the foundation for the establishment and development of the influenza a luciferase reporter virus system,and for the future recombination of reporter viruses,especially H3N2 influenza luciferase reporter virus in vitro high-throughput screening and in vivo animal models the built application provides the possibility.