Construction Of Luciferase-labeled Recombinant Influenza Virus (IAV-Gluc) And Visualization Of Its Respiratory Deposition

Recently,frequent outbreak of influenza A virus brought a serious threat to human health and caused great damage on the economy,and even affected the social stability.Transmission through aerosol and large scope of the infection are the important factors of the world’s pandemic.The H1N1 influenza A outbreak in 2009 can spread through aerosol with a strong ability of human-to-human transmission,so the world health organization(WHO)alerted to the highest warning level---6 grade.This event aroused the attention on the aerosol mechanism research of influenza virus.At present,the influenza virus aerosol transmission mechanism is not very clear.There are many factors that can influence the spread of influenza virus aerosol,such as environmental factor,host respiratory sialic acid related receptor types,key sites of amino acid sequence,antiviral drugs in the body,vaccine immunity levels and Respiratory tract deposition efficiency.Monitoring the dynamic change of influenza virus loads within the respiratory tract will provide basic data and technical support for the research of tissue tropism,deposition,replication and clear rules,molecular mechanism and propagation of the disease.The traditional virus deposition research need to use a lot of animals to autopsy at different time points.There is a big biological safety hidden trouble.Live virus markers and visualization research can effectively overcome the above shortcomings and can be real-time,convenient to reflect the virus infection,replication and clear.By reverse genetic technology,we inserted foreign luciferase gene in the end of Rico/8/34(PR8)NA gene and built visualization of recombinant luciferase influenza A(H1N1)virus in order to do the research on animal influenza virus infection and respiratory tract deposition.This method can quickly learn the tissue distribution and load conditions of the virus by testing animal respiratory bioluminescent situation and implement continuous,real-time and dynamic monitoring of animal viral load in vivo with the characteristic of easy handling,visual result and rapid qualification and quantitation.This study contained four parts:Firstly,we built the luciferase influenza A(H1N1)virus(IAV-Gluc)and accomplished the identification.With A/Puerto Rico/8/34(PR8)strain as the skeleton,we successful built 8 two-way transcription/expression plasmids by reverse genetics technology.Then we transfected the 293 T cells using the 8 plasmids and vaccinated the transfection supernatant in 9-age SPF eggs in order to save the IAV-Gluc.By agglutination test(HA),gene amplification,morphological observation and luciferase expression level test,we identified the successful construction of recombinant luciferase influenza A(H1N1)virus(IAV-Gluc).Secondly,we completed the study of IAV-Gluc biological characteristics.We analyzed and compares the biological characteristics in six aspects: stability,pathogenicity,transmission ability,proliferation,NA activity and receptor binding properties.The results show that P1-P5 generation of IAV-Gluc can express stable luciferase gene expression with the same level.Pathogenicity is about 1/1000 of the wild poisonous PR8.In guinea pig it got 100% of the direct contact and aerosol transmission capacity,consistent with wild strains.In SPF egg the proliferation ability is about 1/20 of the wild strains,and no obvious difference was found about the proliferation curve.there is no significant difference about the neuraminidase activity,which Cannot influence the ability of the virus.Both of the IAV-Gluc and wild strains show a completely alpha 2,3 sialic acid receptor binding capacity.The results show that the IAV-Gluc retained the biological characteristics of wild strains except the weakened pathogenicity.Thirdly,we completed the visualmonitoring of IAV-Gluc deposition in the respiratory tract.Combining technology of imaging in vitro,we analyzed the dynamic process of virus deposition,replication and clear in mice.After Intranasal infection by IAV-Gluc,we used IVIS SPECTRUM(Imaging system in vitro)to monitor the viral load in the body for six consecutive days.The bioluminescent signal can be detected 24 h post infection and reached the peak level 48 h post infection.The Bioluminescent sources were the lungs and trachea in mice.There was a strong positive linear relationship between the fluorescence intensity and virus degree in lung.At the same time,we established the bioluminescent rapid method for the identification of viral infection,which can directly evaluate viral load in live animals.Fourthly,research on the IAV-Glucdeposition and replication within the respiratory tract under different ways of infection.After Challenged with the same dose of the virus in two different ways,intranasal inoculation(IN)and aerosol exposure(AR).Results showed that the intranasal inoculation group of guinea pigs had more virus deposition in the lungs(12.27%),and aerosol exposure group of guinea pigs had only 1.34% of the virus deposition in the lung,but it could make much more rapid replication within 48 hours.Combining the results of small animals living imaging system and traditional virus titer detection technique,we further confirmed certain differences between intranasal inoculation and natural route of aerosol expoure.Intranasal inoculation caused the distribution of the virus was dotted in the lungs,and aerosol exposure caused the distribution of the virus was evenly scattered in the lungs,which led to the increasing of the efficiency of viral replication,The replication efficiency of the AR group reached 11073 times of the original deposition amount in 48 hours,but the replication efficiency of the IN group was only 15.4 times of the original deposition amount in 24 hours.Aerosol exposure way is more advantageous to the replication of the virus in the lungs and has a higher efficiency of infection compared with Intranasal way.We built the luciferase influenza a(H1N1)virus(IAV-Gluc)and accomplished the identification about stability,pathogenicity,transmission ability,proliferation,NA activity and receptor binding properties,which showed that the IAV-Gluc retained the biological characteristics of wild strains except the weakened pathogenicity.The bioluminescent signal can be detected after IAV-Gluc infection and there was a strong positive relationship between the fluorescence intensity and virus degree.Aerosol exposure way is more advantageous to the replication of the virus in the lungs and has a higher efficiency of infection compared with intranasal way.This study can not onlybenefit the further evaluation of influenza A virus deposition in vivo and propagation lawin vitro,research on pathogenic molecular mechanism,antiviral drug screening and the development of candidate vaccine strain,but also provide technical reference for the researches on the other subtype of the influenza virus.