Preliminary Study On The Oncolytic Activity Of Enterovirus D68 And Establishment Of A New Type Of Neutralization Experiment

Cancer is the second largest non-communicable disease in humans.In 2015,more than 8.8 million people died of cancer worldwide.Whether in developed or developing countries,the incidence and mortality of cancer have increased year by year,which has had a tremendous impact on human production and life,as well as the social economy.At present,the main treatment methods for cancer still focus on surgical resection,physical radiation therapy and chemical radiation therapy,but the efficacy is still not satisfactory.In recent years,as oncolytic virus based on human herpes simplex virus(HSV-1)has shown a good effect in the treatment of melanoma tumors,the development of novel oncolytic virus therapy has received great attention.Enteroviruses are important targets for the development of novel oncolytic viruses.At present,there are many new oncolytic viruses based on enteroviruses being developed.Enterovirus D68(EV-D68)is an enterovirus that causes mainly upper respiratory tract infections and has a milder overall symptoms and has good potential for the development of oncolytic viruses.This study carried out the feasibility study of the development of novel oncolytic viruses based on EV-D68,systematically analyzed the killing efficacy of EV-D68 against different human tumor cells,and provided an important basis for the development of novel oncolytic virus therapy.This study first analyzed the in vitro cytotoxicity of EV-D68 against 92 different human tumor cells.The results showed that EV-D68 had a good in vitro killing effect on a variety of different human tumor cells,among which the killing effect on lung cancer,liver cancer,prostate cancer,lymphoma and some lymphatic leukemia cells was better.The oncolytic ability of cancer-like has not been reported.In this study,lymphoma and lymphoid leukemia cells were selected for preliminary exploration of the in vitro and in vivo oncolytic effects of EV-D68.Based on the previous study,this study examined the EV-D68 receptor sialic acid on the surface of 15 lymphoma and leukemia cells.The results showed that the high expression of sialic acid in lymphoma and leukemia cells may be the reason of EV-D68 killing lymphoma and leukemia cells.Further,in this study,CCK-8 kit was used to test the in vitro survival rate of lymphoma and leukemia cells infected with EV-D68.According to different survival rates,the cells were divided into three different groups based on sensitivity.Differently,this paper selected RAJI,THP-1 and HEL from three types of lymphomas and leukemias to evaluate the effects of oncolysis in mice,and the results were similar to those in vitro.Finally,in order to explore the mechanism of EV-D68 on lymphoma and leukemia,the study continued to use RAJI,THP-1 and HEL for related experiments.The results showed that after infection with EV-D68,PARP and Caspase3 shear occurred in RAJI cells.The flow cytometry analysis showed that Annexin V was positive,indicating that RAJI cells showed significant apoptosis,whereas THP-1 and HEL with weaker killing effects were negative.After the apoptosis inhibitor Z-VAD-fink was introduced to inhibit apoptosis,the oncolytic effect of EV-D68 on RAJI cells was significantly decreased.This shows that the possible oncolytic mechanism of EV-D68 is to kill tumor cells by inducing apoptosis.In addition,the detection of viral load and virus-neutralizing antibodies in patients undergoing oncolytic viruses is an important part of evaluating the efficacy of oncolytic viruses.At present,the virus detection and neutralizing antibody detection of enteroviruses still rely on traditional RT-PCR methods and CPE cytopathic methods.These methods are cumbersome,time-consuming,subjective,and not suitable for high-throughput rapid testing.In order to apply EV-D68 to tumor treatment services better,this study used hybridoma technology to screen and obtain monoclonal antibody 15C5 that efficiently recognized EV-D68 virus,and established EV-D68 detection method based on enzyme-linked immunospot assay(ELISPOT).The method can reduce the time took in detection from 5-7 days by the traditional CPE method to 1 day.Under the premise of satisfying the accuracy,this method has the advantages of rapidness,convenience,objectiveness,high throughput,and the like.The quality control of this future EV-D68 oncolytic virus is of great significance.In summary,this article applies EV-D68 to the study of tumor treatment for the first time and finds that it has a good killing effect on lymphoma and leukemia cells,and has initially explored the relationship between EV-D68 oncolysis and apoptosis.Finally,in order to better detect the viral load and neutralizing antibody levels in patients in the future clinical application of EV-D68,this study established a new EV-D68 virus titer and detection method for neutralizing antibodies based on ELISPOT.