Oncolytic Effect Of Newcastle Disease Virus F48E9 Strain To Colorectal Cancer And Establishment Of Reverse Genetic System For F48E9

Some research reports showed that the number of new cancers in China continued to rise,and growth trend of colon cancer patients was obvious.At present,colon cancer is mainly treated by surgical resection,supplemented with radiotherapy,chemotherapy or immunotherapy.After the surgery,metastasis and recurrence of colon cancer is easy to happen.Immunotherapy is one of the most promising approaches for treatment of cancer.It was selected as one of the "Top Ten Scientific Breakthroughs of 2013" by Science.Oncolytic therapy is one of the important approaches of immunotherapy.Oncolytic viruses can exert their inhibitory or elimination effects through direct infection of tumor cells and activation of the immune system.Some virus strains had been used for clinical trial to treat tumors and shown good therapeutic effects,and one of the modified HSV oncolytic viruses had been officially listed in the United States.NDV is a RNA virus that mainly infects poultry and birds.Occasionally,the human was infected with NDV and got a conjunctivitis or mild flu symptoms.Since the 1960s,there had been some attenuated NDV such as 73-T,MTH-68,PV701,HUJ,LaSota and others to carry out Phase I and Phase II clinical trial,but failed to go on Phase III.It may suggest that the existing attenuated NDV strains are not sufficiently effective,and it is necessary to develop some better NDV oncolytic virus strains.Newcastle disease F48E9 strain is a standard velogenic strain in China,there were few reports that F48E9 strain was used for oncolytic research.Hu Lihua et al.had compared the oncolytic effect of NDV virulent strain F48E9 and attenuated strain LaSota on colorectal cancer cell line LS1747 in vitro.It shown that F48E9 strain had a potential for treating tumor.However,this study did not evaluate F48E9 oncolytic effect in vivo and side effects of F48E9 on BALB/c mice.Herein we evaluated the oncolytic effect of NDV virulent F48E9 strain on colorectal cancer model in vitro and in vivo,and evaluated the side effect of NDV F48E9 strain on BALB/c mice by intravenous injection.The results showed that F48E9 at an MOI of 0.1 showed an obvious inhibition on the growth of CT26 cells.With the increase in MOI,its inhibitory effect on cell growth was significantly enhanced;The growth of murine CT26 tumor subcutaneously transplanted in BALB/c mice was significantly inhibited by F48E9 strain at 2×107 pfu/mL.The average survival time of the virus-treated group was 38.4 days,and PBS-treated group was 15.2 days.Immunohistochemical experiment showed that F48E9 significantly inhibited the proliferation of CT26 cells of transplanted tumor in mice;Safety evaluation of F48E9 strain by intravenous injection in normal BALB/c mice showed that F48E9 had no significant side effects.The above results suggested that Newcastle disease F48E9 strain could be used as a candidate for further oncolytic therapy research.In order to enhance the oncolytic activity of Newcastle disease F48E9 strain,we tried to construct the reverse genetic system for genetic modification of the F48E9 strain.Firstly,the F48E9 strain genome was sequenced,then sub-genomic of each genome fragment was cloned into the pblue vector respectively,and each fragment was then constructed into the same pCI-neo vector to obtain the F48E9 strain full-length cDNA genomic clone.The three helper plasmids pCI-L,pCI-NP and pCI-P of the F48E9 strain were also cloned into the pCI-neo vector,and co-transfected into BHK21 cells together with pCI-F48EP clone,the recombinant virus was identified by hemagglutination and plaque formation assay.The results shown that the cell lysate transfected with the plasmids had 1 unit of hemagglutination but was not able to form plaques.In conclusion,this study evaluated the oncolytic efficacy of wild-type Newcastle disease F48E9 strain for colorectal cancer cells,and performed a safety analysis of F48E9 strain in BALB/c mice.It was found that F48E9 could significantly inhibit growth of colorectal cancer cells in vitro and had obvious oncolytic effect in vivo,and had no obvious side effects in BALB/c mice.This study also attempted to construct the reverse genetic system of F48E9 strain for genome modification.For now,F48E9 full-length antisense cDNA genomic and three helper protein sequence had been cloned into pCI-neo vector.After several attempts of transfection,no infectious viral rF48E9 virus particles were obtained,and further optimization of the transfection system was needed.