Establishment Of An In Vivo Bioluminescent Imaging Model Of Influenza A Virus Infection And Its Applications In Antiviral Evaluation Of Traditional Chinese Medicines

ObjectiveAnimal models are invaluable in studying viral replication in vivo,the pathogenesis of viral infection,the host immune response,and the efficacy of antiviral interventions.Despite their utility,animal models have been constrained by the inability to monitor viral replication dynamics in real time.Cohorts of animals are infected and euthanized,and viral load or immune responses are measured in predetermined tissues.A growing list of viruses have been engineered to express luciferase and exploit this technology,enabling rapid measures of viral load over time（i.e.,longitudinal measurements）,tissue distribution,interhost transmission,and the impact of therapeutic treatments in animal models.In this study,we used molecular cloning and reverse genetics techniques to construct a recombinant influenza reporter virus inserted with a luciferin reporter gene.Based on this,we established a robust and sensitive in vivo imaging model of influenza virus-infected mice.Applied to the process of evaluating traditional Chinese medicine against influenza virus,The"in vivo imaging technology"can realise non-invasive and real-time observation of virus replication dynamics in mice,and sensitively reflect the influence of drug intervention on virus replication in vivo,reduce the demand of experimental animals,and improve the experimental flow.At the same time,this study further explored the mechanism of Chinese medicine with anti-influenza virus activity,in order to discover new anti-influenza drugs.Methods1.Construction of recombinant influenza reporter plasmid with luciferase reporter gene.The recombinant influenza reporter plasmid（pPolI-NS-Fluc）with luciferase reporter was constructed by amplifying the target fragment of the reporter gene and inserting the NS fragment at the appropriate position.The main experimental procedures included primer design,target fragment amplification,agarose gel electrophoresis,PCR product recovery,restriction endonuclease digestion,vector-target fragment ligation,receptive cell transformation,plasmid extraction and positive cloning sequencing.2.Packaging of recombinant reporter virus with luciferase reporter gene.The recombinant influenza reporter plasmid pPolI-NS-luc was co-transfected into 293T cells and MDCK cells with the other seven influenza virus plasmids p DZ-PB2,p DZ-PB1,p DZ-PA,p DZ-HA,p DZ-NA,p DZ-M and p DZ-NP by reverse genetics.After cytopathic changes,the supernatant was collected,centrifuged and virus was amplified in the allantoic cavity of 9-day-old SPF chicken embryos.3.Evaluation of in vitro and in vivo properties of recombinant reporter viruses.First,the growth curve,replication capacity and median lethal dose of the recombinant reporter virus were measured at the cell and mouse level.Genome stability was assessed after continuous passage,including virus titer,reporter signal expression,RNA genome integrity detection by electrophoresis and deep genome sequencing.At the same time,different positive drugs（ribavirin,barloxavir）were used to evaluate the feasibility and sensitivity of the recombinant reporter virus as an in vitro efficacy evaluation system.4.In vivo imaging model of influenza-infected mice was established.The optimal time of image acquisition after substrate injection was optimized;the correlation between the intensity of bioluminescence and the viral titer of the lung tissue was determined.After infection,the mouse lungs were continuously imaged and the dynamic changes of virus infection in the mouse lung tissue were characterised by bioluminescence signals.At the same time,oseltamivir,ribavirin and Lianhua Qingwen granules were selected as positive agents to evaluate the sensitivity,stability and feasibility of the in vivo imaging system as a screening system for antiviral drugs.5.The anti-influenza activity of single herbal medicine and Chinese patent medicine was evaluated.Influenza virus activity was evaluated using in vivo imaging model.The anti-influenza effect of traditional Chinese medicine was evaluated by comparing the intensity of lung bioluminescence between the experimental group and the negative-positive group.6.Antiviral activity evaluation of Chinese medicine against H1N1 in vitro.The recombinant luciferase reporter system was used to verify the anti-influenza virus effect of the medicine and to detect the viral inhibition and cytotoxicity of the medicine at different concentrations.The half effective concentration(IC₅₀)and half toxic concentration(CC₅₀)of the traditional Chinese medicine were fitted.Immunofluorescence was used to detect its inhibitory effect on wild-type influenza virus.The broad-spectrum antiviral activity of medicine against different subtypes of influenza virus strain A/PR/8/34（H1N1）,A H1N1（pdm09）,A/Brisbane/10/2007（H3N2）and influenza B strain IBV-Yamagata was tested by virus titer reduction assay.To determine the general stage of action and mechanism of action of the medicine,a single cycle replication time experiment was performed.7.Antiviral activity evaluation of medicine against H1N1 in vivo.Wild-type influenza virus A/PR/8/34（H1N1）was used to determine the effects of medicine on mortality and body weight of lethal(5LD₅₀)infected mice.The change of virus titer in lung tissue after infection was detected by RT-PCR in a non-lethal（0.8LD50）infection model.The anti-inflammatory and immunomodulatory effects of the drug were assessed by measuring the expression of proinflammatory cytokines IL-1β,TNF-αand IFN-γin the lungs of infected mice.Results1.For the PR8-NS-Gluc reporter virus,the optimised plasmid pPolI-NS ^CE1-Gluc was constructed based on the"balance compensation strategy"of the previous experiments（in the NS fragment 3’NCR region,the promoter compensation mutation CE1（G3A/C8U）was introduced）.PR8-NS^CE1-Gluc reporter virus was packaged.The expression of the Gluc reporter gene was increased more than 10-fold.However,no luminescence signal was detected by in vivo imaging of infected mice due to skin tissue absorption or instrument sensitivity.2.pPolI-NS-Fluc was successfully constructed by means of molecular cloning technology.To prevent the insertion of the large reporter gene Fluc from interfering with the subsequent packaging process of IAV,the promoter compensatory mutation CE1（G3A/C8U）was introduced into the NS fragment 3’NCR region of the pPolI-NS-Fluc plasmid based on the"balance compensation strategy".Alternatively,the promoter compensation mutant CE2（G3A/U5C/C8U）with stronger promoter compensation ability increased the ability to tolerance foreign genes.Two reporter plasmids pPolI-NS^CE1-Fluc and pPolI-NS^CE2-Fluc carrying compensation mutations were successfully constructed.3.Insertion of the large reporter gene Fluc prevented the successful packaging of pPolI-NS-Fluc into a recombinant reporter virus;The two reporter plasmids carrying the promoter compensation mutation can be successfully packaged into the IAV genome because the compensation mutation improves the replication ability of the modified fragment.Following reverse genetics,two recombinant reporter viruses,PR8-NS^CE1-Fluc and PR8-NS^CE2-Fluc,with the ability to replicate and express the reporter gene have been successfully packaged.4.In the replication process of the two recombinant viruses,CE1 and CE2 could enhance the replication ability of damaged NS fragments.The genome of the recombinant reporter virus PR8-NS^CE1-Fluc was found to be unstable.The inserted reporter gene would be lost with the passage of the viral chicken embryo.The virus was not used for further experimental studies.PR8-NS^CE2-Fluc maintained viral replication and genome stability at least in the P5generation.It was able to replicate effectively in vitro with stable reporter gene expression and caused disease in mice.At the same time,PR8-NS^CE2-Fluc was reported to be sensitive to several potent anti-influenza drugs and can be used as a tool to evaluate antiviral efficacy in vitro.5.Based on the PR8-NS^CE2-Fluc reporter virus,a robust imaging model of influenza-infected mice was established.At least 60 min after substrate injection,Fluc signal imaging data remained stable.This model can be used to monitor the spatial distribution and temporal progression of viral infection in the same animal in real time and to visualise the temporal course of viral infection.At the same time,the bioluminescence signal can be used to quantify lung viral load,as the relative strength of the collected bioluminescence signal correlates well with viral replication.Positive drugs were used to verify the sensitivity of the in vivo imaging model,this demonstrated that the model can be used to evaluate the activity of anti-influenza compounds.The bioluminescence signals can be quantified quickly and easily.6.In vivo imaging model was used to evaluate the anti-influenza activity of 14 herbs and 3Chinese medicines.The results showed that Jinhua Qinggan granules has anti-influenza virus activity.7.Study on the antiviral activity of Jinhua Qinggan Granules in vitro and in vivo.JHQG showed anti-influenza activity against PR8-NS^CE2-Fluc,the corresponding IC₅₀ value was0.379 mg/m L and CC₅₀ value was 5.826 mg/m L.Immunofluorescence assay showed that JHQG was also able to inhibit wild-type influenza virus in a dose-dependent manner.Virus titer reduction experiments showed that JHQG had broad-spectrum antiviral activity against influenza A viruses（A H1N1（pdm09）,A/PR/8/34（H1N1）,A/Brisbane/10/2007/（H3N2））and influenza B viruses（IBV-Yamagata）.Single-cycle dosing experiments showed that JHQG mainly targets the post-invasion phase of the influenza virus life cycle to play an antiviral role.In mice,JHQG can prolong survival and improve quality of life in the lethal model of viral infection.In the sublethal model of virus infection,JHQG can significantly reduce the titer of influenza virus in mouse lung tissue,reduce the m RNA expression of pro-inflammatory cytokines（TNF-α,IL-1β）and immunoregulatory factors IFN-γin lung tissue of influenza A virus-infected mice,and reduce the inflammatory response in the lungs of influenza A virus-infected mice.Ameliorate acute lung injury caused by influenza virus infection.Conclusions1.For the recombinant reporter virus PR8-NS-Gluc,increasing the expression of the reporter gene did not significantly improve the sensitivity of in vivo imaging models in mice.2.Based on the"balance compensation strategy",the promoter compensation CE2（G3A/U5C/C8U）was introduced into the 3’NCR region of the NS-modified fragment to increase the capacity to accommodate foreign genes.In this study,the recombinant reporter virus PR8-NS^CE2-Fluc carrying the firefly luciferase reporter gene was successfully constructed.3.Recombinant PR8-NS^CE2-Fluc maintains viral titer and genome stability for at least 5consecutive generations in chicken embryos.It has cellular replication capacity and stable reporter gene expression which can be used for in vitro evaluation of antiviral drugs.It is pathogenic in mice and can cause acute lung injury in mice.4.In vivo imaging model of influenza in mice can be established using the recombinant reporter virus PR8-NS^CE2-Fluc.The signal of this model is stable,and the bioluminescence signal can be used to detect and quantify the viral load in the lungs of mice.The tissue distribution and time course of influenza virus infection can also be visualised.This system can be used for in vivo evaluation of the activity of traditional Chinese medicine against influenza and for screening of antiviral drugs that have an indirect role through metabolites or immune regulation.5.Jinhua Qinggan Granules have broad-spectrum antiviral activity.By acting on the post-invasion part of the influenza virus life cycle.It can reduce influenza virus titers in lung tissue of mice,reduce lung inflammation after influenza virus infection,and improve acute lung injury caused by influenza virus infection.