A Preliminary Study On The Oncolytic Effect Of Human Influenza Virus On Tumor Cells And Construction Of Influenza A Virus With NS1 Fragment Deletion

Oncolytic viruses(OVs)are viruses that treat tumors.They can target,infect,replicate and kill cancer cells in human cancer cells without affecting normal tissue cells.OVs can simultaneously induce tumor immune activation in the body,and further achieve the therapeutic effect of anti-tumor immunity.Objective: Influenza virus is one of the earliest viruses used for oncolytic research,more and more studies have shown that influenza virus has good oncolytic potential.However,the oncolytic effects of different types or strains of influenza virus on different tumor cells have not been systematically evaluated.Therefore,in this paper,by detecting two human influenza A viruses A/PR/8/34(PR8),A/WSN/33(WSN)and two influenza B viruses B/Victoria/12048/2019(BV),B/ Yamagata/58/2019(BY)screened out strains and tumor cells for subsequent experiments based on the difference in the oncolytic ability of different tumor cells.On this basis,the reverse genetics of influenza virus was used to analyze NS gene of different transformations were carried out,the biological characteristics of different viruses were evaluated.Methods: In this experiment,four strains of influenza viruses PR8,WSN,BV and BY were cultured stably in chicken embryos.Cell viability of human colon cancer cell HT29,HCT116,human lung cancer cell A549,H1975,H1299 and normal lung cell MRC-5 infected with different MOI was determined by celltiter-Gl O method.The replication curve of the virus in different tumor cells was detected by Q-PCR.After selecting the PR8 strain with high replication ability in HT29 of colon cancer cells,The expression differences of endogenous trypsin in tumor cells were detected by Q-PCR and Wb.Finally,apoptosis of HT29 cells induced by PR8 was detected by TUNEL assay.On this basis,we constructed multiple NS1 protein deficient influenza virus strains using PR8 as the skeleton by reverse genetics and detected the genetic characteristics of the viruses.Results: 1.Influenza viruses PR8,WSN,BV,BY could be amplified and stably passaged on chicken embryos and maintained a high titer.2.Influenza A virus PR8 strain has obvious inhibitory effect on colon cancer cells HT29.3.Influenza B virus has weak oncolytic ability to the above tumor cells.4.The endogenous trypsin in tumor cell HT29 has high expression,which may be the reason why influenza virus is sensitive to HT29 cells.5.Influenza virus PR8 strain can obviously induce the apoptosis of colon cancer cells HT29.6.The attenuated influenza virus strains PR8-del NS1 and PR8-NS1-80 can be rescued by reverse genetics,but cannot be stably passaged.Conclusions: Influenza virus has the potential to become an OVs.Influenza A virus PR8 strain can infect tumor cells and inhibit the proliferation of colon cancer cells HT29;by studying replication kinetics of influenza virus,PR8 has stronger replication ability than the other three viruses.The high expression of endogenous trypsin in HT29 may be the reason for the better oncolytic effect of PR8.The above results indicate that influenza A virus PR8 strain is suitable as a candidate virus for oncolytic therapy of colon cancer cell HT29,which lays a certain foundation for further research.