Preliminary Study On The Oncolytic Activity Of Newcastle Disease Virus Strain F48E9

Cancer has been the first leading cause of death in the world.The overall survival rate of cancer patients has been significantly improved by surgery,radiotherapy and chemotherapy.However,for patients with advanced cancer and distant metastasis,there is still lack of effective treatment measures.Therefore,it is necessary to explore more effective methods for tumor therapy.Oncolytic virus is a potential treatment for malignant tumors because of its high oncolytic efficiency,specificity and safety as well as its limited side effect.Newcastle disease virus(NDV)is a negative-sense,single strand RNA avian virus with high pathogenicity and lethality.It belongs to the genus Avulavirus in the family Paramyxoviridae.In 1950s NDV was first found to inhibit gastric cancer metastases,since then many studies have proved that NDV has the potential to kill tumor cells specifically and can also stimulate a variety of immune system to enhance antitumor immune response.It has been historically considered as a complementary and alternative medicine(CAM)by the national cancer institute(NCI)in the US.The research of abroad for NDV mainly focus on strains of clinical trials such as 73-T,MTH-68,HUJ and PV701.The most common strain in Chinese studies is La Sota.a lentogenic strain.There is a few research about virulent strain F48E9' s anti-tumor spectrum and ability which was isolated in China.In this connection,this study investigated the effectiveness of NDV strain F48E9 in the treatment of various cancer,inhibitory effects of F48E9 on nasopharyngeal carcinoma(CNEl),liver cancer(HepG2)and breast cancer(MCF7)in nude mice in vivo,as well as its safety.In addition,for improving the virus titration efficiency,this study also prepared specific NP monoclonal antibody and established a double-antibody sandwich ELISA assay.Compare with the traditional plaque formation assay for virus titration its operation is complicated and time-consuming.The main findings are as follows:(1)Optimized a NDV culture system using Vero cell and obtained the purified,1.2×108 pfu/mL NDV F48E9 by plaque purification and cell culture.Used CCK-8 agent to detect the oncolytic activity of F48E9 to 16 tumor cell lines in vitro including liver cancer,lung cancer,cervical cancer,nasopharyngeal carcinoma and so on.The results showed that F48E9 is cytotoxic to almost all these tumor cells but with varying degrees of ability.F48E9 can replicate in many tumor cells and cause cytopathic effect.The study also found F48E9 can induce tumor cell apoptosis which showed dose-dependent manner by Annexin V-FITC/PI staining and flow cytometry analysis.(2)The study established the model of CNE1 1 HepG2 and MCF7 cells xenografts in nude mouse.After injected F48E9 intratumorally,it can be observed that the tumor growth of mice treated F48E9 virus were slower than mice treated control.F48E9 can inhibite tumor growth in vivo without side-effect.Compared with the control group,mice with virus infection are healthy and no obvious organic lesions.(3)For measuring the viral titer more quickly and conveniently,the study cloned NP gene of F48E9 and successfully expressed recombinant NP protein.Purified recombinant NP protein was used to immunize mice and obtained 7 monoclonal antibodies.The best antibody pair was screened that 3C10 uesd as coated antibody,4E7 used as detection antiboby and Developed a double-antibody sandwich ELISA assay.(4)Optimizing the parameters of this assay and standard curve,linearity range,detection limit,sensitivity were determined.The linear range of standard curve was 4.6x 105 pfu/mL?7.5x 106 pfu/mL,the coefficient correlation was 0.9972,the limit of detection was 4.1 X 105 pfu/mL,the sensitivity was 2.47 X 105 pfu/mL.This method has a good correlation with plaque formation assay.In summary,NDV F48E9 strain possesses powerful anti-tumor potency.And this study identified two NP mAb which could be employed to establish a double-antibody sandwich ELISA assay for detecting virus titer.