Screening Tool For Chinese Medicine Against Influenza Virus Was Constructed By Reverse Genetics

BackgroundAccording to the statistics of the World Health Organization,there are about 1billion people infected by influenza worldwide,and about 300,000 to 500,000 people die every year.There are four types of influenza,namely influenza A B C and D（discovered in 2011）,among which influenza A is the most pathogenic and infectious,with the highest mortality rate.At present,the methods of treating influenza virus include inoculation of influenza vaccine and the application of antiviral drugs（including traditional Chinese medicine against influenza virus）.As influenza virus is RNA virus,it has a strong mutation ability,which is easy to lead to antigen drift and antigen transformation,making the vaccine ineffective and the virus resistant to anti-influenza drugs.Therefore,more drugs,such as traditional Chinese medicine,need to be screened in order to cope with the possible emergence of influenza pandemic in the future.At present,MTT method is the most common method for drug screening.However,this method has low reproducibility,complicated experimental procedures and long time consuming,so it needs to be replaced by a more direct and efficient method that can be used for mass drug screening.In this project,we use reverse genetics to rescue influenza A virus carrying m Cherry gene,which can provide a powerful tool for screening anti-influenza Chinese medicine.Influenza can be regarded as a disease in traditional Chinese medicine.In ancient times,Chinese medicine played a very important role in the treatment of diseases.And last year’s novel corona virus,with the application of traditional Chinese medicine,it is greatly reduced mortality rate.Fruccinia chinensis,for verbena plants Fruccinia chinensis dry mature fruit,evacuation of wind heat,Qingli head effect,for the common medicine in the prescription of traditional Chinese medicine cold.However,there are few studies on its antiviral effect.In this paper,water extract of Fructus was selected to verify whether it has antiviral effect on the new H1N1influenza.Objective（1）Using reverse genetics to construct a new H1N1 strain carrying m Cherry red fluorescent reporter gene;A novel H1N1 mouse adaptation strain was produced by BABL/C mice and a mouse adaptation strain carrying m Cherry gene was constructed.（2）Verify whether the recombinant virus carrying m Cherry gene can stably exist and successfully express red fluorescence through nucleic acid,protein and fluorescence methods,and then explore the characteristics of the virus from the polymerase activity and animal pathogenicity.（3）To preliminarily explore whether the water extract of Fructus chinensis has the effect of resisting influenza virus.Methods:（1）Reverse genetics was used to construct the plasmid PA-m Cherry to rescue the new H1N1 recombinant influenza virus carrying the m Cherry gene,and the same method was used to produce the recombinant virus carrying the m Cherry gene of the H1N1 mouse adaptive strain.（2）The gene stability and the expression of reporter genes of the recombinant influenza virus were verified by PCR,western blot and indirect immunofluorescence.The virus growth curve and luciferase report were used to detect and measure the MLD₅₀of the virus to mice to explore the characteristics of the virus.（3）The antiviral effect of aqueous extract of Fructus on Influenza A（H1N1）virus was studied by thiazolium blue staining method（MTT method）.The recombinant virus carrying m Cherry gene was used as a screening tool to study the antiviral effect of Fructus chinensis by fluorescence method.Results（1）Construction of recombinant virus carrying m Cherry geneThe method of reverse genetics was used to find the appropriate packaging signal site in the PA gene fragment of A/Guangdong/GLW/2018（H1N1）（GLW-WT）virus.The m Cherry was inserted into PA,and the influenza A virus with the ability of carrying red fluorescent gene,expressing red fluorescent protein and replicative passage was successfully prepared.Then,virus GLW-MA with pathogenicity to mice was isolated and purified through the passage of the virus in mice.Red fluorescent protein was successfully expressed by inserting m Cherry into PA of GLW-MA.This provides a powerful tool for in vitro and in vivo screening of antiviral drugs.（2）The study of virus propertiesVirus growth curve,nucleic acid,protein,indirect immunofluorescence and other methods proved that the recombinant virus carrying m Cherry gene could accommodate m Cherry gene,and successfully expressed red fluorescence on MDCK.The mutant of GLW-MA strain of the new H1N1 virus was detected by luciferase report.The three mutant sites of PA I61M E351K G631S could increase the polymerase activity of the polymerase complex.Mutations in PA and HA may be the molecular factors responsible for the development of mouse adaptations by GLW-MA strains.GLW-WT（H1N1）virus was not pathogenic to mice,and GLW-MA and GLW-MA-m Cherry were both pathogenic to mice.（3）To explore the antiviral effect of water extract of Fructus chinensisThe IC₅₀value of water extract against GLW-WT virus was 7.78±1.21ug/m L byMTT assay.The results of progeny virus production reduction method showed that the progeny virus production decreased gradually with the increase of drug concentration.Using GLW-WT-m Cherry as a screening tool,the fluorescence expression of GLW-WT-m Cherry virus in cells decreased with the increase of drug concentration.ConclusionRecombine influenza viruses GLW-MA-m Cherry and GLW-WT-m Cherry were successfully constructed with m Cherry gene by reverse genetics.Recombinant influenza virus carrying m Cherry gene was used as a screening tool to investigate the anti-influenza effect of water extract of Fructus chinensis.