Establishment And Applications Of An In Vivo Imaging Mouse Model Of Influenza Subtype H3N2 Virus Infection

ObjectiveIn this study,X31（H3N2）virus and Firefly Luciferase（Fluc）reporter gene were combined,a live tracer imaging model of influenza A H3N2 mice that can observe the virus infection in vivo in real time was constructed,Chinese herbal extracts and traditional Chinese patent medicines and simple preparations were taken as research objects to evaluate the activity of anti influenza A virus（IAV）,which provides a new method for research on attenuated live vaccines,antiviral drugs,and other related technologies.Method1.The reporter virus inserted into Fluc was constructed and evaluated in vitro.According to the construction strategy of 6:2 genetic heavy ligand,six fragments of PR8-NS^CE2-Fluc reporter virus preserved in our laboratory were used as skeletons,and two fragments of Hong Kong flu A/Hong Kong/1/1968（H3N2）mice adapted to the virus strain,namely hemagglutinin（HA）and neuraminidase（NA）,were added.The recombinant plasmids were co-transfected into the mixture of 293T-MDCK cells,so the influenza reporter virus X31-NS^CE2-Fluc with luciferase gene was saved.The relationship between the replication ability of recombinant X31-Fluc,the expression of luciferase and viral load was evaluated on the cell level.The structures of the three tested compounds（CBS1194,ING 16-13 and ING 16-24）all have a common pharmacophore-imidazole[1,2-a]pyridine.The anti-viral activity of the three compounds was evaluated in vitro,and their 50%inhibition concentration(IC₅₀)against the X31-Fluc reporter virus was calculated.The Z’Factor was calculated,and the feasibility of virus used to evaluate antiviral drug was evaluated.2.Imaging model of mice infected with H3N2 subtype influenza virus in vivo was established and optimized.The pathogenicity of X31-Fluc virus in mice was determined,and the Median lethal dose(LD₅₀)was calculated.Mice were infected with different doses of X31-Fluc virus,and on the 1st,2nd,3rd,5th and 8th day after-infection,bioluminescence imaging（BLI）in vivo was detected by the small animal imaging system to establish an in the mice imaging model of influenza mice.R² was calculated by evaluating the in vivo characteristics of recombinant virus X31-Fluc and fitting the linear relationship between pulmonary virus titer and lung luciferase activity.The optimized volume of intranasal inoculation virus was determined to select the infection scheme of mice,and the difference of intra-group variation was calculated.The BLI method was used to evaluate the positive drugs in vivo to verify the effect of viral infection dose on the efficacy of drugs,and to evaluate the antiviral effects of Oseltamivir and Baloxavir.3.The in vivo imaging model of H3N2 subtype influenza virus infected mice is applied in the evalution of traditional Chinese medicine（TCM）,vaccines and small-molecule compounds.An imaging model of influenza mice was established to provide a“novel method”for evaluating the antiviral activity of TCM.Firstly,the model was used to evaluate the effect of TCM on influenza H3N2 subtype reporting virus in vivo.Determine whether seven kinds of proprietary TCM have significant inhibitory effect on influenza H3N2 strain in mice;further evaluate the effective TCM in vitro and calculate IC₅₀.Secondly,to evaluate the immune efficacy of live attenuated vaccine（M1-PTD）on influenza mice,to evaluate the lowest dose of complete protection produced by different doses of M1-PTD vaccine in mice,and to evaluate whether there is broad-spectrum immunogenicity to two subtypes of influenza mice after immunization with M1-PTD vaccine.Finally,the imaging model of influenza mice was established to determine whether the three novel antiviral compounds which can effectively inhibit influenza A H3N2 virus in vitro also have good inhibitory activity in vivo.Results1.Successfully constructed the reporter virus X31-Fluc and evaluated the replication kinetics in vitro.Chicken embryo amplification experiments showed that after five passages,the expression level of reporter gene Fluc was still robust,and the reporter virus was genetically stable;multi-cycle replication experiments showed that compared with the wild virus X31,the replication of X31-Fluc decreased,and the peak virus titer decreased by about one log.there was a positive correlation between virus titer and luciferase expression in infected cells.Three novel compounds（CBS1194,ING 16-23,and ING16-24）that may target Group 2 specific influenza hemagglutinin were evaluated by X31-Fluc in vitro.It was found that they all significantly inhibited the reported virus X31-Fluc with IC₅₀ values of 15.6μM,34.29μM and 21.28μM,respectively.The calculated Z’Factor is 0.77（>0.5）,which shows that the detection method has high repeatability,good reliability and stability.The reported virus X31-Fluc can be used to evaluate antiviral drugs against influenza A（H3N2）.2.The tracer model of luciferase reporter virus in vivo was successfully established and optimized.It is reported that mice infected with the virus X31-Fluc virus have pathogenicity in vivo,and LD₅₀ is about 35230 TCID₅₀/m L.Wild type X31 virus have higher virulence,LD₅₀ value 10.39 TCID₅₀/m L.Using BLI method,it was found that the virus was mainly distributed throughout the lung and nasal cavity.BLI dynamics showed that a strong BLI signal was detected on the first day,and the signal began to decrease after reaching the peak on the second day,and there was almost no signal on the 8th day,indicating the replication,the diffusion and clearance of the virus into the body.There was a positive correlation between lung signal and challenge dose(10²-10⁵ TCID₅₀)in different dose groups,r²=0.798,indicating that the increase of virus dose was accompanied by the increase of BLI expression signal.The initial infection efficiency was relatively high in nasal infected mice inoculated with 50μL virus,and the intra-group variation of lung signal in mice was 0.15.X31-Fluc virus was used to evaluate positive drugs in vivo,and the therapeutic effect of Baloxavir was better than that of Oseltamivir at 30 mg/kg dose.Compared with 1000 TCID₅₀ low virus dose group,Oseltamivir had a preferable inhibitory effect on X31-Fluc in mice challenged with a high virus infection dose(10⁴ TCID₅₀).3.The in vivo imaging model of H3N2 subtype influenza virus infected mice was successfully applied.The efficacy of seven kinds of proprietary Chinese’s medicines in vivo were evaluated by BLI method.The results showed that after preventive treatment with Yu-ping-feng granule（YPF）,the replication of reported virus X31-Fluc was inhibited on the 6th day of infection,indicating that YPF promoted virus clearance in the later stage of virus infection.The in vitro activity study showed that YPF had the antiviral effect on the H3N2 subtype influenza virus strain（X31-Fluc）,and the IC₅₀ value was 22.16 mg/m L.Vaccine evaluation experiments showed that immunization with M1-PTD could induce dose-dependent protection against influenza A virus（IAV）infection in mice.1000 TCID₅₀M1-PTD provides complete protection against PR8-Fluc virus infection.Mice immunized with 100 and 10 TCID₅₀ vaccines were partially protected.10000 TCID₅₀ M1-PTD vaccine provide complete protection to mice infected with different IAV strains（PR8-Fluc and X31-Fluc viruses）.Live attenuated influenza vaccine M1-PTD has the potential to become a“universal influenza vaccine”.Finally,the structure-activity analysis of three novel small molecular compounds showed that moderate doses of ING 16-24 or CBS1194 compounds had certain potential in inhibiting X31-Fluc virus in vivo.ConclusionIn this study,we produced a recombinant A H3N2 influenza reporter virus carrying Fluc gene,which can be used to evaluate antiviral drugs in vitro.CBS1194,an inhibitor targeting Group 2 specific influenza hemagglutinin,was identified in our laboratory in the early stages.It was found that ING 16-23 and ING 16-24 compounds had the same pharmacophore（imidazole[1,2-a]pyridine）with CBS1194 compound.The antiviral activity of the three compounds was evaluated by the reporter virus in vitro,and the results showed that all the compounds could inhibit reporter virus.The pathogenicity of the virus to mice was identified by evaluating the virulence of X31-Fluc reporter virus in vivo.In this study,the BLI method was utilized to construct a live imaging model of H3N2subtype influenza virus infected mice for the first time,which can track the virus load in mice in real-time.When the volume of rhinovirus is 50μL,the initial infection efficiency of reported virus X31-Fluc is the highest,therefore the established in vivo imaging model of H3N2 subtype influenza virus infected mice was optimized.The imaging model provides a novel method for evaluating the activity of TCM,immunogenicity of vaccine,structure-activity analysis of compounds and so on.It is found that Yu-ping-feng granule promotes virus clearance in the later stage of influenza A（H3N2）infection,which may be related to enhanced immunity;M1-PTD live attenuated influenza vaccine has efficacy against different IAV subtypes（PR8-Fluc and X31-Fluc）;compounds ING 16-24 or CBS1194 can be used as a new starting point for structure-activity analysis to synthesize more effective small-molecule drugs.