Construction Of Recombinant NDV-rAnh/EGFP And Investigation Of Its Tumor Inhibitory Ability

Cancer is a major cause of deaths in humans. In clinical cancer therapy, the limited efficacy and toxicities of current chemotherapies and radiotherapies have provided an impetus for the search of new therapeutics. Newcastle disease virus (NDV) is an avian paramyxovirus with a negative-stranded RNA genome that belongs to the genus Avulavirus within the Paramyxovirinae subfamily of the Paramyxoviridae family. It has a natural preference for replicating in tumor cells but not in normal cells. Therefore, NDV is currently under investigations for oncolytic virotherapy. Reverse genetics manipulation technique now allows genetic manipulation of this virus to improve its cancer killing ability and safety.Anhinga virus was originally isolated from a captive bird in a zoological park in Florida.It is classified among the mesogenic viruses. There is no research about Anhinga strain used in cancer treatment currently.This study was designed to investigate the effectiveness of a genetically engineered NDV (Anhinga strain) in the treatment of cancer. The EGFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the HN genes and L genes to construct a full-length cDNA clone of NDV with EGFP. This plasmid was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into BSRT7/5 cell which stably expressing T7 RNA polymerase. The rescued virus NDV-rAnh/EGFP was first propagated in embryo eggs and the allantoic fluid was harvested. The appearance of EGFP expression in NDV-rAnh/EGFP infected cells confirmed that the recombinant NDV with heterologous gene was rescued successfully. The rescued virus could stably express the heterologous gene for fifteen passages in embryo eggs. The identification of replication kinetics in HepG-2 cells of NDV-rAnh/EGFP shows that the insertion of EGFP did not affect the replication ability of the recombinant virus.NDV-rAnh/EGFP was tested in vitro for suppressing different human cancer cells by MTT assay after infection with different viral titers. The data showed that the NDV-rAnh/EGFP could inhibit the growth of many tumor cells, but the inhibitory effects were different for each cell. This cancer killing was dose-related and correlated with viral replication ability in different tumor cells.To test NDV-rAnh/EGFP suppressing ability in vivo,we established the H22 hepatic carcinoma solid tumor model and H22 ascites tumor model in Kuming mice. The two models were treat intraperitoneally and intratumorally with NDV-rAnh/EGFP. The results showed that virus could effective elongate the survival time of the ascites tumor model mouse and suppress the tumor growth rate of solid tumor model mouse. In conclusion, we successfully rescued the NDV-rAnh/EGFP strain by reverse genetics technology. Our data indicate that NDV-rAnh/EGFP strain can kill various tumor cells in vitro on different level. The recombinant virus also exert obvious tumor suppression ability in vivo. This study established the platform for using recombinant NDV as avirus vaccine vector for cancer therapy.