Construction And Preliminary Application Of H7N9 Avian Influenza Fluorescent Reporter Viruses

Since the emergence of H7N9 influenza virus in 2013,as of May 2020,it has caused 1,568 human infections and serve economic losses to the poultry industry of China.This virus has been posing a huge threat to the health of human and chicken as well.There are many studies that have revealed the characteristic of the epidemiology and cross species transmission of the virus.However,its pathogenic mechanism in both chickens and mammals remains to be fully understand.In order to explore the pathogenic mechanism of H7N9 avian influenza virus,we selected two strains of H7N9 influenza virus A/chicken/Guangdong/SD008/2017（abbreviated as CK/SD008）and A/chicken/Guangdong/SD008/2017（PB2/627K）（abbreviated as CK/SD008-PB2/627K）as models to construct report viruses.The report viruses were then used to evaluated the infection of H7N9 virus in mice and the influence of GSDME molecule on the replication of the virus in vivo.First,we inserted the Venus or Apple reporter gene into the NS gene segment of CK/SD008 virus to construct recombinant plasmids pHH21-NS-Venus and pHH21-NS-Apple.Using the 12 plasmid reverse genetic operating system,four fluorescent reporter viruses SD008-627E-Venus,SD008-627E-Apple,SD008-627K-Venus,and SD008-627K-Apple were constructed.The reporter viruses were used to infect A549 cells after passaging in chicken embryos for 2 times and mice for 3 times,and then the cells were examined with an inverted fluorescence microscope.The results showed that all of the four virus-infected cells expressed fluorescent proteins.To compare the pathogenicity of the reporter viruses and their parents,C57BL/6 mice were infected with four reporter viruses and their parental viruses.The mice’s lethal dose(MLD₅₀)and organ viral loading were tested.The results showed that the MLD₅₀ and organ virus content of the four reporter viruses were similar to that of the parental viruses.MDCK cells were infected with the mouse adaptive reporter virus SD008-627K-Venus（mSD008-627K-Venus）,and then the number of fluorescent spots and plaques were counted using an inverted fluorescence microscope and with naked eye,respectively.The results showed that the number the fluorescent spots was similar to the number of plaques.Mice were infected with mSD008-627K-Venus,and their lung cells were collected 3 days post the infection to separate the CD45 and CD11b positive cells by a flow cytometry.The results showed that parenchymal cells（CD45）were the major cell type infected by the virus and the infection with the virus lead to an increase in host lung leukocytes（CD45⁺）,of which mononuclear macrophages and neutral Granulocytes（CD11b⁺）took the majority.The above results indicate that we have constructed successfully four H7N9 reporter viruses that are similar in pathogenicity with their parental viral strains and express the fluorescent proteins stably.The reporter viruses can be used to study the infection and pathogenic mechanism of influenza（H7N9）viruses.GSDME molecule is nuclear membrane-binding proteins and functions as a factor leading to cell pyrolysis.To explore the effect of GSDME molecules on the replication of H7N9 highly pathogenic influenza virus,we prepared an GSDME knockdown A549 cell line and an GSDME over expression RAW264.7 cell line,and the mouse adaptive reporting virus SD008-627K-Venus（mSD008-627K-Venus）was used to infect the both cell lines.The infection efficiency,the cellular compartmental distribution of GSDME,and the expression of PB2 were analyzed by assays of flow cytometry,laser confocal microscope,western blotting and fluorescence quantitative microscope,respectively.The results showed that the knockdown of GSDME decreased dramatically the infection efficiency of H7N9 viruses in the A549 cells,while an increase of which was observed in the GSDME over expression RAW264.7 cells.The GSDME was observed distributing around the nuclear membrane of the cells.The expressions of PB2 and NS1 in GSDME knockdown cells were lower than that in the GSDME overexpression cells.These results indicate that the overexpression of GSDME increases the replication of H7N9 influenza viruses in the infected cells,and the GSDME molecules distributed on the nuclear membrane affect the content of influenza virus PB2 gene in the nucleus.In this study,we have constructed successfully four H7N9 reporter viruses,which are similar in terms of infections and pathogenicity with their parental strains.The reporter virus was used to explore the mechanism of GSDME molecule promoting the replication process of the influenza（H7N9）virus.This study provides with powerful tools and concrete clues for the studies on the infection and pathogenicity of the H7N9 influenza viruses.