Construction And Visual Research Of Influenza Fluorescent Reporter Viruses

Influenza A virus（IAV）is an important pathogen that exists widely all over the world.It can infect A variety of hosts and cause respiratory diseases such as livestock,waterfowl and humans.Due to the frequent mutation of IAVs,pandemics occur every once in a while,including four outbreaks in the past 100 years,which not only cause huge economic losses,but also seriously threaten human life and health,and even affect social stability.Traditional virus research in animal experiments requires the use of a large number of experimental animals,high costs in all aspects.And fluorescent report viruses can not damage under the condition of the animal body,of the viral load in the continuous and real-time dynamic monitoring,to understand biological processes of virus in animals,including virus infection,tissue distribution and viral load,etc.,thus has qualitative quantitative results of easy operation,intuitive,quick,etc.In this study,we used reverse genetics to construct the luciferase reporter virus by inserting the luciferase gene Nluc（Nanoluc luciferase）into the NS1 fragment 3’end of the NS gene of H1N1 influenza virus A/Puertorico/8/34（PR8）;through the rescued recombinant fluorescence Purification and passage of luciferase reporter virus,determination of EID₅₀ and TCID₅₀,determination of in vivo and in vitro proliferation capacity,and mouse pathogenicity test to evaluate its biological characteristics;use of recombinant luciferase reporter virus and small animal in vivo imaging system for virus propagation in vivo Visualization research;using recombinant luciferase reporter virus to infect mice and gavage anti-influenza positive drugs for treatment to evaluate the feasibility of reporter virus for high-throughput screening of drugs.The results showed that:The constructed and rescued IVA recombinant luciferase reporter virus was named PR8-NLuc.PR8-NLuc can be stably inherited to F5generation.PR8-NLuc was proliferated in SPF chicken embryos and MDCK cells,and the highest average titers were 10^7.68 EID₅₀ and 10^4.38EID₅₀,which were lower than the average titers of 10^8.78EID₅₀ and 10^6.06EID₅₀ of wild-type strains（P<0.05）,but the growth curve was similar to that of wild-type strains.Its titer was positively correlated with luciferase activity.The lethal half dose of PR8-NLuc to mice was5.83log₁₀EID₅₀,which was higher than that of wild-type strain 2.17log₁₀EID₅₀（P<0.05）.In mice infected with PR8-NLuc,obvious fluorescence signal could be detected on the 2nd day after infection,the fluorescence signal reached the peak between 2d and 4d,and still could be detected on the 6th day after infection.The mice infected with PR8-NLuc were treated with drugs.Compared with the untreated mice,the fluorescence signal decreased on the 2nd day of infection,the fluorescence signal decreased significantly on the 4th day,and almost no fluorescence signal was detected on the 6th day.The results indicate:In this study,a luciferase reporter virus PR8-NLuc was successfully constructed,which could be stably inherited to F5 generation.The virulence and proliferation of PR8-NLuc were weaker than that of the wild-type strain,but the growth curve of PR8-NLuc was similar to that of the wild-type strain,and the biological characteristics of the wild-type strain were retained to some extent.The titer of PR8-NLuc is proportional to luciferase activity,and the detection time of PR8-NLuc is better than that of conventional detection methods such as viral titration.It has the possibility of being used in real-time,dynamic and visual monitoring of viral load and rapid screening of high-throughput drugs.