Screening Of Potent NDV Strains Against Liver Cancer Cells

Objective:Newcastle disease virus(NDV)belongs to Avulavirus genera of the Paramyxoviridae family,which are enveloped viruses containing single-stranded negative-sense RNA.It was reported that NDV could infect various human tumor cells,multiplying and replicating independently and selectively kill them,which was contributed to the treatment of cancer patients. The former study demonstrated that the anti-tumor capacity of NDV was associated with their virulences.NDV was divided into lytic and nonlytic strain, in which the lytic strain is more powerful than the nonlytic one in anti-tumor effect.Studies from abroad figured out that 73-T,PV 701 and MTH-68/H strains were lytic strains.But,in China,a nonlytic NDV strain LaSota strain was chose for the treatment of some cancers.Till now,none of document is found about the anti-cancer trial by using lytic NDV strains in China.Therefore we carried out this experiment in which 17 NDV strains were identified, expecting to find a highly lytic anti-liver tumor NDV strain,which would provide us with some new clues for the biological therapy of the liver cancer.Materials and Methods:Eleven NDV strains isolated in Jiangxi Panyang Lake,one NDV Strain isolated in HK and five NDV strains isolated in Guangxi(kindly offered by Professor Y.Guan,Joint Influenza Research Center in Shantou University Medical College and HongKong University,Faculty of Medicine)were selected for this research.Those NDV were inoculated in egg,incubated in 35℃for 48 hours for virus amplification.Allantoic fluids containing viruses were harvested after testing haemagglutination(HA)titre of virus,and then stored at-80℃.The cytotoxic effect of selected NDV strains on the three liver cancer cell lines (HepG-2,SMMC-7721,BEL-7404)and on one strain of normal liver cell line (HL-7702)were performed by using microculture tetrazolium(MTT)assay,two NDV strains with relative high killing potency were selected and propagated in the HepG-2 cell line.After 48-72 hours,viruses in the media were harvested by centrifugation and purified by plaque assay.The HA test was conducted on the purified virus to determine the HA titre.The MTT assay,Plaque assay and TCID50 were carried out to detect the cytotoxic effect of the purified virus on three liver cancer cell lines.Two NDV strains were selected for the further genetic analysis.Viral RNAs were extracted from virus-infected allantoic fluids.After reverse transcription,cDNA was amplified by using specific primer for F gene.PCR product was purified and then inserted into a pGEM-T vector.and then transformed into EPI-100 strain.Positive clone containing fusion glycoprotein (F)gene was selected and sequenced.At last,compared with the sequences of standard NDV strains in national gene database,the homology,hydrophobicity, antigenicity,and phylogeny of F genes were analyzed.Results:1.All 17 selected NDV strains showed more significant pathological changes in three passaged liver-cancer cell lines(HepG-2 lines and SMMC-772 lines and BEL-7404 lines)than a normal liver cells in human.(HL-7702 lines).2.The cytotoxic changes in three passaged liver-cancer cell lines for 17 selected NDV strains became more obvious with increase of virus concentration. In the meantime,cytotoxic effect of these NDV strains was correlated with the virus exposure time on the cells.3.NDV 7793 strain and NDV D817 strain had more significant cytopathic effect on three liver cancer cell lines were confirmed and selected,which were inoculated in cell lines for 72 hours,showed about 80%lethality of liver cancer cells and less 5%lethality of human normal cells respectively.4.Both NDV 7793 and D817 strains appeared to replicate much more efficiently in human cancer cells than they did in normal human liver cell.Its ability to replicate is up to 256-512 times faster in liver cancer ceils than in human normal cells.5.There were no significant statistical differences in the cytotoxic effect between two viruses on the liver cancer cells line in vivo.6.NDV strains passaged in liver cancer cells obtained more powerful anti-tumor ability.7.Compared with 8 standard NDV strains,the nucleotided and amino acid sequence identities of F gene of 17 NDV strains ranged from 79.8%to 92.4% and 89.7%to 95.5%respectively.Phylogenetic analysis indicated that NDV 7793 strain was closely related to Ulster strains which is belonged togenotypeâ… , and both NDV D817 strain and Iraq -AG-68 strain belonged to genotypeâ…¥, they locate in the same sublineage.8.Analysis of antigenicity of F protein cleavage of NDV 7793 strain indicates that NDV 7793 strain differs from NDV La Sota strain.9.The typical amino acid sequence in the protein lytic site of F protein of NDV 7793 strain is ¹¹²G-K-Q-G-R-L¹¹⁷,the feacture of lower virulent NDV strain with F protein cleavage sequence.White NDV D817 strain possesses the feacture of high virulent NDV strain with F protein cleavage sequence ¹¹²R-R-Q-K-R-F¹¹⁷.Thus combining with analysis for hydrophobicity and antigenicity,we can concluded that NDV 7793 strain is attenuated virulent strain in terms of molecular characteristics and NDV D817 strain is virulent one.Conclusions:1.To 17 selected NDV stains,their ability to replicate and kill tumor cells without injuring normal cells make it possible to be applied to petential cancer biological therapy.NDV 7793 strain and NDV D817 strain possesses more powerful anti-tumor potency among 17 selected NDV stains.2.NDV 7793 strain is attenuated virulent strain.It belongs to genotypeâ… , a nonlytic strain.While NDV D817 strain is high virulent strain which belongs to genotypeâ…¥,a lytic strain.3.NDV 7793 strain differs from NDV La Sota strain commonly used in anti-cancer trials.4.In respect to the safity and effectiveness for biological treatment,NDV 7793 strain is a more appropriate stain in anti-tumor therapy,accounting for its potent effect on tumor cells wihout harming human normal cells,also for its attenuated virulent feacture.